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Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase
Theresa A. Hill1, Jean Broadhvest2, Robert K. Kuzoff3, and Charles S. Gasser4
Section of Molecular and Cellular Biology, University of California, Davis, CA, 95616
1Current address: USDA ARS WRRC-GGD, 800 Buchanan St., Albany, CA 94710 2Current address: Bayer BioScience N.V., Technologiepark 38, 9052 Ghent, Belgium 3Current address: Department of Biological Sciences, University of Wisconsin, Whitewater, WI, 53190 4Corresponding author: Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Ave., Davis, CA 95616, Tel. 530-752-1013. Fax 530-752-3085 E-mail [email protected]. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AY254472.
Genetics: Published Articles Ahead of Print, published on July 18, 2006 as 10.1534/genetics.106.060657
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Running title: SHORT INTEGUMENTS2 is a mitochondrial GTPase
Key words: growth, ovule, cell division, plant development, signal transduction
Charles S. Gasser
Section of Molecular and Cellular Biology
University of California, Davis
1 Shields Ave. Davis, CA 95616
Tel. 530-752-1013
Fax 530-752-3085
e-mail : [email protected]
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ABSTRACT
The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short
integuments due to early cessation of cell division in these structures. SIN2 was isolated
and encodes a putative GTPase sharing features found in the novel DAR GTPase family.
DAR proteins share a signature DAR motif and a unique arrangement of the four
conserved GTPase G motifs. We find that DAR GTPases are present in all examined
prokaryotes and eukaryotes, and that they have diversified into four paralogous lineages
in higher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases, have
been found to localize to mitochondria and are related to eubacterial proteins that
facilitate essential steps in biogenesis of the large ribosomal subunit. We propose a
similar role for SIN2 in mitochondria. A sin2 insertional allele has ovule effects similar
to sin2-1, but more pronounced pleiotropic effects on vegetative and floral development.
The diverse developmental effects of the mitochondrial SIN2 GTPase support a
mitochondrial role in the regulation of multiple developmental pathways.
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INTRODUCTION
Arabidopsis ovules are a useful model system for studying developmental mechanisms.
Arabidopsis ovules initiate on the inner surface of immature carpels as relatively
featureless finger-like primordia. As they elongate, three different regions become
specialized and undergo morphogenesis and cellular differentiation (ROBINSON-BEERS et
al. 1992). The distal-most region, the nucellus, is the site of meiosis and embryo sac
formation. The central chalazal region is the site of the most visible morphogenic changes
as it gives rise to two appendages, the inner and outer integuments. The basal region
elongates through division and coordinated expansion of cells forming the funiculus, a
supporting stalk. During this process, the developing ovule becomes bilaterally
symmetrical as a result of differential growth that causes the funiculus to curve toward
the base of the carpel and the outer integument to curve toward the carpel apex. At
maturity, both integuments have grown to enclose the nucellus and form a terminal
micropylar opening (Figure 1A). Mutations altering ovule morphogenesis may also
disrupt other plant developmental pathways and the relative morphological simplicity of
ovules can facilitate overall understanding of the underlying biochemical or molecular
processes.
Numerous genes affecting growth and patterning of ovules have been identified via
mutagenesis and cloning (SCHNEITZ 1999; SKINNER et al. 2004). Several of the genes
regulating Arabidopsis ovule development manifest their effects through alterations in the
pattern or progress of cell division. These genes encode proteins with a variety of
biochemical functions (SKINNER et al. 2004). For example, mutations in
AINTEGUMENTA (ANT) and INNER NO OUTER (INO), encoding AP2 and YABBY-
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domain transcription factors, respectively, result in a complete absence of both
integuments or only the outer integument (BAKER et al. 1997; ELLIOTT et al. 1996;
GAISER et al. 1995; KLUCHER et al. 1996; VILLANUEVA et al. 1999). TSO1 encodes a
novel nuclear protein required for proper orientation of cell elongation and cytokinesis
during floral organ and integument development (HAUSER et al. 2000; HAUSER et al.
1998; SONG et al. 2000). Severe mutations in the HUELLENLOS (HLL) gene, encoding a
mitochondrial L14 ribosomal subunit, lead to an arrest in ovule growth and degeneration
of the apical regions of the primordia, revealing a role for mitochondrial activity in the
process of ovule growth (SCHNEITZ et al. 1998; SKINNER et al. 2001). Among other
floral effects, reduced activity of the putative protein kinase TOUSLED (TSL) causes
short outer and protruding inner integuments (ROE et al. 1997a; ROE et al. 1997b; ROE et
al. 1993). The variety of protein classes involved in ovule growth implies complex
regulation of this process at the levels of transcription, signal transduction and
metabolism.
SHORT INTEGUMENTS 2 (SIN2) is required for sustaining cell divisions during
integument development (BROADHVEST et al. 2000). At anthesis, sin2-1 ovules have
fewer integument cells than wild type, leaving their nucelli exposed (Figure 1B). sin2-1
also has subtle pleiotropic effects on flower development. The gynoecia of sin2-1
mutants generally have a cleft stigma and occasionally bear an outgrowth on the
corresponding valve. Additionally, in sin2-1 sepals marginal cells appear to be absent or
highly reduced in number. These pleiotropic effects suggest that SIN2 facilitates several
morphogenic pathways.
Double mutants with sin2-1 revealed functional relationships between SIN2 and other
ovule development genes (BROADHVEST et al. 2000). ant-5 sin2-1 double mutants were
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indistinguishable from ant-5 mutants, but ant and sin2 had similar synergistic interactions
with hll. Both ant hll and sin2-1 hll double mutants exhibited a reduced number of
ovules and an earlier abortion of primordia development than hll single mutants. The
ovule effects of sin2-1 were additive with the cell expansion defects of tso1-3. Thus
SIN2 may act downstream of ANT in a common pathway with HLL, and in parallel to
TSO1 to promote cell division during integument growth (BROADHVEST et al. 2000).
In order to better understand regulation of cell division during organ formation we
have identified and characterized the SIN2 gene and isolated a second allele, sin2-2.
SIN2 encodes a putative GTPase of a relatively uncharacterized subclass, termed the
DAR GTPases on the basis of a conserved aspartate-alanine-arginine (DAR) motif and
other conserved features (FU et al. 1998). Some DAR GTPases have been found to be
important for cell division in bacteria, fungi, and human stem cell lines, where they are
associated with assembly or subcellular transport of ribosomal subunits (BASSLER et al.
