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Cloning and molecular characterization of vff1 gene encoding Forisomes of Vicia faba
Von der Fakultät für Mathematik, Informatik and Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation
vorgelegt von
M. Sc. Maria Eugenia Fontanellaz
aus Rosario, Argentinien
Berichter: Universitätsprofessor Dr. rer. nat. R. Fischer Universitätsprofessor Dr. rer. nat. D. Prüfer Tag der mündlichen Prüfung: 7. Dezember 2006
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Contents ___________________________________________________________________________
I
I INTRODUCTION
I.1 Phloem structure and function 1
I.1.1 Sieve element-companion cell complex 1
I.1.2 P-Proteins and regulation of phloem transport 2
I.1.3 Forisomes 5
I.1.4 Ca2+-binding motifs and secondary protein structures 7
I.1.4.1 EF-hand 7
I.1.4.2 Coiled-coil domains 8
I.2 Promoter sequences and regulation of transcription 9
I.2.1 Phloem-specific promoters 9
I.3 Aim of this thesis 12
II MATERIALS AND METHODS 14
II.1 Materials 14
II.1.1 Chemicals and consumables 14
II.1.2 Enzymes and reaction kits 14
II.1.3 Buffers, media and solutions 15
II.1.4 Matrices and membranes 15
II.1.5 Primary antibodies, secondary antibodies and substrates 15
II.1.6 Biological material 15
II.1.6.1 Bacterial strains 15
II.1.6.2 Plants 16
II.1.6.3 Animals 16
II.1.7 Vectors 16
II.1.7.1 Bacterial expression vector 16
II.1.7.2 Plant expression vectors 17
II.1.8 Oligonucleotides 17
II.1.9 Equipment and applications 18
II.2 Methods 20
II.2.1 Plant material and isolation of Forisomes 20
II.2.2 Peptides sequencing 20
II.2.3 Isolation of genomic plant DNA 21
Contents ___________________________________________________________________________
II
II.2.4 RNA isolation 21
II.2.5 Libraries construction and screening strategies 22
II.2.5.1 cDNA expression library 22
II.2.5.2 cDNA libraries 22
II.2.6 PCR technologies 24
II.2.6.1 Cloning of 5’ and 3’ ends of full-length cDNAs using
RACE-PCR 24
II.2.6.2 PCR amplification of cDNA 25
II.2.6.3 Identification of introns and genomic DNA cloning by
LD PCR 26
II.2.6.4 Genome Walking 26
II.2.7 Recombinant DNA technologies 27
II.2.7.1 Isolation of plasmid DNA from E.coli 27
II.2.7.2 Quantification of DNA 27
II.2.7.3 PCR amplification 27
II.2.7.4 Agarose gel electrophoresis of DNA 28
II.2.7.5 Preparative agarose gel electrophoresis 28
II.2.7.6 DNA sequence analysis 29
II.2.8 Preparation and transformation of E. coli 29
II.2.8.1 Preparation of electro-competent E. coli 29
II.2.8.2 Transformation of E. coli by electroporation 29
II.2.8.3 Culturing of E. coli and glycerol stock preparation 29
II.2.9 Preparation and transformation of Agrobacterium tumefaciens 30
II.2.9.1 Preparation of electro-competent Agrobacterium cells 30
II.2.9.2 Transformation of Agrobacterium by electroporation 30
II.2.9.3 Determination of the efficiency of recombinant bacteria
transformation 31
II.2.9.4 Growth of recombinant A. tumefaciens and preparation
of glycerol stocks 31
II.2.10 Generation and characterisation of transgenic plants 31
II.2.10.1 Transient transformation of tobacco leaves 31
II.2.10.2 Preparation of recombinant Agrobacteria 31
II.2.10.3 Vacuum infiltration of tobacco leaves 32
II.2.10.4 Stable transformation of tobacco plants 32
Contents ___________________________________________________________________________
III
II.2.10.5 Growth of N. tabacum cv. Petite Havana SR1 33
II.2.10.6 Measurement of GUS activity and histochemical analysis 33
II.2.11 Expression and purification of recombinant proteins 34
II.2.11.1 Expression and purification of Forisome-GST fusion
proteins from E. coli 34
II.2.11.2 Fermentation at 4-liter scale 35
II.2.12 Protein analysis 36
II.2.12.1 Quantification of proteins 36
II.2.12.2 SDS-PAGE and Coomassie brillant blue staining 36
II.2.12.3 2D-PAGE analysis 37
II.2.12.4 Immunoblot analysis 38
II.2.13 Polyclonal antibodies production 38
II.2.13.1 Mouse immunization 38
II.2.13.2 Chicken immunization 39
II.2.13.3 Rabbit antisera 39
II.2.14 Determination of antisera titers 39
II.2.15 Confocal immunofluorescence microscopy 41
III RESULTS 41
III.1 Molecular characterization of Forisome genes 41
III.1.1 Analysis of isolated Vicia faba Forisomes by SDS-PAA
gel electrophoresis 41
III.1.2 Generation and characterization of Forisome-specific
polyclonal antibodies 41
III.1.2.1 Immunoblot analysis of isolated Vicia faba Forisomes 41
III.1.2.2 Immunoblot analysis of isolated Vicia faba Forisomes
on 2D-gel electrophoresis 42
III.1.3 Screening of cDNA Libraries 43
III.1.4 Immunoscreening of cDNA expression library 45
III.1.5 PCR screening of cDNA expression library 46
III.1.6 Cloning full-length Forisome cDNA 46
III.1.7 Isolation of total RNA from Vicia faba 51
III.1.8 Identification of the transcription start sites for vff1 52
III.1.9 Molecular characterisation of the vff1 genomic clone 53
Contents ___________________________________________________________________________
IV
III.2 Molecular cloning and characterization of the vff1 promoter 54
III.2.1 Construction of genome walking libraries 54
III.2.2 Identification and cloning of vff1 gene 5’-flanking region 54
III.2.3 Potential regulatory sequences in the vff1 promoter 56
III.2.4 Characterization of vff1 promoter in transgenic tobacco 58
III.2.4.1 Cloning of vff1 promoter into the pTRAk-GUS vector 58
III.2.4.2 Expression of vff1 Promoter-GUS fusions in tobacco
plants 60
III.2.4.2.1 Tissue specificity of vff1 promoter in transgenic
plants 60
III.2.4.2.2 Developmental regulation of vff1 promoter in
transgenic plants 63
III.2.4.3 Deletion analysis of vff1 promoter in transiently
transformed tobacco leaves 65
III.3 Bacterial expression and characterization of VFF1 66
III.3.1 Cloning of vff1 into the bacterial expression vector
pGEX-5X-3 66
III.3.2 Bacterial expression and purification of VFF1 66
III.3.3 Characterization of GST-VFF1 fusion protein by
immunoreativity toward Forisome-specific mouse and
chicken antisera 68
III.4 Immunological characterization of native and recombinant
Forisomes 70
III.4.1 Reactivity of polyclonal anti-GST-VFF1 antibodies to isolated
Forisomes 70
III.4.2 Characterization of native Forisomes by immunofluorescence 71
III.4.2.1 Reactivity of Forisome-specific polyclonal antibodies to
native Forisomes 71
III.4.2.2 Reactivity of VFF1-specific polyclonal antibodies to native
Forisomes 73
IV DISCUSSION 75
IV.1 Molecular characterization of Forisome genes 75
IV.1.1 Analysis of Vicia faba Forisome subunits 75
Contents ___________________________________________________________________________
V
IV.1.2 Cloning and characterization of vff1 gene 76
IV.1.2.1 Screening of cDNA libraries 76
IV.1.2.2 Database search and cloning of vff1 gene 78
IV.1.2.3 Sequence analysis of vff1 gene 78
IV.1.2.4 Motifs identified within the vff1 gene 79
IV.1.3 Immunological evidence of vff1 gene encoding a Forisome
protein 82
IV.1.3.1 Expression and purification of recombinant VFF1
protein 82
IV.1.3.2 Immunoreactivity of Vicia faba Forisome-specific
polyclonal antibodies to recombinant VFF1 proteins
and native Vicia faba Forisomes 83
IV.1.3.3 Immunoreactivity of VFF1-specific polyclonal antibodies
to recombinant VFF1 proteins and native Vicia faba
Forisomes 84
IV.2 Molecular characterization of vff1 promoter 85
IV.2.1 Sequence analysis of vff1 promoter 85
IV.2.2 Phloem-specific expression pattern driven by vff1 promoter 86
IV.2.3 Differential expression pattern driven by vff1 promoter in the
sieve element-companion cell complex 88
IV.2.4 Developmental expression pattern of vff1 promoter in different
maturation stages 90
IV.2.4.1 Vff1 promoter expression pattern in roots of N. tabacum 90
IV.2.4.2 Vff1 promoter expression pattern in leaves of N. tabacum 91
IV.2.5 Vff1 promoter activation during the sink-source transition 92
IV.2.6 Vff1 promoter expression induced upon wounding 93
IV.2.7 Deletion analysis 94
IV.3 Conclusion and future prospects 95
V SUMMARY 97
VI REFERENCES 99
VII APPENDICES 116
VII.1 List of abbreviations 116
Contents ___________________________________________________________________________
VI
VII.2 Schematic presentation of vectors maps 119
VII.3 Figures 121
VII.4 Tables 122
Chapter I Introduction ___________________________________________________________________________
1
I Introduction
I.1 Phloem structure and function
In higher vascular plants, phloem is a highly specialized tissue for long distance transport of
metabolites and signaling components. In contrast, water and inorganic nutrients flow from
roots to shoots and leaves in the xylem. Topologically, xylem flux takes an apoplastic, i.e.,
extracellular path, as the xylem vessels consist of the lignified walls of dead cells. On the
other hand, products of photosynthesis travel from source to sink tissues in the sieve tubes of
the phloem, following a symplastic pathway (Behnke and Sjölund, 1990). Moreover, a wide
variety of other material is also transported through the phloem, including proteins, amino
acids, solutes, viruses, and various signaling molecules.
Sieve tubes consist of sieve elements (SEs), terminally differentiated cells that undergo
exceptional cytoplasmic reorganization to become functional phloem conductive cells capable
of long-distance translocation. Mature sieve elements are characteristically devoid of most of
the intracellular organelles, nuclei, central vacuole, Golgi bodies and dictyosomes (Oparka
and Turgeon, 1999; Cronshaw, 1981) as well as most of the ribosomes and microtubules that
are lost during the course of differentiation (Sjölund, 1997). Microfilaments also vanish
during sieve element maturation (Parthasarathy and Pesacreta, 1980). Reorganization of the
endomembrane system results from degeneration of the tonoplast and dictyosomes and
changes in the endoplasmic reticulum (Sjölund and Shih, 1983).
Having a common boundary, arranged end-to-end sieve elements develop numerous pores in
the cell walls between them. Plasmodesmata (PD) connecting SEs across the end walls
become greatly modified giving rise to the sieve plates that are characteristic of this tissue
(Behnke, 1989). Sieve plates ensure cytoplasmic continuity of the entire sieve-tube network,
enabling pressure-driven mass flow throughout the plant body (Knoblauch and van Bel, 1998;
van Bel et al., 2002). According to the mass flow concept of phloem transport (Münch, 1930),
the transport of materials through phloem sieve tubes is passive, nonselective, and driven
entirely by pressure gradients that are maintained by active loading of photosynthetic products
in source tissue and unloading of materials in sink tissue.
I.1.1 Sieve element-companion cell complex
After the first description of the SE by Hartig (1837) the typical sieve element/companion cell
complex of the phloems, became center of numerous investigations. Typically, each sieve
element in angiosperms is accompanied by one or more companion cells (CCs), which
interact intimately with the sieve element and play a crucial role in regulating phloem loading
and unloading and in the turnover of sieve element proteins and other components (Oparka
and Turgeon, 1999). Sieve elements and companion cells are derived from unequal
Chapter I Introduction ___________________________________________________________________________
2
longitudinal division of a single fusiform mother cell (Esau, 1969). During the course of
differentiation, cytoplasmic degeneration include a disintegration of the nucleus together with
the loss of ribosomes which hinders its protein biosynthesis (Thompson and Schulz, 1999).
Thus, the cell that becomes the sieve element, gradually lose genetic and metabolic control.
As a consequence, these elongated conducting elements of the phloem, the SEs, become
intimately (structurally, developmentally, and functionally) associated with adjacent
companion cells between which are capable of exchanging information via specialized pore
plasmodesmata units (PPUs; van Bel and Kempers, 1996). These PPUs are different from
other plasmodesmata. The lateral (axial) plasmodesmata that connect the SE and CC (PPUs)
are always branched on the CC side of the shared wall only, thereby comprising one broader
pore-like channel at the sieve element side while several narrower branching out at the
companion cell side (Esau and Thorsch, 1985). The PPUs form a continuity between the sieve
element plasma membrane and that of the companion cell, hence, connecting their
cytoplasms. Because of these intimate structural and functional connections between SE and
CC, the SE-CC complex is frequently viewed as a single functional entity within the phloem.
During the differentiation of phloem sieve elements, the endoplasmic reticulum undergoes
unique modifications to form the sieve element reticulum (SER; Sjölund and Shih, 1983)
which persists in mature, functioning sieve tubes. Cisternae of the SER lack ribosomes and
are restricted to the periphery of the sieve element at late stages of development. Some of the
SER are seen as single cisternae in close contact with the sieve element plasma membrane.
Probably the companion cell ER is also in contact with the sieve element reticulum held
together by the PPUs. Both the PPUs and the SER possibly ensure the protein transport of the
companion cell to the sieve element (Fisher et al., 1992; Imlau et al., 1999).
I.1.2 P-Proteins and regulation of phloem transport
More than a hundred proteins were detected in phloem exudates of Cucurbita maxima,
Triticum aestivum, Ricinus communis and Oryza sativa (Eschrich and Heyser, 1975; Fisher et
al., 1992; Nakamura et al., 1993; Sakuth et al., 1993). These phloem proteins were considered
to maintain the physiological functions of sieve tubes, such as sieve plate occlusion, signal
transduction, redox regulation, and phloem loading, among others (Hayashi et al., 2000).
Mature sieve elements retain a modified endoplasmic reticulum (ER), mitochondria,
numerous types of proteins, and specialized sieve element plastids. Most of these components
are arranged along the lateral walls of the sieve element and collectively constitute a system
known as the parietal layer (Ehlers et al., 2000). Based on electron micrographs and
descriptions of plastids from the sieve elements of many different species, two types of sieve
element plastids are distinguished (Behnke, 1991a). S-type plastids contain only starch
inclusions, while P-type plastids contain mainly protein inclusions along with some starch
inclusions. Furthermore, crystalloid proteins were present in these sieve elements. Behnke
Chapter I Introduction ___________________________________________________________________________
3
(1991b) referred to “nondispersive” versus “dispersive” protein bodies, also called P-proteins
or structural sieve element proteins. These terms arose because of changes that are observed
during sieve element ontogeny. Early in the maturation process, many protein bodies are often
observed, some of which disperse as the sieve element matures and some that remain
unchanged in mature sieve elements. Early stages of sieve element differentiation are
characterized by the appearance of structurally distinct cytoplasmic proteins, in reference to
the P-proteins. Ultrastructural investigations originally defined P-proteins as idiosyncratic
components of the structural architecture of sieve elements (Cronshaw and Esau, 1967).
Depending upon the plant species, P-proteins form fibrillar, tubular, or crystalline inclusions
whose accumulation and structural state appear to be strictly controlled during differentiation.
Figure I-1 In vivo structure of sieve elements in Vicia faba (Knoblauch and van Bel, 1998). Sections of two sieve tubes with CCs, mostly in staggered position are depicted. SEs and CCs are mostly connected through numerous pore plasmodesma units. P-plastids, mitochondria, and ER are parietally positioned and evenly distributed. Parietal proteins are locally aggregated. Parietal proteins and ER are sometimes located on the sieve plates or the margins of the sieve pores but do not impede mass flow. A large spindle-shaped crystalline protein cluster rests close to the sieve plate. The massive protein body is a specialty of the Fabaceae. C, callose; CC, companion cell; CP, crystalline protein body; CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; P and Pl, plastids; PP, parietal protein; PPU, pore plasmodesma unit; SE, sieve element; SP, sieve plate; V, vacuole.
There are many shapes of non-dispersive fibrous and crystalloid protein bodies, also called P-
proteins, which may be quite large and are often observed in the lumen of sieve elements
(Figure I-1). Electron micrographic images sometimes show masses of fibrous or amorphous
protein located directly in front of or within the pores of sieve plates and that appear to block
transport through the pores. To date, the large crystalloid P-protein bodies that occur
exclusively in Fabaceae were classified as being non-dispersive (Behnke, 1991b).
Confocal laser scanning microscopy (CLSM) allowed the direct visualization of fluorescent
dyes moving in sieve tubes of V. faba (Knoblauch and van Bel, 1998), which provided
definitive evidence of unimpeded mass flow in intact plants. This study also reported that P-
Chapter I Introduction ___________________________________________________________________________
4
type plastids in V. faba actually exploded upon injury of sieve elements, releasing their
protein contents, which, together with the dispersed crystalloid protein, rapidly occluded the
sieve plate pores. Furthermore, it was observed that crystalloid P-proteins of V. faba rapidly
disperse and occlude sieve plate pores after injury or osmotic shock (Knoblauch et al., 2001).
This process is rapidly reversible and controlled by calcium fluxes. Protein from the large
crystalloid protein bodies in V. faba sieve elements dispersed to plug the sieve plate pores
after injury from micropipette injection (Figure I-2) or osmotic shock induced by various
osmolytes.
Figure I-2 Conformational change of crystalline P-protein bodies in Vicia faba as a response to injury (Knoblauch et al., 2001). Crystalloid P-protein bodies were observed in CDMFDA-stained phloem tissue of broad bean (V. faba) by CLSM. (A) Condensed conformation before insertion of a micropipette (not visible; tip diameter of 2 mm). (B) Dispersed conformation after injury. Micropipette insertion triggers the transformation of the dense, elongate crystalloid into a sieve tube plug. Asterisks, crystalline P-protein bodies; CC, companion cell.
Addition of the chelating agent EDTA completely prevented crystalloid P-protein dispersal,
and repeated exchanges of Ca2+ and EDTA-containing media induced the alternate dispersal
and reassembly, respectively, of crystalloid P-proteins in injured sieve tubes. Furthermore, it
was also observed that the Ca2+ response could be mimicked partly by unphysiological pH
values (Knoblauch et al., 2003). Plugging of sieve plates to maintain turgor pressure within
the sieve tube after injury to a sieve element is the most generally accepted role for these
proteins, although other functions in pathogen and pest defence have been proposed (Read
and Northcote, 1983).
It has been suggested that not only these crystalloid P-proteins but also P-protein originating
from all sources within V. faba sieve elements (e.g., P-plastids, parietal proteins, and larger
crystalloid P-proteins) takes part in the occlusion of sieve plate pores after injury to cells
(Knoblauch and van Bel, 1998). Moreover, proteins in V. faba are much more sensitive to
perturbation than are the P-type plastids. Unlike the reversibility of crystalloid protein
dispersal, explosion and dispersal of the P-type plastids appears to be irreversible. Dispersal
Chapter I Introduction ___________________________________________________________________________
5
of parietal proteins after injury has also been observed and was found to be more sensitive to
perturbation than was the explosion of P-type plastids (Knoblauch and van Bel, 1998).
P-protein, as a structural entity, has been observed in sieve elements of all dicotyledons
examined (Evert, 1990) and in the majority of monocotyledons, although conspicuously
absent in families such as the Poaceae (Eleftheriou, 1990). The lack of P-protein also appears
to be a consistent feature of gymnosperms (Schulz, 1990) and seedless vascular plants.
Large crystalloid P-proteins are a particular characteristic of the Fabaceae (legumes). Analysis
sieve elements of Urtica dioica (Urticaceae) and Rubus fruticosus (Rosaceae) in response to
wounding showed that the P-protein bodies present in these species failed to disperse and
occlude sieve pores, even when severely damaged in the presence of free Ca2+. P-type plastids
also appear to have somewhat limited distribution. Proteinaceous P-type plastids were
observed in just 64 of 382 dicot families studied by Behnke (1991a); the majority of families
contained starch-filled S-type plastids.
I.1.3 Forisomes
The conformational regulatory properties of the crystalline P-proteins in Vicia faba sieve
elements are the most remarkable in the field of long-distance transport in plants. Their
reversible change between a dispersed and a condensed condition adjusts the permeability of
the sieve plates and disposed the authors to give to these protein bodies the name Forisome
(lat. foris: gate, greek soma: body; Knoblauch et al., 2003).
In contrast to P-plastids that are long-establish organelles in the SEs of dicotyledons (Bhenke,
1981), Forisomes are not organelles in the strict sense, as they are not separated from the
cytosol by a membrane (Knoblauch and Peters, 2004). In vivo studies using a transient
fluorescence dye on the dispersed conformation adopted by the Forisomes (Knoblauch et al.,
2001) evidenced the presence of a distinctive protein body instead of a complete dissolution
into unordered fibrils observed by electronmicrographs. This implies that Forisomes consist
of fibrils that are organized quite differently in the condensed (longitudinally expanded) and
the dispersed (longitudinally contracted) state, however, held together after the induced
conformational change. In the latter, the fibrils form an irregular, loose network.
In addition, electronmicrographs of condensed Forisomes showed longitudinally arranged
fibrils with a perfectly regular perpendicular cross-striation (wavelength 12 to 15 nm)
(Knoblauch et al., 2001). This striation is always visible in longitudinal sections of expanded
Forisomes, regardless of the plane of section (radial or tangential). Therefore, the protein(s) in
expanded Forisomes must be organized in a series of transversal planes. The regular striation
pattern consistently has been found in earlier studies (Lawton, 1978a; Arsanto, 1982), and the
disordered appearance of Forisomes in the plug-forming state was documented as well.
Lacking any knowledge of the dynamics of Forisomes, these two conditions had been
Chapter I Introduction ___________________________________________________________________________
6
interpreted as different stages of sieve element differentiation (Wergin and Newcomb, 1970;
Palevitz and Newcomb, 1971; Arsanto, 1982), or as preparation artefacts of little
physiological significance (Fisher, 1975; Lawton, 1978b). However, in the context of recent
findings (Knoblauch and Peters, 2004), available ultrastructural data rather seem to document
a remarkable example of rigorously regulated, rapid, and reversible molecular self-assembly.
The parallelization of fibrils and the establishment of the perpendicular striation during
Forisome expansion point to a lateral co-alignment of at least two types of structural domains
that alternate on each fibril. These domains might differ in physicochemical parameters such
as hydrophobic or electrical charge patterns, causing them to organize in alternating planes
that are oriented normal to the Forisome axis. If so, fibril co-alignment might actually play a
role in driving Forisome expansion. A paradigmatic example of such a process can be found
in nematode sperm cells, in which pH-controlled lateral interactions between MSP (major
sperm protein) subfilaments initiate the assembly of MSP filaments and filament bundles.
Notably, the reversible lateral coalignment of MSP filaments appears to provide the driving
force for nematode sperm motility (Bottino et al., 2001).
Further in vitro studies in Forisomes isolated from individual cells by micro-dissection
indicated that Ca2+ above a threshold of less than 100 nM induces a reversible doubling of
Forisome diameter and a reduction of Forisome length by almost one third (Knoblauch et al.,
2003). This anisotropic response results in a decrease of aspect ratio from 10 to 3 combined
with a more than threefold volume increase, which is the geometric basis for sieve tube
plugging. Contraction/expansion cycles can be induced in vitro by exchanging the incubation
medium. Therefore, no soluble factors are required for Forisome action, implying also that
reversible depolymerization, an important mechanism of motility in many cell types
(Mogilner and Oster, 2003), does not occur. Both Ca2+-induced contraction and chelator-
mediated expansion can be completed in less than 0.1 sec. Forisomes can be fixed between
pairs of glass fibers to form Forisome-powered micro-forceps. In such setups, they develop
forces of up to 0.1 µN in both contraction and expansion (Knoblauch et al., 2003).
Calcium is an important component of many signal transduction pathways, and calcium
regulation has been implicated in phloem function. Knoblauch et al. (2001) showed that an
influx of calcium into legume sieve elements stimulates the rapid and reversible dispersal of
crystalloid P-protein to occlude sieve plate pores. The concentration of free calcium in sieve
tubes of R. communis has been found to be significantly higher than that in surrounding tissue
(Brauer et al., 1998), and calcium-dependent protein kinases have been detected in rice
phloem sap (Nakamura et al., 1993). Volk and Franceschi (2000) showed evidence of a
calcium channel in the sieve element plasma membrane of tobacco and of the aquatic plant
Pistia stratiotes using immunolabeling with antibodies to a calcium channel protein. These
authors proposed that calcium channels become activated during wounding or pathogen
attack, facilitating calcium influx into phloem tissues. McEuen et al. (1981) detected a
Chapter I Introduction ___________________________________________________________________________
7
calcium binding protein distinct from calmodulin in phloem exudates of C. maxima and
speculated that it might be associated with P-protein function.
On the other hand, given that in general the presence of ATP is a requirement for the majority
of motor-like biomolecules, it become evident that Forisomes might represent a previously
unknown class of mechano-proteins since their reversible conformational change is not driven
by ATP but energized by changes of the free Ca2+ concentration or pH variations. ATP-driven
motor proteins, the actuator of living cells, possess promising characteristics in micro- and
nanodevices, however, their dependence on a strictly defined chemical environment can be
disadvantageous. ATP-independency, together with a periodic reversible motion, are difficult
properties to ascertain. These two remarkable features are found in Forisomes, making them
one of the most promising proteins in the field of biomimetic engineering.
The wide range of applications for biomimetic smart materials underscored the need for the
molecular and biological investigation of the Forisome described in the available work.
I.1.4 Ca2+-binding motifs and secondary protein structures
I.1.4.1 EF-hand
The EF-hand is the most frequent motif found in Ca2+-binding proteins (Nakayama and
Kretsinger, 1994; Muranyi and Finn, 2001). The EF-hand family is a large class of Ca2+-
binding proteins that contain homologous Ca2+-binding sites within a characteristic helix-
loop-helix motif (da Silva and Reinach, 1991; Kawasaki and Kretsinger, 1994; Falke et al.,
1994; Chazin, 1995). EF-hands are generally found back-to-back in anti-parallel pairs with β-
sheet-like hydrogen bonding occurring between the loops of the coupled sites. These coupled
sites are often found to have cooperative metal ion binding, as in the case of calmodulin.
Within this characteristic helix-loop-helix motif, the Ca2+ is enclosed in the loop between two
α-helices, in a pentagonal, bipiramidal configuration.
Proteins containing the EF-hand Ca2+-binding motif, such as calmodulin and calcineurin B,
function as regulators of various cellular processes that are sensitive to Ca2+ (Nakayama and
Kretsinger, 1994; Kawasaki and Kretsinger, 1995; Ikura, 1996; Polans et al., 1996; Schafer
and Heizmann, 1996). Many essential cellular processes, such as cell cycle control, nucleotide
metabolism and signal transduction, are tightly regulated by calcium. In certain Ca2+-binding
proteins, regulatory domains undergo large conformational changes upon ion binding, and
this response modulates their overall intramolecular conformation and their interaction with
other proteins, including aggregation and dissociation (Rashidi, H.H. et al., 1999; Nakashima,
K. et al., 1999; Pal, G.P. et al., 2001).
Despite the dominance of the EF-hand motifs, other known Ca2+-binding motifs (Muranyi, A.
and Finn, B.E., 2001; Swairjo, M.A. and Seation, B.A., 1994; Nalefski, E.A. and Falke, J.J.,
1996; Rizo, J. and Südhof, T.C., 1998; Weis, W.I., 1996) are found in Ca2+-binding proteins.
Chapter I Introduction ___________________________________________________________________________
8
I.1.4.2 Coiled-coil domains
The rodlike α-helical coiled-coil is one of the simplest yet most common structural motifs
occurring in proteins. Consisting of two to five α-helices twisted into a left-handed supercoil,
the occurrence of this structure is well documented, occurring in a wide variety of proteins
including motor proteins, DNA binding proteins, extracellular proteins, and viral fusion
proteins (Lupas, 1996; Kohn et al., 1997; Burkhard et al., 2001). The presence of a
continuous interface of hydrophobic amino acids along the length of the helices provides a
major source of stability to the fold as the hydrophobics pack in a knobs-into-holes fashion
shielded from the bulk solvent (Crick, 1953). The pattern of repeating hydrophobic residues at
positions a and d of the heptad repeat (denoted abcdefg) that are responsible for coiled-coil
formation was first identified by Hodges et al. (1972) from the amino acid sequence of
tropomyosin. Later, a dimerization domain of a family of transcription factors, the leucine
zipper (Landschulz et al., 1988), was also described comprising this structural motif.
Figure I-3 Structures of parallel coiled coils (Müller, K.M. et al., 2000). (A) Helical wheel diagram of a parallel, dimeric coiled coil illustrating the disposition of the characteristic heptad repeat. (B-C) X-ray crystal structures of dimeric and trimeric GCN4 leucine zipper variants. The backbone and interface residues are shown superimposed on the ribbon representation of the helices.
Chapter I Introduction ___________________________________________________________________________
9
The coiled-coil domain is responsible for oligomerization of protein subunits and concomitant
folding of the proteins. The versatility of coiled coils for oligomerization derives from their
diversity of oligomeric structures. Coiled coils are gently twisted, ropelike bundles containing
two to five α-helices in parallel or antiparallel orientation. The N and C termini of the helices
are easily accessible, facilitating linkage to other proteins. Parallel dimers and trimers are by
far the most commonly observed coiled coils (Figure I-3).
I.2 Promoter sequences and regulation of transcription
The regulation of gene expression at the level of transcription is a key control point affecting
gene expression in response to a variety of extra- and intracellular signals, during
developmental processes and for tissue specificity. In eukaryotes, there are tens of thousands
of protein-coding genes, each of which has its unique program of transcription. The cis-acting
DNA sequences that encode these transcriptional programs include transcriptional enhancers,
proximal promoters, and core promoters also called minimal promoters. A typical eukaryotic
promoter consists of a minimal promoter and upstream cis-elements. The minimal promoter is
essentially a TATA box, an A/T-rich region located about 30 nucleotides (nt) upstream of the
transcription start site where RNA polymerase II, TATA-binding protein (TBP), and TBP-
associated factors (TAFs) bind to initiate transcription. However, minimal promoters alone
have no transcriptional activity. The cis-elements, to which tissue-specific or development-
specific transcription factors bind, individually or in combination, determine the spatio-
temporal expression pattern of a promoter at the transcriptional level. Enhancers and proximal
promoters are recognized by sequence-specific DNA-binding proteins that regulate
transcription (Blackwood and Kadonaga, 1998; Lee and Young, 2000; Lemon and Tjian,
2000; Malik and Roeder, 2000). Enhancers are often located many kilobase pairs (kbp)
upstream or downstream of the transcription start site, whereas proximal promoters are
typically within a couple hundred base pairs (bp) of the start site. Core promoters encompass
the transcription start site and specify the site of transcription initiation by the basal
transcriptional machinery (Orphanides et al., 1996; Smale, 1997; White, 2001). The core
promoter is at a unique and important position in the transcription process, as it is the eventual
target of the action of the many sequence-specific factors and co-regulators that control the
transcriptional activity of each gene.
I.2.1 Phloem-specific promoters
Regulation of transcription is achieved by the activity of multiple proteins that bind to
regulatory elements, many of which are upstream of the promoters and alter basal rates of
transcription initiation and/or elongation (Roeder, R.G., 1991; Yankulov, K. et al., 1994). To
understand the mechanisms of tissue-specific and constitutive gene expression in plants, a
Chapter I Introduction ___________________________________________________________________________
10
number of promoters and transcription factors have been studied in recent years (Benfey, P.
N. and Chua, N.-H., 1989; Leyva, A. et al., 1992; Fujiwara, T. and Beachy, R. N., 1994;
Suzuki, M, 1995; Faktor, O. et al., 1996; Yin, Y. et al., 1997b; Ringli, C. and Keller, B.,
1998; Niggeweg, R. et al., 2000). It was shown that constitutive promoters, such as the
Cauliflower mosaic virus 35S promoter (CaMV 35S; Odell et al., 1985), a double-stranded
DNA virus belonging to the Caulimoviridae family, and the promoter from Cassava vein
mosaic virus (CsVMV; Verdaguer, B. et al., 1998), a plant pararetrovirus from the
Caulimoviridae family, are modular in organization and multiple cis-elements. These
elements, with specific transcription factors, apparently interact in an additive and/or
synergistic manner to confer gene expression in all plant tissues. Similarly, tissue-specific
promoters contain multiple elements that contribute to promoter activity in both positive and
negative ways (Leyva, A. et al., 1992; Fujiwara, T. and Beachy, R. N., 1994; Suzuki, M. et
al., 1995; Ellerstrom, M. et al., 1996; Ringli, C. and Keller, B., 1998; Hauffe, K. D. et al.,
1993). Among tissue-specific promoters, several promoters from plants (Yang and Russell,
1990; Brears et al., 1991; DeWitt et al., 1991; Ohta et al., 1991; Martin et al., 1993; Hérouart
et al., 1994; Shi et al., 1994; Truernit and Sauer, 1995; Tornero et al., 1996), Agrobacterium
(Schmülling et al., 1989; Sugaya et al., 1989; Guevara-García et al., 1993) and viruses
(Medberry et al., 1992; Bhattacharyya-Pakrasi et al., 1993; Rohde et al., 1995) have been
reported to drive expression of reporter genes in the phloem of transgenic plants, thus
designated as phloem-specific promoters. These tissue-specific promoters include the
glutamine synthetase 3A (GS3A) gene promoter (Brears et al., 1991), the Arabidopsis H+-
ATPase isoform 3 (AHA3) gene promoter (DeWitt et al., 1991), the Agrobacterium rolC
promoter (Schmülling et al., 1989), the Arabidopsis sucrose synthase gene (Asusl) promoter
(Martin et al., 1993), the maize sucrose synthase-1 (Shl) gene promoter (Werr et al., 1985;
Yang and Russell, 1990) and the rice tungro bacilliform virus (RTBV) promoter
(Bhattacharyya-Pakrasi et aL, 1993; Yin and Beachy, 1995).
Brears et al. (1991) found that when the GS3A promoter is deleted to nucleotide -132 relative
to the transcriptional start site, it can still direct tissue-specific expression, and a 17 bp
imperfect palindrome was identified as a putative cis-element by virtue of the binding of a
nuclear protein complex (Brears et al., 1991). Later on, Hehn and Rohde (1998) identified a
phloem specific motif of 13 bp in length which has been described to be highly conserved
among four phloem-specific promoters, originating from a pea glutamine synthase gene
(GS3A), coconut foliar decay virus (CFDV), rice tungro bacilliform virus (RTBV), and an
Agrobacterium rhizogenes rolC gene as well as the a Robinia pseudoacacia inner-bark lectin
gene (Rplec2; Yoshida et al., 2002).
Furthermore, the rice tungro bacilliformbadnavirus (RTBV) promoter (Qu, R.D. et al., 1991;
Hay, J. M. et al., 1991) and the transcription factors that interact with it served as a model
system to study plant tissue-specific gene expression. The virus, which replicates solely in
phloem tissues, has a single promoter that is active in transfected protoplasts and is phloem-
Chapter I Introduction ___________________________________________________________________________
11
specific in transgenic rice plants (Yin, Y. and Beachy, R. N., 1995; Yin, Y. et al., 1997a;
Chen, G. et al., 1994; Klöti, A. et al., 1999). Within the promoter sequence, multiple cis-
elements, including motifs associated with tissue-specific expression like GATA box (Lam E
et al., 1989), were identified as being required for phloem-specific gene expression (Yin, Y.
and Beachy, R. N., 1995; Yin, Y. et al., 1997b; He, X. et al., 2000; Meisel, L. and Lam, E.,
1997). The GATA A(N)3GATA motif is known to be important for phloem-specific gene
expression not only from the rice tungro bacilliform virus RTBV promoter (Yin et al., 1997a)
but also the same motif was found in the promoters of GS3A (Brears et al., 1991), the
Arabidopsis plasma membrane H+-ATPase gene (AHA3; DeWitt et al., 1991), a potato
invertase gene (Hedley et al., 2000) and the Rplec2 (Yoshida et al., 2002).
The 13bp and GATA motifs are conserved between virus and plant promoters, implying an
important role for these motifs for gene expression in the phloem.
Here, the cloning of a promoter sequence for the vff1 gene encoding Forisomes in Vicia faba
is described, and its expression pattern using promoter-reporter fusion in transgenic plants
further analysed.
Chapter I Introduction ___________________________________________________________________________
12
I.3 Aim of the thesis
The striking feature of Forisomes is the reversible and fast conformational change together
with their ATP-independency which make these structures interesting in the field of
nanotechnology biomaterials. Knoblauch et al. (2001) have reported that these conformational
changes are energized by modifications of the free Ca2+ concentration or pH variations.
Several other non-ATP based motors have been proposed, such as DNA-based nanoactuators
(Yurke, B. et al., 2000), flagella motors (Berg, H. C., 1974) and viral protein linear motors
(Dubey, A. et al., 2003). However, whereas all these have dimensions of the order of several
tens of nanometres, Forisomes are macroscopic assemblies whose size is in the order of
micrometers. Hence, although they could not be used to develop devices at the nanoscale,
they are easy to manipulate using current state-of-technology tools and their ‘technological
readiness’ is therefore higher than that of other non-ATP-based motors.
Taken together, these unique characteristics of Forisomes built up the framework of this
thesis. Hence, the objective of the present study was to clone the gene/s encoding Forisomes
in Vicia faba and to identify specific calcium-binding motives. Subsequent recombinant
expression of the isolated gene/s with their concomitant molecular and biochemical
characterization were employed to confirm gene-struture-funtion relationships. Finally, the
promoter sequences regulating the expression of Forisome proteins were identified and
studied in greater detail.
A schematic overview of this thesis is presented in Figure I-4.
Chapter I Introduction ___________________________________________________________________________
13
Generation and characterization of Forisome-specific
polyclonal Abs
Isolation of Vicia faba Forisomes
Peptides sequencing
Amplification of vff1 full-length cDNA and genomic sequence
Cloning of vff1 full-length cDNA in
bacterial expression vectors
Expression and purification of VFF1-
GST proteins
Generation and characterization of VFF1-GST-specific
polyclonal Abs
Immunological evidence of vff1 gene encoding a Forisome
protein
Amplification of vff1 promoter sequence
Cloning of vff1 promoter into plant expression vectors
Generation of transgenic
plants expressing GUS under control of
vff1 promoter
Differential expression pattern driven by vff1
promoter
Figure I-4 Schematic outline of the thesis
Chapter II Material and Methods ___________________________________________________________________________
14
II Material and Methods
II.1 Material
II.1.1 Chemicals and consumables
The chemicals used throughout the work were purchased from the following companies:
Amersham Bioscience (Freiburg, D), Axis-Shield PoC (Oslo, Norway), Boehringer
Mannheim (Mannheim, D), Duchefa (Haarlem, NL), Fluka (Neu-Ulm, D), Gibco BRL
(Eggenstein, D), Invitrogen (Karlsruhe, D), Merck (Darmstadt, D), Molecular Probes (Leiden,
NL), New England Biolabs (Frankfurt, D), Roche (Mannheim, D), Roth (Karlsruhe, D), Serva
(Heidelberg, D), and Sigma-Aldrich (Taufkirchen, D).
The consumables were from: Amicon (Witten), BioRad (München, D), Biozym (Hess.
Oldendorf, D), Eppendorf (Hamburg, D), Genetix (New Milton, UK), Greiner (Solingen, D),
Kodak (Stuttgart), MilliPore (Schwalbach, D), Qiagen (Hilden, D), Schleicher&Schuell
(Dassel, D), Whatman (Bender & Hobein, Bruchsal, D) and Zeiss (Oberkochem).
II.1.2 Enzymes and reaction kits
Restriction enzymes either from New England Biolabs (Schwalbach) or GibcoBRL were used
for DNA digestion. ExpandTM high fidelity DNA Taq polymerase from Roche or Advantage®
2 Polymerase Mix from BD Biosciences Clontech were used for PCR amplification. TaqDNA
polymerase produced at the Fraunhofer IME (Aachen, D) was used for check-PCR
amplification.
The following kits were used:
Oligotex® mRNA isolation kit from Qiagen,
QIAprep® Spin Mini/Midiprep kit from Qiagen,
QIAquick® gel extraction kit from Qiagen,
QIAquick® PCR purification kit from Qiagen,
MinEluteTM PCR purification kit from Qiagen,
RNeasy mini kit from Qiagen,
SMART TM PCR cDNA Synthesis kit from BD Biosciences Clontech,
SMARTTM RACE cDNA Amplification Kit from BD Biosciences Clontech,
SUPERSCRIPTTM First-Strand Synthesis System for RT-PCR from Invitrogen,
Universal GenomeWalkerTM kit from BD Biosciences Clontech,
ECL Advance Western blotting detection kit from Amersham Biosciences,
Nucleon Phytopure Plant DNA extraction kit from Amersham Biosciences,
PCR-Select TM cDNA Subtraction kit from BD Biosciences Clontech.
Chapter II Material and Methods ___________________________________________________________________________
15
II.1.3 Buffers, media and solutions
All standard solutions, buffers, and media were prepared according to Sambrook et al. (1989),
Ausubel et al. (1995) and Coligan et al. (1995). Compositions of otherwise special media and
solutions or buffers are listed at the end of the respective method section. Media for bacterial
and plant tissue cultures were sterilized by autoclaving (121oC/1-2 bar). Thermolabile
components were filter sterilised by passing through a 0.2 µm filter (Millipore) and added to
the autoclaved media or buffer after they were cooled down to 60-50°C.
II.1.4 Matrices and membranes
Glutathione sepharose 4B from AmershamPharmacia Biotech was used for purification of
GST fusion proteins (II.2.11).
HybondTM-C nitrocellulose membrane (0.45µm) from Amersham Life Science and Whatman
no.1 paper from Whatman (Maidstone, England) were used in immunoblot analysis
(II.2.12.4).
II.1.5 Primary antibodies, secondary antibodies and substrates
Mouse anti-GST monoclonal antibody provided by Dr. Michael Monecke (RWTH Aachen,
Institut für Biologie VII, Germany) was used for analyses of GST and GST fusion protein
expression. Alkaline phosphatase (AP) or horseradish peroxidase (HRP)-conjugated to goat
anti-mouse IgG (H+L, Fc), rabbit anti-chicken (Fc) and goat anti-rabbit (Fc) (Dianova)
antibodies were used as secondary antibody in immunoblot analysis (II.2.12.4) and ELISA
(II.2.14). NBT/BCIP (BioRad) and pNPP (BioRad) or ABTS-H2O2 [0.5 mg of 2,2’-azinobis
(3-ethylbenzthiazolinesulfonicacid) (ABTS)/ml, 0.002% H2O2, 0.1 M citrate-phosphate
buffer (pH 4.3)] were used as substrate for detection of immobilized proteins in immunoblot
(II.2.12.4) and ELISA (II.2.14), respectively.
Goat anti-mouse Alexa Fluor® 488 (Molecular Probes) was used as secondary antibody in
immunofluorescence studies.
II.1.6 Biological material
II.1.6.1 Bacterial strains
E. coli strains DH5α and XL1-Blue were used as a host cells for all intermediate cloning
constructs. The strain BL21(λDE3) was used for expression of VFF1-GST-fusion proteins
(II.2.11).
Chapter II Material and Methods ___________________________________________________________________________
16
Agrobacterium tumefaciens GV 3101::pMP90RK (gentr, kanr), rifr (Koncz and Schell, 1986)
was used for Agrobacterium-mediated gene transfer into tobacco leaves (II.2.9) (Table II-1).
Table II-1 Name, suppliers and genotypes of bacterial strains used throughout this thesis
Strain Source Genotype
DH5α Ausubel et al., 1995 F- (f80d Lac 2∆M15) ∆(LacZYA-argF) U169end A1 rec1 hsdR17(rk
- mk+) deoR thi-1
supE44 gyrA96 relA1 λ-
BL21(λDE3) Novagen F- ompT hsdSB (rB – mB -) gal dcm (DE3)
XL1-Blue Stratagene recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB laclIq Z∆M15 Tn10 (Tetr)]
Agrobacterium Koncz and Schell, pMP90RK GmR, KmR, Rif R
tumefaciens 1986
GV 3101
II.1.6.2 Plants
Nicotiana tabacum L. cv. Petite Havana SR1 was used for transient protein expression after
vacuum infiltration of recombinant Agrobacteria (II.2.10.3) and for generation of stable
transformed plants (II.2.10.4).
II.1.6.3 Animals
6-8 weeks old female BALB/c mice as well as White Leghorn chicken about 12 weeks old
were used for immunization (II.2.13.1 and II.2.13.2) with isolated Forisomes from Vicia faba.
II.1.7 Vectors
II.1.7.1 Bacterial expression vector
pGEX-5X-3 from Amersham Pharmacia Biotech, modified at the multiple cloning site by
insertion of a NcoI site was used for subcloning the vff1 gene, together with partial fragments
(amino/carboxy terminus, center domains), and amplification of the resulting plasmids in E.
coli DH5α. After confirmation of the DNA sequences, pGEX-VFF1 along with the plasmids
harbouring the different protein domains were introduced into E. coli BL21 and expression of
the GST-fusion proteins was carried out.
Chapter II Material and Methods ___________________________________________________________________________
17
II.1.7.2 Plant expression vectors
pUC103 - GUS (K. Fritze, MPIZ, Köln, Deutschland) containing the GUS-term construct
(III.2.4.1) was used for subcloning of the GUS-term reporter gene.
pTRA (Thomas Rademacher, Institut für Biologie VII, RWTH Aachen, Germany) is a
optimized plant expression vector containing the Cauliflower mosaic virus (CaMV) 35S
promoter with duplicated enhancer region (35SS) or the phloem-specific promoter of the
Coconut foliar decay virus (CFDV) and the pA35S untranslated region from Cauliflower
mosaic virus (CaMV). A matrix attachment region was introduced to improve transcription.
This binary vector was used for subcloning the vff1 promoter (III 2.4.1) as well as series of
thirteen truncated vff1 promoter fragments (III 2.4.3) in replacement of the 35SS promoter.
Schematic presentation of the vector maps are presented in the Appendix VII.2.
II.1.8 Oligonucleotides
Oligonucleotides used for sequence analysis and amplification of DNA are listed below
(Tables II-2, II-3, II-4) . All oligonucleotides were synthesized by Metabion International AG
(Martinsried, Germany).
Table II-2 Primers used for PCR amplification in deletion analysis of vff1 Promoter in Vicia faba
forward f-P-pTRAk-1 5′ -TTG GCG CGC CTT GAC TTG TAG ATA TGT TG - 3′
f-P-pTRAk-2 5′ -TGG GCG CGC CTC TAT AAA CGT TAA TGT TTG - 3′
f-P-pTRAk-3 5′ -TTG GCG CGC CTG AAA CAA CTC AAA CTT ATA - 3′
f-P-pTRAk-4 5′ -TTG GCG CGC CCA GTG GTG ACT CTT GCT ATC- 3′
f-P-pTRAk-5 5′ -TTG GCG CGC CCG GAT GAA AAT GGT ACT AAC- 3′
f-P-pTRAk-6 5′ -TTG GCG CGC CCT ACA TTC TCA ACG ATG CGA G - 3′
f-P-pTRAk-7 5′ -TTG GCG CGC CTT GTA TAC ATT CTC AAC GAT G - 3′
f-P-pTRAk-8 5′ -TTG GCG CGC CCA AAC ATT TTG TAT ACA TTC - 3′
f-P-pTRAk-9 5′ -TTG GCG CGC CAT ATA GTA CTA TCT AAA GG - 3′
f-P-pTRAk-10 5′ -TTG GCG CGC CTC CTC GAT GAC CCT TCA ATG- 3′
f-P-pTRAk-11 5′ -TTG GCG CGC CAT AAA ACA CTT TGC ACA CTT G - 3′
f-P-pTRAk-12 5′ -TTG GCG CGC CGT AAT AAC ATA TGA TAT TTA AA - 3′
f-P-pTRAk-13 5′ -TTG GCG CGC CAT TTC TCC TTC ATT TTT ATA TTT - 3′
reverse r-P- pTRAk 5′ -CAT GCC ATG GTG ACT CAA ATT TCA GAG AA - 3′
Chapter II Material and Methods ___________________________________________________________________________
18
Table II-3 Primers used for cDNA amplification in RACE-PCR
5’-RACE-GSP1 5′ -CTC ACC TCC ATC ACA CGT CCA CAA GTA GGA TTT GG - 3′
5’-RACE-GSP2 5′ -ACT CTT CAA TGC TCT GAA CCG TTC CTT GCG GAT ATC GTC - 3′
5’-RACE-GSP3 5′ -CAC AAT GGG GAT CCA CAA AAT CTT GAA GTC TTC TTT CTT - 3′
5’-RACE-GSP4 5′ -TGG GCT TAT CGA AGA TCT TCC TGC GGT TAA ATA AAT CGT - 3′
5’-RACE-GSP5 5′ -GGA ATT TCC AAT TGG GTC TGT TGC TGT GAC CCT ATT AAG T - 3′
3’-RACE-GSP1 5′ -GGT GTA GAA AGG AAG AAA CAA AAC AAG AAG CAT CAA G - 3′
Table II-4 Primers used for PCR amplification and subclone Vicia faba forisome cDNA, N-terminal, C-terminal and central domains
Namec Sequencea Product size (kb) b
vff1 fw: 5’- CAT GCC ATG GGA ATG TCC TTT TCT AAC TCA -3’ 2.1
bw: 5’- TAA AGC GGC CGC AAC ACC AAA GTT ATT TGG -3’
N- vff1 fw: 5’- CAT GCC ATG GGA ATG TCC TTT TCT AAC TCA -3’ 1.2
bw: 5’- TAA AGC GGC CGC GTT CCT TGC GGA TAT CGT -3’
M- vff1 fw: 5’- CAT GCC ATG GGT TCC TTG AGA CAA CTG AAT -3’ 1.1
bw: 5’- TAA AGC GGC CGC ACG TTT CAG AGT CTC GTG -3’
C- vff1 fw: 5’- CAT GCC ATG GGA TTC AGA GCA TTG AAG AGT -3’ 0.9
bw: 5’- TAA AGC GGC CGC AAC ACC AAA GTT ATT TGG -3’
a letters in bold represent restriction endonuclease sites (NcoI and NotI) introduced into each primer to allow efficient cloning of the PCR product into the appropriate vector. b The product size is that resulting from PCR using the matching set of sense and antisense primers with the corresponding template. c Designation of the oligonucleotide as “fw” indicates the sense primer, whereas “bw” indicates the antisense primer. vff1, N-vff1, C- vff1 and M-vff1 stand for full-lengh forisome cDNA, NH3-terminal, COOH-terminal and central-domains respectively.
II.1.9 Equipment and applications
BioBench stainless steel reactor (Applikon Dependable Instruments, Schiedam, The
Netherlands).
Biomek 2000 with 384 High Density Replica Tool (HDRT) (Beckmann Coulter, Fullerton,
US).
Cameras and scanner: MP4 (Polaroid, Cambridge, MA, USA). E.A.S.Y 429K camera
(Herolab, Wiesloch), Arcus II Scanner with AGFA FotoLook 3.5 Software (AGFA, Köln),
Fujifilm LAS-1000 (Fujifilm, Tokyo, Japan).
Chapter II Material and Methods ___________________________________________________________________________
19
Centrifuges: AvantiTM 30, AvantiTM 20 and AvantiTMJ-25 (Beckman, California, USA),
Biofuge A (Heraeus, Hanau), Sigma 3-10 and Sigma 4-10 (Sigma, St. Louis, Missouri, USA),
RC5C and RC5B plus (Sorval instruments, Du Pont, Bad Homburg). Rotors: F0650, F2402H,
JLA 8.1000, JLA 10.500 and JA 25.50 (Beckman), #1140 and #11222 (Sigma), RLA-300,
SS-34 and GS-3 (Du Pont).
Confocal Fluorescence Microscope: Leica TCS SP (Leica, Wetzlar, Germany).
DNA gel electrophoresis apparatus: wide mini and mini cells for DNA agarose
electrophoresis and power supplies (BioRad).
DNA sequencer: ABI Prism 3730 DNA Analyzer and BigDyeTM cycle sequencing
terminator chemistry apparatus (Applied Biosystems, Foster, CA, USA).
Electroporation apparatus: “Gene pulserTM”, “Pulse controller” unit, Extender unit
(BioRad) and 0.2 cm cuvettes (BioRad).
Fluorescent Image Analyzer: Phosphorimager FLA-3000 (Fujifilm, Tokyo, Japan).
InnovaTM 4340 incubator shaker (New Brunswick Scientific, Nürtingen).
Light Microscope: Leica DMLFS (Leica, Wetzlar, Germany) with a JVC camera TK-C1360
(Tokio, Japan).
PCR Thermocyclers: Primus and Primus 96 plus (MWG-Biotech).
Photometer: Eppendorf biophotometer with Printer DPU 414 (Eppendorf, Hamburg), and
multi-channel spectrophotometer Spectromax 340 (Molecular Devices, Sunnyvale,
California).
Probe sonicator: Bandelin Sonoplus sonicator UW2070 with Titan Microtip MS72
(Bandelin Electronic, Berlin).
Protein gel electrophoresis and electroblotting equipment: Mini-PROTEAN IIITM
electrophoresis system (Bio-Rad), Mini Trans-Blot Cell (Bio-Rad), Gel Air, Dryer (Bio-Rad).
QPix picking/gridding robot (Genetix, New Milton, UK)
Software: Windows NT 4.0 operating system (Microsoft); Microsoft Office 2000
(Microsoft); Adobe Photoshop 6.0 (Adobe); Chromas; Origin 6.0 (Data analysis and technical
graphics, Microcal Software, Inc.); Align IR, version 1.2 (LI-COR); GCG (Wisconsin
Package v10.2 TM, Genetic Computer Group Madison, Wisc.).
Speed-Vac centrifuge: Eppendorf Concentrator 5301 (Eppendorf)
UV-Transilluminators: wavelength 302 nm and UVT-20M (Herolab). UV-chamber (Bio-
Rad).
Vibrating-blade Microtome: Leica VT 1000S (Leica Microsystems Wetzlar GmbH,
Germany).
Chapter II Material and Methods ___________________________________________________________________________
20
II.2 Methods
All experiments related to the genetic engineering were performed according to the
regulations of “S1-Richtlinien” and were officially approved by the Regierungspräsidium des
Landes NRW” (RP-Nr.: 23.203.2 AC 12, 21/95) and “BGA” [AZ 521-K-1-8/98:AI3-
04/1/0866/88 (S1) and 55.8867/-4/93 (greenhouse)].
General recombinant DNA techniques, i.e. PCI (phenol/chloroform/isoamyl alcohol) and CI
(chloroform/isoamyl alcohol) extraction, DNA precipitation, restriction enzyme digestion,
DNA ligation, DNA agarose gel electrophoresis, were performed according to the standard
protocols described in Sambrook et al. (1989) and Ausubel et al. (1995).
II.2.1 Plant material and isolation of Forisomes
Vicia faba L. cultivar Witkiem major (Nunhem Zaden, Haelen, The Netherlands) were grown
in a greenhouse at 20 °C and a 14/10 h light/dark period (daylight plus additional lamp light).
The first three internodes of up to 60 plants were excised 6 weeks after germination. The rind
was pulled off the central wood cylinder in two strips; the phloem tissue was then scratched
off these strips with scalpels and transferred to Ca2+-free medium (50 mM KCl, 10 mM, 10
mM of either HEPES or Tris/HCl, pH 7.3). The collected tissue was frozen in liquid nitrogen,
homogenized in a mortar, thawed in Ca2+-free medium, and passed through a 60-µm-mesh
filter. The filtrate was centrifuged at 2,500g and 20 °C for 30 min. The pellet was resuspended
in 11.25 ml Ca2+-free medium and 3.75 ml of an 80% Nycodenz® (Axis-Shield PoC, Oslo,
Norway) solution were added. Using another 15 ml of the Nycodenz® solution, a density
gradient (20 to 80% Nycodenz®) was produced with a custom-built gradient mixer. The
gradient was centrifuged at 150,000g and 20 °C for 3 h in an ultracentrifuge (OTD75B
Sorvall, Wilmington, Delaware, USA). Forisomes accumulated in a layer corresponding to
roughly 1.2 gml–1. This layer was extracted and washed by adding a threefold volume of Ca2+-
free medium. The sample was centrifuged at 2,500g and 20 °C for 30 min, and the pellet
containing the Forisomes was resuspended in a small volume of medium. With the aim of
avoiding the presence of membrane-bound proteins, which normally contaminate the isolation
of Forisomes, the sample was washed two times by adding a threefold volume of 1 mM Na2-
EDTA, 0,1% (v/v) Triton X-100 medium. The sample was centrifuged at 2,500g and 20 °C
for 10 min after each washing step and the pellet was resuspended in 500 µl of 1xPBS.
II.2.2 Peptides sequencing
A sample of the isolated Forisomes was separated by SDS-PAGE (sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; II.2.12.2). Coomassie blue staining demonstrated two
Chapter II Material and Methods ___________________________________________________________________________
21
predominant bands migrating at ~ 70kDa. These bands were excised from the gel, digested
with trypsin and analyzed by peptide mass fingerprinting following nanoLC-ESI-MS/MS and
MALDI-TOF-MS. Peptide sequencing was done by different companies (Proteome Factory
AG, Berlin; TopLab, Munich; Fraunhofer IME, Aachen) and the sequences obtained were
subject to comparison against a Medicago truncatula public EST data base (http://medicago.
toulouse.inra.fr/Mt/EST).
II.2.3 Isolation of genomic plant DNA
Genomic DNA was isolated from young Vicia faba leaves using the Nucleon Phytopure Plant
DNA extraction Kit (Amersham).
II.2.4 RNA isolation
Total RNA was isolated from a powdered pool of young leaves and stems using hot borate
method with modifications, as described by Moser et al. (2004). 1g of the plant tissue was
extracted in 3.5 ml of pre-warmed (80°C) RNA extraction buffer. The homogenate was mixed
thoroughly, incubated at 80°C for 5 min and then distributed in 2 ml microcentrifuge tubes
(1.4 ml of sample per tube). To inactivate contaminating ribonucleases, 1 mg of proteinase K
(2.2 mg of proteinase K for 1 g of tissue) was added following incubation at 42°C for 1 h.
After adding 2 M KCl to final concentration of 160 mM, the samples were kept on ice for 45
min. The samples were centrifuged at 13,000 rpm at 8°C for 15 min to enable pelleting of
insoluble particulates. The supernatant was then transferred to a new 2 ml microcentrifuged
tube, where 8 M LiCl to a final concentration of 2 M was added to allow RNA precipitation.
The samples were incubated on ice at 4°C overnight. The precipitated RNA was obtained by
centrifugation at 13,000 rpm at 8°C for 25 min. After discarding the supernatant, the RNA
pellet was washed with 1 ml of ice cold 2 M LiCl and then centrifuged at 13,000 rpm and 8°C
for 15 min. The RNA pellet was then resuspended with 600 µl of 10 mM Tris-HCl (pH 7.5)
and mixed thoroughly at room temperature. For removal of polysaccharides, 1/10 vol. of 2 M
potassium acetate (pH 5.5) was added to each sample following incubation for 10 min on ice.
The samples were centrifuged at 13,000 rpm and 8°C for 15 min. The supernatant was
transferred into new 1.5-ml microcentrifuge tubes, mixed with 0.9 vol of cold (4°C)
isopropanol and then stored at -20°C for 1 h to allow RNA precipitation. As a final step, the
RNA precipitate was centrifuged at 13,000 rpm and 8°C for 25 min. After washing the
obtained RNA pellet with 1 ml of cold (4°C) 80% (v/v) ethanol and centrifugation at 13,000
rpm and 8°C for 10 min, the samples were dried out in ice under sterile flow cabinet. The
RNA was resuspended in 30-50 µl of RNase-free water, pooled together in a new 1.5-ml
microcentrifuge tube and stored at -80°C. The mRNA was isolated using the Oligotex mRNA
isolation kit (Qiagen USA, Valencia, CA).
Chapter II Material and Methods ___________________________________________________________________________
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RNA extraction buffer: 0.2 M sodium tetraborate decahydrate, 0.03 M EDTA (pH 8.0), 1%
(w/v) SDS, 1% (w/v) deoxycholate acid sodium salt, 2% (v/v) β-
mercaptoethanol, 0.5% (w/v) spermidine, 2% (w/v) PVP (mol wt
40000), 1% (w/v) IGEPAL.
II.2.5 Libraries construction and screening strategies
II.2.5.1 cDNA expression library
A custom cDNA Expression Library of Vicia faba plant tips constructed in λTriplEX2TM
phagemid was purchased from Clontech (Cat. # CS1009u, BD Biosciences Clontech, Palo
Alto, USA). Total RNA was isolated from plant tips using the acid-guanidinium-phenol-
chloroform method (Sambrook et al., 1989) and 5µg of poly(A)+ RNA was purified using
Oligotex mRNA isolation kit (Qiagen USA, Valencia, CA). A library of 3.6x106 independent
clones was obtained with an average insert size of 1.5 kb and an insert size range between 0.5-
3.8 kb.
Library screening
The amplified cDNA expression library was subjected to immuno and PCR screenings as
described in the protocol suggested by the manufacturer (BD Biosciences Clontech, Palo
Alto, CA).
Polyclonal antiserum from chicken and mice was raised against isolated Forisomes (II.2.1).
Absorption of non-specific antibodies that react with E. coli proteins was carried out using a
lysate of E. coli XL-1Blue as described in the protocols provided with the picoBlue
immunoscreening kit (Stratagene). These pre-absorbed sera (dil. 1:30000) were used for
immunoscreening of the cDNA Expression Library using species-specific, horseradish
peroxidase-conjugated goat anti-mouse IgG (H+L, Fc) or rabbit anti-chicken (Fc) (Dianova)
antibodies as secondary antibody.
PCR screening of the amplified cDNA expression library was carried out using a mixture of
primers designed according to the Forisome sequenced peptides (II.2.2).
II.2.5.2 cDNA libraries
Construction of subtracted cDNA library
PCR-select cDNA subtraction was employed using PCR-SelectTM cDNA Subtraction Kit (BD
Biosciences Clontech, Palo Alto, CA) according to the manufacturer's protocol. The tester
(samples prepared from Vicia faba plant leaves) and driver (samples from the Vicia faba root
tips) cDNA were reverse transcribed from 2 µg mRNA of leaves and root tips, respectively,
Chapter II Material and Methods ___________________________________________________________________________
23
digested with RsaI and then ligated to different adaptors. Two rounds of hybridization and
PCR amplification were performed to enrich the differentially expressed sequences.
Construction of not subtracted cDNA library
Total RNA was isolated from Vicia faba young root tips by the acid-guanidinium-phenol-
chloroform method (Sambrook et al., 1989) and used for cDNA library construction
following the SMART TM PCR cDNA Synthesis Kit protocol (BD Biosciences Clontech, Palo
Alto, CA).
The subtracted and non subtracted cDNAs were inserted directly into the TOPO TA Cloning
strategy (InvitrogenTM) followed by TOP10F´ transformation (InvitrogenTM), producing the
resultant cDNA libraries.
Both unamplified libraries were plated onto LB agar 230 mm x 230 mm Vented QTray with
cover plates (Cat. # X6023, Genetix, New Milton, UK) containing LB agar, 50 µg/ml
kanamycin, spreaded with 40 µl of 100 mM IPTG in addition to 40 µl of 40 mg/ml X-gal on
each LB plate) and grown at 37oC overnight. Using a QPix (Genetix, New Milton, UK)
picking/gridding robot a blue/white screening was carried out and the white colonies were
arrayed into 384-well microtiter plates (384 Well Low Profile Microplate, flat bottom with
cover, Cat.#X6001, Genetix, New Milton, UK) containing LB medium supplemented with
freezing mix [0.4 mM Mg SO4, 1.5 mM Na3-citrate, 6.8 mM (NH4)2SO4, 3.6% (v/v) glycerol,
13 mM KH2PO4, 27 mM K2HPO4, pH 7.0]. Bacteria were grown in microtitre plates wells at
37oC overnight and the clones were gridded onto 222 mm x 222 mm Nylon filters membranes
(Hybond-N+, Amersham) in duplicate pattern for DNA hybridizations.
cDNA libraries screening
Hybridization screening was carried out on subtracted and non-subtracted Vicia faba cDNA
libraries using mixtures of isotope labeled ([γ32P]ATP) oligonucleotides designed according to
Forisome sequenced peptides (II.2.2) as probes.
Approximately 30,000 clones from the subtracted cDNA library and 20,000 clones from the
not subtracted cDNA library were transferred using a Biomek 2000 with 384 High Density
Replica Tool (HDRT) (Beckmann Coulter, Fullerton, US) to Hybond-N+ (Amersham)
membranes placed over LB-Kan-X-Gal plates and incubated at 37°C overnight. After 30 min
of incubation positioned face up onto Whatman 3MM paper soaked with denaturation buffer
(0.5 M NaOH; 1.5 M NaCl), the membranes were washed with neutralization buffer (1.5
NaCl; 0.5 M Tris-HCl pH 7.2) for another 5 min. Following softly cleaning of the cells debris
with a paper, the membranes were washed with 2XSSC buffer and air dried on Whatman
Chapter II Material and Methods ___________________________________________________________________________
24
3MM paper. The nylon filters were exposed at 50 mJoule under UV cross linker (GS Gene
Linker UV Chamber, Bio-Rad). Prehybridization was carried out with hybridization buffer
[5X SSC, 10X Denhardt solution, 1% (w/v) SDS and 10 µg/ml of denatured salmon sperm
DNA (Boehringer, Mannheim)] at 48°C for 1 h with gentle shaking in waterbath and the
membranes were hybridized overnight in the same conditions upon addition of the labeled
probes in 300 µl of hybridization buffer (90°C). After hybridization, membranes were washed
by gently shaking twice for 10 min in 2XSSC containing 0.1% (w/v) SDS at RT and another
two times at 48°C. Radioactivity on the membranes was detected by placing the membranes
on a support, sealing them with Saran wrap and then exposing to imaging plates for 12 h.