2001; BIALKOWSKA and KURLANDZKA 2002; KALLSTROM et al. 2003; MATSUO et al.
2006; MORIMOTO et al. 2002; SAVEANU et al. 2001; TSAI and MCKAY 2002; UICKER et
al. 2006). We found SIN2 to localize to mitochondria, and hypothesize a function in
mitochondrial ribosome assembly. In conjunction with hll, sin2 mutants provide an
attractive system with which to study the role of mitochondrial function in the
development of a multicellular organism.
MATERIALS AND METHODS
Plant material: Plants were grown on soil as previously described (KRANZ and
KIRCHHEIM 1987; ROBINSON-BEERS et al. 1992). Some plants were germinated on 1%
agar containing 1% sucrose, 1X Murashige and Skoog salts and 1X Gamborg's B-5
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vitamins (GAMBORG et al. 1968; MURASHIGE and SKOOG 1962). The Students t-test was
used to determine if mutant plants were significantly different from wild type plants in
germination rate, number of days to flowering, number of rosette leaves produced, leaf
production rates, and number of axillary meristems elongation. Plant transformation
vectors and methods were as described (MEISTER et al. 2002).
Genetic mapping and complementation: Using a mapping population generated by
crossing sin2-1 (Landsberg erecta, Ler) with wild type Columbia (Co-3), SIN2 was
previously localized to chromosome 2 between the cleaved amplified polymorphic
sequence (CAPS) m429 and simple sequence length polymorphism (SSLP) AthBIO2
(BROADHVEST et al. 2000). Of 31 chromosomes that had a recombination event between
m429 and AthBIO2, four recombination points were between SIN2 and the SSLP marker
F13H10 Indel2 (Cereon accession CER448978; The Arabidopsis Information Resource
(TAIR) http://www.arabidopsis.org/Cereon/) and one recombination was between SIN2
and a ClaI RFLP within T11A7.16 (At2G41740). This region is spanned by three
overlapping bacterial artificial chromosome (BAC) clones (T11A7, T1K18, and T32G6;
CHOI et al. 1995). Cosmid subclones were generated from BAC T1K18 by partial
digestion with Sau3AI and insertion of 12 – 25 kb fragments in pOCA28-15 (ESHED et al.
1999). The overlapping cosmids cJBT1K18.136, cJBT1K18.105 and CJBT1K18.107
were identified and assembled into a contig using hybridization and fingerprint data.
These cosmids spanned the annotated genes At2G41600.1 to At2G41730.1, and were
transformed into plants from the sin2-1 mapping population. The genotype of transgenic
plants at the SIN2 locus was determined with the F13H10 Indel2 SSLP and the DdeI
CAPS marker 6D20R in At2G42090.1.
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Molecular methods: Coding regions for genes within the complementing cosmid
(cJBT1K18.136) were isolated from sin2-1 homozygous and Ler plants by reverse
transcription polymerase chain reaction (RT-PCR) using gene specific primers and
SuperscriptII reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNAs were
sequenced by Davis Sequencing, Davis CA. Sequences were compared using
Sequencher (Gene Codes Corp., Ann Arbor, MI). Genomic subclones including each of
these genes along with the sequences 5' and 3' extending into the next closest genes were
isolated from cJBT1K18.136 and transformed into plants from the sin2-1 mapping
population. For At2g41670 (SIN2) a 4.0 kb EcoRI fragment was isolated and inserted
into these same sites in pBJ97 (GLEAVE 1992) creating pTH13. At2g42670 was removed
from pTH13 on a NotI fragment and inserted in the same site in pMLBART (GLEAVE
1992) for plant transformation. The 5' end of the SIN2 cDNA was determined by
sequencing clones obtained with the GeneRacer 5'RACE System (Invitrogen, Carlsbad,
CA) using primers specific to SIN2. The full-length cDNA was inserted as an
Spe1/BamHI fragment into XbaI/BamHI digested pMON999 (MEISTER et al. 2002)
creating a P35S::SIN2::NOS3’ transcriptional fusion (pTH52). The cDNA was also
attached to 5.6 kb SIN2 5’-flanking region using a common NruI site in the first exon to
create pTH64. The gene fusions in pTH52 and pTH64 were transferred to pMLBART
(GLEAVE 1992) on NotI fragments for plant transformation.
Primers designed to amplify the SIN2 genomic region, SIN2KOF2 (GAT GGG TTA
TTA CGA TTT GGG CAG TTA TT) and SIN2KOR (CAG TTT CAG GGA CAT CGT
CAA GGA TAA AG), were used to screen the Wisconsin α-population of insertion lines
for additional sin2 alleles as described by Krysan et al. (1999). The insertion site in sin2-
2 was determined by sequencing PCR products using SIN2 primers on either side of the
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insertion and the T-DNA JL202 primer. The SIN2KOR and JL202 primers (KRYSAN et
al. 1999) were used to detect the sin2-2 insertion in segregating populations.
An RT-PCR product generated using synthetic primers T32G6.19F and SIN2GR on
first strand cDNA from Ler inflorescence poly-A+ RNA was cloned into the pCR2.1
vector (Invitrogen, Carlsbad, CA) forming pTH19. The insert was sequenced and found
to include the entire SIN2 coding region with no mutations except for those intentionally
introduced to remove the stop codon. This cDNA was mobilized on an SpeI/BamHI
fragment and inserted into pRJM86 (MEISTER et al. 2002) at the same sites to form a
fusion with the green fluorescent protein (GFP 1.1.15; SCHUMACHER et al. 1999) coding
region. The an SpeI/KpnI fragment containing the SIN2:GFP fusion protein coding
region was inserted between the CaMV 35S promoter and nopaline synthase
polyadenlyation region (NOS3’) in pMON999 (MEISTER et al. 2002). A NotI fragment
consisting of P35S::SIN2:GFP NOS3’ was inserted into the NotI site of the plant
transformation vector pMLBART (GLEAVE 1992) and transformed into plants.
Microscopy: Samples were prepared for scanning electron microscopy and images
were acquired and edited as described (BROADHVEST et al. 2000). Samples were fixed
for light microscopy as described by Baum and Rost (1996). For viewing petal veins,
following fixation, flowers were cleared overnight in a saturated aqueous chlorylhydrate
solution. Petals were removed from the flowers and observed with a Ziess Axioplan
(Ziess Inc., Oberkochen, Germany) microscope using dark field optics. GFP localization
was determined using transgenic SIN2:GFP T2 seedlings which were stained for 1 hr in a
1X MS solution containing the mitochondrial specific stain MitoFluor Red589
(Molecular Probes, Eugene, OR) at 500 nM and were visualized on a Zeiss (Oberkochen,
Germany) Axioscope microscope using fluorescein isothiocyanate and
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tetramethylrhodamine isothiocyanate filter sets respectively. Images were recorded using
the Openlab system (Improvision, Inc., Lexington, MA) and were adjusted for contrast in
Photoshop 6.0 (Adobe Systems, Inc., San Jose, CA).