Hybridization signals were detected by a phosphorimager (FLA-3000, FUJI).
100X Denhardt solution: 2% (w/v) Ficoll, 2% (w/v) PVP and 2% (w/v) BSA
II.2.6 PCR technologies
II.2.6.1 Cloning of 5’ and 3’ ends of full-length cDNAs using RACE-PCR
3’- and 5’- RACE-Ready cDNA populations were synthesized from mRNA using BD
SMARTTM RACE cDNA amplification kit (BD Biosciences Clontech, Palo Alto, CA).
Primer design and PCR for detection of expression of hypothetical Forisome gene
Gene-specific primers 3’-RACE-GSP1 and 5’-RACE-GSP1 were designed based on an EST
fragment from Medicago truncatula containing the identified Forisome peptides. These
primers were used for the first round of 3’- and 5’- RACE-PCR, repectively. The criteria for
primer design was that they should be 23 to 28 nucleotides long (optimum 25 nucleotides)
having 50% to 70% GC content with melting temperature ~ 70°C, which enables touchdown
PCR. PCR conditions for detection of the gene expression from 3’- and 5’- RACE-Ready
cDNA populations were as follows: 5 cycles of 94°C for 30 sec, 72°C for 3 min, 5 cycles of
94°C for 30 sec, 70°C for 30 sec, and 72°C for 2 min, 20 cycles of 94°C for 30 sec, 67°C for
30 sec, 72°C for 2 min, followed by 72°C for 10 min. The performance of each PCR reaction
was checked by running 5 µl of each reaction on agarose gels (II.2.7.4), with appropriate
DNA markers.
5’-RACE. The 5’-most Forisome-related sequences were obtained by performing 5’RACE
(rapid amplification of cDNA ends) with a SMART RACE cDNA amplification kit (BD
Biosciences). First-strand cDNA was synthesized from poly(A)+ RNA at 42°C for 1 h with
PowerScript reverse transcriptase by using 5’- RACE CDS primer [T25VN, where N is A, C,
G or T and V is A, G or C]. The SMART II A oligonucleotide (5’-AAGCAGTGGTATCAAC
Chapter II Material and Methods ___________________________________________________________________________
25
GCAGAGTACGCGGG-3’) was included. This oligonucleotide anneals to the C-rich cDNA
3’ tail and serves as a primer for second strand DNA synthesis. After the first PCR
amplification with 5’-RACE-GSP1 primer, different gene-specific backward primers (5’-
RACE-GSP), derived from the hypothetical Forisome sequence obtained, were used to
perform four additional rounds of 5’-RACE-PCR, along with a universal primer (5’-
CTAATA CGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAG T-3’) that can
anneal to the SMART II A oligonucleotide sequence.
3’ RACE. The 3’-most Forisome-related sequences were obtained by performing 3’-RACE
with a SMART RACE cDNA amplification kit. First-strand cDNA was synthesized from
poly(A)+ RNA at 42°C for 1 h with reverse transcriptase by using the oligo(dT)-based 3’-
RACE CDS primer A [AAGCAGTGGTATCAACGCAGAGTAC(T)30VN, where N is A, C,
G, or T and V is A, C, or G]. Different gene-specific forward primers (3’RACE-GSP) were
derived from the hypothetical Forisome sequence obtained in the first round of amplification.
These specific primers were used in the subsequent PCRs along with the universal primer that
can anneal to primer A.
After examination by gel electrophoresis (II.2.7.4), RACE reaction products were cloned into
pCR® 2.1-TOPO® (Invitrogen) (please refer to the appendices VII.2). White colonies were
selected and the verification of inserts was done by colony PCR. PCR conditions were as
follows: 94°C for 4 min, 25 cycles of 94°C for 30 sec, 53°C for 30 sec, 72°C for 1 min, and
72°C for 10 min. Four to five independent clones for each of the 5’- and 3’-RACE products
were sequenced from both ends using generic sequencing primers. The assembled cDNA
sequences were aligned with the Forisome genomic sequence using the Wisconsin Package
v10.2, Genetics Computer Group (GCG), Madison, Wisc.
II.2.6.2 PCR amplification of cDNA
Polymerase chain reaction (PCR) was used for amplification of Forisome gene from a Vicia
faba RACE-Ready cDNA population (II.2.6.1). Double-stranded cDNA was amplified, based
on the protocol of Sambrook et al. (1989), using high fidelity DNA-polymerase and DNA
polymerase buffer from Roche. Two specific primers, 5’-CATGCCATGGGAATGTCCTTTT
CTAACTCA-3’ and 5’-TAAAGCGGCCGCAACACCAAAGTTATTTGG-3’ corresponding
to amino-terminal and carboxyl-terminal regions respectively (III.3.1), were used in the
amplification to introduce the NcoI and NotI restriction sites. The reactions were performed in
0.2 ml PCR reaction tubes (Biozym Diagnostik GmbH, Hessisch Oldendorf), using a DNA
thermal Cycler (MWG). PCR reactions were carried out in a total volume of 50 µl under the
following conditions: 95°C for 2min, 35 cycles of 95°C for 1 min, 53°C for 1 min, 72°C for 2
Chapter II Material and Methods ___________________________________________________________________________
26
min 50 sec, and 72°C for 5 min. The performance of each PCR reaction was checked by
running 5 µl of each reaction on agarose gels (II.2.7.4), with appropriate DNA markers.
II.2.6.3 Identification of introns and genomic DNA cloning by LD PCR
Intron sequences were identified by LD PCR with the Expand Long Template PCR System
(Roche) using 100 ng of genomic DNA as a template; thermocycling was performed at 95°C
for 2 min, 95°C for 10 sec, 53°C for 30 sec and 68°C for 5 min, for 10 cycles; 95°C for 15
sec, 53°C for 30 sec and 68°C for 5 min, for 25 cycles followed by 68°C for 7 min. The
performance of each PCR reaction was checked by running 5 µl of each reaction on agarose
gels (II.2.7.4), with appropriate DNA markers. The amplified fragments were cloned into
pCR® 2.1-TOPO® (Invitrogen) (please refer to the appendices VII.2) and sequenced. Intron
delimitation within genomic sequences was done by comparing with the putative Forisome
cDNA sequence using the Wisconsin Package v10.2, Genetics Computer Group (GCG),
Madison, Wisc.
II.2.6.4 Genome Walking
Genomic DNA was extracted from Vicia faba young leaves as described before and was used
for genome walking experiments. Genome-walking PCR was performed using a Genome
Walker kit (BD Biosciences Clontech Laboratories, Palo Alto, CA) according to the
manufacturer’s manual. Briefly, Vicia faba genomic DNA was digested by DraI, HpaI and
SspI restriction enzymes. Adaptor DNA provided in the kit was ligated to the blunt-end DraI-,
HpaI- and SspI-digested genomic DNA fragments generating three Genome Walking
Libraries. The first PCR was conducted using an adaptor primer (5’-GTAATACGACTCACT
ATAGGGC-3’) provided by the kit and a GSP1 (5’-CACAATGGGGATCCACAAAATCTT
GAA-3’) that was complementary to the 5’ region of Forisome cDNA. Second-round PCR
was done using the nested adaptor primer (5’-ACTATAGGGCACGCGTGGT-3’) and GSP2
(5’-TCTGAACCGTTCCTTGCGGATATCGTC-3’) complementary to the upstream
sequence of Forisome. The conditions used for the first and second PCR were 7 cycles at
94°C for 25 sec and at 72°C for 3 min, 32 cycles at 94°C for 25 sec and at 67°C for 3 min
followed by one cycle at 67°C for 7 min, five cycles at 94°C for 25 sec and at 72°C for 3
min, and 20 cycles at 94°C for 25 sec and at 67°C for 3 min followed by one cycle at 67°C for
7 min, respectively. The DNA amplified by PCR was cloned into pCR® 2.1-TOPO®
(Invitrogen) and was sequenced.
The performance of each PCR reaction was checked by running 5 µl of each reaction on
agarose gels (II.2.7.4), with appropriate DNA markers.
Chapter II Material and Methods ___________________________________________________________________________
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II.2.7 Recombinant DNA technologies
All procedures for nucleic acid manipulation were done according to standard methods
(Sambrook et al., 1989) unless otherwise described.
II.2.7.1 Isolation of plasmid DNA from E.coli
Recombinant plasmid DNA was purified using QIAgen plasmid DNA Mini-and Midi- prep
kits according to the manufacturer’s instructions based on the alkaline lysis method
(Sambrook et al., 1989). Quality and yield of plasmid DNA was examined by
spectrophotometric analysis (II.2.7.2) and agarose gel electrophoresis (II.2.7.4). Isolated
plasmid DNA was stored at -20°C.
II.2.7.2 Quantification of DNA
The DNA concentration was evaluated by optical density (1 OD260nm unit = 50 µg/ml of
double stranded DNA) according to Sambrook and Russell (2000). The DNA purity was
determined by the OD260nm/OD280nm ratio of the measured optical density, which is 1.8 for
pure DNA.
II.2.7.3 PCR amplification
For rapid identification of recombinant E. coli and Agrobacteria control PCR was carried out
to detect plasmids as described by Jesnowski et al. (1995).
The optimal annealing temperature (Tp) of the primers was experimentally optimised or
calculated based on the empiric formula (Wu et al., 1991):
Tp = 22 + 1,46 [2X (G + C) + (A + T)]
PCR reactions were carried out in a total volume of 50 µl as described in table II-5.
Amplification was carried out under the following conditions: initial denaturation at 95°C for
2 min, 35 cycles of denaturation at 95°C for 1 min, primer annealing at 55°C for 1 min,
primer extension at 72°C for 1 min, and final extension at 72°C for 5 min. The annealing
temperature and the time for denaturation were changed according to Tp value of primers and
the length of the target gene. The performance of each PCR reaction was checked by running
5 µl of each reaction on agarose gels (II.2.7.4) with appropriate DNA markers.
Chapter II Material and Methods ___________________________________________________________________________
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Table II-5 PCR reactions
Components Volume Final concentration 10X PCR buffer 5 µl 1X
50 mM MgCl2 1.5 µl 1.5 mM
10 mM dNTPs 1 µl 0.2 mM each
10 pmol forward Primer 0.5-1 µl 10 pmol
10 pmol backward primer 0.5-1 µl 10 pmol
Template DNA 0.5-5 µl 10-100 ng
Taq DNA polymerase (5U/µl) 0.25 µl 1.25 units
dd H2O to 50 µl
II.2.7.4 Agarose gel electrophoresis of DNA
Plasmid DNA, and PCR-fragments were separated in 0.8-1.2% (w/v) agarose gels prepared in
TBE buffer containing 0.1 µg/ml of ethidium bromide. Agarose gels and electrophoresis of
the samples were carried out as described by Sambrook et al., (1989). Known amounts of
DNA molecular markers such as 1 Kb ladder (New England Biolabs), 100 bp ladder (New
England Biolabs) were used for evaluation and determination of DNA concentration and size.
The DNA samples were loaded onto the agarose gels after the addition of DNA loading
buffer. The visualization of the DNA bands and documentation of the gels was performed
using a UV transilluminator at 302 nm and a black and white E.A.S.Y 429K camera (Herolab)
together with a photo printer (Mitsubishi).
TBE buffer (pH 8): 90mM Tris base, 90mM boric acid, 0.2 M EDTA
DNA loading buffer: 1x TBE, 5% (w/v) sucrose, 0.04% (w/v) bromophenol blue, 2% (w/v)
SDS
II.2.7.5 Preparative agarose gel electrophoresis
Preparative gel electrophoresis was used for large scale purification of a particular DNA
fragment from a mixture of DNA fragments after restriction enzyme digestion. The DNA
fragment of interest was excised from the gel on an UV transilluminator with a sterile scalpel.
The DNA extraction was performed with QIAquick gel extraction kit according to the
manufacturer’s instructions. The concentration of recovered DNA was measured by
spectrophotometer or determined by agarose gel electrophoresis and was used in further
experiments.
Chapter II Material and Methods ___________________________________________________________________________
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II.2.7.6 DNA sequence Analysis
The cloned fragments were sequenced by the dideoxy-chain termination method (Sanger et
al., 1977) using the Bigdye Terminator Cycle Sequencing kit v3.1 (ABI Prism 3730 DNA
Analyzer Applied Biosystems) according to the manufacturer’s instructions; sequence data
were analyzed using the Wisconsin Package v10.2, Genetics Computer Group (GCG),
Madison, Wisc.
II.2.8 Preparation and transformation of E. coli
II.2.8.1 Preparation of electro-competent E. coli
E. coli strains DH5α and BL21(DE3) were used to prepare electro-competent cells as
described by Dower et al. (1988). 5 ml LB-broth were inoculated with a single bacterial
colony isolated from an LB plate and incubated at 37°C o/n. Three ml of fresh o/n culture was
transferred into 500 ml of LB broth. The mid-log phase (OD600nm = 0.5-0.8) was reached after
incubating at 37°C with shaking (180 rpm) for 3-4 hours. Then the cells were placed on ice
for 15-20 min. and harvested by centrifugation (3000g/4°C/10 min). A three times washing
step with sterile water followed and the cells were resuspended in ice-cold 10% (v/v) glycerol
to a 300-fold concentration from the original culture volume (at >1010 cells/ml). 40 µl aliquots
were stored at -80°C.
II.2.8.2 Transformation of E. coli by electroporation
50-200 ng of DNA were used for the transformation of freshly thawed electro-competent cells
(II.2.8.1). The mixture was transferred to the bottom of a prechilled (0.2 cm) cuvette. The
electroporation was performed using a BioRad Gene Pulser II® under the following
conditions: capacitance 25 µF, the pulse controller set to 200 Ω and the potential difference
set to 2.50 kV. After application of the pulse, 0.8-1 ml of SOC medium was added and the
cells were incubated at 37°C with shaking for 1 h to allow expression of the antibiotic
resistance genes. Finally, 50-100 µl of each transformation were plated onto prewarmed
selective LB-agar plates and incubated at 37°C o/n.
II.2.8.3 Culturing of E. coli and glycerol stock preparation
Selected positive colonies of all strains were streak on a separate LB agar plate to obtain
individual bacterial colonies. After o/n incubation at 37oC the plates were stored at 4oC for
short periods (less than 2 weeks). LB medium containing the suitable antibiotics and 2%
(w/v) glucose was inoculated with a single recombinant colony of E. coli and grown o/n at
Chapter II Material and Methods ___________________________________________________________________________
30
37°C with vigorous shaking. Bacteria glycerol stocks were prepared by mixing 600 µl of a
fresh overnight culture with 600 µl of 40% (v/v) sterile glycerol and stored at -80°C.
II.2.9 Preparation and transformation of Agrobacterium tumefaciens
II.2.9.1 Preparation of electro-competent Agrobacterium cells
Agrobacterium tumefaciens strain GV3101 was streaked on YEB-agar plate containing 100
µg/ml Rifampicin (Rif) and 25 µg/ml Kanamycin (Km) (YEB-Rif-Km) and grown at 28°C
during 2-3 days). 5 ml of YEB-Rif-Km medium, placed in a 100 ml Erlenmeyer flask, were
inoculated with an isolated colony and incubated at 28°C for two days with continuous
shaking (250 rpm). 1 ml of the culture was transferred into 100 ml of YEB-Rif-Km medium
and grown at 28°C for 15-20 h with shaking (250 rpm) until the OD600nm reached 1-1.5. The
cells were chilled on ice for 15 min and collected by centrifugation at 4000g for 5 min at 4°C.
The bacterial pellet was washed three times with 10 ml of dH2O and resuspended in 500 µl of
sterile 10% (v/v) glycerol. 45 µl-aliquots of the cell suspension were transferred into
prechilled sterile microcentrifugation tubes, frozen immediately in liquid nitrogen and stored
at -80°C.
YEB medium (pH 7.4): 0.5% (w/v) nutrient broth; 0.1% (w/v) yeast extract; 0.5% (w/v)
Bacto-Trypton; 0.5% (w/v) sucrose; sterile 2 mM MgCl2 after
autoclaving.
YEB selection medium: YEB medium supplemented after autoclaving with 50 µg/L
Rifampicin (rif), 50 µg/L Carbenicillin (carb) and 25 µg/L
Kanamycin (kan).
II.2.9.2 Transformation of Agrobacterium by electroporation
An aliquot of electrocompetent Agrobacterium cells (II.2.9.1) was thawed on ice and mixed
with 0.2-1.0 µg of plasmid DNA (II.2.7.1). After incubation on ice for 3 min, the bacteria
cell/DNA mixture was transferred into a prechilled electroporation cuvette (0.2 cm) and
assembled into a safety chamber. An electric pulse (25 µF, 2.5 kV, 200Ω) was applied and the
cells were resuspended in 0.8-1 ml of SOC medium. Following this, the cells were incubated
at 28°C with shaking (250 rpm) for 1 h. Finally, 1-10 µl of the cells were plated on YEB-agar
selection media plates (100 µg/ml Rifampicin, 25 µg/ml Kanamycin and 100 µg/ml
Carbenicillin) and incubated at 28°C for 2-3 days. As a negative control, transformation of
Agrobacterium cells was carried out by replacing the DNA with sterile water.
Chapter II Material and Methods ___________________________________________________________________________
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II.2.9.3 Determination of the efficiency of recombinant bacteria transformation
Known concentrations of supercoiled plasmid pUC19 for E. coli and pSSH1 for A. tumefaciens
cells were used to transform each new batch of prepared competent cells with the aim of
testing the efficiency of transformation. The following transformation rates were obtained;
electro-competent E. coli >108/µg pUC19 and A. tumefaciens >103/µg pSSH1.
II.2.9.4 Growth of recombinant A. tumefaciens and preparation of glycerol stocks
To determine the presence of plasmid, single colonies of A. tumefaciens were examined by
control PCR (II.2.7.3) and the selected positive colonies were inoculated into 10 ml of YEB
selection medium (II.2.9.1). The cells were grown at 28°C for 2-3 days with vigorous shaking
at 250 rpm. After transferring the cell culture to Falcon tubes, the Agrobacteria cells were
spun down by centrifugation at 4000g for 10 min at 15°C. The cell pellet was resuspended in
a 1:1 volume of YEB selection medium and glycerol stock media (GSM). 100 µl-aliquots
were stored at –80°C for further experiments.
glycerol stock medium (GSM): 50% (v/v) glycerol, 100 mM MgSO4, 25 mM Tris pH7.4;
filter sterilised.
II.2.10 Generation and characterisation of transgenic plants
II.2.10.1 Transient transformation of tobacco leaves
Growth of recombinant Agrobacterium (II.2.9.2) and vacuum infiltration of tobacco leaves
was performed as described by Kapila et al. (1997).
II.2.10.2 Preparation of recombinant Agrobacteria
100 ml of YEB selection medium was inoculated with 100 µl of glycerol stock (II.2.9.4) of
the selected recombinant Agrobacteria carrying a plant expression vector. After an overnight
growth at 28°C under vigorous shaking, the culture was centrifuged during 10 min at 5000g
and 15°C. The cells were then resuspended in 250 ml of induction media and incubated at
28°C o/n. The next day the culture was centrifuged (4000g and 15°C for 15 min) and the
bacterial pellet was resuspended in 50 ml of MMA containing 200 µM acetosyringone and
left for induction for 2 h at room temperature. After a 1:10 dilution in MMA medium, the
OD600nm of the bacterial suspension was measure and adjusted to an OD600nm of 1. The diluted
bacterial suspension was used for vacuum infiltration of tobacco leaves (II.2.10.3).
Chapter II Material and Methods ___________________________________________________________________________
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Induction medium (pH 5.6): YEB medium; 10 mM MES. Sterile 2 mM MgSO4, 20 µM
acetosyringone, 100 µg/ml Rifampicin, 100 µg/ml
Carbenicillin, 25 µg/ml Kanamycin were added after
autoclaving.
MMA medium (pH 5.6): 0.43% (w/v) MS-salts (Murashige & Skoog, basic salt
mixture); 10 mM MES; 2% (w/v) sucrose. Sterile 200 mM
acetosyringone was added after autoclaving and directly
before use.
II.2.10.3 Vacuum infiltration of tobacco leaves
For each construct (P-pTRAk-1 to P-pTRAk-13; III.2.4.3) 4 young leaves of N. tabacum cv.
Petite Havana SR1 were immersed in 100 ml of A. tumefaciens suspension. Continuous
vacuum was applied decreasing the pressure to approximately 60 mbar for 15-20 min. The
pressure was then released slowly and the leaves were briefly rinsed in tap water. To incubate
the infiltrated leaves, a plastic tray with moistened Whatman paper no. 1 was used where the
leaves were placed with adaxial side upwards. The plastic tray was sealed with saran wrap to
maintain humidity and placed at 25°C with a 16 h photoperiod for 2-3 days. As control, leaves
were infiltrated with Agrobacteria suspension, which did not contain pTRAk vff1P-GUS
plasmid.
II.2.10.4 Stable transformation of tobacco plants
Recombinant A. tumefaciens transformed with pTRAk-vff1P-GUS plasmid carrying the
putative Forisome promoter was used for the generation of transgenic tobacco plants by leaf
disc transformation. Transgenic T0 plants were regenerated from transformed callus (Fraley et
al., 1983; Horsch et al., 1985). Young leaves from 4-5 week old wild type N. tabacum cv.
Petite Havana SR1 grown on MS medium (Murashige and Skoog, 1962) were cut into 1 cm2
squares and transferred into 50-100 ml Agrobacteria suspension (II.2.10.2). After incubation
at RT for 30 min the leaf pieces were transferred onto sterile wet Whatman 3MM paper in
petri dishes. The petri dishes were then closed with saran wrap and incubated at 26-28°C in
the dark for two days. As following step, the leaf squares were washed with distilled water
containing 100 µg/ml Kanamycin, 200 µg/ml Claforan and 200 µg/ml Betabactyl
(Ticarcillin/Clavulanic acid, 25:1) and transferred onto MS II-plates. The plates were placed
at 25°C in the dark for one week followed by incubation for 2-3 weeks with a 16 h
photoperiod. The newly formed shoots were cut and transferred onto MS III-plates and
cultured for another 10-14 days under the same conditions until roots began to grow. The
small plantlets were transferred into larger sterile pot containing MS III medium and
Chapter II Material and Methods ___________________________________________________________________________
33
incubated at 25°C during 2 weeks (16h photoperiod) before transfer into soil. Young leaves
from regenerated transgenic plants were used for the measurement of GUS activity.
Integration of the chimeric genes into the N. tabacum genome was examined by PCR. In all
tested transgenic plants, the PCR product had the expected size, indicating that the promoter
fragment and the GUS gene were linked in the genomic DNA and that each transgenic plant
contains at least one copy of the respective chimerical gene.
MS medium (pH 5.8): 0.43% (w/v) MS salts (Duchefa, No. M0221); 0.01% (w/v)
Myo-inositol (Serva); 2% (w/v) sucrose; thiamine-HCl 0.4
mg/L; 0.8 % (w/v) agar; 500 µl/L vitamine I.
MS II medium (pH 5.8): MS medium supplemented after autoclaving with sterile
1mg/L BAP (in DMSO, from Sigma); 0.1 mg/L NAA
(Sigma); 100 mg/L Kanamycin; 200 mg/L Claforan; 0.8 %
(w/v) agar; 200 mg/L Betabactyl.
MS III medium (pH 5.8): MS medium supplemented after autoclaving with 100 mg/L
Kanamycin; 200-250 mg/L Claforan; 200-250 mg/L
Betabactyl, 0.8 % (w/v) agar.
Vitamine I solution: 0.4% (w/v) glycin; 0.1% (w/v) nicotinic acid; 0.1% (w/v)
pyridoxine. The vitamine I solution was filter sterilised
through a 0.2 µm filter (Millipore) and stored at 4°C.
II.2.10.5 Growth of N. tabacum cv. Petite Havana SR1
Tobacco plants were grown in ED73 standard soil (Patzer, Sinntal-Jossa) with 0-30% (v/v)
sand under the following conditions: 16 h artificial light, 20-27°C, 10K Lux and 30-65%
humidity. To prevent pollination from other plants, flowers were covered with plastic bags
with micro pores. Mature, dried seeds were stored in paper bags at RT.
II.2.10.6 Measurement of GUS activity and histochemical analysis
Tobacco plants and young T1 seedlings grown from tissue culture were used for the
measurements of GUS activity. For the histochemical analysis, tobacco plants were
transferred to soil and grown in a greenhouse. GUS activities were measured in situ on
greenhouse plants, 40 to 50 cm tall and before flower initiation. Sections of leaves and thin
cross-sections of tobacco leaves, stem, roots and flower were cut by hand with a scalpel blade,
and placed in a small amount of staining buffer in Eppendorf tubes. GUS activity was
histochemically detected according to the method described by Jefferson (1987), but with
Chapter II Material and Methods ___________________________________________________________________________
34
0.5mM of potassium ferri- and ferrocyanide added to limit diffusion of GUS reaction products
(Caissard et al., 1994). Hand-cut fresh tissue sections were subjected to an overnight
incubation at 37°C in staining solution consisted of 1 mM X-gluc (100µl of X-Glucuronide
stock solution diluted in 8 ml of staining buffer). For analysis of young T1 seedlings, whole
plantlets were collected about 1 week after germination and immersed in the buffer containing
X-gluc followed. Slight vacuum was applied before incubation to facilitate substrate
infiltration. After staining, chlorophyll was extracted from photosynthetic tissues with 100%
(v/v) methanol at 65°C for 1 h followed by 70% (v/v) ethanol. Ethanol was changed 2-3 times
over a 24 h period and the tissue samples were incubated at room temperature until they were
bleached and the blue GUS stain were clearly visible. The GUS expression was detected
microscopically by the distinct blue color which results from the enzymatic cleavage of X-
GlcA. Samples were stored at 4°C [in 70% (v/v) ethanol].
X-Glucuronide stock solution: 80 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide
cyclohexylammonium salt (X-GlcA; Sigma-Aldrich,
Munich, Germany) disolved in dimethylformamide. The
stock solution was kept at -20 oC wrapped in plastic foil.
Staining buffer (pH 7.0): 100 mM NaH2PO4; 10 mM NaEDTA; 0.5mM potassium
ferricyanide and 0.5 mM potassium ferrocyanide; 0.1%
(v/v) Triton X-100.
II.2.11 Expression and purification of recombinant proteins
II.2.11.1 Expression and purification of Forisome-GST fusion proteins from E. coli
The expression and purification of GST-fusion proteins were carried out according to a
modified protocol based on Smith (1993).
An isolated colony of E. coli strain BL21(λDE3) (Novagen) transformed with the
recombinant vector pGEX-VFF1 (II.1.7.1) was used for the inoculation of 50 ml 2YT
medium supplied with 1% (w/v) glucose and 100 µg/ml ampicillin. The culture was grown
o/n at 37 oC under vigorous shaking. The recombinant bacteria o/n grown culture was diluted
1:100 into 500 ml of fresh pre-warmed 2YT/amp medium containing 0.1 %(w/v) glucose.
After incubation at 28oC for 6-7 hs until an OD600nm of 0.8-1, the temperature was gradually
reduced to 16oC and the expression of the GST-fusion protein was induced by adding IPTG to
a final concentration of 0.1 mM. The induction of the protein expression was taken place o/n
at 4oC. The following day the cells were harvested by centrifugation at 7700g and 4oC for 10
min. The supernatant was discarded, the bacterial pellet was drained and placed on ice until
resuspension with 25 ml of ice cold 1 X PBS buffer. The resuspended cells were transferred
Chapter II Material and Methods ___________________________________________________________________________
35
into a 50-100 ml plastic beaker and disrupted by sonication on ice 2-3 times for 30 sec (150W
with 30 sec intervals). Triton X-100 20% (v/v) was added to a final concentration of 1% (v/v)
and stirred gently for 30 min to aid in solubilization of the fusion protein. The bacterial lysate
was cleared of cellular debris by centrifugation at 12000g for 10 min at 4oC. The supernatant
was subjected to glutathione affinity chromatography according to the manufacturer’s
instructions (AmershamPharmacia Biotech).
II.2.11.2 Fermentation at 4-liter scale
A fed-batch aerated fermentation of an isolated colony of E. coli strain BL21(λDE3)
(Novagen) transformed with the recombinant vector pGEX-VFF1 (II.1.7.1) was conducted
with a 7-liter BioBench stainless steel reactor (Applikon Dependable Instruments, Schiedam,
The Netherlands). A starter culture was grown in 400 ml of LB medium supplemented with
100 µg/ml ampicillin. After reaching late exponential phase at 37°C and being shaken at 100
rpm, the culture was used for inoculation into the 7-liter vessel containing 3.6 liters of a
starting growing medium. The culture was grown overnight at 37°C and agitated at 500-750
rpm. Once the glucose was exhausted from the starting medium after 13 h (as indicated by a
sudden decrease in the oxygen requirement of the culture), the bacterial culture was
supplemented with 3% (2,12ml/L.h) of feeding medium containing glucose and
MgSO4.7H2O. Upon reaching the active growing phase (OD600nm of 41.85), the temperature
was reduced to 16°C within 2 h (-5°C each 30 min) and the expression of the GST-fusion
protein was induced by adding IPTG to a final concentration of 0.1 mM. Thirty min later a
peristaltic pump started to deliver 2% (1.41 ml/L.h) of the feed medium to avoid the depletion
of glucose. Growth was conducted at 37°C and induction at 16°C with pH maintained at 6.8
throughout the experiment with concentrated NH4OH (25%). Aeration was maintained at 0.5
vvm (vol.broth/vol medium.min) and agitation was controlled at 500 to 750 rpm to maintain
the level of dissolved oxygen above 30% air saturation. After 15 h of expression the cells
were harvested by centrifugation at 10000g and 4°C for 30 min. 289.5g of pellet was
obtained, resuspended in 200 ml of 1XPBS buffer and subject to cell disruption using a
Microfluidizer® processor M-110L (Microfluidics, Newton, MA). Triton X-100 20% (v/v)
was added to a final concentration of 1% (v/v) and stirred gently for 30 min. The GST-fusion
protein was recovered by centrifugation at 12000g for 20 min at 4oC. The supernatant was
subjected to glutathione affinity chromatography according to the manufacturer’s instructions
(Amersham Pharmacia Biotech) and the purified protein was analysed by SDS-PAGE.
Starting growing medium: 122 mM KH2PO4; 35 mM NH4(H2PO4); 0.48 mM
CaCl2.2H2O; 0.5 mM FeSO4.7H2O; 139 mM Glucose; 6.1
mM MgSO4.7H2O; 10.9 mM Citric acid; 0.95mM L-
Chapter II Material and Methods ___________________________________________________________________________
36
arginine.HCl; 0,1% (v/v) Ptm1 trace metals; 0,2 g/l
Methionine and 100 mg/l ampicilin.
The salt solution [KH2PO4, NH4(H2PO4), CaCl.2H2O, FeSO4.7H2O] in the fermentation
apparatus was autoclaved, whereas the additional feed components (glucose, MgSO4.7H2O,
Citric acid, L-arginin.HCl, trace metals, and Methionine) were filter sterilized and added
aseptically prior to inoculation along with 100 µg/ml ampicillin.
Feeding medium: 2.78 M glucose and 60 mM MgSO4.7H2O. The
feeding medium was filter sterilized.
Pichia trace metals solution, PTM1: 24 mM CuSO4.5H2O, 0.53 mM NaI, 19.87 mM
MnSO4. H2O, 0.83 mM Na2MoO4.2H2O, 2.10 mM
CoCl2, 0.15 mM ZnCl2, 0.32 mM H3BO3, 0.23 M
FeSO4.7H2O, 0.82 mM Biotin, 0.5% (v/v) H2SO4.
II.2.12 Protein analysis
II.2.12.1 Quantification of proteins
The estimation of the concentration of isolated Forisomes was determined by SDS-PAA gel
electrophoresis and Coomassie staining comparing with known concentrations of loaded
bovine serum albumin (BSA) as standard.
II.2.12.2 SDS-PAGE and Coomassie brillant blue staining
Protein samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE, stacking
gel: T = 4%, C = 2.6%, pH 6.8; separating gel: T = 12%, C = 2.6%, pH 8.8) (Ausubel et al.,
1995) under 20V/cm for 1 hour. Before loading onto the gel, protein samples were
denaturated in the presence of SDS and β-mercaptoethanol for 5 min at 100°C. A pre-stained
broad range protein marker (P7708S, New England BioLabs Inc.) was used. Protein bands
were revealed by staining with Coomassie brilliant blue or transfer to nitrocellulose
membrane for further analysis (Ausubel et al., 1995). The detection of the bands was
performed by staining the polyacrylamide gel for 30 min and RT with Coomassie staining
solution followed by incubation with destaining solution until the protein bands were clearly
visible.
Chapter II Material and Methods ___________________________________________________________________________
37
Protein loading buffer, 6x: 8% (w/v) SDS, 40% (v/v) glycerol, 200 mM Tris-
HCl (pH6.8), 5% (v/v) β-mercaptoethanol, 0.05%
(w/v) bromophenol blue.
Electrophoresis running buffer(pH 8.3): 25 mM Tris; 192 mM glycin; 0.5% (w/v) SDS.
Coomassie staining solution: 50% (v/v) methanol; 10% (v/v) glacial acetic acid;
0.05% (w/v) Coomassie Brilliant Blue R-250.
Coomassie destaining solution: 5% (v/v) methanol; 7.5% (v/v) glacial acetic acid.
II.2.12.3 2D-PAGE analysis
Isolated Forisomes were quantified and then precipitated by adding 2 volumes of 50 % (w/v)
TCA in acetone, followed by incubation on ice for 1 h. Samples were centrifuged for 45 min
at 15300 rpm and 4°C, pellets were resuspended in 500 µL chilled (-20°C) acetone and
incubated at -20°C for 1 h. Samples were spun again at 15300 rpm and 4°C and resuspended
in the same volume of acetone. All acetone was removed and the pellets left to dry at 30°C.
The pellets were then redissolved in 125 µl of Lysis buffer and loaded onto a 7 cm
immobilized pH 3–10 linear gradient strip (BioRad). Actively rehydratation was carried out
for 16 hours in the IEF-cell (BioRad) at 25Vh. Then, the sample was pipetted into the cleft;
the IEF-strips were applied bubble free and covered with mineral oil. Next morning the
isoelectrofocusing was performed according to the following program: 500V RampR 250Vh,
1000V RampR 500Vh, 8000V RampR 6500 Vh and holding at 500V. After the IEF-run, the
strips were reduced at 20°C by incubation in equilibration buffer I for 15 min and alkylated at
20°C by an incubation in equilibration buffer II for another 15 min. Strips were shortly
washed with H2O and placed on a 12% PA-gel, next to a piece of Whatman paper along with
prestained protein marker, and fixated with 52°C pre-warmed LM-agarose. Second-dimension
electrophoresis was carried out at RT using 120V for 20 min followed by 180V for 40 min
(until the bromophenol blue dye had run off the end of the gel). Protein spots were visualized
by silver staining.
Lysis buffer: 9 M urea, 4% (w/v) CHAPS, 0,4% (w/v) ampholytes 3–10, 65 mM
DTT, 0.1% (w/v) bromophenol blue. The ampholytes, DTT and
bromophenol blue were freshly added before use.
Equilibration buffer I: 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.05 M Tris pH8.8
and 64mM DTT.
Equilibration bufferII: 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.05 M Tris pH8.8
and 2,5% (w/v) iodoacetamid.
Chapter II Material and Methods ___________________________________________________________________________
38
II.2.12.4 Immunoblot analysis
Electrophoretically separated proteins (II.2.12.2) were transferred to Protran BA 85
nitrocellulose membrane (Schleicher&Schuell) for 1 h by electroblotting (250 mA) using the
Mini Trans-blot cell from BioRad. After blotting, the membrane was blocked by shaking for
1h at RT with 5% (w/v) skim milk powder (Roth) dissolved in PBS-T buffer. The
immunodetection was carried out either with Forisome specific mouse antiserum in a dilution
of 1:2000 in 1XPBS buffer, Forisome specific chicken antiserum in a dilution of 1:6000,
GST fusion VFF1 protein specific rabbit antiserum, or monoclonal anti GST antibody raised
in mouse (dilution 1:5000). The appropriate secondary polyclonal antibody couple either to
alkaline phosphatase (AP) or horseradish peroxidase (HRPO) was used for colorimetric or
chemiluminescence detection. Primary antibodies were diluted in blocking buffer while the
secondary antibodies were diluted in 1xPBS buffer. Membranes were revealed with substrate
solution (NBT/BCIP from BioRad or ABTS substrate tablets from Roche). For the
chemiluminescence detection a LAS-1000 (Fujifilm, Tokyo) was used.
Electrotransfer buffer (pH 8.3): 25 mM Tris; 192 mM glycin; 0.5% (w/v) SDS; 20% (v/v)
methanol.
PBS buffer (pH 7.4): 137 mM NaCl; 2.7 mM KCl; 8.1 mM Na2HPO4.12H2O;
1.5 mM KH2PO4
PBST buffer(pH 7.4): 137 mM NaCl; 2.7 mM KCl; 8.1 mM Na2HPO4.12H2O;
1.5 mM KH2PO4; 0.1% (v/v) Tween 20
AP buffer (pH 9.6): 100 mM Tris-HCl, pH 9.6, 100 mM NaCl, 5 mM MgCl2
II.2.13 Polyclonal antibodies production
II.2.13.1 Mouse immunization
Mouse immune sera to Forisomes were prepared by subcutaneous immunization of two
BALB/c mice with 50 µg of isolated Forisomes (II.2.1) previous denaturalization at 100°C for
15 min and emulsified in 40 µl of Freund’s complete adjuvant (GERBU Biochemicals,
Gaiberg). The mice were subsequently boosted three times at 2-week intervals with same
quantity of denaturated Forisomes. For the first and second boost, 20 µl of Freund’s
incomplete adjuvant were used while no adjuvant was added in the last boost. Blood was
taken after the second and last boosts from the tail vein using a 26 gauge needle (1-2 µl) and a
capillary for picking up blood. The blood was diluted 1000-fold with PBS for determination
of antibody titers.
Chapter II Material and Methods ___________________________________________________________________________
39
II.2.13.2 Chicken immunization
Polyclonal antiserum from chicken was raised against isolated Forisomes (II.2.1). For the first
immunization, 100 µg of Forisomes in 500 µl of 1XPBS buffer subject to 100°C for 15 min
were used along with 40 µl of Freund’s complete adjuvant (GERBU Biochemicals, Gaiberg).
Two further injections in the breast muscle were given at three weekly intervals with the same
amount of antigen and 20 µl of Freund’s complete adjuvant. Polyclonal IgY antibodies were
purified from eggs yolk (Polson, A. et al., 1985) ten days after the second injection to
estimate the antibody titers.
II.2.13.3 Rabbit antisera
Rabbit immune sera to full-length recombinant GST-VFF1 were prepared by Eurogentec s.a.
(Liège Science Park, Belgium) following their standard immunization protocol. Purified
fusion protein (II.2.11) was denatured in SDS and electrophoretically separated on a
denaturing 12.5% polyacrylamide gel and subsequent polyacrylamide gel electrophoresis
(PAGE) (II.2.12.2). A single band of recombinant GST-VFF1 fusion protein revealed by
brief staining with Coomassie blue was excised from the gel. The acetic acid from the
Coomassie destaining solution was washed out by rinsing the band slice in destilled water for
2 h at RT and the antigen included in the polyacrylamide gel slice was submitted for
immunization to Eurogentec. Approximately 100 µg of protein was used for immunization
and boosting of a rabbit.
II.2.14 Determination of antisera titers
The determination of polyclonal antibody titers from sera of mice and rabbit was performed
by direct ELISAs using isolated Forisomes (II.2.1) after denaturalization at 100°C for 15 min
(mice antisera determination) and overexpressed GST-VFF1 protein (II.2.11) (rabbit antisera
determination) as antigens. To determine the titer of VFF1 protein specific antibodies,
10 µg/ml of GST-VFF1 protein were coated to high binding ELISA plates. GST (10 µg/ml)
was included as control. In the evaluation of Forisomes specific mice antibodies 10 µg/ml of
antigen were used. Antigens in 0.1 M carbonate buffer (pH 9.6) were coated overnight at 4oC
and blocked with 1% (w/v) BSA in 1XPBS. Serial dilutions of sera samples in blocking
buffer in an appropriate range for the particular analysis were added to the coated plates and
incubated at 37°C for 1.5 hours. After three washes with PBST bound antibodies were
detected by addition of 1:5000 diluted AP-labeled goat anti-rabbit polyclonal antibodies or
HRPO-labeled goat anti-mouse polyclonal antibodies in blocking buffer. The substrates used
for developing of AP labeled secondary antibodies and HRPO conjugated goat anti mouse
antibodies were p-nitrophenyl phosphate (pNPP) (Sigma) and ABTS-H2O2 [0.5 mg of 2,2’-
Chapter II Material and Methods ___________________________________________________________________________
40
azinobis(3-ethylbenzthiazolinesulfonicacid) (ABTS)/ml, 0.002% (v/v) H2O2, 0.1 M citrate-
phosphate buffer (pH 4.3)] (Roche), respectively. ELISA plates were incubated at 37°C for
1 h followed by measurement of the OD at 405 nm with a Spectra Max® 340 microplate
reader (Molecular devices, CA).
The evaluation of chicken polyclonal antisera was carried out by immunoblot (II.2.12.4).
II.2.15 Confocal immunofluorescence microscopy
Vicia faba stems were cut with a razor blade into small sections alongside the phloem and
fixed overnight in fixative solution [4% (w/v) paraformaldehyde, 0.2% (v/v) glutaraldehyde in
0.1 M phosphate buffer, pH 7.4] at 4°C. The stem sections were then subject to washing five
times for 15 min with 0.1 M phosphate buffer (pH 7.4) at 4°C. Thick longitudinal and cross
sections (~30µm) from fixed Vicia faba stems were obtained using a vibratome (Leica VT
1000S) and collected on slides coated (5 min at RT and dried for 1 h at 60°C) with poly-L-
lysine. To improve the penetration of reagents across thick sections, the tissue was dehydrated
and rehydrated through ethanol series. Dehydration was carried out at RT by immersing the
sections in a solution of 50% (v/v) ethanol for 5 min, followed by increasing concentrations of
ethanol [70% (v/v), 96% (v/v) and 100% (v/v)] for 5 min each. Rehydration was performed
through an ethanol series [100% (v/v), 95% (v/v), 70% (v/v), 50% (v/v) for 5 min each] and a
final washing with dH2O. Cell wall digestion was carried out for 1 h with 2% (w/v) cellulose
in 0.1 M phosphate buffer (pH 7.4) followed by a disruption of the cell membrane with 0.5%
(v/v) Triton-100X in the same buffer for 1 h. Non-specific binding sites were blocked with a
solution of 3% (w/v) BSA in 0.1 M phosphate buffer (pH 7.4) buffer for 10 min. Forisome
specific mouse polyclonal antibodies (II.2.13.1) in a dilution 1:2 in 0.1 M phosphate buffer
(pH 7.4) was incubated for 2 h at RT for epitopes recognition. After washing three times with
0.1 M phosphate buffer (pH 7.4) for 5 min, the antigen-antibody binding reaction was
revealed by adding 1:30 fold diluted goat anti-mouse Alexa Fluor® 488 (Molecular Probes) in
0.1 M phosphate buffer (pH 7.4). The sections were then washed first with 0.1 M phosphate
buffer (pH 7.4) and then with distilled water. A confocal fluorescence microscope (Leica TCS
SP) was used for analyses of immunofluorescence.
Phosphate buffer 0.1 M (pH 7.4): 19% (v/v) of 200 mM NaH2PO4.H2O with 81% (v/v) 9.9
mM Na2HPO4.2H2O.
Chapter III Results __________________________________________________________________________
41
III Results
III.1 Molecular characterization of Forisome genes
III.1.1 Analysis of isolated Vicia faba Forisomes by SDS-PAA gel electrophoresis
SDS-PAA gel analysis (Figure III-1) of isolated Vicia faba Forisomes (II.2.1) revealed the
presence of several bands with different molecular weights indicating that Vicia faba
Forisomes might be protein complexes consisting of several subunits. A major band of
approximately 70kDa could be detected as well as several minor bands of lower and higher
molecular weights which might represent dimerization and multimerization derivates. The
70-kDa protein was extracted from the gel, digested with trypsin and analyzed by peptide
mass fingerprinting following nanoLC-ESI-MS/MS and MALDI-TOF-MS (II.2.2).
Figure III-1 SDS-PAGE analysis of isolated Vicia faba Forisomes. Approximately 5µg of isolated V. faba Forisomes (II.2.1) were resolved on 12% (w/v) SDS-PAA gel (II.2.12.2) and stained with Coomassie brilliant blue (II.2.12.2). M: protein marker.
III.1.2 Generation and characterization of Forisome-specific polyclonal antibodies
III.1.2.1 Immunoblot analysis of isolated Vicia faba Forisomes
Polyclonal antisera were raised against native Forisomes (II.2.1) in mouse and chicken
(II.2.13.1 and II.2.13.2). After the third boost, polyclonal antibodies were obtained from sera
and the titer was determined by immunoblot analysis (II.2.12.4). Antigen specificity of
mouse and chicken antisera against isolated V. faba Forisomes is shown in figure III-2A and
B demonstrating a specific binding of mouse antiserum to the major band of approx. 70kDa as
well as another band of slightly lower molecular weight and the upper bands running at >100
kDa resolved by SDS-PAGE analysis. Chicken polyclonal antibodies showed a strong
reaction mainly to the major band of approx. 70kDa while the immunoreativity against the
kDa
62
83
175
47,5
32,5
25
16,5
M FkDa
62
83
175
47,5
32,5
25
16,5
M F
62
83
175
47,5
32,5
25
16,5
M F
Chapter III Results __________________________________________________________________________
42
bands of higher molecular weights was less evident. Degradation products and/or proteins of
lower molecular weights were observed between 50 and 72 kDa. A total protein extraction
from tobacco was used as negative control. No cross hybridization of preimmunized mouse
and chicken antisera to V. faba Forisomes could be detected (data not shown).
Figure III-2 Immunoblot analysis of isolated Vicia faba Forisomes with mouse and chicken antisera. Approximately 5µg of isolated V. faba Forisomes (II.2.1) were separated on a 12% SDS-PAA gel (II.2.12.2) and blotted onto nitrocellulose membrane for immunoblot analysis (II.2.12.4). Blotted Forisome proteins were incubated with mouse (dil. 1:2000) (A) and chicken antisera (dil. 1:5000) (B) for 1 hour followed by addition of a 1:5000 dilution of GAMHRPO(H+L) and RACHRPO polyclonal antibodies, respectively. 1: isolated V. faba Forisomes; 2: tobacco total protein extraction as negative control. Detection was carried out by chemiluminescence using a LAS-1000 (Fujifilm, Tokyo).
III.1.2.2 Immunoblot analysis of isolated Vicia faba Forisomes on 2D-gel
electrophoresis
In order to identify proteins difficult to resolve on Coomassie brilliant blue-stained one-
dimensional PAGE, a two-dimensional-gel electrophoresis was performed (II.2.12.3).
Approximately 10µg of isolated V. faba Forisomes were resolved by this technique (Figure
III-3A). Furthermore, an immunoblot analysis was performed with the aim of characterize
their antigenicity toward Forisome-specific mouse and chicken antisera (Figure III-3 B and
C).
After two-dimensional PAGE, V. faba Forisomes are separated into at least 8 forms spanning
the pI values from 6.0 to 7.5 (Figure III-3A). The major band of ~70kDa observed on one-
dimensional PAGE is composed of two and three proteins running at the same molecular
weight. This heterogeneity might indicate the result of post-translational modifications (e.g.
glycosylations). Despite the observed high degree of complexity of Forisomes preparation, a
simplified pattern appears after immunoblot analysis in which the Forisome proteins
kDa
62
83
175
47,5
32,5
25
16,5
1 2kDa
62
83
175
47,5
32,5
25
16,5
1 2
A B
kDa
62
83
175
47,5
32,5
25
16,5
1 2kDa
62
83
175
47,5
32,5
25
16,5
kDa
62
83
175
47,5
32,5
25
16,5
1 2kDa
62
83
175
47,5
32,5
25
16,5
kDa
62
83
175
47,5
32,5
25
16,5
1 2
A B
Chapter III Results __________________________________________________________________________
43
molecules of higher molecular mass are of less acidic pI, whereas those migrating as lower
molecular mass forms are apparently more acidic. As shown in Figure III-3B, the
immunodetection with mouse polyclonal antibodies revealed the presence of three groups of
proteins running at a molecular weight of ~70kDa. Among two of them the presence of two
distinct isoforms is confirmed. Figure III-3C represents the immunodetection with polyclonal
chicken antibodies. In this case, two groups of proteins migrating as ~70 kDa were observed.
However, in this population the less acidic forms are better represented. Also the 150 kDa
species, resolved into two charge forms, were indicated displaying exclusively basic pI (8–9).
Not only with mouse but also with chicken polyclonal anitbodies the major ~70 kDa band was
observed migrating as barely detectable multiple spots of pI 5-6.
Figure III-3 2D-gel electrophoresis and immunoblot analysis of isolated V. faba Forisomes. Approximately 10µg of isolated V. faba Forisomes (II.2.1) were subjected to 2D-gel electrophoresis analysis (II.2.12.3). (A) pH 3 to 10 gel of V. faba Forisomes. Sample was solubilized and resolved by IEF (II.2.12.3). The second dimension was carried out on a 12% SDS-PAA gel (II.2.12.2) and silver stained (II.2.12.2). (B) Immunoblot analysis with Forisome-specific mouse antiserum (dil. 1:2000) followed by addition of a GAMHRPO(H+L) polyclonal antibodies (dil. 1:5000). (C) Immunoblot analysis with Forisome-specific chicken antiserum (dil. 1:5000) followed by addition of a RACHRPO polyclonal antibodies polyclonal (dil. 1:5000). Detection was carried out by chemiluminescence using a LAS-1000 (Fujifilm, Tokyo).
III.1.3 Screening of cDNA Libraries
For identification of Forisome-encoding genes, subtracted and non-subtracted Vicia faba
derived cDNA libraries (II.2.5.2) were screened by hybridization to specific DNA probes. The
screening of the recombinant E. coli clones in these arrayed libraries was performed as
described previously in the Materials and Methods section (II.2.5.2).
C
50
75
150
50
75
B
150
kDa
50
75
A
150100
pH 3 pH 10
C
50
75
150
C
50
75
150
50
75
B
150
50
75
B
150
kDa
50
75
A
150100
kDa
50
75
A
150100
pH 3 pH 10
Chapter III Results ___________________________________________________________________________
44
DNA probes
Peptide sequences (EVTSV and VMEVSWHYK) (II.2.2) of isolated Forisomes, which had a
relatively low codon degeneracy, were used to design mixtures of oligonucleotide probes for
arrayed Vicia faba cDNA libraries screening. Eight different probes (15-mer of 768-fold
degeneracy corresponding to EVTSV, 27-mer of 1536- fold degeneracy corresponding to
VMEVSWHYK, 24-mer of 768-fold degeneracy corresponding to VMEVSWHY and 24-mer
of 384-fold degeneracy corresponding to MEVSWHYK; Table III-1) were synthesized by
Metabion International AG (Martinsried, Germany) and end-labelled with 30 MCi of
[γ32P]ATP (Harrison, B. and Zimmerman, 1986).
Table III-1 Oligonucleotides used for hybridization screening
Peptide sequence E V T S V (N) 15-mer 5´ GAR GTN CAN WSN GTN 15-mer 5´ NAC NSW NGT NAC YTC
Peptide sequence V M E V S W H Y K 27-mer 5´ GTN ATG GAR GTN WSN TGG CAY TAY AAR 27-mer 5´ YTT RTA RTG CCA NSW NAC YTC CAT NAC 24-mer 5´ GTN ATG GAR GTN WSN TGG CAY TAY 24-mer 5´ RTA RTG CCA NSW NAC YTC CAT NAC 24-mer 5´ ATG GAR GTN WSN TGG CAY TAY AAR 24-mer 5´ YTT RTA RTG CCA NSW NAC YTC CAT
Y = C or T; N = A, G, T, or C; and R = A or G
Figure III-4 Hybridization screening on arrayed Vicia faba cDNA libraries. Subtracted and non-subtracted Vicia faba cDNA libraries were screened by hybridization to ([γ32P]ATP) isotope labelled oligonucleotides. These specific probes were designed according to Forisome peptide sequences (EVTSV and VMEVSWHYK) as previously described (II.2.5.2 and III.1.3). The duplicate pattern of the positive clones is observed.
Chapter III Results ___________________________________________________________________________
45
Positive clones were identified after tertiary screening in both unamplified libraries (Figure
III-4). Approximately 30,000 colonies from the subtracted cDNA library and 20,000 colonies
from the non-subtracted cDNA library were screened and 74 colonies were identified and
characterized. Although 23 of the clones exhibited strong hybridization signals, the sequence
analysis did not contain the identified Forisome peptides. Thus, the allocation of these DNA-
and/or DNA fragments to the Forisome genes was not possible.
III.1.4 Immunoscreening of cDNA expression library
In order to identify cDNAs encoding proteins involved in the constitution of Forisomes, pre-
absorbed sera from immunized chicken and mice (II.2.5.1) were used for immunoscreening of
a cDNA expression library of Vicia faba plant tips (II.2.5.1). Considering that the titer of this
amplified library was determined as 2.8x106 pfu/µl, 20,000 pfu per plate were subjected to
immunoscreening.
Figure III-5 Immunoscreening of Vicia faba cDNA expression library of plant tips. A cDNA expression library was constructed in λTriplEX2TM phagemid and purchased by Clontech (BD Biosciences Clontech, Palo Alto, USA). The induction of the fusion proteins expression was carried out presoaking prenumbered nitrocellulose filters in 10mM IPTG and when plaques were clearly visible, placing a damp filter onto each plate as described in the protocol suggested by the manufacturer (BD Biosciences Clontech). Proteins immobilized on the filters were detected using pre-absorbed serum from immunized chicken as primary antibody and RAC-FcHRP (Dianova) as secondary antibody (dil. 1:5000). Detection was carried out using ECL Advance Western blotting detection kit (II.1.2). (A) IgY pre-ab (phage lysate and Physalis protein extraction) dil. 1:30000. (B) positive control: human β2-microglobulin clone. Immunodetection was carried out with rabbit anti-human β2-microglobulin control antibody at dil. 1:1000 followed by GAR-FcHRP (1:5000). (C) negative control: preimmunized IgY pre-ab (phage lysate and Physalis protein extraction) (dil. 1:30000). (D) negative control: filter treated with RAC-FcHRP alone.
Due to a relative high background obtained during the procedure the pre-absorbed sera from
immunized chicken and mice were subjected to absorption of non-specific antibodies that
react with plant proteins. This was carried as described in established protocols (picoBlue
immunoscreening kit; Stratagene) using a total protein extraction from Physalis floridana.
Initial tests included a control with the secondary antibody alone to check whether it binds
A CB DA CB D
Chapter III Results ___________________________________________________________________________
46
non-specifically and a control in absence of the inducer IPTG to determine the basal level of
bacterial protein expresion which could affect the background; hence, a precise identification
by immunoscreening (data not shown). Depending on the background levels and the signal
strength of the bound antibody only the pre-absorbed serum from immunized chicken was
used as primary antibody. Forty-five positive clones appeared from approximately 100,000
plaques after a tertiary screening (Figure III-5), and the positive λTriplEx2 phagemids were
converted into the pTriplEx2. Subsequently, the nucleotide sequences of inserts displaying
potential open reading frames (ORFs) were analyzed, however, none of the identified
fragments turned out to be unrelated sequences.
III.1.5 PCR screening of cDNA expression library
Since the initial attempts to clone cDNA and/or cDNAs for Vicia faba Forisomes by
immunoscreening with polyclonal antibodies were not successful PCR screening of the cDNA
expression library was carried out. Degenerate primers encoding the peptides sequences
EVTSV and VMEVSWHYK from Vicia faba Forisomes (Table III-1) were used to amplify
and clone sequences that might correspond to genes encoding these proteins in Vicia faba.
Although many attempts using different PCR conditions were performed the identified PCR
products do not show any distinct Forisome-related genes.
III.1.6 Cloning full-length Forisome cDNA
Due to the fact that all previous attempts in isolating full-length Forisome cDNA clones failed
a RACE PCR technique was considered as an alternative cloning strategy. Hence, the rapid
amplification of cDNA ends (RACE) (II.2.6.1) was used to obtain the full-length cDNA
sequence of Forisome, resolve the start of the 5’ untranslated region (5’-UTR), and locate the
poly(A)+ addition site of the gene.
A 5’- RACE was attempted with degenerated oligonucleotides primers designed after the
isolated Forisome peptides sequences (Table III-1). This initial trial was unsuccessful. To
circumvent this problem, a recently published database for Medicago truncatula – the model
plant for Fabaceae – (EST-Library; http://www.medicago.org/genome) was used for further
studies. After aligning the isolated Forisome peptides sequences from Vicia faba (II.2.2) with
this Medicago library patches of amino acids conserved among the two species were outlined
belonging to the same DNA sequence. Nested primers, based on these conserved peptides
(EGFDIAFK and EVTSVN), were used to perform a PCR amplification of genomic Vicia
faba DNA resulting in a 200bp fragment. RACE PCRs, using gene specific primers (GSP)
derived from the hypothetical Forisome sequence obtained, extended the identified 200bp
fragment by additional ~1.2kb in the upstream and downstream direction. The 5’- and 3’-
RACE methods yielded cDNA bands of 1.3 and 0.8 kb, respectively (Figure III-6).
Chapter III Results ___________________________________________________________________________
47
Figure III-6 Agarose gel electrophoresis of 5’- and 3’- RACE PCR amplification products of Vicia faba cDNA. Lanes show the PCR products (II.2.6.1) amplified using different GSP in the RACE PCRs (II.2.6.1). 5µl of each sample were subjected to electrophoresis through a 1.2% (w/v) agarose gel (II.2.7.4). (A) 5’- and 3’- RACE PCRs. (B) reamplification of the 5’-RACE PCR products obtained in the first amplification. M: 1Kb DNA ladder (Invitrogen).
The nucleotide sequences of these bands overlapped with the 200bp initial fragment and
included the 3’ untranslated region of the gene. Genomic Vicia faba DNA, diluted 1:50 to
approximately 100 ng/µl, was used as a control in order to confirm that the amplification
products are derived from cDNA (data not shown). Further 5’-RACE PCRs were carried out
(II.2.6.1) extending the cDNA sequence resulting in retrieval of the 5’ untranslated region
sequence. A high-fidelity enzyme system (II.1.2) was used to reduce potential errors during
5’- and 3’- RACE PCRs. However, to detect possible mutations during PCR amplification,
this reaction was done in triplicate in independent reactions, and the products were sequenced.
All the three RACE sequences matched exactly with each other. The sequences were then
assembled into full length cDNAs. After conceptual translation of the ORF, the start codon
was defined. Among the cDNA sequences obtained, two ORFs were determined (ORF1 and
ORF2) differing from each other in patches of several nucleotides in the amino terminal
amino acids sequences (Figure III-7), representing a putative second Forisome gene.
The full-length Forisome cDNA (Figure III-8) was obtained by reverse transcriptase
polymerase chain reaction (RT-PCR) using primers designed based on the cDNA sequence
flanking the start and stop codons. The amplified sequence was cloned into pCR®2.1-TOPO
(Invitrogen) and sequenced from both directions with 10-12-fold redundancy. A cDNA
sequence of 2053bp was determined revealing the presence of 10 different peptides sequences
belonging to the Vicia faba Forisomes, therefore a putative Forisome gene (vff1, 2056 bp)
encoding a Vicia faba Forisome protein of 79 kDa (VFF1).
1.3 Kb
3‘-RACE 5 ‘-RACE
M 1 2 3 4 M 3 4
1.3 Kb
5‘-RACE BA
0.8 Kb
3‘-RACE 5 ‘-RACE
M 1 2 3 4 M 3 4
1.3 Kb
5‘-RACE
1.3 Kb
3‘-RACE 5 ‘-RACE
M 1 2 3 4 M 3 4
1.3 Kb
5‘-RACE BA
0.8 Kb
3‘-RACE 5 ‘-RACE
M 1 2 3 4 M 3 4
1.3 Kb
5‘-RACE
Chapter III Results ___________________________________________________________________________
48
+1
Reverse transcription RT-PCR conducted using total RNA, which was obtained from Vicia
faba young leaves and stems (III-1.7), confirmed at the same time that the putative vff1 gene
was expressed in this plant tissue.
+1 C12 GA ATGTCCTT TTCTAACTCA ACTGCTGCTG CT...ACTGG CACTTTGGTT F04 GA ATGTCCTT TTCTAACTCA ACTGCTGCTG CT...ACTGG CACTTTGGTT E06 GA ATGTCCTT TTCTAACTCA CCTGCTGCTG CT...ACTGG CACTTTGGTT C07 GA ATGTCCTT TTCTAACTCA CCTGCTGCTG CTGCAACTGG CACTTTGGTT C09 GA ATGTCCTT TTCTAACTCA CCTGCTGCTG CTGCAACTGG CACTTTGGTT D06 GA ATGTCCTT TTCTAACTCA CCTGCTGCTG CTGCAACTGG CACTTTGGTT C12 CAAC...... ...ATGGTGG CAACGGCACT AATAAT...A GT TTGATCCA F04 CAAC...... ...ATGGTGG CAACGGCACT AATAAT...A GT TTGATCCA E06 CAAC...... ...ATGGTGG CAACGGCACT AATAAT...A GT TTGATCCA C07 CAACTTCAAC ATGGTGGTGG CACTGCCACT AATAATAACA GTTTGATCCA C09 CAACTTCAAC ATGGTGGTGG CACTGCCACT AATAATAACA GTTTGATCCA D06 CAACTTCAAC ATGGTGGTGG CACTGCCACT AATAATAACA GTTTGATCCA
Figure III-7 Sequence alignment of 5’- RACE PCR products of Vicia faba cDNA. The arrows at +1 position in the cDNAs sequences represent the first nucleotide of the coding region. Letters in bold indicate alternatives initiation codons. Sequences from the clones C12, F04 and E06 belong to ORF1 while the others represent sequences from ORF2. Figure III-8 RT-PCR amplification product of full- length Forisome Vicia faba cDNA. Total RNA was isolated from Vicia faba young leaves and stems (III-1.7). 10µl of sample were subjected to electrophoresis onto a 1.2% (w/v) agarose gel. Primers were designed based on the cDNA sequence flanking the translational start and stop codons. M: 1Kb DNA ladder (Invitrogen).
2050 bp
M
2050 bp
M
Chapter III Results ___________________________________________________________________________
49
1 MSFSNSPAAA TGTLVQHGGN GTNNSLIQKT ATSSHPHHKA NNYLPNPFEL
51 HDSHILDKVY LTHVTDDEFC DTDIIFDLVS TLILQSNT QI PVTGFKPDFP
101 TLKLISCQ MI TTRSVAHCVH QTTLWILQNL RSYSWDAKAL ITLAAFTLEY
151 GNYLQLNRVT ATDPI GNSLR QLNQIQTRKI STDIPELVNF IVHKLLHLKE
201 WAAWSAEGYD PEDVPALTEA LQEIPVFVYW TIASIVAS TG NLVGVSDYNL
251 SEYRERLSGI VQKLVVHLNN CKLQISYIDD LFNRRKIF DK PKDIVDCLKA
301 LIHHNGADSP QIYEGAIHVK TGLEVFR HKH VLMFISSLDS IEDEISLLNS
351 IYERLQENSK ESIKGFKKED FKILWIPIVN NWDDIRKE RF RALKSGIKWY
401 AVEYFYELPG HRIITDPERI GYIGNPIIPV FNPHGYIT NI DAMDLIFQWG
451 IDAFPFRKSD GIDLTFK WKW LWDVIKKATP GLQVKGDRYI FIYGGTNNKW
501 IQDFTL ELEK IKRHETLKRA DVIIDNYQLG K DDPNRVPSF WIGVERKKQN
551 KKHQEAVDCE IQDIVKSLF C LKRDPQGWVI LSKGQNIKLL GHGEPAYQTL
601 AEFQN WKDRV LEKEGFDIAF KEYYEMKAKE LSGREPCEVV NVDTYSSNVI
651 ATIA CPNPMC GRVMEVSSVH YKCCHRDEPN NFGV
Figure III-9 Vicia faba Forisome-1 (VFF1) predicted amino acid sequence. Comparative analysis of the resulting data enabled direct identification of 10 peptides sequences from Vicia faba Forisomes proteins. Letters in bold indicate alternatives initiation codons for translation. Underlined sequences match to peptides sequenced from purified Vicia faba Forisomes. Red letters indicate a carboxyterminal succession of several cysteines. A coiled-coil motif is represented by reverse shaded letters. The standard single-letter code is used.