Phylogenetic analysis: BLAST (ALTSCHUL et al. 1997) searches were used to
identify sequences in GenBank encoding proteins similar to SIN2 and other Arabidopsis
DAR proteins. A large number of proteins containing G motifs were identified. DAR
proteins (defined as those including a DAR-G4-G1-G2-G3 arrangement of motifs but
which did not have two complete G domains in tandem; FU et al. 1998) uniformly gave
the best matches, followed by ENGA proteins. Parsimony analysis was performed with
PAUP4.0b6 (SWOFFORD 1999) on alignments created using ClustalX (THOMPSON et al.
1997). Three separate alignments were produced using gap opening:substitution costs of
2.5:0.5, 5:0.4 and 10:0.3, and the BLOSUM series of weightings (HENIKOFF and
HENIKOFF 1992). ENGA sequences from the ENGA DAR motif to the second G3 motif
were used as an outgroup. Regions unstable between alignments were detected using
SOAP (LOYTYNOJA and MILINKOVITCH 2001). Sequences less than 70% stable were not
included in the phylogenetic analysis (culling). A second analysis was performed in
which all three alignments, arranged in tandem, were included (elision; WHEELER et al.
1995). In parsimony analysis, characters were weighted using a BLOSUM45 amino acid
substitution matrix, which was generated from the published BLOSUM45 matrix
(HENIKOFF and HENIKOFF 1992) by substituting “15 – n” for each value “n” of the matrix.
This results in all positive values, ranging from 0 to 20. An amino acid to gap transition
was given a cost equal to the highest value in the matrix, 20. Bootstrap values were
calculated from 500 bootstrap replicates of heuristic searches on ten random sequence
additions per bootstrap replicate and tree bisection-reconnection procedure for branch
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swapping. Branches that were not consistent between the culling and elision methods of
character weighting or those that had bootstrap values less than or equal to 50% were
collapsed.
Acession numbers: The GenBank/ EMBL accession number for the SIN2 cDNA is
AY254472. The accession numbers for other protein sequences used in the analysis are:
Arabidopsis DGP2 (CAB40031); Arabidopsis DGP3 (AAD15335); Arabidopsis DGP4
(AAF27009); Arabidopsis DGP5 (AAG52287); Arabidopsis DGP6 (AAD26884);
Arabidopsis DGP7 (AAF22888); Oryza DGP4 (BAB61154); Oryza DGP3 (AU077558);
Oryza DGP1 (AAAA01011536); Oryza DGP2 (AC077693); Medicago DGP6
(BG452200); Hordeum DGP5 (BF619769); Zea DGP6 (AAD41267); Lycopersicon
DGP1 (BG133077); Leishmania DGP3 (CAC32254); Leishmania DGP2 (CAC32261);
Leishmania DGP1 (AAF73086); Bacillus ylqF (F69880); Borrelia DGP (AAC67000);
Vibrio DGP (AAF96431); Streptococcus DGP (AAK75264); Synechocystis DGP
(NP_441269); Nostoc DGP (NP_484788); Methanococcus DGP (G64482); Pyrococcus
DGP (F75050); Pyrobaculum DGP (NP_558827); Human MTG1 (AAH04409); Human
LSG1 (NP_060855); Human nucleostemin (GNL3, NP_055181); Human GNL2 (NGP1,
AAH00107); Human HSR1 (XP_041722); Human GNL3L (NP_061940); Drosophila
DGP2 (Dme1_CG3983, AAF55384); Drosophila DGP1 (Dmel_CG6501; AAF56060);
Drosophila DGP4 (Dme1_CG14788, AAF45628); Drosophila DGP5 (Dme1_CG9320,
AAF53920); Drosophila Ngp (AAF57834); Caenorhabditis DGP4 (C53H9.2,
AAK68267); Caenorhabditis DGP3 (T24970); Caenorhabditis nst-1(CAA88860);
Caenorhabditis DGP1 (Y67D2.4, NM_065016); Saccharomyces LSG1 (NP_011416);
Saccharomyces MTG1 (S55083); Saccharomyces NUG1 (S50464); Saccharomyces
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NOG2 (NP_014451); Treponema ENGA (P96128); Aquifex ENGA (O67749); Borrelia
ENGA (O51461); Rickettsia ENGA (NP_360667)
RESULTS
SIN2 encodes a putative GTPase: The ethylmethanesulfonate-induced sin2-1 allele
was previously mapped to chromosome 2 using a sin2-1 Ler / Col. segregating population
(BROADHVEST et al. 2000). Further analysis of this population localized SIN2 to a region
spanned by three overlapping bacterial artificial chromosome (BAC) clones (T11A7,
T1K18, and T32G6:(CHOI et al. 1995)). Figure 2A illustrates the overlapping cosmid
subclones of T1K18 that were transformed into sin2-1 heterozygous progeny from the
mapping population. Eight of the nine identified homozygous sin2-1 plants (genotyped
with mapping markers flanking the SIN2 locus) carrying cJBT1K18.136 exhibited a
wild-type ovule phenotype, indicating complementation of the mutation. cJBT1K18.136
comprises four complete predicted coding regions (At2g41660, 41670, 41680, and
41690; TAIR, http://www.arabidopsis.org/). Subclones spanning these coding regions
from Ler and sin2-1 plants were sequenced and a single gene, At2g41670, was found to
contain a C to T mutation unique to sin2-1 plants. A 4.0 kb Eco RI fragment spanning
At2g41670 (Figure 2B) was also able to complement the sin2-1 mutation, confirming this
gene as SIN2.
RT-PCR generated a 1.25 kb SIN2 cDNA that derived from the eight exons spanning
2 kb of genomic sequence predicted for At2g41670 and encoded a 386 amino acid
protein (Figure 2B). The 5' end of the SIN2 mRNA was determined by 5' RACE PCR to
be thirty-four bases upstream of the putative translation start codon. The complete
coding region of the cDNA was fused to 5.6 kb of genomic sequence 5’ to the SIN2
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coding region, or to the CaMV35S promoter, and both constructs complemented the sin2-
1 mutation. While we were unable to detect SIN2 transcript by in situ hybridization,
transcript was detected by RT-PCR in all structures assayed (Figure 2C). Fusions of
either the 5.6 or 1.5 kb regions 5’ of the SIN2 coding sequence to the GUS coding region
failed to produce GUS activity sufficient to be detected by histochemical staining in
transgenic plants. These observations indicate that SIN2 is likely expressed at low levels
in a variety of plant organs.
The predicted SIN2 protein contains the four sequence motifs (G1-G4) typical of all
members of the GTPase superfamily (BOURNE et al. 1991). The sin2-1 lesion produces a
change of a conserved serine to phenylalanine at position #150 within the P-loop or
putative GTP binding pocket present in the G1 motif (Figure 2B). Because sin2-1 was
the only mutant allele of this gene we had identified, and since mutations in the G1 motif
can have a gain of function effect (BOURNE et al. 1991), it was not possible to assess if
the effects of sin2-1 on plant development were typical of SIN2 loss of function.
Determination of the SIN2 sequence enabled screening for an insertional allele. A
transgenic line in which a T-DNA had inserted in the seventh exon of SIN2, and
associated with deletion of the SIN2 nucleotides corresponding to amino acids 281 to 290
at the insertion site, was identified in the Wisconsin Knock-Out population (KRYSAN et
al. 1999). This lesion was designated sin2-2 (Figure 2B).
Plants heterozygous for sin2-2 were not visibly different from wild-type siblings,
indicating that sin2-2 is a recessive allele. sin2-2 plants produced ovules similar to those
of sin2-1 mutants with a reduced number of ovule integument cells (Figure 1B and 1C;
BROADHVEST et al. 2000). However, the extent of sin2-2 integument reduction was more
variable, with some sin2-2 pistils including as many as six ovules with a nearly wild-type
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appearance in addition to those with short integuments (Figure 1D). These
morphologically wild-type ovules were usually at the distal end of the carpel. Even with
this variation in ovule phenotype, sin2-2 plants were completely female sterile but male
fertile as were sin2-1 plants. The similarity of the most severe integument phenotype
between the sin2-2 insertional mutant and the sin2-1 point mutant suggests that sin2-1 is
a loss of function allele and SIN2 acts to promote cell divisions in the developing
integuments.
sin2-2 illuminates additional developmental roles for SIN2: sin2-1 and sin2-2
plants displayed both overlapping and allele-specific phenotypes. However, it is
possible that some of the specific phenotypic effects observed in sin2-1 and sin2-2 plants
were linked to their respective genetic backgrounds (Ler and Ws respectively). Mutant
phenotypes were always compared to those of wild-type siblings found in the same
segregating population (unless specified otherwise). A reduction in the expected 1:3
(sin2-1:wt) segregation ratio had been previously noted and was proposed to be due to
reduced viability of sin2-1 plants (BROADHVEST et al. 2000). A greater degree of
reduction was observed in sin2-2 plants. When seeds from sin2-2 heterozygote plants
were sown on soil 15% failed to germinate, an additional 12% failed to survive to
maturity and sin2-2 mutants were underrepresented in the population of plants reaching
maturity (3 of 143 total versus 35 of 143 that would be expected). However, the sin2-2
allele was found at the expected frequency among 70 phenotypically wild-type plants (47
of 140 chromosomes, a 1:2.04 ratio of homozygotes to heterozygotes), consistent with
Mendelian transmission of the allele. On media, the sin2-2 germination defect was
partially rescued, with an overall germination efficiency of 98.5 % (336 of 341) in a
segregating population. The under-representation of sin2-2 mutants when grown on soil
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indicates that sin2-2 plants exhibit a greater reduction in viability on soil than sin2-1
plants.
Vegetative growth rates were also affected in sin2 mutants. Both sin2-1 and sin2-2
mutants exhibited an extended germination period. In a sin2-2 segregating population at
day thirteen of growth on media, eighty-two seedlings had only cotyledons while two
hundred fifty-seven had developed visible rosette leaves. Forty-eight of these seedlings
with two leaves >1 mm and forty-eight bearing only cotyledons were transplanted to soil.
All seedlings without leaves that subsequently matured to flowering were shown to be
homozygous for the sin2-2 allele, demonstrating a slower germination rate for sin2-2
seeds compared to wild-type siblings. Detailed analyses showed that both sin2-1 and
sin2-2 seedlings exhibited slower emergence of radicle, cotyledons and secondary leaves
than their wild-type siblings, with sin2-2 showing the strongest effects in regards to
germination and seedling growth (Table 1). The leaf emergence rate of sin2-2 was
reduced by half, but sin2-1 plants behaved as their wild-type siblings (Table 1). The
sin2-2 plants also appeared darker green than their wild type siblings (Supplemental
Figure 1A at http://www.genetics.org/supplemental/). These alterations in germination,
leaf production and greening suggest the involvement of SIN2 activity in a variety of
growth processes during vegetative development.
In addition to the reduction in integument growth, sin2 mutations affected other
aspects of reproductivedevelopment. The number of rosette leaves produced prior to
flowering was increased in both mutants relative to wild type when grown in continuous
light (Table 1). This alteration of flowering time appeared specific to long day conditions
since sin2-1 mutants grown under short day (12 hrs light) produced a wild-type number
of rosette leaves (12.63 + 1.39 vs. 12.85 + 0.96). sin2-2 mutants also showed reduced
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apical dominance, characterized by a doubling in the number of secondary inflorescences,
while sin2-1 did not show any obvious alterations in apical dominance (Table 1). In
some cases, sin2-1 plants had multiple branches originating from the axil of a single
cauline leaf resulting in a bushy phenotype (Supplemental Figure 1 at
http://www.genetics.org/supplemental/). sin2-2 plants also exhibited a marked increase
in vascular discontinuity within the petals relative to wild type, sometimes exhibiting a
completely isolated vein region (Supplemental Figures. 1 and 2 at
http://www.genetics.org/supplemental/). Taken together the pleiotropic effects of both
sin2 mutant alleles suggest SIN2 may be playing a role in the production, perception, or
response to a variety of developmental signals.