Vff1 cDNA obtained by RT-PCR was aligned against the reconstructed full-length cDNA
sequences from the 3’- and 5’-RACE products verifying the ORF1. However, after several
attempts, the second open reading frame (ORF2) could not be confirmed. The full-length vff1
(2352 bp) cDNA had 57 bp of 5’- untranslated region (5’-UTR), an open reading frame of
2056 bp, and a 3’-untranslated region (3’-UTR) of 239 bp. The open reading frame had two
ATGs within the first 330 bp (Figure III-7 and III-9). The sequence context near the first start
codon had matched well with the sequence context conserved around the initiation codon for
plant genes (Lütcke et al., 1987; Joshi, 1987; Cavener and Ray, 1991). A putative
polyadenylation signal, AAATAA (Heidecker and Messing, 1986), preceded poly(A)+ by 21
bp. The deduced amino acid sequence had 684 amino acid residues (Figure III-9) comprising
10 of the peptides that were sequenced (II.2.2) from purified Vicia faba Forisomes (Figure III-
9). There is no recognizable signal sequence (Emanuelsson et al., 2000) based upon sequence
analyses.
Chapter III Results ___________________________________________________________________________
50
A Coiled-coil motif
F I S S L D S I E D E I S L L N S I Y E R L Q E N S K E S I K
B Amphipatic α-helix
Figure III-10 Representation of coiled-coil motif and amphipatic αααα-helix comprised in the VFF1 protein sequence. (A) heptad repeat (abcdefg) representative of coiled-coil structural motifs depicted in shaded letters. Nonpolar amino acids are indicated in blue letters whereas polar amino acids are represented by red letters. (B) amino acid sequence of the coiled-coil motif (A) depicted as an amphipatic α-helix. Hydrophobic amino acids residues are characterized by blue small boxes, hydrophilic amino acid by red letters
Considering the importance of Ca2+ in the regulation of the comformational change in vitro,
Ca2+-binding motifs where searched within the Forisome cDNA sequence by comparison with
the European Bioinformatics Institute database (www.ebi.ac.uk/InterProScan/). However, no
evidence of the presence of a conserved motif was retrieved. Motif searches against the
PROSITE, Pfam, and Smart databases, after exclusion of patterns with a high probability of
occurrence, did not detect any known motifs. Nevertheless, further attemps to identify well-
a b c d e f g a b c d e f g a b c d e f g
Chapter III Results ___________________________________________________________________________
51
known motifs were performed using computer analysis (http://www.ch.embnet.org/software/
COILS_form.html; Lupas, A., 1996). COILS is a program that compares a sequence to a
database of known parallel two-stranded coiled-coils and derives a similarity score. By
comparing this score to the distribution of scores in globular and coiled-coil proteins, the
program then calculates the probability that the sequence will adopt a coiled-coil
conformation. This analysis revealed another structural motif, an amphipathic α-helix within
the coding region of VFF1 comprising a coiled-coil domain (Figures III-9 and III-10).
The α-helical coiled coil is one of the principal subunit oligomerization motifs in proteins. Its
most characteristic feature is a heptad repeat pattern of primarily apolar residues that
constitute the oligomer interface. The pattern of repeating hydrophobic residues at positions a
and d of the heptad repeat (denoted abcdefg) are responsible for coiled-coil formation. The
presence of a continuous interface of hydrophobic amino acids along the length of the helices
provides a major source of stability to the fold as the hydrophobes pack in a knobs-into-holes
fashion shielded from the bulk solvent (Crick, 1953).
In addition, motif searches against the Smart database, after inclusion of sequences (outlier
homologues) that are often difficult to detect using HMM methodology, retrieved a 100%
identity within the VFF1 amino acid sequence (167-395) to an unknown domain
(d1gw5a_domain; http://smart.embl-heidelberg.de/) which belongs to the ARM superfamily.
Previous studies reported by Huber et al. (1997) indicated that each ARM repeat forms a
trihelical structure that folds into a superhelix, and six ARM repeats are proposed to constitute
a protein interaction domain.
A so-called low-complexity region – ISSLDSIEDEISLL – for which no structure can be
predicted with the algorithms available (Schultz et al., 1998) was also identify within the
VFF1 amino acid sequence (335-348). Wright and Dyson (1999) reported that these low-
complexity regions only acquire a rigid structure (e.g., an α-helix) upon interaction with
another protein partner or oligomerization into a protein complex.
III.1.7 Isolation of total RNA from Vicia faba
Total RNA was isolated from young leaves and stems as described in the material and
methods section (II.2.4). RNA concentration and purity of isolated total RNA was determined
by spectrophotometry. The yield of total RNA isolated ranged between 1-1.5 mg per gram of
biomass. The integrity of the RNA was checked on a 1.2% (w/v) agarose gel (Figure III-11).
The 28S and 18S ribosomal RNA were visible as distinct bands whereas the mRNA was
present as background smear indicating the quality and integrity of the isolated total RNA.
Chapter III Results ___________________________________________________________________________
52
28S
18S
1
Figure III-11 Analysis of total RNA isolated from Vicia faba. Total RNA (II.2.4) was isolated from Vicia faba young leaves and stems and separated on a 1.2% (w/v) agarose gel (II.2.7.4) containing 2.4 M formaldehyde. The major ribosomal RNA species (28S and 18S rRNA) are indicated. 1: 2µl of total RNA.
III.1.8 Identification of the transcription start sites for vff1
To identify the transcription start sites for vff1 gene, a SMART RACE analysis was
performed by using three reverse oligonucleotides derived from the coding region (821, 735
and 637 bp downstream of the start codon) and two adapter primers provided with the kit for
the primary and nested PCR. A weak smear on the agarose gel was observed in the first PCR
with outer primer AP1 and reverse primers GSP1 or GSP2 by using the total RNA from Vicia
faba young leaves and stems (data not shown).
Figure III-12 RACE analysis of the transcription start site of the vff1 gene. Analysis of the nested PCR (II.2.6) products on a 1.2% (w/v) agarose gel (II.2.7.4). Three RACE assays were conducted by using two adapter primers (AP1and AP2) and three vff1 GSPs (GSP1, GSP2, and GSP3, corresponding to nucleotide residues 821–846, 735–756, and 637–662, respectively). In the first RACE analysis, AP1-GSP1 and AP1-GSP2 primer pairs were used in the primary and secondary PCRs, respectively. Total RNA was extracted from Vicia faba young leaves and stems (III.1.7). Nested PCR product was cloned into the pCR®2.1-TOPO vector (Invitrogen), and subsequently verified by sequence analysis (II.2.7.6).
1018
M 1 2
Nested PCR
506
bp
1018
M 1 2
Nested PCR
506
bp
Chapter III Results ___________________________________________________________________________
53
The primary PCR products were used as template for two different nested PCRs by using AP2
and GSP2, or GSP3. Only one single product was obtained from both nested PCRs (Figure
III-12). This result indicates that vff1 transcript may have one single translational site,
although it does not rule out the possibility that another transcription site is located closely to
the first one that PCR products were inseparable on the agarose gel electrophoresis. The 5’-
RACE product was cloned into pCR®2.1-TOPO (Invitrogen). The sequencing of 10 different
clones, randomly selected, revealed that they share the same sequence (data not shown). The
first base of this product was therefore assigned position -57, and it is located 57 bp upstream
of VFF1 start codon.
III.1.9 Molecular characterisation of the vff1 genomic clone
Application of LD PCR (II.2.6.3) on Vicia faba genomic DNA using sense and antisense
primers derived from the cDNA resulted in isolation of two genomic fragments of ~3 and 4.5
kb (Figure III-13).
Figure III-13 LD PCR on Vicia faba genomic DNA using primers derived from the cDNA. Genomic fragments of ~3 and 4,5 kb were amplified from 250-500 ng of Vicia faba genomic DNA (II.2.3) with sense and antisense primers derived from the Forisome cDNA. 5µl of each sample were analyzed on a 1.2% (w/v) agarose gel (II.2.7.4). Numbers indicates the PCR products samples amplified using different PCR conditions (Expand Long Template PCR System, Roche). 1: optimized buffers for amplification of fragments larger than 15 kb, 2: from 12 – 15 kb, and, 0.5 – 12 kb, respectively. M: 1Kb DNA ladder (Invitrogen).
The ~3 kb band was selected for sequence analysis and compared to the cDNA sequences
obtained from analysis of the RACE products. The entire genomic sequence of the Forisome
gene (vff1) was obtained by PCR displaying a 2710-bp fragment. Sequencing of the resulting
clones followed by the alignment of the cDNA and the genomic sequences of the vff1gene
revealed the presence of six introns flanked by GT and AG canonical intron border sequences.
These intervening sequences are located in the coding region and their lengths are 201, 92, 82,
89, 113, and 83 bp, respectively (sequences not shown). Sequences of the exons are identical
M 1 2 3bp
40723054
M 1 2 3bp
40723054
Chapter III Results ___________________________________________________________________________
54
to sequences obtained with cDNA. The ORFs of the Forisome cDNA and genomic sequences
were resolved. The translated amino acid sequence predicted a protein of 684 residues with a
molecular mass of 79-kDa and an isoelectric point (pI) of 6.79. Three N-glycosylation sites
and several protein kinase phosphorylation sites were predicted by protein sequence analysis
using the PROSITE algorithm.
III.2 Molecular cloning and characterization of the vff1- promoter
III.2.1 Construction of genome walking libraries
Genomic DNA extracted from Vicia faba young leaves was digested with DraI, HpaI, MscI
and SspI restriction enzymes. Short adaptor DNA sequences provided in the Genome Walker
kit (II.1.2) were ligated to the blunt-end DraI-, HpaI-, MscI- and SspI-digested genomic DNA
fragments and generating four Genome Walking Libraries (Figure III-14)
Figure III-14 Analysis of Vicia faba genomic DNA digestion. A very high average molecular weight indicates the high quality of the isolated Vicia faba genomic DNA necessary in the construction of the GenomeWalker Libraries. Separate aliquots of genomic DNA were thoroughly digested with five different restriction enzymes that recognize a 6-base site, leaving blunt ends (in this case, DraI, HpaI, MscI, SspI and EcoRV). Three of these restrictions enzymes generated adequate Genome Walking Libraries. 5µl of each sample were analyzed on a 1.2% (w/v) agarose gel. DraI: DraI library, HpaI: HpaI library, MscI: MscI library, SspI: SspI library, EcoRV: digestion with EcoRV, control: HDL-EcoR V library provided with the BD GenomeWalker Human Kit, M: 1Kb DNA ladder (Invitrogen).
III.2.2 Identification and cloning of vff1 gene 5’-flanking region
While attempting to isolate and clone the genomic 5’-flanking sequence of vff1 using
Genome-Walking PCR (Clontech Laboratories, Palo Alto, CA) (II.2.6.4) coupled with a
combination of adapter and gene specific primers (GSP; Figure III-15A), a portion of a
putative vff1 promoter was cloned as follows. Primers (GSP1 and GSP2) were designed based
DraI HpaI MscI SspI EcoRV M controlDraI HpaI MscI SspI EcoRV M control
Chapter III Results ___________________________________________________________________________
55
GSP2
GSP2
GSP1
on the 5’ end of the Forisome cDNA. The first PCR amplification of genomic DNA, using
GSP1 and an adaptor specific primer, produced two DNA fragments from four genomic DNA
libraries, with lengths ranging from 0.7 to 2.7 kbp (Figure III-16). A second PCR
amplification of the first-round PCR product with nested GSP2 and adaptor primers yielded
relatively shorter DNA fragments that were cloned and sequenced. The GSP2 primer
sequence at the 3’ end of this fragment flanked DNA sequence identical to the 5’ region of
vff1 cDNA. A putative TATA box (Figure III-15B, square) and a transcription initiation site
(Figure III-15B, asterisk) were predicted in the 5’-upstream region of this DNA fragment by
the Neural Network Promoter Prediction (http://www.fruitfly.org/seq tools/promoter. html;
Reese and Eeckman, 1995). These results initially suggested that the isolated fragment
contained the promoter regions of vff1.
A
TCTCTGAAATTTGAGTCATCATGTCCTTTTCTAACTCACCTGCTGCTGCTACTGGCACTTT
GGTTCAACATGGTGGCAACGGCACTAATAATAGTTTGATCCAAAAGACTGCCACTTCCTCA
CATCCACACCACAAAGCTAATAATTACTTGCCAAATCCATTTGAACTTCATGATTCTCACA
TTTTGGACAAAGTCTATCTCACTCATGTCACGGATGATGAATTTTGTGATACTGATATCAT
TTTCGACCTTGTCTCCACTCTTATACTTCAGAGTAATACACAGATTCCTGTCACCGGTTTC
AAACCAGATTTTCCTACATTGAAGCTGATTTCTTGTCAGATGATAACTACACGTAGTGTTG
CACACTGCGTTCACCAAACAACACTATGGATTCTCCAAAACCTGAGATCCTACTCTTGGGA
TGCAAAAGCACTTATAACTCTAGCTGCATTCACTTTGGAGTATGGAAACTATTTGCAACTT
AATAGGGTCACAGC…
B
…gttttgataagaataaaataataactttagtttcattacacacttccatt ccttgacaca
tggtatcaaatcaaacgtctcttaaactctcatgcattcttccacactata aataagttat
gcaacttcaccaaccaatcc*ACCATTCTTAAACCAAACCTAAGAAGTGTT ACTACTTCTC
TGAAATTTGAGTCATCATGTCCTTTTCTAACTCACCTGCTGCTGCTACTGGCACTTTGGTT
CAACATGGTGGCAACGGCACTAATAATAGTTTGATCCAAAAGACTGCCACTTCCTCACATC
CACACCACAAA…
Figure III-15 Nucleotide sequence of the 5’-flanking region of the vff1 gene. A. Sequence of the 5’ region of vff1 cDNA. The first ATG in the coding region is highlighted by reverse shading. The 5’-untranslated region is underlined. The sequences used to design GSP1 and GSP2 for genome-walking PCR are shown in bold letters, and their positions are indicated by arrows. B. DNA sequence of a portion of the 2.7kb product obtained by genome-walking PCR. The predicted TATA-box and transcription initiation site are highlighted by a square and an asterisk, respectively. The promoter sequence is shown in small letters and the untranslated region in the putative cDNA is underlined. The entire 5’-upstream sequence of the promoter region is not shown.
Chapter III Results ___________________________________________________________________________
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Figure III-16 Primary and secondary BD GenomeWalker™ PCR. Primary and secondary (nested) PCR was performed using BD Advantage 2 Polymerase Mix (II.1.2) and the cycling parameters as described in the manufacture’s protocol. The GSP1 and GSP2 primers used were designed based on the 5’ end of the Forisome cDNA and are described in Figure III-15. DraI: DraI Library, HpaI: HpaI Library, MscI: MscI Library, SspI: SspI Library, M: 1 kb DNA ladder (Invitrogen), 1: positive control HDL-EcoRV amplified using positive control primers PCP1 and PCP2 provided with the BD GenomeWalker Human Kit (II.1.2), 2: negative control: not adding DNA library.
III.2.3 Potential regulatory sequences in the vff1 promoter
A search for cis-acting elements in the 5’-upstream region of vff1 (-2070 to +1) using plant
cis-acting regulatory DNA elements (http://www.dna.affrc.go.jp/PLACE/signalscan.html;
Higo et al., 1999) indicated that the putative promoter contained several characteristic
elements of eukaryote promoters, including motifs associated with tissue specific expression
like CAAT-BOX1 ( Shirsat et al., 1989) and GATA box (Lam, E. et al., 1989) (Figure III-17)
but no elements that appeared to contribute to expression in phloem were retrieved.
Nevertheless, a sequence comparison of the vff1 promoter and other phloem-specific
promoters revealed another motif in the vff1 promoter that was homologous to sequences
conserved among several other phloem-specific promoters. The first element identified was a
GATA motif known to be important for phloem-specific gene expression from the rice tungro
bacilliform virus RTBV promoter (Yin et al., 1997a). The same motif was found in the
promoters of GS3A (Brears et al., 1991), the Arabidopsis plasma membrane H+-ATPase gene
(AHA3; DeWitt et al., 1991), a potato invertase gene (Hedley et al., 2000) and the Rplec2
(Yoshida et al., 2002). Sequences matching the consensus sequence of the GATA motif,
A(N)3GATA, were found at –1788 (ATATGATA), –497 (AATGGATA) and –154 (ATATG
ATA) in the vff1 promoter (Table III-2).
DraI HpaI MscI SspI M 1 2 M DraI HpaI MscI SspI 1 2
Primary PCR Secondary PCR
3054
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bp
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Table III-2 Sequence comparison of GATA motifs present in the vff1 promoter and other phloem-specifc promoters
Promoter Sequence Position Origin vff1 ATATGATA –1788 Vicia faba AATGGATA –553 ATATGATA –210 Rplec2 ACATGATA –561 Robinia pseudoacacia AATTGATA –224 ATTTGATA –186 RTBV AGAAGATA –143 RTBV GS3A ATTAGATA –589, –557, –366 Pisum sativum AAGTGATA –201 AGTAGATA –115 AHA3 ACATGATA –294 Arabidopsis thaliana AGACGATA –166 AGATGATA –126 Nucleotides conserved between the promoters are underlined. Positions of sequences are indicated relative to the transcription start sites.
Another phloem specific motif of 13 bp in length was identified which has been described to
be highly conserved among four phloem-specific promoters (Hehn and Rohde, 1998),
originating from a pea glutamine synthase gene (GS3A), coconut foliar decay virus (CFDV),
rice tungro bacilliform virus (RTBV), and an Agrobacterium rhizogenes rolC gene as well as
the a Robinia pseudoacacia inner-bark lectin gene (Rplec2; Yoshida et al., 2002). In the vff1
promoter, a sequence that showed 69% homology with the 13 bp motif of the CFDV, GS3A
and RTBV promoters was located 524 bp upstream of a TATA box (Table III-3). Taken all
together, the occurrence of several elements specific for expression in the phloem is
noteworthy.
Table III-3 Sequence comparison of the 13 bp motifs present in the vff1 promoter and other phloem-specific promoters
Promoter Sequence Distance from Origin TATA box (bp) vff1 ATAAGGAGGAACA 524 Vicia faba CFDV ATAAGAACGAATC 169 CFDV GS3A ATAAGACAGAATC 255 Pisum sativum RTBV TTAAGTACGAATC 129 RTBV Rplec2 ATTAGAAAGAAAA 238 Robinia pseudoacacia rolC TTAAGTACAGACA 123 Agrobacterium rhizogenes
Nucleotides conserved in at least four promoters are underlined.
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CAAT BOX1
GATA BOX
Asc I
Nco I
TATA BOX
GGCGCGCCTTACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTAACTATAAAACTTTTTTCTTCATCAAAAA
CCCTAAAAACAATTTCTCCTTCATTTTTATATTTTATGCATAATTATTATTGTAATTCATATTCAATGGTTTGTA ATAATAATTAGTACTTTATTGCTTTCTTCCCCCTATTCACAAAATTGTACCCCTCATTCCTAAAGCGTTGTAATA ACATATGATATTTAAATTCTGCAATTTATCGCTTTCGAGTAATTAGGATTTGCATGACAAGACTAAAACCAAACC ATAGAAAACCATAATTCAAAGATAACATATATGAAAATAAAACACTTTGCACACTTGCACCTCTAGGGTAACTCA CACTTATTGCCTCGGATATAAACAGTCATGTCTCTCGAATGTGAGATGTTTTAGCAAAGACCCTCGATGAAAGAT CGTAGTTCCTCGATGACCCTTCAATGTTGTCTTCGGTAAACGATCTTAGTCCCTTATGACAATTTCTAAAACATC TTACAAGTTGTCTTCTATGACTGATGATATGGTTCTCTATCAATCAATGGTAATATAGTACTATCTAAAGGAGGA TGAAAATAACAAAGACTTTTTGAGTAGGTAGATTGCTCGTAATTGCTTGCTCACAAACAAAATCATTCCACACCT CACTTTCAAAATACTTTCCAAAACAAACATTTTGTATACATTCTCAACGATAAGAGAACCATTACAAAATTGCTT ACATTGACATTTTCAAAGCATCTTTCCAAAACACTCTTTTTAACAAATATTTTCAAAACTAACACTTTGTATACA TTCTCAACGATGCGAGAATCATTGCAAAGTTTTCTAAACTTAGGCGTTTTCAAAATATTTTCAAACTTACACAAA CACAAAGTATTTCAAAGTGAGCTAGCAATTAAGTGTACTACATTCTCAACGATGCGAGAATCATTGCAAAGTTTT CTAAACTTAGGCGTTTTCAAAATATTTTCAAACTTACACAAAAACAAAGTATTTCAAAGTGAGCTAGCAATTAAG TGTACTACGGATGAAAATGGTACTAACACATTCATTTTTCATAACCTACCCCGAACTCAAGAAAATTCTCAAAAG GGGGTATTTCGTATTCTTTTTACCCTTTTCTTTATTAGATAAAAGAAAAGTCAGTGGTGACTCTTGCTATCCGCA ATATTTGCGATAAAACACAAAACAATCAAGTCAATTCTCCCACTATATTATGCTATTATATTTAGTTTNTAAGA CATCATTTTTCACAACAATAGAAATGAAACAACTCAAACTTATATAACCAAACCATGCAACAAATTGATGGCTAT AATGTAAACACAAAATCTCTCTTTCATAAGTTCACCAAAAAATACTCACGTATTTTCAATTCCATTTTCTATAAA CGTTAATGTTTGTCCTTTTGTCTCTCTCCACATAAGGAGGAACATTTATGTGGACCAAATATTTAAAGGGTATGA TTCCTTATATAATATTTAATAATGGATAGTATGATTCTTGACTTGTAGATATGTTGATTCAATCTATAAGACAAA ATCTAAAATAAGACAAAATTTGAAGAACTAATTAATAAATAAAATAAAATTTGATTCGATTAAAACCATCGGTAC TTTCCTTCAATAATATAGTTATATAAAAAAATGAAATAGTAGTAATAGTATATGTCTATCTCTTTACTCTTCCCA AAAGCTGAGGGTTGGCATTATTCTCTTGATAGAGTTGCTTCAATTAATTAACGTAAAGGAATGGGGTATGTTAGA ACAACAAATCATTGCAGCGAGTTTCACGTTTTGAGACCATATGCATTAGATTCAGATGCTACTATATGATAACAT TGTCTTTTGTTTTGATAAGAATAAAATAATAACTTTAGTTTCATTACACACTTCCATTCCTTGACACATGGTATC AAATCAAACGTCTCTTAAACTCTCATGCATTCTTCCACACTATAAATAAGTTATGCAACTTCACCAACCAATCC* accattcttaaaccaaacctaagaagtgttactac ttctctgaaatttgagtcaccATGGTGACTCAAATTTCAGAGAA Figure III-17 Nucleotide sequence of vff1 promoter and the 5’-flanking region of the vff1 gene. Two genomic DNA fragments, corresponding to the vff1 gene 5’-flanking region, were amplified by a PCR-mediated genome-walking technique (II.2.6.4) using DraI, HpaI, MscI and SspI digested Vicia faba genomic libraries, respectively. Both DNA fragments were cloned into pCR®2.1-TOPO (Invitrogen) vector and sequenced (II.2.7.6). The DNA sequence of the 2.7kb product is shown. The ATG start codon is highlighted by reverse shading and the 5’UTR, obtained by RACE analysis, is indicated in small letters. The predicted TATA-box and transcription initiation site are highlighted by a square and an asterisk, respectively. Putative cis-elements, CAAT Box1 and GATA motif-like elements are boxed and the GATA and 13 bp phloem-specific-element-like motifs are indicated in red letters. Nucleotides conserved between phloem specific GATA (Table III-2) and 13bp motifs are indicated in bold letters (Table III-3). The sequences used to design specific primers for cloning the putative promoter sequence (II.2.6.4) are highlighted in bold, and their positions are indicated by arrows.
III.2.4 Characterization of vff1 promoter in transgenic tobacco
III.2.4.1 Cloning of vff1 promoter into the pTRAk-GUS vector
To characterize tissue specificity of vff1 promoter expression, the 5’-upstream region of this
gene (-2070 to +1) was cloned into a promoter-less vector encoding a GUS gene to create a
reporter vector designated pTRAk-vff1P:GUS (Figure III-18). The vff1 promoter sequence in
the 5’-upstream region of vff1 gene (-2070 to +1) was amplified using forward (5’-
Chapter III Results ___________________________________________________________________________
59
TTGGCGCGCCTTGACTTGTAGATATGTTG-3’) and reverse (5’-TTGGCGCGCCTCTAT
AAACGTTAATGTTTG-3’) primers, which contained AscI and NcoI restriction enzyme sites
at their 5’ ends. The obtained PCR products were digested with AscI and NcoI and cloned into
the shuttle vector pTRAk-GUS containing the uidA (GUS) gene. The pTRAk-GUS vector
was constructed as follows: the GUS-terminator cassette (~2,1 kb) was excised from pUC103-
GUS with NcoI and XbaI and subsequently subcloned into NcoI and XbaI sites in pTRAktrfP
binary vector (Thomas Rademacher, Institut für Biologie VII, RWTH Aachen, Germany)
replacing the DSRed gene. The CaMV 35SS promoter was removed from the pTRAktrfP
vector by excision with AscI and NcoI restriction enzymes and replaced with the putative vff1
promoter in order to drive the expression of the GUS gene.
Figure III-18 Strategy for cloning the vff1 promoter-Gus expression cassette into pTRAk. Promoter fragment was ligated upstream of the Gus coding sequence as transcriptional fusion. P35S: 35S promoter from CaMV with duplicated enhancer; pA35S: 3' UTR of CaMV; GUS: uidA coding region.
The correct sequence of the construct was confirmed by sequence analysis (II.2.7.6) and the
plasmid DNA was introduced into Agrobacterium tumefaciens strain GV3101 by
electroporation (II.2.9.2). A. tumefaciens harbouring this reporter-gene cassette were used for
transformation of N. tabacum cv. Petite Havana SR1 plants (II.2.10). A plant expression
vector pTRAk containing the uidA gene driven by a doubled enhanced constitutive 35S
promoter from the Cauliflower mosaic virus (CaMV; Kay et al., 1987) was used as positive
control. The obtained recombinant clones were analyzed for the presence of the cDNA insert
AscI
3’
3’ pTRAk-35SS:DSRed NcoI XbaI AscI
5’ Dsred pA35S 35SS
3’ pUC 103:GUS NcoI XbaI
5’ GUS pA35S 35SS
vff1P
pTRAk-35SS:GUS NcoI XbaI 5’
GUS pA35S 35SS
AscI
3’ pTRAk-vff1P:GUS NcoI XbaI
5’ GUS pA35S vff1P
AscI
NcoI/XbaI digestion
AscI/NcoI digestion PCR amplification followed by AscI/NcoI digestion
AscI NcoI
Chapter III Results ___________________________________________________________________________
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by colony PCR with vector- and vff1 promoter- specific primers and glycerol stocks (II.2.9.4)
were prepared for both recombinant A. tumefaciens strains.
III.2.4.2 Expression of vff1 Promoter-GUS fusions in tobacco plants
Vectors encoding the uidA gene driven by the vff1 promoter were transferred via
Agrobacterium transformation into N. tabacum as a model species. The promoter activity of
the GUS fusions was evaluated in stably transformed kanamycin-resistant plants by
histochemical staining. GUS activity was assayed on sections of leaves and thin cross-
sections of tobacco leaves, stem, roots and flowers (II.2.10.6), and confirmed at different
stages of development, either on plantlets grown in vitro or on greenhouse-grown mature
plants. The primary transformants showed detectable levels of GUS activity. The same pattern
of cell-specific expression for vff1P-GUS in transgenic tobacco plants was observed for three
independently transformed plants. In addition to regenerated T0 plants, the selfed T1 progeny
(five plants for each transformant) were similarly tested for their GUS expression patterns and
the same results were obtained. Hence, it was demonstrated that GUS expression dictated by
the vff1 promoter in transgenic tobacco plants was transmitted to the next generation. The
integration of the transgenes in the plant genome was further confirmed by PCR on individual
T1 progeny plants using primers directed against the vff1 promoter (data not shown).
III.2.4.2.1 Tissue specificity of vff1 promoter in transgenic plants
Histochemical staining for GUS activity showed that the developmental expression of vff1
promoter-GUS fusions was concentrated in the vascular tissues of all transgenic tobacco
organs, including leaves, stems, roots and reproductive organs. A similar vascular pattern was
observed both on plants grown in vitro and in the greenhouse, at different stages of
development (data not shown).
The expression of GUS activity was observed in stems of vff1P-GUS transformed tobacco
plants (Fig. III-19A, B and C). A special feature of tobacco stem is that it contains two groups
of phloem tissues – the internal and external phloem – which are located along the two sides
of the xylem. Most other dicotyledonous plants only have external phloem. Stems sections
from first internodes of 6-week-old vff1P-GUS tobacco plants showed GUS activity restricted
to both types of phloem cells but not in any other cell types of the stem, including epidermis,
xylem, and ground tissue cells of pith and cortex (Fig. III-19A). At a higher magnification
(Fig. III-19B), the cellular localization of GUS activity was found to be highly specific. A
group of three to four cells residing at the center of phloem tissue clusters were highly stained
whereas the adjacent phloem parenchyma cells and vascular cambium cells show little or no
stain. The remaining cell types of the vascular cylinder, including xylem fiber, xylem
parenchyma cells, pith parenchyma cells, pericyle, and endodermis cells were not stained.
Chapter III Results ___________________________________________________________________________
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It was observed that two to three pairs of sieve elements and companion cells were localized
at the center of each phloem tissue bundle, and GUS activity was only visible in the
companion cells (Fig. III-19C). In addition, a faint GUS activity was also observed in some
sieve elements of young phloem tissue (data not shown).
Figure III-19 Expression of vff1 promoter-GUS fusions in transgenic tobacco. Histochemical analysis of GUS activity in transgenic tobacco plants harbouring the 2070-bp vff1 promoter::GUS construct revealed a vascular-specific acivity of the vff1 promoter. A, thin-cross-section of stem indicating the GUS activity in the adaxial (adp) and abaxial (abp) layer of cortex (internal and external phloem, respectively); B, higher magnification of a cryocross-section of stem with a similar pattern of staining in vascular cylinder; C, companion cell (CC)-specific activity of the vff1 promoter. The GUS activity is absent from all other cells including sieve element (SE) and phloem parenchyma (PP). D, leaf sections showing a phloem specific expression of the reporter gene in the midrib (mr) and lateral veins (lv); E, higher magnification (x4) view of the leaf. F, cryocross-section (50µm) of petiole showing the GUS activity in the adaxial (adp) and abaxial (abp) layer of cortex. For cytological observations, after GUS staining with X-gluc and clearing in 70% ethanol (II.2.10.6), stained sections were visualized with a Leica microscope Typ:DMLFS (Leica, Wetzlar, Germany) with a JVC camera Typ: TK-C1360 (Tokio, Japan). Pi, pith; C, cortex; vc, vascular cambium; e, epidermis.
Phloem cell-specific expression of vff1::GUS activity was also observed in flowers in the
inflorescence meristem and young floral buds (Fig. III-20A). In young flowers and at the
distal tip of floral organs the reporter gene expression was also evident (data not shown). In
later stages, expression was localized to developing ovules (Fig. III-20B).
X
ad P
ab P
mr
lv
lv
mr
PP
SE
CC
X
B C
D E F
ad P
ab P
X
A
X
ad P
ab P
PiC
vc
e
lv
X
ad P
ab P
mr
lv
lv
mr
PP
SE
CC
X
B C
D E F
ad P
ab P
X
A
X
ad P
ab P
PiC
vc
e
X
ad P
ab P
mr
lv
lv
mr
PP
SE
CC
X
B C
D E F
ad P
ab P
X
A
X
ad P
ab P
PiC
vc
e
lv
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Figure III-20 Expression patterns of vff1 promoter-GUS fusions in transgenic tobacco flowers. Histochemical analysis of GUS activity (II.2.10.6) in transgenic tobacco flowers (II.2.10.4) revealed a phloem cell-specific expression of the vff1 promoter. A and B, thin-cross sections (II.2.10.6) of flower organs. The GUS activity is localized in the inflorescence meristem (A) and in the developing ovules (B). Stained sections were visualized with a Leica microscope Typ:DMLFS (Leica, Wetzlar, Germany) with a JVC camera Typ: TK-C1360 (Tokio, Japan).
To analyse the expression pattern in more detail, promoter-GUS studies were performed on T1
vff1P-GUS tobacco seedlings. After kanamycin selection of primary transformants (T0) the
plants were cultivated in the greenhouse. Self-fertilized seeds were collected from T0 plants
and were germinated on MS medium with kanamycin and the seedlings (T1 progeny) were
grown in vitro. GUS enzyme activity could be detected in different tissues of the seedlings but
not in cotyledon tissue, (Fig III-21A). Vff1::GUS roots showed the strongest staining for GUS
activity in dividing meristematic tissues (Fig. III-21D). As root development proceeded
through the zones of elongation and maturation, GUS activity become progressively restricted
to the stele. GUS activity was significantly reduced but still restricted to vascular tissues in
older roots (data not shown). In contrast, strong GUS activity was clearly detected in
vff1::GUS tobacco during the early stages of lateral root development. In lateral roots of those
plants, GUS activity was detected in the meristematic and elongation zones of vascular tissue
(Fig. III-21C, E and F).