SIN2 is a member of a family of atypical GTPases: Database searches indicated
that SIN2 shares several conserved features, including the unique conserved aspartate-
alanine-arginine (DAR) motif, with a relatively uncharacterized family of GTPases, the
DAR GTPases (FU et al. 1998). We performed similarity searches of all publicly
available sequence databases and identified DAR proteins from bacteria, archaea, yeast,
plants and animals. Sequence alignments generated from sixty-one representatives of the
identified proteins revealed additional unreported conserved features. Figure 3 shows
that sequence and order conservation was present within the four G motifs (in the non-
canonical order: G4-G1-G2-G3 (REYNAUD et al. 2005)), the DAR motif and a previously
unreported C-terminal motif. In addition to its unique position, the G4 motif showed the
greatest intersequence variability among the G motifs. However, the typical G4 motif
found in all GTPase superfamily proteins, (Z)4NKXD (were Z is any nonpolar or
hydrophobic amino acid residue and X is any amino acid residue), was found in 80% of
these DAR proteins and seven of the ten deviations from this consensus consisted of
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conservative lysine to arginine or aspartate to glutamate substitutions. The DAR proteins
were also found to invariably have a lysine or arginine one to three amino acids preceding
the four hydrophobic or nonpolar amino acids of the G4 motif. Examination of the DAR
motif region revealed two additional conserved amino acid residues, aspartate (D) and
proline (P), flanking the DAR sequence, and several positions generally consisting of
nonpolar or hydrophobic residues expanding the DAR consensus to D(Z)3XZXDARXP.
The DAR motif was always found N-terminal to the four G motifs in our sample. C-
terminal to the G motifs, a loosely conserved sixth motif, L(X)5G, was also identified.
This additional motif, as well as the DAR motif, is likely to be involved in specific
protein-protein interactions.
While each prokaryotic genome examined included only a single DAR gene, multiple
DAR genes were observed in eukaryotes, with seven members found in Arabidopsis
(SIN2 and DAR GTPases 2–7 (DGP2–7)). Maximum parsimony analysis was used to
evaluate the relationships between fifty-one of the identified DAR proteins selected to
represent the breadth of the entire class. The resulting tree indicated that eukaryotic and
eubacterial DAR proteins fall into four distinct clades, which we have designated 1 – 4 in
Figure 4A. Each clade was found to include representatives from all major groups of
multicellular crown eukaryotes; plants, animals and fungi. Each clade also included at
least one Arabidopsis DGP. This analysis was not sufficient to resolve the relationships
between the archeal proteins and the rest of the DAR proteins or between clades 1–4.
All eubacterial DAR proteins and SIN2 were included in clade 1. Also included in
this clade were the Arabidopsis DGP2 (At4g10650) and DGP3 (At4g02790) proteins as
well as proteins from rice, humans, Drosophila, Caenorhabditis, and Saccharomyces.
Interestingly, some of the non-plant clade 1 proteins were shown to localize to
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mitochondria (BARRIENTOS et al. 2003). SIN2 and DGP2, along with their respective
rice orthologs Oryza SIN2 and Oryza DGP2, appeared to be more closely related to each
other than to any other DAR proteins, suggesting that SIN2 and DGP2 resulted from a
duplication postdating the divergence of plants from other eukaryotes. Thus, it is possible
that these proteins share overlapping functions. DGP3, which includes a predicted
chloroplast transit peptide (data not shown), was placed with high bootstrap support into
a small subgroup within clade 1 containing the cyanobacterial DAR proteins.
Accordingly, DGP3 may play a role in the chloroplast that is analogous to that of SIN2.
DGP4 (At3g07050), DGP5 (At1g52980) and DGP6/7 (At2g27200/At1g08410) were
found in clades 2, 3 and 4 respectively, each of which also includes plant, animal and
fungal representatives, but no bacterial proteins. The clade 4 proteins DGP6 and DGP7
were 72% identical and likely resulted from a relatively recent duplication event. Only
one clade 4 protein was found from each of the other plant species represented in the
sequence databases. Proteins within a particular clade exhibited increased amino acid
conservation among residues flanking each of the six described motifs. Clade 2–4
proteins were longer at both their N- and C-termini than clade 1 proteins. The N-terminal
extensions had conserved features within each group. However, the sequences C-
terminal to the LXG motif were highly variable both among and within groups.
Families of GTPases have been defined by similarities among G1 and G2 sequence
motifs (CALDON et al. 2001). Our database searches and a previous study (REYNAUD et
al. 2005) indicated that the DAR subfamily shares some sequence conservation with
members of the Era family of GTPases, which include Era, EngA, and ThdF/TrmE and
which are found in all eubacteria (CALDON et al. 2001). A comparison of the SIN2 G1
and G2 motifs with the same regions from E. coli Era family members showed that the
- 19 -
G1 sequences GXPNVGKS are conserved between SIN2 and Era family members
(Figure 4B). The secondary structure predicted from the DAR consensus sequence was
compared with the E. coli EngA and Era crystal structures (CHEN et al. 1999; ROBINSON
et al. 2002). All the G motifs of the DAR proteins were predicted to lie within similar
structural regions of EngA and Era (Figure 4D). Within this family, the DAR GTPases
share the greatest sequence similarity with EngA proteins. The G2 sequence PGXT was
found in both SIN2 and EngA (Figure 4B; CALDON et al. 2001) and several EngA
proteins were found of have a D(X)6DAR motif 29 amino acids N-terminal to a G4-G1-
G2-G3 segment (Figure 4C). This is similar to the 22 amino acid distance between the
SIN2 DAR and G4 motifs. In addition, the predicted DAR protein consensus structure
had similar features to the region of EngA from its DAR-like motif to the C-terminal G3
(Figure 4D). These traits suggest SIN2 and other DAR GTPases may be derived from an
EngA-like progenitor.
SIN2 localizes to mitochondria: The Sacchromyces DAR proteins have been found
to localize to different cellular compartments, including the nucleus, cytoplasm and
mitochondria (BARRIENTOS et al. 2003; BASSLER et al. 2001). The sub-cellular
localization of SIN2 was evaluated in transgenic Arabidopsis plants expressing a
SIN2:green fluorescent protein (GFP) fusion protein. The localization of the fused GFP
moiety was determined in the roots, hypocotyls, cotyledons and trichomes of T2
seedlings. Most cells showed a punctate GFP fluorescence pattern that co-localized with
fluorescent stained mitochondria indicative of a mitochondrial localization of the SIN2
protein in planta (Figure 5). This is consistent with the reported localization of those
Sacchromyces and Human DAR proteins that group within the SIN2 clade 1
(BARRIENTOS et al. 2003).
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DISCUSSION
SIN2 is a member of a bacterial/organellar subclass of the DAR GTPase family:
Sequence similarity analyses show that SIN2 includes the DAR motif and reveal a non-
canonical G motif order in the DAR GTPases with the G4 motif preceding G1 through
G3 (Figure 3; FU et al. 1998). Structural similarities were found between the G domains
of DAR proteins and the E. coli Era GTPases, including presence of conserved residues
in the G1 and G2 motifs (Figure 4B), suggesting that the DAR family is closely related to
the Era family (CALDON et al. 2001).
Interestingly, members of the EngA subclass of Era proteins have two canonical G1-
G4 GTPase domains in tandem (Figure 4D; MEHR et al. 2000) and, therefore, contain the
G4-G1-G2-G3 arrangement observed in DAR proteins. In addition, several EngA
proteins include a recognizable D(X)6DAR sequence motif at a position relative to the
first G4 motif that is similar to that seen in DAR proteins (Figure 4C). Notably, there is
additional amino acid conservation between EngA and DAR proteins within the effector-
binding G2 motif (Figure 4B). This level of conservation between EngA and DAR
proteins suggests the two classes derive from a common ancestor that included the
duplication of the canonical G1-G4 GTPase domains and a DAR motif, and that the DAR
proteins derived from this common ancestor through truncation (Figure 4B-4D).
Eukaryotic DAR proteins resolve into four moderately to well supported clades (1-4),
suggesting a early expansion of the gene family (Figure 4A; REYNAUD et al. 2005). In
contrast, eubacterial DAR GTPases are confined to clade 1, along with SIN2, DPG2,
DGP3 and other eukaryotic representatives. This association is supported by another
recent phylogenetic analysis (REYNAUD et al. 2005). These results suggest that the
- 21 -
eukaryotic proteins in this clade could have been acquired in an early endosymbiotic
event or other horizontal gene transfer. The first possibility is consistent with the
mitochondrial localization of SIN2 (this work) and of its putative orthologs in yeast and
mammals (BARRIENTOS et al. 2003). The sequence similarity and close association
between SIN2, DGP2 and plant orthologs in the phylogram indicates that DGP2 is also
likely a mitochondrial protein that derived from a recent gene duplication event. In
contrast, DGP3 and a rice ortholog associate with the DAR proteins from known bacterial
relatives of chloroplasts (Figure 4) and include putative chloroplast transit peptides (data
not shown). Thus, DGP3 likely represents the chloroplast DAR ortholog in higher plants,
as has been previously hypothesized (REYNAUD et al. 2005). The structure of clade 1
therefore suggests that each of the two endosymbiotic events related to plant evolution,
acquisition of mitochondria and plastids, brought with them specific DAR GTPase
lineages.
DAR proteins are important for ribosomal assembly in many subcellular
compartments: Recent reports shed light on the biological function of some of the clade
1 proteins that are putative orthologs of SIN2. B. subtilis YlqF (Figure 4A) was shown to
facilitate an essential step in assembly of the large ribosomal subunit (MATSUO et al.
2006; UICKER et al. 2006). Cells deficient in YlqF accumulate incomplete 50S ribosomal
subunits lacking the L16 and L25 protein components (MATSUO et al. 2006; UICKER et
al. 2006). Evidence of participation of the GTPase activity of YlqF in the assembly
process is provided by the stimulation of YlqF GTPase activity in the presence of 50S
subunits, and an association of YlqF with 50S subunits upon addition of a non-
hydrolysable GTP analog (MATSUO et al. 2006). Functional studies on humans and
Saccharomyces MTG1 proteins indicate conservation of the ribosome assembly function
- 22 -
for these proteins in the eubacterial-derived mitochondria. Saccharomyces MTG1 was
shown to be important for translation in mitochondria and a role in assembly of the
mitochondrial large ribosomal subunit was hypothesized (BARRIENTOS et al. 2003). It
was further shown that the human MTG1 protein could partially complement the
Saccharomyces mtg1 mutant, indicating that the ribosome biogenesis function is likely
also conserved in animals (BARRIENTOS et al. 2003).
Other diverse DAR proteins have also been found to participate in ribosome
biogenesis in other sub cellular compartments. The Saccharomyces clade 2 protein
NUG1 appears to be involved in ribosome assembly in the nucleus and transport through
nuclear pores (BASSLER et al. 2001; NISSAN et al. 2002). The Saccharomyces clade 3
protein NOG2 has functions similar to NUG1 and in addition may be involved in mRNA
processing (BASSLER et al. 2001; BIALKOWSKA and KURLANDZKA 2002; SAVEANU et al.
2001). LSG1, the Saccharomyces clade 4 protein, is located in the cytoplasm and is also
important for ribosome transport through nuclear pores (KALLSTROM et al. 2003; NISSAN
et al. 2002). This indicates conservation of a ribosome biogenesis function among
divergent DAR GTPase in the subcellular compartments where they reside.
The reported functions of SIN2 orthologs as well as the mitochondrial localization of
SIN2 and of its closest eukaryotic orthologs suggests strongly that SIN2 is a DAR
GTPase that functions in mitochondrial ribosome assembly in Arabidopsis. SIN2 mRNA
was present in all tested plant parts as would be expected for a mitochondrial protein.
Effects of SIN2 reduced activity in Arabidopsis are consistent with a role in
supporting mitochondrial translation: At the molecular level, the mutation found in the
sin2-1 allele would generate a protein with a single amino acid substitution in the GTP
loop, the GTP-binding motif of the GTPase. Mutations in this motif were identified in
- 23 -
other GTPases and led to gain or loss of function phenotypes (BOURNE et al. 1991).
However, the phenotypic overlap observed between the sin2-1 and the insertional sin2-2
allele suggests that both alleles cause loss of function or a decrease in SIN2 protein
activity.
Mitochondria are involved in biochemical energy utilization and one would expect
lower energy levels to translate into reduced or retarded growth. The reduced growth
observed in the integuments of sin2 ovules resulted from a decrease in the number of cell
divisions in these structures (BROADHVEST et al. 2000, this study). This reduced growth
could result from insufficient energy production due to compromised mitochondrial
activity. Indeed, we have previously characterized the phenotypic effects of mutations in
HLL, which encodes a mitochondrial ribosomal protein homologous to the L14 proteins
of Eubacteria (SKINNER et al. 2001). hll mutations result in a dramatic reduction in ovule
growth, apparently due to compromised translation in mitochondria (SCHNEITZ 1999;
SKINNER et al. 2001). The combination of sin2-1 and hll-1 mutations is synergistic,
leading to almost complete loss of ovule primordial growth (BROADHVEST et al. 2000).