Strong GUS activity was also detected in the first leaves and shoot apical meristem of
vff1::GUS seedling suggesting a preferential expression of vff1 promoter in developing
vascular tissue and especially in young phloem tissue of tobacco plants. Examination of the
root-shoot transition zone of vff1::GUS plants showed that GUS activity in the stele continue
as vascular bundles emerged in the stem (Fig. III-21B).
These results indicate (Fig. III-19-21) that the vff1 promoter directs a phloem-specific
expression of the GUS gene in transgenic tobacco plants. However, it is important to show
that the results are not due to differential staining anomalies. For comparison, transgenic
tobacco plants transformed with the same GUS gene but with a different plant gene promoter
were tested under identical conditions for the GUS assay. As expected, control transgenic
plants obtained with the p35SS::GUS construct exhibited GUS activity in all organs and
A B
Chapter III Results __________________________________________________________________________
63
tissues (data not shown), including cortex, phloem, xylem, pith tissue, trichomes and
mesophyll cells in aerial and root organs as previously reported (Odell et al., 1985). The
experiments confirm that the phloem cell expression pattern observed for vff1P-GUS
transgenic plants is highly specific and is not due to some unknown factors that may
differentially affect activity staining in our GUS assay.
Figure III-21 Vff1 promoter expression pattern in vff1P::GUS transgenic plants. Histochemical localisation of GUS activity in vff1P::GUS N. tabacum seedlings. GUS activity is indicated in transgenic tissue by an indigo dye precipitate after staining with X-Gluc. (A). twelve-day-old tobacco seedling grown on MS medium with staining in the leaves and at the base of cotyledons. (B) shoot apex of a 12-day-old seedling at higher magnification. (C) GUS staining throughout root apical meristem and lateral root. (D) Root apical meristem GUS activity in the meristematic and elongation zones. (E) GUS activity in lateral roots from seedlings. (F) GUS activity in growing lateral root. am, root apical meristem; c, cotyledon; e, elongation zone; l, leaf; lr, lateral root; m, meristematic zone; s, shoot.
III.2.4.2.2 Developmental regulation of vff1 promoter in transgenic plants
To determine if the vff1 promoter activity is developmentally regulated, T1 vff1P-GUS tobacco
seedlings were used to determine the expression pattern at different maturation stages.
In developing leaves there is a basipetal gradient in maturity, the distal end being more mature
than the base. The first evidence of GUS staining was visible near the leaf base, where the
tissue was more immature (Figure III-22). In young leaves, minor vein staining began at the
lamina base and progressed towards the tip as the leaves aged, in an opposite pattern as the
sink-source transition of photoassimilate transport (Turgeon, 1989). With increasing maturity,
staining becomes progressively restricted to regions of class-I, then to class-II veins, and to
A
B F D E
l
c
e
m
e
s
C
am
lr
A
B F D E
l
c
e
m
e
s
C
am
lr
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isolated regions of class-III veins. Staining became progressively less intense in the more
distal (mature) regions of the leaf. The onset of the sink/source transition in expanding leaves
seemed to delimit the staining towards the apical portion of the leaf (Figure III-22).
Figure III-22 Expression pattern of vff1 promoter in immature veins of leaves. A progressive and opposite, basipetal (tip-to-base) staining pattern in midrib and minor veins is evident in the three developing leaves, the most mature of which is stained in only the base while the following in maturity is stained to approximately the midway point. Basipetal maturation is a characteristic of features associated with the sink-to-source transition in leaves. Leaf tissue near the tip of a growing leaf that has completed the sink-source transition shows no staining. am: apical meristem of root; c: cotyledon; l1: first pair of leaves; l2: staining pattern in sink leaf; l3: staining pattern of immature veins in the proximal region of a leaf undergoing the sink-source transition.; l4: leaf in a late stage of the sink-source transition. Figure III-23 Expression pattern of vff1 promoter in veins of mature tobacco leaves. A, mature leaf tissue indicating a most apparent staining in class-I and class-II veins. The dye can also be seen in some patches of class-III veins. B, in class-III veins the staining is mainly localized in the junctions with class-IV veins (arrow). Class-V veins are not stained. Stained sections were visualized with a Leica microscope Typ:DMLFS (Leica, Wetzlar, Germany) with a JVC camera Typ: TK-C1360 (Tokio, Japan).
c
l4
l2
l3
l1
amc
l4
l2
l3
l1
am
I II
III
II
III
IV
V
A B
I II
III
II
I II
III
II
III
IV
V
III
IV
V
A B
Chapter III Results __________________________________________________________________________
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1 2 3 4 5 6 7 8 9 10 11 12 13 M
3054
506
bp
In mature leaves GUS activity was only detected in class-I (midrib) veins, and class-II veins
(branching from the midrib) (Figure III-23A). Class-III veins, which define large segments of
the vein network, were often stained. Staining was first evident in isolated patches of class-III
veins, often at branch points where class-IV veins merged with them (Figure III-23B). Class-
V veins being the most extensive in the leaf did not stain (Figure III-23B). Staining decreased
in intensity toward the finest veins. No staining was detected in mesophyll cells.
It was also noted that damaged tissue demonstrated intense staining in the wounded area,
presumably because GUS enzyme was released from the minor veins into the apoplast (data
not shown).
III.2.4.3 Deletion analysis of vff1 promoter in transiently transformed tobacco leaves
The resulting GUS activity in transgenic tobacco plants revealed that the DNA fragment
display phloem-specific promoter activity (III.2.4.2.1; Fig. III-19-21). This result suggests
that the cell-specific elements may be present in this sequence. To localize the responsible
elements/motifs for phloem-specific promoter activity, a series of thirteen truncated promoter
fragments (P-pTRAk-1 to P-pTRAk-13), starting from nucleotide -2070 to nucleotide -543 of
the upstream sequence, and ending in the first ATG of the coding region, were prepared by
PCR (Fig. III-24). The correct sequences of the promoter fragments were confirmed by
sequence analysis (II.2.7.6) and cloned unidirectional into a promoter-less GUS reporter
vector, pTRAk-GUS, and the fusion promoter-GUS constructs were transiently expressed in
Nicotiana tabacum SR1 plants (II.2.10.1) with the phloem-specific Coconut Foliar Decay
Virus (CFDV) promoter (Hehn und Rohde, 1998) as the positive control. Three to five
independent transient expressions were performed. Nevertheless, among all the PCR-
generated fragments there was no substantial difference in GUS activities.
Figure III-24 PCR amplification of truncated vff1 promoter-GUS fusions fragments. Different lengths of the vff1 promoter, starting from nucleotide -2070 to nucleotide -543 of the upstream sequence, and ending in a 5’ position relative to the ATG start codon of the vff1 coding region, were ligated with the uidA gene. Lanes 1-13: PCR amplification products (II.2.7.3) of the promoter fragments using P-pTRAk-1 to P-pTRAk-13 specific primers (Table II-2), separated on a 1.2% (w/v) agarose gel (II.2.7.4). Lane M: 1Kb DNA ladder (Invitrogen).
Chapter III Results ___________________________________________________________________________
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III.3 Bacterial expression and characterization of VFF1
III.3.1 Cloning of vff1 into the bacterial expression vector pGEX-5X-3
For the production of recombinant VFF1 protein in bacteria the entire vff1 cDNA (III-1.6) as
well as partial fragments (amino/carboxy terminus, center part) were amplified by PCR from
a Vicia faba RACE-Ready cDNA population (II.2.6.1). Oligonucleotides specific to the 5' and
3' end of vff1 (II.2.6.2) were used to obtain its full-length cDNA. Oligonucleotides used in the
PCR amplification of the N-terminal (+1 to 1317bp), center- (649 to 1707bp) and C-terminal
(1317 to 2056bp) domains as well as of the entire vff1 cDNA are shown in Table II-4 (II.1.8).
Restriction sites NcoI and NotI were introduced into the forward and reverse oligonucleotides
for cloning into pGEX-5X-3 bacterial expression vector (II.1.7.1 and II.2.6.2). The PCR
products were subsequently ligated into the NcoI/NotI sites of pGEX-5X-3 vector and verified
by sequence analysis (II 2.7.6).
III.3.2 Bacterial expression and purification of VFF1
In order to obtain VFF1 protein for immunization, the amplified sequences were fused to the
C-terminus of GST and expressed in E. coli BL21λDE3 (II.2.11.1) (Fig. III-25).
Figure III-25 SDS-PAGE and immunoblot analysis of affinity purified GST fusion proteins. Vff1 cDNA as well as its amino/carboxy terminus and center domains were subcloned into the pGEX-5X-3 vector (II.1.7.1 and II.2.6.2) and expressed in the E. coli strain BL21(λDE3) upon induction with IPTG. The cytoplasmic overexpressed fusion proteins were purified by glutathione affinity chromatography. Protein were separated on a 12 % (w/v) SDS-PAA gel and stained with Coomassie brilliant blue (A) (II.2.12.2) or blotted onto nitrocellulose membrane (B) (II.2.12.4). Immunodetection was carried out using 1:5000 diluted anti GST monoclonal antibody and GAMAP polyclonal antibody (1:5000). Detection was performed with NBT/BCIP for 1 min at RT. M: 5 µl of Prestained Protein Marker. 1: 10 µl of GST-VFF1-C-domain elution fraction; 2: 10 µl of GST-VFF1-M-domain elution fraction; 3: 10 µl of GST-VFF1-N-domain elution fraction and 4: 10 µl of full-length VFF1 elution fraction. N-domain: N-terminal protein domain, C-domain: C-terminal protein domain, M-domain: central potein domain.
VFF1
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The GST fusion proteins contained a Factor Xa cleavage site for removal of the GST fusion
part. When protein expression was induced with 1 mM IPTG at 30°C most of the fusion
proteins were produced as insoluble inclusion bodies (data not shown). To improve the
solubility of recombinant VFF1 protein (and subdomains) the following conditions were
employed: i. increase of culture volume to 4l, ii. decrease of IPTG concentration to 0.1 mM,
iii. decrease in the growth and induction temperatures from 30°C to 16°C after induction and
iv. increase of induction time to 12 hs. Another attempt to improve the yield of overexpressed
VFF1 was a step-wise inactivation of the proteases by increasing the temperature to 42°C for
1h, performed to minimize the degradation of the fusion proteins. Nevertheless, there was no
significant increase in the yield of the overexpressed fusion proteins even seeming to induce
the formation of inclusion bodies (data not shown).
The analysis of the elution fractions by SDS-PAGE (Figure III-25A) revealed the presence of
bands which sizes corresponded with the expected molecular weights of 104, 70, 67 and 60,3
kDa of the GST fusion full-length, N-terminal, C-terminal and center protein domains,
respectively.
Following the elution steps, a significant amount of the overexpressed fusion protein
remained bound to the matrix (Figure III-26, line 2). However, affinity purified fusion
proteins showed high purity in Coomassie-stained SDS-polyacrylamide gels (Figure III-26).
After purification, the elution fractions were dialyzed overnight in 1XPBS buffer and the final
protein concentration was determined by SDS-PAGE (II.2.12.2) using BSA of known
concentration as a standard (data not shown). The amount of purified GST-VFF1 ranged
between 0.7-100 µg per L culture volumes. Cleavage of the Forisome fusion protein from the
GST domain with Factor Xa was not successful probably due to an almost complete
degradation of the overexpressed protein (data not shown).
Figure III-26 SDS-PAGE analysis of affinity purified GST fusion VFF1. Affinity purified VFF1-GST fusion protein obtained from the GST expression system was resolved on 12% (w/v) SDS-PAA gel followed by staining with Coomassie brilliant blue (II.2.12.2). 1: 5 µl of sonified E. coli BL21(λDE3) cells containing the pGEX-5x-3 plasmid harboring VFF1-GST; 2: 5 µl of Glutathione sepharose 4B from AmershamPharmacia Biotech used for purification of GST fusion proteins; 3: 10 µl of the first elution fraction of VFF1-GST fusion protein; 4: 10 µl of the second elution fraction of VFF1-GST fusion protein; M: Prestained Protein Marker.
M 1 2 3 4
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Figure III-27 SDS-PAGE and immunoblot analysis of affinity purified recombinant VFF1. VFF1 cDNA was subcloned into the pGEX-5x-3 vector and overexpressed at a 4-liter fermentation scale (II.2.11.2) in the E. coli strain BL21(λDE3) upon induction with IPTG. The expressed fusion protein was purified using a Glutathione sepharose matrix. Proteins were separated on a 12 % (w/v) SDS-PAA gel and either stained with Coomassie brilliant blue (A) (II.2.12.2) or blotted onto nitrocellulose membrane (B) (II.2.12.4). Immunodetection was carried out using 1:5000 diluted anti GST monoclonal antibody and GAMAP polyclonal antibody (1:5000). Detection was done with NBT/BCIP for 1 min at RT. P: 5 µl of pellet; SN: 5 µl of cytoplasmic extract; FT: 5 µl of flow trough; W: 10 µl of wash fractions; C: 5 µl of matrix; E1, E2, E3, E4: 10 µl of elution fractions; M,c: 5 µl of Prestained Protein Marker together with 100 ng of GST protein as control.
With the aim to increase the yield of recombinant VFF1 protein fermentation at 4-liter scale
was carried out (II.2.11.2). The result of SDS-PAGE and immunoblot analysis of the elution
fractions of the affinity purified VFF1 are shown in Figure III-27 (A) and (B), respectively,
and revealed the presence of a band of approximately 100 kDa corresponding to the
calculated molecular weight of 104 kDa. A significant amount of other co-eluted bands were
also present on the Coomassie stained SDS-PAA gel. The most prominent co-eluted bands
run in a range between the molecular sizes of 24 and 33 kDa. These co-eluted proteins were
detected by immunoblot analysis demonstrating that these proteins probably representing
degradation forms of the recombinant VFF1 protein. As GST is known to induce dimerization
of proteins (Lim et al., 1994), upper bands were also immuno detected.
Attempts to purify soluble GST-tagged VFF1 (MW 104 kDa) at a fermentation scale with the
above mentioned parameters gave no improvements (Figure III-27). Therefore, gel slices from
the corresponding single band of recombinant GST-VFF1 fusion protein band were excised
from the SDS-PAA gel and send for polyclonal antibodies production in rabbits to
EUROGENTEC (Seraing, Belgium).
III.3.3 Characterization of GST-VFF1 fusion protein by immunoreativity toward
Forisome-specific mouse and chicken antisera
To assess whether the recombinant VFF1 proteins were recognized by the Forisome-specific
mouse and chicken antisera an immunoblot analysis was performed. Hence, the different
bacterial expressed VFF1 fragments together with 5 µg of isolated Vicia faba Forisomes as
P SN FT W C M,c E1 E2 E3 E4 P SN FT W C M,c E1 E2 E3 E4
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Chapter III Results ___________________________________________________________________________
69
positive control were used to assay their immunoreactivity. None of both preimmune sera
recognized either the recombinant fusion proteins or the isolated Forisome proteins from
Vicia faba (data not shown).
As shown in Figure III-28A, only co-eluted proteins were detected by immunoblot analysis
with the mouse antiserum. These proteins turned out to be not degraded forms of the GST-
VFF1 but unrelated bacterial contaminants as confirmed by the presence of the same pattern
of bands in a cytoplasmic extract of E. coli (data not shown). However, the immunoblot
analysis with Forisome-specific chicken antiserum indeed revealed the presence of bands at
the expected sizes of 104, 70, 67 and 60,3 kDa for the GST fusion VFF1 and N-terminal, C-
terminal and center- protein domains, respectively. It was also observed a strong reaction
against low molecular weight fusion proteins (Figure II-28 B, lanes 1 and 2). Nevertheless, no
cross hybridization of the Forisome-specific chicken antiserum to GST proteins could be
detected (data not shown) demonstrating that these proteins were degraded or perhaps
truncated forms of VFF1 recombinant protein. This result indicates that the Forisome-specific
chicken antibodies are recognizing epitopes present on the recombinant protein suggesting
that the isolated vff1 gene may encode one of the proteins involved in the constitution of Vicia
faba Forisomes.
Figure III-28 Characterization of GST-VFF1 fusion protein by immunoreactivity against Forisome-specific mouse and chicken antisera. GST fusion VFF1 together with the N-terminal, C-terminal and center- protein fragments of vff1 cDNA were subcloned into the pGEX-5x-3 vector and overexpressed at a 4-liter fermentation scale (II.2.11.2) in the E. coli strain BL21(λDE3). Proteins were separated on a 12 % (w/v) SDS-PAA gel (II.2.12.2) and electroblotted onto a nitrocellulose membrane (II.2.12.4). (A) Immunoreactivity of recombinant proteins toward Forisome-specific mouse antisera (dil. 1:2000). Immunodetection was performed with GAMAP(H+L) polyclonal antibodies (dil. 1:5000). (B) Immunoreactivity of the recombinant proteins toward Forisome-specific chicken antisera. Immunodetection was performed with RACAP(Fc) polyclonal antibodies. Detection was done with NBT/BCIP for 2 min at RT. 1: 10 µl of full-length VFF1 elution fraction; 2: 10 µl of GST-VFF1-N-domain elution fraction; 3: 10 µl of GST-VFF1-M-domain elution fraction; 4: 10 µl of GST-VFF1-C-domain elution fraction; F: 5µg of isolated Forisomes from Vicia faba; M: 5 µl of Prestained Protein Marker.
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Chapter III Results ___________________________________________________________________________
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III.4 Immunological characterization of native and recombinant Forisomes
III.4.1 Reactivity of polyclonal anti-GST-VFF1 antibodies to isolated Forisomes
Preliminary immunoblot analysis (III.3.3) confirmed that polyclonal Forisome-specific
antibodies recognized epitopes located on both, isolated Vicia faba Forisomes and
recombinant VFF1 protein. As an attempt to demonstrate that the protein encoded in the vff1
gene is present in the Forisome protein-complex, rabbit polyclonal antibodies raised against
the recombinant protein were subjected to immunoblot analysis against native Vicia faba
Forisomes. Since the attempts to purify soluble GST-VFF1 gave no improvements and the
cleavage of the Forisome fusion protein from the GST domain with Factor Xa resulted in a
significant loss of protein, gel slices from the corresponding single band were excised from
SDS-PAA gel. These denatured antigens were send for polyclonal antibodies production in
rabbits to EUROGENTEC (Seraing, Belgium).
Figure III-29 Immunoblot analysis of Vicia faba isolated Forisomes and recombinant VFF1 fusion protein using VFF1 specific rabbit antiserum. Specific binding of VFF1 rabbit anitserum (II.2.13.3) to isolated Vicia faba Forisomes (II.2.1) and overexpressed VFF1 (II.2.11.1) was characterized by immunoblot analysis. Proteins and either stained with Coomassie brilliant blue (II.2.12.2) (A) or blotted onto nitrocellulose membrane (II.2.12.4) followed by immunoblot analysis (II.2.12.4) (B). Immunodetection was carried out using 1:500 diluted anti GST-VFF1 rabbit antiserum and GAR(Fc)AP polyclonal antibody (1:5000). Detection was done with NBT/BCIP. 1: 10µl of cytoplasmic extract without IPTG induction; 2: 5µl of cytoplasmic extract after IPTG induction; E: 10µl of overexpressed VFF1elution fraction; F: 5µg of isolated Forisomes from Vicia faba; M: 5µl of Prestained Protein Marker; GST: 100 ng of GST protein as negative control.
To assess whether the recombinant VFF1 indeed expressed in the E. coli strain BL21(λDE3)
only upon IPTG induction, an SDS-PAGE analysis was performed. A sample of cytoplasmic
bacterial extract before and after IPTG induction was subjected to gel electrophoresis
confirming that there was no basal expression of the recombinant protein.
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Chapter III Results ___________________________________________________________________________
71
The result shown in Figure III-29 confirmed that the VFF1 rabbit antiserum binds highly
specific to the isolated Vicia faba Forisome protein confirming that the vff1 gene encodes a
protein involved in the constitution of Forisomes. A striking observation was the absence of a
positive signal against the recombinant fusion protein used as the antigen for immunization.
This fact raised compelling questions whether the epitopes recognized on the isolated
Forisomes are exposed in the bacterial expressed protein when running through the gel.
Presumably, the VFF1-specific antibody was unable to interact with the adopted conformation
of the fusion protein under reducing conditions.
Cross-reactivity to GST could not be excluded since a low binding activity to the GST antigen
was observed. This result shows that the fusion partner elicited a response during the
immunization. Nevertheless, the presence of specific polyclonal antibodies against VFF1
dominated the immunoresponse. The pre immune serum recognized neither the recombinant
fusion proteins nor the isolated Forisome proteins from Vicia faba (data not shown).
It is interesting to note that antibodies raised against the recombinant protein detected the
>100-kDa bands revealed by Coomassie staining (Figure III-1) as well as the additional bands
in the range 50-70 kDa. Since the vff1 sequence predict a protein of 79 kDa, all higher
molecular weight bands are likely to be multimers representing partially assembled Forisomes
that are stable in the presence of SDS and under reducing conditions. Such stability has been
reported for several other multimeric proteins (Grigorian et. al, 2005 and Peaper et. al, 2005).
However, it might sound paradoxical that considering that a major protein band at ~70 kDa
was used to generate the original peptide sequences, this antibodies recognized a strong band
with a molecular weight lower than 70kDa. This might indicate that most of the epitopes
within the 70-kDa protein are masked (e.g. by post-translational modifications) and only
become accessible for the antibodies recognition in the multimeric protein derivates or protein
bands of lower molecular weight (which might represent degradation products of the 70-kDa
band).
III.4.2 Characterization of native Forisomes by immunofluorescence
III.4.2.1 Reactivity of Forisome-specific polyclonal antibodies to native Forisomes
Since preliminary immunoblot analysis (III.1.2) confirmed that polyclonal mouse and chicken
Forisome-specific antibodies recognized epitopes located on denatured Vicia faba Forisomes,
confocal immunofluorescence microscopy (II.2.15) was performed to evaluate their antigen
specificity toward native Vicia faba Forisomes. As shown in Figure III-30A and E, epitopes
located over the entire surface of Forisomes could be recognized by both polyclonal
antibodies.
As previously reported (Knoblauch et al., 2001), Vicia faba Forisomes undergo a reversible
condense-disperse conformational change in response to variations in free Ca+2 concentration.
Chapter III Results ___________________________________________________________________________
72
Figure III-30 Confocal immunofluorescence analysis of Vicia faba native Forisomes using polyclonal Forisome-specific mouse and chicken antibodies. Specific binding of mouse and chicken polyclonal antibodies (II.2.13.1; II.2.13.2) to native Vicia faba Forisomes was characterized by immunofluorescence. Both Forisome-specific antisera in a dilution 1:250 in 1xPBS buffer (pH 7.4) were incubated for 2h at RT for epitopes recognition. Immunodetection was carried out using goat anti-mouse (dil 1:500) or goat anti-chicken (dil 1:500) Alexa Fluor® 488 (Molecular Probes) according to the first antibody. A confocal fluorescence microscope (Leica TCS SP) was used to obtain immunofluorescence. A, immunodetection of native Forisomes in absence of Ca+2 (condense conformation) with Forisome-specific chicken antiserum; B, immunodetection of Forisomes in presence of 20mM Ca+2 (disperse conformation) with Forisome-specific chicken antiserum; C, immunodetection with chicken preimmune serum used as negative control; D, immunodetection with GAC Alexa Fluor® 488 as primary antibody; E, immunodetection of native Forisomes in absence of Ca+2 (condense conformation) with Forisome-specific mouse antiserum; F, immunodetection of Forisomes in presence of 20 mM Ca+2 (disperse conformation) with Forisome-specific mouse antiserum; G, immunodetection with mouse preimmune serum used as negative control; D, immunodetection with GAM Alexa Fluor® 488 as primary antibody.
E F
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Chapter III Results ___________________________________________________________________________
73
To examine whether these polyclonal antibodies could recognize epitopes exposed in the
dispersed structure of the Forisomes, immunofluorescence microscopy in presence in 20 mM
Ca+2 was carried out. Isolated Forisomes from Vicia faba (II.2.1) were used as antigens.
Taking the advantage of a high adherence of Forisomes after centrifugation at 4000 rpm, the
use of microtiter plates enables washing and incubation steps and therefore avoiding a
considerable loss of the antigen.
The dispersed conformation of the Forisomes was not completely achieved in all protein
bodies due to the incubation with a suitable media for the antibodies. However, a transitional
state toward the dispersed conformation could be observed. It is interesting to note a fibrilar
pattern evidenced on the dispersed conformation of Forisomes after incubation with
Forisome-specific-chicken antibodies (Figure III-30B). The same experiment was performed
using Forisome-specific mouse antibodies; though a difference in the recognition pattern in
presence and absence of Ca+2 using this antiserum could not be confirmed. No cross-reactivity
with pre-immune sera and secondary antibodies used as negative controls was observed
(Figure III-30C, D, G and H).
III.4.2.2 Reactivity of VFF1-specific polyclonal antibodies to native Forisomes
Immunoblot analysis confirmed the antigen specificity of VFF1-specific rabbit antiserum to
denatured Forisomes (III.4.1). A further insight in the characterization of Vicia faba
Forisomes is the determination of whether the native protein can be recognized by polyclonal
antibodies raised against the recombinant VFF1. Thus, immunofluorescence microscopy
(II.2.15) was performed as previously described (III.4.2.1).
Figure III-31 Confocal immunofluorescence analysis of Vicia faba native Forisomes using polyclonal VFF1-specific antibodies. Specific binding of polyclonal antibodies raised against recombinant VFF1 proteins to native Vicia faba Forisomes was characterized by immunofluorescence. VFF1-specific antisera in a dilution 1:250 in 1xPBS buffer (pH 7.4) were incubated for 2h at RT for epitopes recognition. Immunodetection was carried out using goat anti-rabbit (dil 1:500) Alexa Fluor® 488 (Molecular Probes). A confocal fluorescence microscope (Leica TCS SP) was used to obtain immunofluorescence. A, immunodetection of native Forisomes in absence of Ca+2 (condense conformation) with VFF1-specific antiserum; B, immunodetection of Forisomes in presence of 20 mM Ca+2 (disperse conformation ) with VFF1-specific chicken antiserum.
A BAA BB
Chapter III Results ___________________________________________________________________________
74
As shown in figure III-31, the same pattern of antibody specificity toward epitopes located
over the entire surface of Forisomes was observed. No cross-reactivity with pre-immune sera
and secondary antibodies used as negative controls was detected (data not shown).
Chapter IV Discussion ___________________________________________________________________________
75
IV Discussion
IV.1 Molecular characterization of Forisome genes
IV.1.1 Analysis of Vicia faba Forisomes subunits
Isolation of V. faba Forisomes (II.2.1) and subsequent SDS-PAGE analysis (Figure III-1)
revealed the presence of several protein bands of different molecular weight. A major band of
approximately 70 kDa was observed together with several minor bands of lower and higher
molecular weights. Within the range of 50-70 kDa, the detected bands might suggests the
presence of different subunits or degradation products of the main (~70 kDa) band, whereas
the higher molecular weights bands might denote dimerization and multimerization derivates.
The reduction of the acrylamide concentration from 12% to 7% enabled the separation of the
main ~70kDa protein into two different bands of slight lower molecular weights with the
larger subunit quantitatively stronger represented. Moreover, complete fractionation into three
individual subunits could be achieved after addition of Ca2+ (data not shown). Nevertheless,
the possibility cannot be excluded that one or several of these three bands in the SDS-PAA
gel might indicate the presence of proteins with the same running properties. This
interpretation seemed reasonable since some sequenced peptides generated from the ~70kDa
large subunit of V. faba Forisomes (II.2.2) could not be found within the VFF1 amino acid
sequence. Alternatively, these lower molecular weight bands may reflect the presence of
additional subunits which comprised the protein complex but where sequences are yet
unknown.
Further experiments were performed with the aim of identify proteins difficult to resolve on
Coomassie blue-stained one-dimensional PAGE. Isolated Forisomes from V. faba were
subjected to two-dimensional PAGE and different forms spanning the pI values from 6.0 to
7.5 (Figure III-3A) were observed, which is in agreement with the isoelectric point (pI) of
6.79 predicted for the vff1 translated amino acid sequence (III.1.9).
The major band of ~70kDa observed on one-dimensional PAGE is composed of possibly two
or three proteins running at the same molecular weight. After immunoblot analysis (III.1.2.2),
a defined pattern was revealed in which the Forisome subunits of higher molecular mass are
of less acidic pI, whereas those migrating as lower molecular mass forms are apparently more
acidic. This heterogenicity among the spots might indicate the result of post-translational
modifications such as glycosylation and/or phosphorylation. It is commonly assumed that a
variety of forms are predominantly accounted for by the differences in the amount of sugar
moieties imparting different charges in the attached N-glycans. Some heterogeneity of
molecular mass is also introduced by differences in the number and size of N-glycans
attached. This can be in part correlated with the observed N-glycosylation and protein kinase
phosphorylation sites predicted by protein sequence analysis (III.1.9). Studies using
Chapter IV Discussion ___________________________________________________________________________
76
antibodies against these post-translational modifications combined with antibodies raised
against the recombinant subunits, might be required to gain more insight into the Forisomes
structure.
IV.1.2 Cloning and characterization of vff1 gene
IV.1.2.1 Screening of cDNA libraries
Attempts to identify Forisome-encoding genes by screening either subtracted or non-
subtracted Vicia faba derived cDNA libraries (III.1.3) retrieved no successful results.
It has been previously reported that the percentage of clones in a subtracted library that
corresponds to differentially expressed mRNAs varies considerably: as high as 95%
(Diatchenko, L. et al., 1996), a midrange of 40–60% (Gurskaya, N. G. et al., 1996; von Stain,
O.D. et al., 1997), and as low as 5% (Diatchenko, L. et al., 1998). In most cases, the
subtraction method greatly enriches differentially expressed genes. Nevertheless, the
subtracted sample might still contain some cDNAs that correspond to mRNAs common to
both the tester and driver samples. Although this background may depend somewhat on the
quality of RNA purification and the performance of the particular subtraction, it mainly arises
when very few mRNA species are differentially expressed in tester and driver, as it might be
the case here.
In general, a limited set of differentially expressed mRNAs and low quantitative difference in
expression means higher background, even if a good enrichment of differentially expressed
cDNAs is obtained. Since the background was moderately high, the possibility of picking
random clones from the subtracted library for Northern blot analysis was considered.
However, this procedure would have been time-consuming and inefficient. Therefore, a
differential screening step was performed instead to minimize background before performing
a Northern blot analysis.
There are two approaches for differentially screening a subtracted library. The first is to
hybridize the subtracted library with 32P-labeled probes synthesized as first-strand cDNA
from tester and driver (Hedrick et al., 1984; Sakaguchi et al., 1986). Clones corresponding to
differentially expressed mRNAs will hybridize only with the tester probe, and not with the
driver probe. Although this approach is widely used, it has one major disadvantage since only
cDNA molecules corresponding to highly abundant mRNAs (i.e., mRNAs which constitute
more than about 0.2% of the total cDNA in the probe) will produce detectable hybridization
signals (Wang, Z. and Brown, D., 1991). Therefore, clones corresponding to low-abundance
differentially expressed mRNAs will not be detected by this screening procedure. Hence, an
alternative approach was considered.
The second alternative approach bypasses the problem of losing low-abundance sequences. In
this method, the subtracted library is hybridized with forward- and reverse-subtracted cDNA
Chapter IV Discussion ___________________________________________________________________________
77
probes (Lukyanov et al., 1996; Wang, Z and Brown, D., 1991). To construct reverse-
subtracted probes, subtractive hybridization was performed with the original tester cDNA as a
driver and the driver cDNA as a tester. Clones representing mRNAs that are truly
differentially expressed hybridize only with the forward-subtracted probe, whereas clones that
hybridize with the reverse-subtracted probe may be considered as background. Although a
good ratio of signal to background was ensured among the clones which exhibited strong
hybridization signals, the sequence analysis did not contain the identified Forisome peptides.
Assuming that Forisomes were expressed in young phloem tissue, a subtracted cDNA library
was constructed using samples prepared from Vicia faba plant leaves as tester in combination
with samples from the Vicia faba root tips as driver (II.2.5.2). Later, it has been observed that
although during root elongation, vff1 promoter expression pattern was restricted to the
meristematic and elongation zones at root tips, in the tips of fully developed roots vff1
expression was no longer detectable (IV.2.4.1). These results indicate that the Vicia faba
subtracted cDNA library might not be representative as among root tips used as drivers the
level of expression of these specific mRNAs was not determined. Thus, a very low
quantitative difference in expression of Forisome mRNAs might have been obtained between
driver and tester cDNAs.