This could be due to the combination of effects of the two mutations leading to even
more severe deficit in mitochondrial function, at least during ovule development.
Interestingly, the ant mutation was found to be epistatic to sin2-1 but synergistic with hll
leading to a similar but somewhat less severe phenotype in comparison to the sin2-1 hll-1
double mutant (BROADHVEST et al. 2000). This supports a hypothesis that ANT is
needed to promote growth and HLL is needed for growth to actually occur. SIN2 would
then facilitate the role of HLL in ovule and integument growth. Thus, this suggests that
HLL is more important for mitochondrial activity than SIN2, at least during ovule
- 24 -
development, consistent with the proposed structural and assembly roles for the two
proteins with respect to the large ribosomal subunit.
Many of the other phenotypic effects observed for sin2 Arabidopsis plants such as
slower growth, reduced pollen formation and reduced organ size, are consistent with a
general decrease in energy metabolism that would be expected for plants with reduced
mitochondrial activity. However, more complex phenotypes observed also suggest a role
for mitochondria in integration of signaling pathways. For example, maintenance of
axillary bud dormancy and continuity of vascular tissue are both under control of auxin-
related pathways (CHATFIELD et al. 2000; HARDTKE and BERLETH 1998). In addition,
inhibition of auxin transport or mutation of the auxin response factor gene ETTIN causes
differential effects along the length of the gynoecium similar to the gradation of effects
observed for sin2-2 (NEMHAUSER et al. 2000). Thus, some aspects of SIN2 action may
reflect an interaction between phytohormone signaling and mitochondrial function. The
broad range of effects that sin2 mutations have on plant growth and architecture is
consistent with the SIN2 GTPase regulating mitochondrial activity in a variety of
developmental pathways within the plant.
The observation that available sin2 mutants are not more seriously growth deficient
suggests that significant mitochondrial function likely persists in these mutant plants.
This implies either that SIN2 activity is not always required for assembly of the
mitochondrial translational apparatus, or that a redundant activity exists. Such a
redundant activity could be supplied by DGP2, which our sequence and phylogenic
studies have identified as a late-branching paralog of SIN2. DGP2 could have
overlapping function with SIN2 and could partially compensate for the reduction or loss
of SIN2 activity in the sin2 mutants. Preliminary analyses of Arabidopsis dgp2 mutants
- 25 -
show that this gene is functional and seem to affect whole plant architecture (Theresa
Hill, unpublished results). This would parallel the observed redundancy for HLL and the
paralogous HULLENLOS PARALOG (HLP) gene (SKINNER et al. 2001). More studies
will be needed to evaluate the role of DGP2 in mitochondria and putative paralogous
interactions with SIN2.
Complexity in mitochondrial modulation of growth pathways: In plants,
mitochondria have been shown to be important for male fertility, appropriate expression
of floral homeotic genes, and programmed cell death (CHRISTENSEN et al. 2002;
HOEBERICHTS and WOLTERING 2003; LEINO et al. 2003; LINKE et al. 2003). Work in our
laboratory has shown that at least two ribosomal proteins targeted to the mitochondria,
HLL and SIN2, are necessary for ovule development and both also affect regulation of
other developmental pathways in Arabidopsis. Interestingly, both of these genes have
functional paralogs in Arabidopsis, DGP2, and HLP, respectively. Together regulation of
these genes could allow for modulation of mitochondrial activity to facilitate differential
growth necessary for morphogenesis. The added complexity that cell mitochondrial
number can vary in a tissue and developmental dependant manner provide additional
plasticity for mitochondria integrated pathways. SIN2 and HLL, along with their
paralogs, offer an entry point to understand the regulation and impact of mitochondrial
protein synthesis on plant development.
- 26 -
ACKNOWLEDMENTS
The authors would like to thank Chris Roxas and Roderick Kumimoto for dedicated
assistance in plant care and genotyping, Yuval Eshed for the gift of pOCA28-15, the
National Science Foundation Arabidopsis Biological Resource Center at Ohio State for
Arabidopsis DNA clones, and members of the Gasser and Bowman Labs for helpful
discussions. This work was supported by National Science Foundation Grant IBN-
0079434 and by a United States Department of Agriculture National Research Initiative
Competitive Grants Program award 2001-35304-09989.
- 27 -
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FIGURE LEGENDS
FIGURE 1. Scanning electron micrographs of stage 3-VI ovules (anthesis, stages from
Schneitz et al., (1995)). (A) Wild-type Ler; (B) sin2-1; (C) and (D) sin2-2 plants. In
(D) the gradation in severity of effects of sin2-2 from the base to the apex of the carpel is
apparent. f, funiculus; ii, inner integument; n, nucellus; oi, outer integument. Bars are 50
µm (A-C) and 100 µm (D).
FIGURE 2. Identification and expression of the SIN2 gene. (A) The chromosomal region
surrounding the SIN2 locus with the molecular markers used to map SIN2 shown above,
and the number of recombination events between markers found in 918 F2 plants
indicated below. The BAC T1K18 spans part of the region between the closest flanking
markers. Cosmid subclones .105, .136 and .107 derived from T1K18 and used in the
complementation test are illustrated. (B) The 4.0 kb EcoRI fragment spanning a single
gene, At2G41670, derived from cJBT1K18.136, that was able to complement the sin2-1
mutant phenotype. The exons in this region and the transcriptional start site as
determined by RT PCR are shown as boxes and a bent arrow respectively. Arrows
indicate the positions of primers used to screen T-DNA lines for additional sin2 alleles.
The position of the missense (sin2-1) and insertional (sin2-2) mutations are indicated.
Numbering is relative to the translational start site. (C) RT-PCR with total RNA from
several plant parts used as template shows that SIN2 RNA was present in all structures
assayed.
- 32 -
FIGURE 3. DAR protein alignment. An alignment of the seven Arabidopsis DAR
proteins, SIN2 and DGP2-7, and the human SIN2 ortholog, Human DGP1, highlights the
six conserved motifs of this family. Residues found in all of these proteins are shown in
white on black. These include amino acids within the GTPase motifs, G1-G4, and the
DAR motif, labeled and underscored below each sequence. There is also a region C-
terminal to the recognizable GTPase motifs which appears loosely conserved and
includes a L(X)5G motif. Amino acids conserved between SIN2 and the closely related
proteins DGP2 (38% identity, 57% similarity) and Human DGP1 (31% identity, 47%
similarity) are highlighted in grey. Residues conserved between SIN2 and DGP2 or
HsDGP1 are shown in black or white letters respectively. The positions corresponding to
the sin2-1 S150F transition and the sin2-2 insertion are indicated above the sequence.