An alternative explanation for these results, and which encompasses also the non-subtracted
library, may be the fact that during the hybridization screen of both libraries, the probes used
were designed according to sequenced peptides which afterwards could not be allocated
within the identified VFF1 sequence. This may imply the presence of proteins, in the
sequenced samples, with the same running properties as the main ~70kDa band. Hence, some
peptides might have been sequenced from proteins isolated along with Forisomes and which
may not be present in the cDNA libraries. Nevertheless, this interpretation does not exclude
the possibility that sequenced peptides absent from VFF1 might be present in still unidentified
Forisome proteins. Hence, the observed results might also suggest a low stability of the
Forisome specific mRNAs and therefore making the construction of representative libraries
impossible.
Furthermore, immunoscreening (III.1.4) and PCR screening (III.1.5) of cDNA expression
library of Vicia faba plant tips was performed. Nevertheless, neither the identification of
cDNAs encoding antigenic Forisome fusion proteins nor the allocation of DNA- and/or DNA
fragments to the Forisome genes was possible.
These results might suggest, as described above, the construction of a not representative
cDNA library due to a low stability of the Forisome specific mRNAs. This interpretation may
therefore also explain the unsuccessful attempts in screening of this cDNA expression library.
In addition, it is of interest to note that degenerate primers were used in the PCR screening of
this Vicia faba cDNA expression library (III.1.5). Degenerate primers provide a tool to search
for more than one gene isoform at a time in a reverse-genetic strategy raising the possibility of
Chapter IV Discussion ___________________________________________________________________________
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amplifying and cloning sequences that might correspond to genes encoding Forisome proteins
in Vicia faba. However, the unsuccessful attempts presented here indicate that, in this case,
the use of degenerated primers was not a suitable method.
Other possible reasons could be considered to explain the inability to generate amplification
products. First, the overlay of the protein subunits in the SDS-PAA gel electrophoresis and, as
a consequence, the identification of peptide sequences of different subunits and/or proteins.
Second, the different length of amino acids residues of the generated peptide sequences with
the concomitant strong deviation among the annealing temperatures of the designed
degenerated oligonucleotides used. Third, it seemed likely that the failure to amplify these
genes might be attributed to the unawareness of the localization of the peptides to each other
within the polypeptide chain that resulted in the placement of primers in regions of the
predicted gene that are actually too close in the final transcript for an adequate PCR
amplification. Finally, the shortness and degeneration of the derived oligonucleotides could
not ensure the use of suitable high annealing temperatures as a condition which enables
touchdown PCR.
IV.1.2.2 Database search and cloning of vff1 gene
Peptide sequences obtained by sequencing the isolated ~70-kDa protein band from Vicia faba
Forisomes were used for screening public databases (II.2.2). Interestingly, two of the peptide
sequences were nearly 100% identical to parts of a M. truncatula 478-bp EST (expressed
sequence tag) from the MENS database (Medicago EST Navigation System) encoding a
partial sequence of 157 amino acids. Based on this sequence information specific primers
were designed and the cDNA sequence along with the full-length genomic sequence could be
cloned by 5´ and 3´ RACE (III.1.6) and LD PCR (III.1.9). Computer analysis of the genomic
and cDNA sequences revealed the presence of a 2710-bp open reading frame, interrupted by
six introns flanked by GT and AG canonical intron border sequences. The identified ORF
encodes a protein of ~ 79-kDa. The resulting cDNA clone was designated vff1 (protein VFF1)
and its deduced amino acid sequence revealed the presence of 10 different peptides sequences
belonging to the Vicia faba Forisome. Nevertheless, many of the sequenced peptides could
not be allocated within the identified cDNA clone, which might indicate the presence of yet
not yet identified additional Forisome subunits.
IV.1.2.3 Sequence analysis of vff1 gene
Given that neither the alignment of the vff1 gene against the completely sequenced genome of
Arabidopsis thaliana nor against the genome of Oryza sativa retrieved any information, a
sequence comparison with known protein databases was performed. The identification of
significantly homologous genes and/or proteins could not be achieved. The strongest
Chapter IV Discussion ___________________________________________________________________________
79
homology with approx. 29% identity to VFF1 showed the amino acid sequence of a protein
(At3g01680; www.ncbi.nlm.nih.gov/BLAST) from A. thaliana, which function is yet
unknown. While the entire sequence exhibited only very small homology with the vff1
Forisome gene, a carboxyterminal stretch of several cysteines could be identified encoded by
both clones (Figure III-9). These specific amino acid residues might be involved in the
stabilization of the protein complex by the formation of disulfide bridges of the side chains,
although formation of covalent S-S bridges is rarely found in intracellular proteins.
Experiments performed in parallel to the vff1 gene cloning, revealed homologous cDNA
clones for two further Fabaceae plants, Medicago truncatula (mtf1, 1944 bp; MTF1, 74 kDa)
(Noll, G., personal report) and Canavalia gladiata (cgf1, 2007 bp; CGF1, 77 kDa) (Ashoub,
A., personal report). These clones were of similar length and shared protein sequence identity
of ~61%.
Perhaps the most noticeable feature of Forisomes is their existence only within the Fabaceae.
Molecular analysis of the structure of this novel protein will lead to a better characterization
of this unique protein.
IV.1.2.4 The different motifs identified within the vff1 gene
Despite the importance given to Ca2+ in the Forisome regulation, no structural features such as
Ca2+-binding domains (e.g. EF-hands; Nakayama, S. and Kretsinger, H., 1994; Muranyi, A.
and Finn, B.E., 2001; Swairjo, M.A. and Seation, B.A., 1994; Nalefski, E.A. and Falke, J.J.,
1996; Rizo, J. and Südhof, T.C., 1998; Weis, W.I., 1996) could be predicted for VFF1.
Nevertheless, computer analysis (http://www.ch.embnet.org/software/COILS_form.html;
Lupas, A., 1996) revealed another structural motif, an amphipathic α-helix within the coding
region of VFF1 comprising a coiled-coil domain (Figure III-10). The coiled-coil domain is
responsible for oligomerization of protein subunits and concomitant folding of the proteins. It
has been described for the first time in the intermediate filament keratin protein (McArthur,
1943; Burkhard et al., 2001). Later, a dimerization domain of a family of transcription factors,
the leucine zipper (Landschulz et al., 1988), was also described comprising this structural
motif. Some amphipatic helices are arranged as interwined helices that are termed as coiled
coils or super-helices. Although structurally simple, coiled coils have the ability to form a
variety of different assemblies ranging from dimers to pentamers (Lupas, A. N. and Gruber,
M., 2005; Walshaw, J. and Woolfson, D. N., 2001).
Furthermore, coiled coils can form homomers or heteromers with their chains arranged in a
parallel or antiparallel fashion (Lupas, 1992). Sequences of parallel left-handed coiled coils
are characterized by a seven-residue periodicity (heptad repeat), with the occurrence of apolar
residues preferentially in the first (a) and fourth (d) positions of the ‘heptad’ (Lupas, A.,
1996). The interaction between these apolar amino acids denote the formation of the
Chapter IV Discussion ___________________________________________________________________________
80
hydrophobic core, which induces coiled coils (Crick, 1953; Lupas, 1996). Charged amino acid
residues, frequently at positions outside of the hydrophobic core, can affect upon changes of
the pH in the surrounding medium (Suzuki et al., 1997; Krylov et al., 1998), after
phosphorylation of the AA side chains (Szilak, L. et al., 1997; Liang, W. et al., 1999) and
after interaction with ions (Farah, C.S. and Reinach, F.C., 1999), the hydrophobic interaction
of the coiled-coil domains. It is also generally acknowledged that the detailed packing
geometry of hydrophobic core residues correlates with the oligomerization state (Harbury, P.
B. et al., 1993; Harbury, P. B. et al., 1994).
The 21 AA coiled-coil domain of the VFF1 is relatively short, however, this has been already
described for some other globular proteins with less than 20 AA (Lupas, 1996; Müller, K. et
al., 2000; Kohn, W. and Hodges, R., 1998; Rieker, J. and Hu, J., 2000; Cho, C. et al., 2004;
Eckert, D. and Kim, P., 2001; Ryadnov, M. et al., 2003; Petka, W. et al., 1998). Moreover
and in contrast to the characterized apolar amino acid residues found in the first (a) and
fourth (d) positions of the heptad pattern, in the VFF1 coiled-coil domain a polar amino acid
residue is present within one of the heptads. In this context, it has been shown that distinct
coiled-coil ‘trigger sites’ within heptad-repeat-containing amino acid sequences might be
necessary to mediate coiled coil formation (Steinmetz, M.O. et al., 1998; Kammerer, R.A. et
al., 1998; Frank, S. et al., 2000). As a hallmark, the coiled-coil trigger site of the actin-
bundling protein cortexillin I contains a distinct inter- and intrahelical salt-bridge pattern,
which includes charged residues even at core positions (Burkhard, P. et al., 2000a). Other
recent results suggest that ionic interactions are not only important for proper alignment,
orientation and selectivity of coiled coils, as shown previously (Kohn, W.D. et al., 1995;
O’Shea, E.K. et al., 1992), but can also contribute considerably to stability of coiled coils
(Burkhard, P. et al., 2000b; Moll, J.R. et al., 2000; Krylov, D. et al., 1998; Spek, E.J. et al.,
1998), thereby modulating assembly of coiled coils in a pH-dependent manner (Suzuki, K. et
al., 1997; Bullough, P.A. et al., 1994).
Taken together, this interpretation raises the possibility that the coiled-coil motive found in
VFF1 proteins might be involved in the Ca2+ dependent reaction of the Forisomes or that
additional and yet unidentified subunits are responsible for the conformational change.
Further analysis of the VFF1 amino acid sequence was performed, nevertheless, no well-
known motives that appeared to contribute to the secondary structure of the protein were
retrieved. Instead, a low-complexity region (III.1.6) was identified for which no structure can
be predicted with the algorithms available (Schultz et al., 1998). One general idea about the
functional relevance of these low-complexity regions is that they only acquire a rigid structure
(e.g., an α-helix) upon interaction with another protein partner or oligomerization into a
protein complex (Wright and Dyson, 1999). This has been reported for soluble N-
ethylmaleimide-sensitive factor attachment protein receptor, which play a role in vesicle
Chapter IV Discussion ___________________________________________________________________________
81
docking and fusion, and which only form an α-helix when they bind to their interaction
partner (Fasshauer et al., 1997; Jahn and Sudhof, 1999).
In addition, it is of interest to note that motif searches against the Smart database, after
inclusion of sequences (outlier homologues) that are often difficult to detect using HMM
methodology, retrieved a 100% identity within the VFF1 amino acid sequence (167-395) to
an unknown domain (d1gw5a_domain; http://smart.embl-heidelberg.de/) which belongs to the
ARM superfamily.
ARM repeats are short 42-amino acid motifs that were first identified in the fruitfly
(Drosophila melanogaster) segment polarity protein, armadillo (Riggleman et al., 1989).
ARM repeats have been subsequently identified in a wide range of eukaryotic proteins, and
these proteins interact with numerous other proteins via their ARM repeats, resulting in the
regulation of a variety of cellular processes (Hatzfeld, M., 1999). Based on the crystal
structure of the mammalian armadillo homolog, β-catenin, each ARM repeat forms a
trihelical structure that folds into a superhelix, and six ARM repeats are proposed to constitute
a protein interaction domain (Huber et al., 1997). Within the VFF1 sequence only one ARM
repeat was identified. Despite this, the highly divergence, not only between proteins but also
between repeats within the same protein in this family, suggest that there are likely other
ARM repeats in the VFF1 sequence that have not been detected. This is evident in the
arrangement of ARM repeats for closely related proteins where a featureless region is present
between ARM repeats in one protein but not in the other relative and the fact that ARM
repeats tend to be tandemly repeated (Mudgil, Y. et al., 2004).
Based on the three-dimensional structure of β-catenin, six ARM repeats have been predicted
to form the basic superhelical structure of the protein interaction domain (Huber et al., 1997).
If this is the typical requirement, then the predicted VFF1 protein with fewer ARM repeats
may has some undetected repeats or it may has gained new functions based on these fewer
ARM repeats (Andrade, M. A. et al., 2001; Meyers, B. C. et al., 1998).
As described above and apart from the data presented here, a succession of several cysteine
residues could also be identified at the carboxy-terminal domain (IV.1.2.3). The reversible
conformational change of Forisomes from the condensed into the dispersed state after
addition of Ca2+ leads not to a complete dissolution of the protein complexes. Forisomes still
remains as distinctive bodies (Knoblauch et al., 2003). A function of the cysteine residues
with the stabilization of the protein complex by the formation of disulfide bridges of the side
chains would be conceivable. Possibly these covalent bonds serve as the linkage of the
individual subunits holding them together and facilitating, after Ca2+ withdrawal, the back
folding of the Forisome into the arranged condensed conformation.
Taken together, from the characterization of the different motifs identified within the VFF1
sequence, it is becoming clear that this protein comprise several structural features related to
helices and coiled-coil motive formation. These structural characteristics might indicate a
Chapter IV Discussion ___________________________________________________________________________
82
possible Ca2+ dependent reaction of the Forisomes. However, future work focusing on the
identification of additional Forisome genes and their molecular characterization are needed
for the elucidation of mechanisms that influence their conformational change.
IV.1.3 Immunological evidence of vff1 gene encoding a Forisome protein
IV.1.3.1 Expression and purification of recombinant VFF1 protein
After cloning and sequence analysis of vff1 gene, bacterial expression of the recombinant
protein was performed with the aim to obtain antigens for immunization. Vff1 cDNA was
genetically fused to the carboxyl-terminal end of GST (III.3.1). Using GST as fusion protein
allowed one step affinity purification of the recombinant overexpressed proteins under native
conditions (Frangioni and Neel, 1993).
Overexpression of the GST-VFF1 fusion protein resulted in formation of inclusion bodies.
Thus, for altering amounts of soluble and correctly folded protein, the cultivation and
induction conditions had to be optimized. Slightly larger quantities of soluble GST-VFF1
fusion protein were obtained by increasing the culture volume, decreasing the IPTG
concentration, increasing the time and reducing the temperature to 16°C after induction. The
positive effect of low temperature to reduce inclusion bodies formation has been
demonstrated in many studies (Bishia et al., 1987; Schein, 1989; Burton et al., 1991). The
rationale behind was based on the assumption that the rate of protein folding is only slightly
affected at temperatures between 15 to 20°C, whereas the rates of transcription and
translation, being biochemical reactions, is substantially decreased. This, in turn provides
sufficient time for protein folding, yielding active proteins and avoiding the formation of
inactive protein aggregates, i.e., inclusion bodies, without reducing the final yield of the target
protein (Oppenheim et al., 1996). Nevertheless and contrary to this expectation, the
improvement in protein expression was not entirely sufficient.
On the other hand, the yield of purified VFF1 cleaved from GST domain with Factor Xa was
unfortunately very low due to an almost complete degradation of the overexpressed protein
even though affinity purified proteins showed high purity in SDS PAGE (data not shown).
Hence, production of recombinant fusion protein was carried out in a 4-liter scale
fermentation, but again, no soluble GST-VFF1 fusion protein could be obtained even using
the optimized conditions described above. This might indicate that formation of VFF1
inclusion bodies was not affected by the expression conditions used. The large size of the
GST-VFF1 fusion protein (104 kDa) could affect the proper refolding of the fusion protein as
reported in other studies (Hackenbeck et al., 1998). In consequence, GST-VFF1 fusion
proteins were isolated from the SDS-PAA gel in large quantities and used for the production
of specific polyclonal antibodies in rabbits.
Chapter IV Discussion ___________________________________________________________________________
83
IV.1.3.2 Immunoreactivity of Vicia faba Forisome-specific polyclonal antibodies to
recombinant VFF1 proteins and native Vicia faba Forisomes
Bacterial expressed VFF1 along with isolated Vicia faba Forisomes as a positive control were
used to assay their immunoreactivity toward Forisome-specific mouse and chicken antisera.
The results shown in Figure III-28B indicated that Forisome-specific chicken antiserum
recognized the overexpressed recombinant VFF1 protein confirming its presence in Vicia faba
Forisomes.
Immunoblot analysis with Forisome-specific mouse antiserum could not reveal the presence
of bands at the expected sizes whereby immunodetection of isolated Forisomes shows a
remarkably strong signal (Figure III-28A). This might indicate that mouse antiserum
recognize different epitopes not present in the recombinant protein, however, exposed on the
isolated Vicia faba Forisomes. In this respect, it would be also conceivable that
conformational epitopes absent from the bacterial expressed protein might have been
responsible for the immunoresponse in mouse and that these same epitopes could be stable in
the presence of SDS and under reducing conditions. Such stability has been reported for
several other multimeric proteins (Grigorian, A. L., et al., 2005; Salahpour, A. et al., 2003;
Peaper, D. et al., 2005). It could, nevertheless, either be discriminated whether the absence of
specific signal is caused by a weak interaction not detectable by immunoblot analysis.
Furthermore, confocal immunofluorescence analysis of Vicia faba native Forisomes using
polyclonal Forisome-specific mouse and chicken antibodies was performed (Figure III-30). It
could be observed that whereby epitopes located over the entire surface of the Forisomes
could be recognized by both polyclonal antibodies, a different pattern was observed in
presence of Ca2+. Knoblauch, M. et al. (2001) have reported that Vicia faba Forisomes
undergo a reversible condense-disperse conformational change in response to variations in
free Ca+2 concentration. Despite the immunoreaction of Forisome-specific polyclonal chicken
antibodies evidenced a fibrilar pattern on the dispersed conformation of Forisomes (Figure
III-30B), Forisome-specific mouse antibodies showed only reactivity against epitopes located
on the surface of the protein bodies. This observation might indicate that epitopes hidden
inside the Forisomes could be exposed as a result of the change in conformation due to a
variation in Ca+2 concentration thus enabling antibodies binding. These epitopes might be
recognized by Forisome-specific chicken antiserum but not by mouse polyclonal antibodies.
On the other hand, conformational epitopes possibly present inside the condensed state of the
Forisomes are lost during the transition to the disperse state with the concomitant loss of
immunoreactivity towards the mouse polyclonal antibodies.
This interpretation may also explain the specific reaction of the mouse polyclonal antibodies
observed against the protein bands of higher molecular weight which were weakly detected by
the specific chicken antiserum (Figure III-28). It seems conceivable that not only the main
band of ~70kDa is recognized by the mouse polyclonal antibodies but also some interactions
Chapter IV Discussion ___________________________________________________________________________
84
between subunits of Forisomes which in turn, are not present in the dispersed conformation.
These interactions may build up the conformational epitopes explained above. This
interpretation might indicate the possibility of homo and heterodimers and/or multimers
forming the Forisome structure. In addition, this is supported by the observation that some
sequenced peptides from isolated V. faba Forisomes were not found within the VFF1 amino
acid sequence confirming that this is not the only subunit present in the Forisomes.
Taken together, it can be assumed that the Forisome-specific chicken polyclonal antibodies
recognize mainly the ~70kDa subunit whereas the Forisome-specific mouse polyclonal
antibodies were raised against not only this protein but also against conformational epitopes
determined by interactions between the ~70kDa protein and other protein subunits comprised
in the condensed Forisome structure stabilized even in presence of SDS and under reducing
conditions.
The fact that no immunodetection could be observed for Forisome-specific mouse antiserum
within the dispersed conformational state of the Forisomes, favours the idea of a
heteromultimer protein. Nevertheless, other, as yet unidentified Forisome protein sequences
might also be required to confirm this hypothesis.
IV.1.3.3 Immunoreactivity of VFF1-specific polyclonal antibodies to recombinant
VFF1 proteins and native Vicia faba Forisomes
From the results presented in Figure III-29 it becomes clear that the VFF1 rabbit antiserum
binds highly specific to the isolated Vicia faba Forisome proteins confirming that the vff1
gene encodes a Forisome protein.
Seemingly contradictory but nonetheless evident was the absence of a positive signal against
the recombinant fusion protein used as antigen for the immunization. An explanation for this
could be that the recognized epitopes are shielded from the VFF1-specific antibodies by the
adopted conformation of the fusion protein under the reducing conditions used in the SDS-
PAA gel electrophoresis.
It is, however, interesting to note that antibodies raised against the recombinant VFF1 protein
detected the >100kDa bands revealed by Coomassie staining (Figure III-1) as well as the
additional bands in the range 50-70kDa. Since the vff1 sequence predicted a protein of 79kDa,
all higher molecular weight bands are thus likely to be multimers representing partially
assembled Forisomes stable in the presence of SDS and under reducing conditions in
agreement with what was explained above. Nevertheless, it might sound paradoxical that
considering that the major protein band at ~70kDa was used to generate the original peptide
sequences and to identify vff1, this antibody recognized a strong band with a molecular weight
lower than 70kDa. This might indicate that most of the epitopes within the 70kDa protein are
masked (e.g. by post-translational modifications) and only become accessible for the
Chapter IV Discussion ___________________________________________________________________________
85
antibodies recognition in the multimeric protein derivates or protein bands of lower molecular
weight (which might represent degradation products of the 70-kDa band).
Confocal immunofluorescense revealed that native V. faba Forisomes are detected by the
specific anti-VFF1 polyclonal antibodies (Figure III-31). Adhesion of co-purified particles on
the Forisome surface, along with a non-specific reactivity of the antibodies to these, can be
not completely excluded. However, the preimmune serum did not show comparable
fluorescence intensity. Moreover, the immunodetection pattern towards the condensed and
dispersed conformation of native Forisomes resembled that one observed with Forisome-
specific chicken antiserum. In this respect, it is worthwhile to note that presumably, the VFF1
protein is highly represented inside the Forisome structure, which is supported by the strong
immunodetection in presence of Ca+2.
IV.2 Molecular characterization of vff1 promoter
The putative promoter of the vff1 gene was isolated from genomic DNA by genome walking
(III.2.2) and its activity as well as tissue specificity was confirmed in transgenic tobacco
plants using GUS as a reporter system (III.2.4). In addition, sequence analysis of the promoter
region of vff1 gene revealed the presence of two conserved motifs which have already been
reported for several phloem-specific promoters (III.2.3). Taken together, these results indicate
that the 5′-upstream region of vff1 functions as a promoter (designated vff1P) that directs
preferential expression of the uidA gene to the phloem of transgenic tobacco plants. Besides
the tissue-specificity (III.2.4.2.1) a spatial and temporal regulation of vff1P was observed
during development (III.2.4.2.2) which seems to be in consistent with the expression of
Forisomes mainly in the early stage of phloem development.
IV.2.1 Sequence analysis of vff1 promoter
Sequence comparison of the vff1 promoter and other phloem-specific promoters revealed the
presence of two shared motifs, the 13bp and GATA motifs. The involvement of these motifs
to phloem-specific gene expression was demonstrated by loss-of-function analysis that
introduced point mutations (Yin et al., 1997a) or deletions (Ohta et al., 1991; Hehn and
Rohde, 1998) into each promoter. The 13 bp and GATA motifs are conserved between virus
and plant promoters, implying an important role for these motifs for gene expression in the
phloem.
Hehn and Rohde (1998) identified a highly conserved motif ‘ATAAGAACGAATC’ (13-bp
motif) involved in activity and phloem-specificity when they compared the Coconut Foliar
Decay Virus (CFDV) promoter sequence with other phloem-specific promoters from Rice
Tungro Bacilliform Virus (RTBV), Commelia yellow mottle virus (CoYMV), rolC of
Agrobacterium rhizogenes and the pea gultamine synthase gene GS3A. Another vascular
Chapter IV Discussion ___________________________________________________________________________
86
tissue-specific promoter from a rice GRP gene (Osgrp-2) was also described to contain this
conserved sequence (Liu et al., 2003). In all these promoters, this motif is localized upstream
of the TATA box. A similar motif with high homology to the above-mentioned 13bp
conserved sequence was also found 524 bp upstream of the TATA box in the vff1 promoter
and 37 bp upstream of TATA box in the C. maxima PP2 promoter (Thompson and Larkins,
1996). These findings imply that the 13-bp motif might be in part responsible for the specific
activity of the vff1 promoter, although function of other sequences in phloem-specificity and
enhancement of the promoter activity could not be ruled out. Further dissection of the vff1
promoter is required to confirm whether these motifs and/or other elements are involved in
gene expression in the phloem.
Several other promoters from plants (Yang and Russell, 1990; Brears et al., 1991; DeWitt et
al., 1991; Ohta et al., 1991; Martin et al., 1993; Hérouart et al., 1994; Shi et al., 1994;
Truernit and Sauer, 1995; Tornero et al., 1996), Agrobacterium (Schmülling et al., 1989;
Sugaya et al., 1989; Guevara-García et al., 1993) and viruses (Medberry et al., 1992;
Bhattacharyya-Pakrasi et al., 1993; Rohde et al., 1995) have been reported to drive expression
of reporter genes in the phloem of transgenic plants. These promoters differ in the degree of
specificity of gene expression. Among them, there are promoters that direct gene expression
in phloem parenchyma cells in addition to sieve elements and companion cells. The
Arabidopsis plasma membrane H+-ATPase gene promoter (DeWitt et al., 1991) is an
example of this type. Other promoters direct gene expression in phloem and additional cells of
other tissues. For example, the Commelina yellow mottle virus promoter drives gene
expression in phloem along with weak expression in xylem parenchyma cells in transgenic
tobacco (Medberry et al., 1992). The sporamin A gSPO-A1 gene promoter directs gene
expression predominantly in internal phloem, but also, to a lesser extent, in pith parenchyma
cells of transgenic tobacco (Ohta et al., 1991). However, the most stringent promoters direct
gene expression only in sieve elements and companion cells of the phloem. Maize and rice
sucrose synthase promoters (Yang and Russell, 1990; Shi et al., 1994) are typical examples of
this type. The vff1 promoter was assigned to the last specificity group, since expression of
GUS was detected exclusively in companion cells and sieve elements of vff1P-GUS
transgenic tobacco plants.
IV.2.2 Phloem-specific expression pattern driven by vff1 promoter
In order to study tissue specificity the β-glucuronidase reporter gene was placed under the
control of the full-length vff1 promoter and examined for promoter activity in N. tabacum.
Transient assays in detached and vacuum-infiltrated tobacco leaves were performed for rapid
verification of the promoter activity (Kapila et al., 1997; Fischer et al., 1999). Although
transient gene expression may not be fully comparable to results obtained with transgenic
plants, it can provide a rapid and reliable method to evaluate promoter expression since there
Chapter IV Discussion ___________________________________________________________________________
87
is no transgene integration and expression levels might be influenced by position effects
(Kapila et al., 1997). Nevertheless, despite some GUS expression could be observed, these
results were not reproducible. As it was confirmed in further experiments, the synthesis of
Forisomes is taking place during the early stages of sieve element development. Moreover,
the subsequent differentiation of the sieve element from the companion cell would only imply
the delivery of subunits of the Forisome, which indicate a diminished level in protein
synthesis. Therefore, the low levels of GUS expression in mature vacuum-infiltrated leaves
denoted an inadequate method for promoter studies.
As an alternative approach to study the vff1 promoter activity, transient expression in isolated
protoplast was carried out; however, this method gave again low and non reproducible levels
of activity. Considering that vascular tissues are highly specialized tissue, activation of
phloem-specific promoters probably requires specific transcription factors. The activity of
such promoters would therefore be expected to be poor in cell cultures or in protoplasts, in
which, even though not devoid of phloem cells, only some few phloem derived protoplasts are
present. This could explain the low activity observed using the vff1 promoter when tested in
transient expression assays in protoplasts (Zhan et al., 1993; Dry et al., 2000). Hence, stable
transformation of N. tabacum plants was used for further analysis.
With the stable integration of T-DNA encoding the uidA gene driven by the vff1 promoter,
transgenic N. tabacum plants were generated. Histochemical assays of GUS activity
demonstrated that in stably transformed tobacco plants, the vff1 promoter conferred a phloem-
specific expression pattern in stems, leaves, flowers and roots. The expression of the uidA
gene was confined to internal and external phloem of stems and leaves (Figure III-19). GUS
activity was also detected in flowers (Figure III-20) and in the phloem of roots (Figure III-21).
In flowers, the staining was localized to the inflorescence meristem and young floral buds
(Fig. III-20A). Roots of these transgenic plants showed the strongest staining for GUS activity
restricted to dividing meristematic tissues (Fig. III-21D). As the histochemical assays were
performed under reducing conditions to prevent diffusion of the GUS product (Guivarc’h et
al., 1996), the accumulation accounted essentially for expression of vff1 promoter in these
tissues.
A chimeric promoter consisting of the double enhancer sequence from the Cauliflower
Mosaic Virus (CaMV) 35S promoter fused upstream to the uidA gene was used as positive
control. When compared with this enhanced CaMV 35S promoter (Kay et al., 1987), the level
of GUS activity driven by vff1 promoter was moderate. This may reflect the low proportion of
phloem tissues in plant organs. Indeed, the phloem is only a small fraction of organ tissues,
representing only 0.4% of leaf volume (Sjölund, 1997).
Taken together, the above data showed that the vff1 promoter drives highly specific
expression of the GUS gene in the phloem of transgenic tobacco plants. The phloem-specific
expression coincides with the occurrence of cis-acting elements detected within the vff1
Chapter IV Discussion ___________________________________________________________________________
88
promoter sequence (III.2.3) that are similar to phloem-specific elements found on several
promoters from plants, Agrobacterium and viruses (IV.2.1).
Furthermore, these results indicate that there may be similar transcription factors in tobacco
and V. faba that regulate expression of the promoter. It is known that some plant promoters
retain specific expression patterns in different plant species (Chen, G. et al., 1994; Klöti, A. et
al., 1999; Jefferson, R. A. et al., 1987; Katagiri, F. et al., 1992; Medberry, S. L. and
Olszewiki, N. E. 1993; John, M. and Petersen, M., 1994) while others do not (Gallusci, P. et
al., 1994; Medberry, S. L. et al., 1992).
IV.2.3 Differential expression pattern driven by vff1 promoter in the sieve element-
companion cell complex
Histochemical analysis of Vff1P-GUS plants showed that the expression pattern driven by the
vff1 promoter was confined to companion cells (Figure III-19) of mature transgenic N.
tabacum leaves. In addition, it was also observed a slight GUS activity in some sieve
elements of young phloem tissue (data not shown). Since Forisomes are found within sieve
elements and considering that enucleated sieve elements are thought to be incapable of protein
synthesis, the specific vff1 promoter expression in companion cell suggested that VFF1, a
subunit of the Forisomes, might be synthesized in companion cells and transported into sieve
elements through plasmodesmata and functional folded in the sieve elements.
Experiments performed in vitro, showed that reversible transitions between a condensed and a
dispersed state of Forisomes can be repeated several times by addition of Ca2+ and/or EDTA
(Knoblauch et al., 2001). However, it was likewise observed that after some reaction cycles
the conformation of the protein bodies in the condensed and/or dispersed condition is no
longer possible. This indicates that the Forisomes might undergo a loss in functionality due to
alterations in their structure. In addition, the fibrilar structure of the Forisomes together with
their reported function involved in the formation of slime plugs upon wounding of the sieve
elements (Knoblauch and van Bel, 1998; Knoblauch et al., 2001), leads to the assumption that
one or several subunits are involved in the structure of the Forisomes. Possibly, the
accumulation of structural P-protein fibres observed by electron microscopy of crystalline P-
proteins (Arsanto, 1982) could refer to an evolutive connection and the possibility that both
structures are assembled by the same subunits. The function of these two phloem proteins in
plugging the sieves elements upon wounding would explain the protein biosynthesis of
individual subunits in the companion cells and their subsequent transport to the sieve
elements for the repair of the proteins. Hence, the delivery of one or several subunits of the
Forisome after differentiation of the sieve element from the companion cell would be
conceivable reinforcing the hypothesis of the transport of VFF1 by the PPUs in the sieve
elements, as described for several proteins (Fisher et al., 1992).
Chapter IV Discussion ___________________________________________________________________________
89
Based on experiments using transgenic plants, snowdrop lectin and GFP have been reported
to traffic into sieve elements in N. tabacum (Shi et al. 1994, Imlau et al., 1999). Moreover,
Fukuda et al. (2005) reported that GUS, which has a larger molecular mass (68 kDa) than
snowdrop lectin (12 kDa) or GFP (27 kDa) enters sieve elements in rice. This is interesting
since the β-glucuronidase gene expression product has a similar molecular mass (68 kDa) to
the Vicia faba Forisome protein of ~70 kDa (VFF1) suggesting the possibility of being
transported to the sieve elements by an analogous process. Large proteins were suggested to
traffick selectively from CC to SE (Fisher and Cash-Clark, 2000; Oparka and Santa Cruz,
2000; Wu et al., 2002), with mechanisms of selectivity that are unknown. It was previously
reported that the selective traffic of a protein from CC to SE, is presumably mediated by
molecular recognition (Itaya, A. et al., 2002). Such selective protein traffic may account for
the presence of proteins of 60 to 200 kD in the phloem exudate of many plant species
(Balachandran et al., 1997; Hayashi et al., 2000). In some cases, a protein may be anchored to
membranes and cleavage of the protein may precede active traffic from CC to SE
(Xoconostle-Cázares et al., 2000).
In addition, previous microinjection experiments demonstrated that 10 kDa, but not 40 kDa,
fluorescent dextran could move through plasmodesmata between sieve elements and
companion cells of Vicia faba (Kempers and van Bel, 1997). In O. sativa, direct injection
experiments produced similar results: that 3 kDa dextran could move through the
plasmodesmata between sieve elements and companion cells, but 42 kDa dextran could not
(Fujimaki et al., 2000). Dextrans of 3, 10 and 40 kDa have Stokes’ radii of approximately 1.2,
2.0 and 4.3 nm, respectively (Fisher and Cash-Clark, 2000), suggesting that the size exclusion
limit of the sieve element-companion cell boundary is at least 1.2 nm in V. faba and 2.0 nm in
O. sativa. However, these results do not exclude the possibility of a different mechanism of
transport taking place in V. faba which might facilitate the movement of larger molecules
between companion cells and sieves elements. In this respect, it is worthwhile to cite the work
of Fukuda et al. (2005) who showed that GUS, with a Stokes’ radius of 3.3 nm (Fisher and
Cash-Clark, 2000), could enter the sieve elements. Some phloem proteins, like C. maxima
PP2 and CmPP16, R. communis glutaredoxin and cystatin, and O. sativa TRXh, were reported
to increase the size exclusion limit of mesophyll plasmodesmata and facilitate their own cell-
to-cell movement after microinjection (Balachandran et al., 1997; Ishiwatari et al., 1998;
Xoconostle-Cázares et al., 1999). To explain the cell-to-cell movement of proteins larger than
the size exclusion limit of plasmodesmata, protein unfolding was proposed. Large proteins
like GUS might be unfolded prior to moving through plasmodesmata and then refolded in the
sieve elements. Indeed, homologues of two chaperones, rubisco-subunit-binding protein and
cyclophilin, have been detected in sieve-tube exudates from T. aestivum, O. sativa, Yucca
filamentosa, C. maxima, Robinia pseudoacacia and Tilia platyphyllos (Schobert et al., 1998).
Moreover, a small heat-shock protein has been detected in phloem sap from O. sativa (Fukuda
et al., 2004). Microinjection experiments on N. tabacum mesophyll cells revealed that TRXh
Chapter IV Discussion ___________________________________________________________________________
90
increased the size exclusion limit of plasmodesmata and modified their own cell-to-cell
movement, and that certain structural motifs of TRXh are necessary for cell-to-cell movement
of this protein (Ishiwatari et al., 1998).
In summary, it is suggested that the efficient transport of the proteins comprising the
Forisomes from companion cells to sieve elements might need specific structural motifs,
which are recognized by plasmodesmata carriers, and modify cell-to-cell movements of these
proteins. Nevertheless, further analysis of VFF1 by microinjection experiments in V. faba
and/or immunolocalization will be required to clarify how this protein is delivered to the sieve
elements.
On the other hand, the expression of VFF1 in sieve elements of young phloem tissue might be
in part correlated with the presumption of the presence of promoter activity in the early stages
of sieve elements differentiation. Indeed, the presence of crystalline P-proteins (Forisomes) in
the early stages of sieve elements development has been reported (Esau, 1971). Besides,
several authors (Wergin and Newcomb, 1970; Wergin et al., 1975) observed condensed
crystalline P-proteins in differentiating sieve elements of the Fabaceae. This accumulation of
fibril material in the proximity of ribosomes in immature sieve elements represents therefore
the preliminary stages of the Forisomes. Thus, the observed GUS expression in the sieve
elements of young phloem tissue of transgenic N. tabacum might be reflective of the activity
of the identified vff1 promoter in the differentiating sieve elements still capable of protein
biosynthesis.
The expression of different fluorescent reporter enzymes (e.g. GFP, YFP, DSRed) under
control of the vff1 promoter will give the possibility to gain more insight into the promoter
activity in vivo.
IV.2.4 Developmental expression pattern of vff1 promoter in different maturation stages
In transgenic tobacco the vff1 promoter directed GUS expression in the phloem cells of the
vasculature in leaves, stems, flowers and roots indicating as highly tissue-specific regulation.
To determine if the promoter expression pattern is developmentally regulated, promoter-GUS
studies were performed on T1 vff1P-GUS tobacco seedlings.
IV.2.4.1 Vff1 promoter expression pattern in roots of N. tabacum
In transgenic vff1P-uidA tobacco plants, expression of β-glucuronidase in developing phloem
tissue and, especially, in the meristematic and elongation zones at root tips of germinating T1
seedlings was detected (Fig. III-21D). During root elongation, this expression pattern was
restricted to the meristematic and elongation zones at root tips. In the tips of fully developed
Chapter IV Discussion ___________________________________________________________________________
91
roots, vff1 expression was no longer detectable in accordance to the cession of developing
phloem synthesis.
The most apical 2mm of the root tip, which showed strong β-glucuronidase activity, is a
region of the root that includes the root meristem, the elongation zone, and the differentiation
zone where lateral root formation is initiated (Schiefelbein and Benfey, 1994; Dolan and
Davies, 2004). The primary root meristem has many cells undergoing rapid cell division
(Beeckman et al., 2001; Birnbaum et al., 2003). It has also been observed (van der Weele et
al., 2003) that the root tip contains two distinct zones with constant elongation rates, lower in
the meristematic zone (0 to 0.5 mm from the tip) and much higher in the elongation zone (0.5
to 1.2 mm from the tip). In the differentiation zone, the growth rate decreases to zero.
Some roots are genetically determined to have the capacity to undergo additional radial
growth, but other roots do not. Only dicotyledons have this capacity and it is more likely to
occur in thicker, lower-order lateral roots. This radial growth is called secondary growth to
distinguish it from primary growth at the root apex. Secondary growth occurs when new
meristematic tissue forms in a ring around the vascular cylinder of roots and produces new
xylem inwards and new phloem outwards (Dean, G. et al., 2004). This would explain the
GUS expression observed in growing lateral root (Fig. III-21C, E and F). Thus, during the
early stages of lateral root formation, vff1 promoter expression seemed to be restricted to the
developing phloem tissue (Fig. III-21F).
In summary, these results provide convincing evidence that indicate not only a spatial but also
a temporal regulation of the expression pattern driven by the vff1 promoter in roots of tobacco
plants.
IV.2.4.2 Vff1 promoter expression pattern in leaves of N. tabacum
Strong GUS activity was also detected in the first leaves and shoot apical meristem of
vff1P::GUS seedling suggesting a preferential expression of vff1 promoter in developing
vascular tissue and especially in young phloem tissue of tobacco plants. In addition, a
particular expression pattern of GUS expression was observed in the veins of tobacco leaves
(Fig. III-23).
The leaf vein classification of dicotyledonous plants is based largely on anatomical
observations (e.g., Avery, 1933). In anatomical terms, minor veins do not have rib tissue that
protrude beneath the surface of the lamina (Esau, 1965). However, from a physiological and
developmental perspective, minor veins are those that are immature in sink leaves and do not
participate in phloem unloading (Turgeon, 1987; Roberts et al., 1997) but mature during the
sink-source transition (Turgeon and Webb, 1976). The anatomical, developmental, and
functional roles of leaf venation have been well studied in tobacco. The veins of tobacco
Chapter IV Discussion ___________________________________________________________________________
92
leaves have been subdivided into classes, based on cell numbers rather than branching pattern,
which can be misleading (Ding et al., 1988).
GUS expression was detected in the phloem of developing leaves (Fig. III-22). In mature
leaves GUS activity was only detected in class-I (midrib) veins, and class-II veins (branching
from the midrib) (Figure III-23A). In class-III veins, staining was first evident in isolated
patches, often at branch points where class-IV veins merged with them (Figure III-23B). For
class-V veins no staining was observed (Figure III-23B). Staining decreased in intensity
toward the finest veins. This finding was interesting since in developing leaves there is a
basipetal gradient in maturity, the distal end being more mature than the base. As a leaf
develops, the class I and II veins undergo differentiation whereas class III veins have not yet
differentiated. In sink tissue, class IV and V veins are still immature. The phloem in these
veins is still undifferentiated (Turgeon and Webb, 1976), class IV and V veins mature
progressively toward the finest ramifications of the vein network (Turgeon and Webb, 1976),
beginning at the junction of the class III veins (Roberts et al., 1997). According to this, it
became obvious that these results confirmed a developmental expression pattern of vff1
promoter.
It seems likely that structural vein maturation is developmentally programmed (Wright et al.,
2003). The activation of the vff1 promoter within the veins of tobacco suggests that the
promoter responds to a regulatory system. Moreover, the reduced staining in late veins
development stages suggests that phloem differentiation may affect the vff1 promoter
regulation. Nevertheless, the nature of these signals, and whether the vff1 promoter responds
to the same conserved signals in different plant species, remains to be determined.
Taken together, the overall temporal and tissue-specific regulation of the vff1 promoter are
indicative of the expression of Forisomes mainly in the first stage of phloem development,
which is in turn related to the biological function of these proteins in the sieve elements of V.
faba as part of a defence mechanism against the loss of assimilates upon wounding
(Knoblauch, M. and Peters, W., 2004). Nevertheless, since the vff1 promoter was introduced
into tobacco, further studies are required to access whether the expression pattern of the vff1P-
GUS construct correlates with phloem development in V. faba.
Moreover, experiments combining GUS expression and in situ RNA hybridization would be
of interest to gain insight in the development profile of the expression pattern driven by the
vff1 promoter.
IV.2.5 Vff1 promoter activation during the sink-source transition
In cotyledons, which act solely as sources, and in the first formed leaves, a functional minor
vein network has not formed by the time the tissue becomes a source (Turgeon and Webb,
Chapter IV Discussion ___________________________________________________________________________
93
1976; Turgeon, 1989; Wright et al., 2003). In sink tobacco leaves, it has been suggested that
the earliest differentiation takes place in the external phloem at the tip of the midrib and that
maturation progresses basipetally (Avery, 1933). However, other reports claim that
maturation of the midrib and major veins occurs acropetally (Esau, 1960; Turgeon and Webb,
1973). Although the present work does not provide any information regarding the structural
maturation of the major veins, it does demonstrate an acropetal progression in the vff1
promoter expression pattern.
Taken together, this can be correlated with the observed absence of staining in cotyledons and
first pair of leaves in vff1P::GUS N. tabacum seedlings whereas it was evident an acropetal
vff1 promoter expression pattern in leaves undergoing the sink-source transition (Fig. III-22).
This might be a consequence of a down-regulation of vff1 promoter expression as the sink-
source transition progresses. The cessation of GUS expression in source tissue of vff1P::GUS
plants undergoing the sink-source transition, suggests that activation of the vff1 promoter is an
accurate marker for the sink-source transition in tobacco leaves.
During the sink-source transition, the minor veins became structurally mature (Turgeon and
Webb, 1973; Turgeon, 1989). Because subunits of Forisomes are thought to be expressed
within the companion cells of mature veins, it could be argued that its expression is linked to
structural maturation of the minor veins, reflecting once again the temporal regulation of the
expression of vff1 promoter.
However, further experiments aiming to identify the factors controlling vff1 promoter
activation and vein maturation, will be required to clarify how putative control points in the
sink-source transition could regulate the vff1 promoter expression pattern in developing
leaves.
IV.2.6 Vff1 promoter expression induced upon wounding
Analyses of vff1 promoter-GUS plants reproducibly gave very strong GUS staining at all sites
where leaves or stem sections were cut before the incubation with the staining solution. This
result suggested that the activity of the vff1 promoter driving this GUS expression might be
enhanced upon wounding however, up to date, convincing evidence is lacking that this is
indeed the case. Therefore, to exclude the possibility that this increased GUS activity at the
sites of wounding may simply result from a better diffusion of substrate into the plant tissue,
cell-free extracts (Roberts et al., 1989; Bauer et al., 1993) should be prepared from untreated
control tissues and from tissues that had been wounded. GUS activities could be determined
in these extracts using the GUS substrate 4-methylumbelliferyl-β-d-glucuronide (MUG),
which yields a fluorescent product after hydrolysis by GUS. Whether GUS-derived
fluorescence indicates an induced expression upon wounding in vff1P::GUS N. tabacum,
would be determined by a comparison to wild-type plants. The absence of GUS-derived
Chapter IV Discussion ___________________________________________________________________________
94
fluorescence in the extracts from wild-type plants, from unwounded or wounded tissues, in
contrast to significant GUS activities in transgenic plants might reflect a wounding
enhancement in the promoter expression.
In addition, to confirm these data analysis of potential changes in GUS mRNA levels in tissue
of tobacco transgenic plants at different times after wounding would be required.
IV.2.7 Deletion analysis
Experiments using 5'-upstream deletion mutants cloned unidirectional into a promoter-less
GUS reporter vector, were conducted to define the region of the vff1 promoter involved in its
organ-, cell-specific and developmentally regulated expression pattern. Even after several
attempts, it was not possible to obtain conclusive evidence. As mentioned above, a striking
feature of the vff1 promoter is its tissue-specific expression together with its developmental
regulation. This interpretation may explain the absence of substantial differences in GUS
activities in the analysis of vff1 promoter in transiently transformed tobacco leaves. Assuming
that the synthesis of Forisomes is taking place during the early stages of sieve element
development and that the subsequent differentiation of the sieve element from the companion
cell imply only the delivery of subunits of the Forisome, thus evoking the possibility of a
diminished protein synthesis, the low levels of GUS expression in mature vacuum-infiltrated
leaves denote an inadequate method for promoter studies. In addition, experiments performed
in protoplasts showed a low GUS activity driven by vff1 promoter which excludes the
possibility of using it as a suitable method.
An alternative approach will be the investigation of the regulation of the vff1 promoter
through the characterization of stable transformed tobacco plants.
It is interesting to note that, though many phloem-specific promoters have been characterized
from vascular plants, plant viruses and phytopathogenic bacteria, the unique advantages of
using phloem-specific promoters derived from plant genes, are attractive in plant
biotechnology (e.g. for the targeted expression of molecules in the phloem against vascular
pests or pathogens). Therefore, the vff1 promoter could be of practical value in
biotechnological applications, especially for genetic engineering of tobacco and possibly other
dicotyledonous plants when the expression of a foreign gene is required mainly in the phloem.
Chapter IV Discussion ___________________________________________________________________________
95
IV.3 Conclusion and future prospects
Previous studies indicated that Forisomes perform mechanical work by deforming
anisotropically without direct interaction with a chemical energy supply such as ATP. It has
also been reported that they are extremely sensitive to their regulative agent, Ca2+, but are
structurally stable if kept under appropriate conditions (Knoblauch et al., 2003). It has been
proposed that, as the response can be evoked alternatively by changes of pH, it is possible to
drive Forisome contraction and curvature electrically by electrotitration. Because Forisomes
consist of protein, they lend themselves to biomolecular engineering, opening the possibility
to create variants of format and functionality. Owing to this combination of desirable
properties, Forisome proteins appear to provide a useful paradigm of peptide actuators in the
rapidly evolving fields of biomimetic robotics, microfluidics and microscale engineering.
Data presented in this study demonstrated the identification and characterization of a cDNA
clone encoding a Vicia faba Forisome protein (VFF1). From the characterization of the
different motifs identified within the aminoacid VFF1 sequence, it was evident that this
protein comprises several structural features related to helices and coiled-coil motive
formation. These structural characteristics, correlated with the observed Ca2+ response in vitro
(Knoblauch et al., 2003), indicated a possible Ca2+ dependent reaction of the Forisomes in
which the VFF1 protein could be involved. Nevertheless, another possibility to explain the
comformational change undergone as a response to this divalent cation is that as yet
unidentified proteins, with known Ca2+-binding domains or pH-responsive domains, might
contribute to the structure of Forisomes. Therefore, further experiments to look at protein
interactions within Forisomes, e.g. cross linking of Forisomes followed by tryptic digest and
mass spectrometry, will provide additional information on Forisome structure.
Considering technical applicability, it would appear desirable if Forisome action could be
controlled. Accordingly, it will be crucial to provide complete and precise data for their
protein composition. The question arises whether there are additional proteins which are
involved in the structure of the Forisomes, however a probable interaction of such proteins
with the VFF1 might lead to their identification. Therefore, a yeast-2-hybrid-system (Fields,
S. and Song, O., 1989) could be used.
Additionally, cloning and recombinant expression of the isolated vff1 gene permitted the
generation of VFF1-specific polyclonal antibodies. Furthermore, Forisome-specific
polyclonal antibodies were obtained. By means of western blot analysis together with
confocal immunofluorescence analysis, immunological evidence of vff1 gene encoding a
Forisome protein is reported here.
Future work will focus on the identification of additional Forisome genes and their molecular
characterization. Expression of potential yet unidentified Forisomes genes will also permit the
Chapter IV Discussion ___________________________________________________________________________
96
production of specific antibodies to provide new insights into the structure of the Forisome
and to uncover some unifying principal underlying their conformational change and related
biological function. Furthermore, an accurate analysis of the antibody specificity would be
conceivable by means of electron micrographs, after embedding of the Forisome and
immunogold detection. As a complete analysis of the potentially further Forisomes genes was
not possible within the time frame of this PhD thesis, the forthcoming cloning and
characterization of these proteins will provide information on their involvement in the
structure of the Forisomes. Cloning and characterization of the vff1 gene described here has
opened up this important opportunity.
Within this thesis, the isolation and characterization of vff1 promoter is also reported. Its
activity as well as tissue specificity was confirmed in transgenic N. tabacum plants using
GUS as a reporter system. In addition, sequence analysis of the promoter region of vff1 gene
revealed the presence of two conserved motifs which have already been reported for several
phloem-specific promoters. Significantly, these findings indicate that the 5′-upstream region
of vff1 functions as a promoter that drives preferential expression of the uidA gene to the
phloem of transgenic tobacco plants. Besides the tissue-specificity a spatial and temporal
regulation of vff1 promoter observed during development provide evidence of the expression
of Forisomes mainly in the first stage of phloem development, which is in turn related to the
biological function of these proteins in the sieve elements of V. faba as part of a defence
mechanism against the loss of assimilates upon wounding (Knoblauch and Peters, 2004).
Nevertheless, since the vff1 promoter was introduced into tobacco, Vicia faba in planta
transformation will be interesting to carry out to access whether the expression pattern of the
vff1P-GUS construct correlates with phloem development in this Fabaceae.
These results also indicated that there may be similar transcription factors in tobacco and V.
faba that regulate expression of the promoter. Central to both basic and applied molecular
biology is the identification of cis elements that contributes to the expression of promoters.
Accordingly, experiments using 5'-upstream deletion mutants were conducted to define the
region of the vff1 promoter involved in its organ-, cell-specific and developmentally regulated
expression pattern. Nevertheless, it was not possible to obtain conclusive evidence. An
alternative approach will be the investigation of the regulation of the vff1 promoter through
the characterization of stable transformed tobacco plants. Moreover, experiments combining
GUS expression and in situ RNA hybridization will be crucial to gain insight in the
development profile of the expression pattern driven by the vff1 promoter.
In addition, the expression of different fluorescent reporter enzymes (e.g. GFP, YFP, DSRed)
under control of the vff1 promoter will give the possibility to gain further insight into the
promoter activity in vivo.
Chapter V Summary ___________________________________________________________________________
97
V Summary
Forisomes are phloem-specific proteins of the family of the Fabaceae which undergo a rapid,
reversible and ATP-independent comformational change when stimulated by variations in
Ca2+ levels or pH. These proteins, present in the sieve elements, switch instantaneously
between a dispersed and a condensed conformation, allowing them to control the stream of
photo-assimilates in the phloem after wounding by sealing the conducting tubes.
Isolation of Forisomes from Vicia faba permitted the sequencing of peptides from which
specific oligonucleotides were designed for library screening. In addition, polyclonal
Forisome-specific antibodies were obtained. First attempts to identify Forisome-encoding
genes were performed by hybridization screening of subtracted and non-subtracted Vicia faba
derived cDNA libraries, however, no successful results could be retrieved. In an alternative
approach, immunoscreening along with PCR screening of cDNA expression library of Vicia
faba plant tips was carried out. Neither the identification of cDNAs encoding antigenic
Forisome fusion proteins nor the allocation of DNA- and/or DNA fragments to the Forisome
genes was possible.
Aligning the isolated Forisome peptide sequences from Vicia faba with a recently published
EST-library for Medicago truncatula – the model plant for Fabaceae – enabled the
identification of patches of amino acids conserved among the two species belonging to the
same DNA sequence. This important finding provided the basis for the isolation of genes
encoding Forisome proteins. Hence, the rapid amplification of cDNA ends (RACE) together
with genome walking was used to obtain the full-length cDNA as well as the genomic
sequence of the Forisome gene vff1.
Sequence analysis of the amplified vff1 was performed and even thought no well-known Ca2+-
binding motif could be identified, different domains which might be involved in Ca2+
dependent reaction of the Forisomes were recognized within the vff1 gene.
Cloning and recombinant expression of the isolated vff1 gene in E. coli permitted the
generation of VFF1-specific polyclonal antibodies. Immunological evidence of the isolated
vff1 gene encoding a Forisome protein was obtained by means of western blot analysis and
confocal immunofluorescence techniques.
Further experiments were performed with the aim of identifying and cloning the vff1 gene 5’-
flanking region. The putative promoter of the vff1 gene was isolated from genomic DNA by
genome walking and its activity as well as tissue specificity was confirmed in transgenic
tobacco plants using GUS as a reporter system. Sequence analysis revealed two conserved
motifs shared with several previously reported phloem-specific promoters. Taken together,
these results indicate that the 5′-upstream region of vff1 functions as a promoter (designated
vff1P) that directs preferential expression of the uidA gene to the phloem of transgenic
Chapter V Summary ___________________________________________________________________________
98
tobacco plants. Besides the tissue-specificity a spatial and temporal regulation of vff1P was
observed during development of transgenic tobacco seedlings.
Chapter VI References ___________________________________________________________________________
99
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VII Appendices
VII.1 List of abbreviations
ABTS 2,2’-Azino-di-(3-ethylbenzthiazoline sulphonate)
Ab Antibody
Ag Antigen
Amp Ampicillin
AP Alkaline phosphatase
ATP Adenosine triphosphate
BCIP/NBT 5-bromo-4-chloro-3-indolyl phosphate/p-nitrobluetetrazolium chloride
bp Base pair
BSA Bovine serum albumin
CaMV 35S 35S promoter from the Cauliflower mosaic virus
carb Carbenicillin
CC Companion cell
CDMFDA 5[6]-carboxy-4,5’-dimethylfluorescein diacetate
cDNA Complementary DNA
CFDV Coconut Foliar Decay Virus
CI Chloroform/isoamyl alcohol (24:1)
CLSM Confocal Laser Scanning Microscopy
cv. Cultivar
Cys Cysteine
dil. Dilution
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleoside triphosphate
DSRed Discosoma-Red
DTT 1,4-dithiothreitol
E. coli Escherichia coli
EDTA Ethylene diaminether tetra-acetic acid
e.g. exempli gratia
ELISA Enzyme-linked immunosorbent assay
ER Endoplasmatic reticulum
Chapter VII Appendices ____________________________________________________________________________________________________
117
EtOH Ethanol
g Relative centrifugal force (RCF)
GFP Green fluorescent protein
GSM Agrobacteria glycerol stock media
GSP Gene specific primers
GST Glutathione S-transferase
GUS ß-Glucuronidase
h Hour(s)
HCl Hydrochloride
HRP Horse radish peroxidase
i.e. id est
IPTG Isopropyl β-D-thiogalactopyranoside
kb Kilobase pair
kDa Kilodalton
Km Kanamycin
L Litre
LB Luria-Bertani medium
M Molarity
MALDI-TOF Matrix-assisted laser desorption ionization-time of flight
MES 2-(N-morpholino)-ethanesulphonic acid
min Minute(s)
MgSO4 Magnesium sulfate
Mr Molecular mass
mRNA Messenger RNA
MS Murashige and Skoog medium
MW Molecular weight
NaCl Sodium chloride
nos Gene of the nopaline synthase
Nt Nucleotide
OD Optical density
o/n Overnight
ORF Open reading frame
Ori Origin of replication
PBS Phosphate buffered saline
Chapter VII Appendices ____________________________________________________________________________________________________
118
PBST Phosphate buffered saline, 0,1% (v/v) tween-20
PCR Polymerase chain reaction
PEG Polyethylenglycol
pH A logarithmic measure of hydrogen ion concentration
pNPP p-nitrophenyl phosphate
poly A Polyadenylation signal
PPUs Pore plasmodesmata units
Rif Rifampicilin
RNA Ribonucleic acid
RNase Ribonuclease
rpm Rounds per minute
RT Room temperature
S Segment; small
SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SE Sieve element
sec seconds
SER Sieve element endoplasmic reticulum
Taq Thermus aquaticus
TB Terrific broth
TBE Tri- buffered saline electrophoresis buffer
TCA Trichloroacetic acid
TEMED N,N,N′, N′-tetramethylene-ethylenediamine
TMV Tobacco mosaic virus
Tris Tris(hydroxymethyl)aminomethane
UTR untranslated region
Ω Omega leader region of TMV
V Volt; variable region
v/v Volume per volume
w/v Weight per volume
w/w Weight per weight
YFP yellow fluorescent protein
Chapter VII Appendices ____________________________________________________________________________________________________
119
VII.2 Schematic presentation of vectors maps
A
B
Chapter VII Appendices ____________________________________________________________________________________________________
120
C
D Figure VII-1 Schematic presentation of the vector maps. A: pCR® 2.1-TOPO® (Invitrogen), the nucleotide sequence of the multiple cloning site is indicated, B: pGEX-5x-3, C,D: pTRA.
Chapter VII Appendices ____________________________________________________________________________________________________
121
VII.3 Figures
Figure I-1 In vivo structure of sieve elements in Vicia faba 3
Figure I-2 Comformational change of crystalline P-protein bodies in Vicia faba
as a response to injury 4
Figure I-3 Structures of parallel coiled coils 8
Figure I-4 Schematic outline of the thesis 13
Figure III-1 SDS-PAGE analysis of isolated Vicia faba Forisomes 41
Figure III-2 Immunoblot analysis of isolated Vicia faba Forisomes with mouse and
chicken antisera 42
Figure III-3 2D-gel electrophoresis and immunoblot analysis of isolated Vicia faba
Forisomes 43
Figure III-4 Hybridization screening on arrayed Vicia faba cDNA libraries 44
Figure III-5 Immunoscreening of Vicia faba cDNA expression library of plant tips 45
Figure III-6 Agarose Gel Electrophoresis of 5’- and 3’- RACE PCR amplification
products of Vicia faba cDNA 47
Figure III-7 Sequence alignment of 5’- RACE PCR products of Vicia faba cDNA 48
Figure III-8 RT-PCR amplification product of full-length Forisome Vicia faba cDNA 48
Figure III-9 Vicia faba Forisome-1 (VFF1) predicted amino acid sequence 49
Figure III-10 Representation of coiled-coil motif and amphipatic α-helix comprised
in the VFF1 protein sequence 50
Figure III-11 Analysis of total RNA isolated from Vicia faba 52
Figure III-12 RACE analysis of the transcription start site of the vff1 gene 52
Figure III-13 LD PCR on Vicia faba genomic DNA using primers derived from the
cDNA 53
Figure III-14 Analysis of Vicia faba genomic DNA digestion 54
Figure III-15 Nucleotide sequence of the 5’-flanking region of the vff1 gene 55
Figure III-16 Primary and secondary BD GenomeWalker™ PCR 56
Figure III-17 Nucleotide sequence of vff1- promoter and the 5’-flanking region of
the vff1 gene 58
Figure III-18 Strategy for cloning the vff1 promoter-Gus expression cassette into
pTRAk 59
Figure III-19 Expression of vff1 promoter-GUS fusions in transgenic tobacco 61
Chapter VII Appendices ____________________________________________________________________________________________________
122
Figure III-20 Expression patterns of vff1 promoter-GUS fusions in transgenic tobacco
flowers 62
Figure III-21 Vff1 promoter expression pattern in vff1P::GUS transgenic plants 63
Figure III-22 Expression pattern of vff1 promoter in immature veins of leaves 64
Figure III-23 Expression pattern of vff1 promoter in veins of mature tobacco leaves 64
Figure III-24 PCR amplification of truncated vff1 promoter-GUS fusions fragments 65
Figure III-25 SDS-PAGE and immunoblot analysis of affinity purified GST fusion
proteins 66
Figure III-26 SDS-PAGE analysis of affinity purified GST fusion VFF1 67
Figure III-27 SDS-PAGE and immunoblot analysis of affinity purified recombinant
VFF1 68
Figure III-28 Characterization of GST-VFF1 fusion protein by immunoreativity
against Forisome-specific mouse and chicken antisera 69
Figure III-29 Immunoblot analysis of Vicia faba isolated Forisomes and recombinant
VFF1 fusion protein using VFF1 specific rabbit antiserum 70
Figure III-30 Confocal immunofluorescence analysis of Vicia faba native Forisomes
using polyclonal Forisome-specific mouse and chicken antibodies 72
Figure III-31 Confocal immunofluorescence analysis of Vicia faba native Forisomes
using polyclonal VFF1-specific 73
Figure VII-1 Schematic representation of vector maps 120
VII.4 Tables
Table II-1 Name, suppliers and genotypes of bacterial strains used throughout
this thesis 16
Table II-2 Primers used for PCR amplification in deletion analysis of vff1 Promoter
in Vicia faba 17
Table II-3 Primers used for cDNA amplification in RACE-PCR 18
Table II-4 Primers used for PCR amplification and subclone Vicia faba forisome
cDNA, N-terminal, C-terminal and central domains 18
Table II-5 PCR reactions 28
Table III-1 Oligonucleotides used for hybridization screening 44
Table III-2 Sequence comparison of GATA motifs present in the vff1promoter and
other phloem-specifc promoters 57
Chapter VII Appendices ____________________________________________________________________________________________________
123
Table III-3 Sequence comparison of the 13 bp motifs present in the vff1 promoter
and other phloem-specific promoters 57
Acknowledgements
First of all, I want to express my gratitude to Prof. Dr. Rainer Fischer for giving me the
opportunity to work in the Fraunhofer Institut für Molekularbiologie und Angewandte
Oekologie IME in Aachen and reviewing my thesis.
Prof. Dr. Dirk Prüfer for entrusting me with this project, for all the valuable suggestions and
support during my work, his critical reading of my thesis, and for agreening to be co-
examiner.
Dr. Hannah Jaag, my gratitude to her for her guidance throughout my doctoral work, and very
much for being of great help on my arrival in Aachen.
It is my pleasure to acknowledge the constructive discussions, motivating enthusiasm and
encouragement of Dr. Michael Knoblauch during the course of this project.
Furthermore, I would like to thank Dr. Winfried Peters for his scientific acumen and critical
thinking.
Sincere thanks are due to my colleagues not only in Aachen but also in Giessen and
Schmallenberg, for their cooperation and kindness.
I thank Dr. Jost Muth for the sequencing and interesting discussions.
Special thanks go to Eva Aguado for her precious help in tissue culture and plant
maintenance.
Most of all, I thank my family for their support, patience and understanding. This work would
certainly not have been possible without them.
I thank the friendship of all those who became an important part of my life during these years
and their seemingly unlimited belief in me.
And last but not least, my very special thanks go to those who having most closely
experienced and shared all the ups and downs that came along during this time, managed to
make my life easier and funnier.
____________________________________________________________________________________________________
Lebenslauf
Persönliche Daten
Name: María Eugenia Fontanellaz
Geburtsdatum: 25.09.1973
Geburstort: Rosario, Argentinien
Familienstand: Ledig
Staatsangehörigkeit: argentinisch
Ausbildung
1986-1990 Instituto Politécnico Superior Gral. San Martín Gymnasium,
Rosario, Argentinien
1991-2001 Fakultät für Biochemische und Phamazeutische
Wissenschaften, Rosario, Argentinien
Abschluß: Master of Sciences
Thema der Arbeit: “Functional properties of whey protein
concentrate“
4/2002-2/2006 Promotion am IME Fraunhofer Institut für Molekularbiologie
und Angewandte Oekologie Aachen unter Anleitung von Prof.
Dr. Rainer Fischer
Berufserfahrung
4/2002-12/2005 Wissenschaftlicher Angestellter am IME Fraunhofer Institut für
Molekularbiologie und Angewandte Oekologie Aachen
1/2006-6/2006 Wissenschaftliche Angestellter am Institut für Biologie VII der
RWTH Aachen
Seit 7/2006 Wissenschaftlicher Angestellter (Post-Doc) am Institut für
Biochemie und Biotechnologie der Pflanzen Westfälische
Wilhelms-Universität Münster