FIGURE 4. Phylogenetic analysis of DAR GTPases. (A) The single tree generated by
parsimony analysis of a culled alignment was modified to collapse branches that were not
supported in both culling and elision analyses by bootstrap values greater that 50%. Four
main clades are resolved, designated clades 1-4. Eukaryotes are represented in each clade.
Prokaryotic sequences are found in clade 1 and two orphan branches (including
Methanococcus and Pyrobaculus sequences). Arabidopsis proteins are highlighted with
grey boxes with SIN2 outlined in black. Numbers on the branches indicate bootstrap
values based on 500 bootstrap replicates. Branch lengths are relative to distance as
calculated using the transformed BLOSUM45 matrix. Clade designations are shown
above the respective branches. Proteins without previous names were given “DGP”
designations. (B) to (D) DAR proteins share conserved sequences and secondary
- 33 -
structure with Era family members. (B) An alignment of the G1 and G2 motifs of SIN2,
E. coli Era, ThdF, EngA1 and EngA2 demonstrates that the SIN2 G1 motif is similar to
that of Era family members (Era, ThdF, and EngA). (C) A region of EngA similar to a
DAR motif was found between EngA1 G3 and EngA1 G4. (D) A comparison of the
clade 1 consensus secondary structure predicted by the Jpred server (CUFF et al. 1998)
with that derived from the E. coli EngA and E. coli Era crystal structures (CHEN et al.
1999; ROBINSON et al. 2002). EngA structure (arrorws and cylinders indicate ß-sheet and
α-helix regions, respectively) appears to derive from a direct repeat of two Era-like units.
The DAR GTPases, represented by SIN2, resemble the central (G4 to G3) region of those
EngA proteins that include DAR motifs.
FIGURE 5. Localization of SIN2:GFP in Arabidopsis. Black and white images of
Arabidopsis hypocotyl cells transformed with 35S:SIN2:GFP. Fluorescence appears
white. (A) Green fluorescent protein is localized to small particles within the cells which
correspond to mitochondria stained with MitoFluor 589 dye shown in B.
- 34 -
TABLE 1
Growth measurements
Days to
Radicle
Emergence
Days to
Cotyledon
emergence
Days to
Two Leaves
Leaf Emergence
(days/leaf)
Days to Bolting Number of
Rosette Leaves
Number of
Active Branches
SIN2
(Ler)
2.2 + 1.2
(57)
4.7 + 1.5
(57)
10.7 + 0.8
(57)
2.8
R2 = 0.98
29.6 + 3.4 (57) 5.4 + 1.0 (57) 3.0 + 0.6 (17)
sin2-1
(Ler)
2.6 + 0.7
(10)
7.2 + 2.3
(10)**
12.2 + 2.2
(10)
2.2
R2 = 0.99
34.6 + 1.9
(14)***
7.6 + 1.4
(14)***
3.2 + 0.7
(12)
SIN2
(Ws)
1.1 + 0.2
(39)
3.1 + 0.6
(39)
9.1 + 0.4
(39)
2.2
R2 = 0.98
24.5 + 1.7 (36) 6.1 + 0.9
(36)
4.1 + 0.7
(16)
sin2-2
(Ws)
3.7 + 1.5
(23)***
9.1 + 1.8
(23)***
19.2 + 5.5
(23) ***
5.1
R2 = 0.98
49.4 + 9.2
(19)***
7.6 + 2.3 (13)^ 10.4 + 2.4
(11)***
Numbers represent the mean + the standard deviation of the mean. Statistically significant differences between wt-1 and sin2-1 or wt-2
and sin2-2 are designated ^ p < 0.05, * p < 0.01, ** p < 0.001, *** p < 0.0001 where p values are determined by the students T-test.
EcoRI(-1813)
sin2-1S150 >F
sin2-2Δ aa 280-291
EcoRI(+2217)
B
m429 BIO2F13H10Indel2
SIN2T11A7.16
18 1 8
T1K18
.136.105 .107
Figure 2
A
4
C
Figure 3.
DGP3 MFAQ-----------------LAPSP------------SPSPANVSHFVHRRTFRQCTYAVSAS--------------------------------LPTANSSRPPPIQANIIVGGKDLNLDVTRKDDSIG----------FKLEANE------- 77 DGP4 MVKRSK---------------KSKSK----------RVTLKQKHKVLKKVKEHHKKKAKDAKKLG----------------------LHRKPRVEKDPGIPNDWPFKEQELKALEVRRARALEEIEQKKEARKE--------RAKKRK------- 93 DGP5 MVKKEKKANVSGKPKHSLDANRADGKKKTTETRSKSTVNRLKMYKTRPKR-NAGGKILSNEYQSKELPNSRIAPDRRWFGNTRVVNQKELEYFREELQTKMSSNYNVILKERKLPMSLLTDNKKQSRVHLLDMEPFQDAFGRKTKRKRPKLVASD 154 DGP6 MGKNEK---------------TSLGR---------ALVKHHNHMIQETKEKGKSYKDQHKKVLESVTEVSDID------------AIIEQAEEAERLFAIHHDSATPVPINMDTGSSSSGITAKEWKEQRMREEALHASS-LQVPRRPHWTPKMN 118 DGP7 MGKSEK---------------TSLGR---------SLVKHHNHMIQESKDKGKYYKNLQKKVLESVTEVSDID------------AIIEQAEEAERLYTINHSSSTPLSINLDTNSSSSVIAAEEWREQQKIEEALHASS-LQVPRRPPWTPEMS 118
SIN2 ---------------MVMMLKKTVKKGLIGGMSFAKDAGKINWFPGHMAAATRAIRNRLKLSDLVIEVRDARIPLSS-ANE-DLQ--SQMS-AKRRIIALNKKDLANPNVLNKWTRHFESS--------------------------------KQ 103 DGP2 ---------------MATAKTWKIAREIGDAVIKASRNPNRRWYGPHMAAAVRAISERIPLVDFVLEIRDARIPLSS-EYE-LLRKFSPLP-SKR-IIVLNKMELADPLELKKCIDYFEERN--------------------------------Y 104HsDAR1 -------------MRLTPRALCSAAQAAWRENFPLCGRDVARWFPGHMAKGLKKMQSSLKLVDCIIEVHDARIPLSG-RNPLFQETLG----LKPHLLVLNKMDLADLTEQQKIMQHLEGEG-------------------------------LK 127 DGP3 -DEIDWMNLESD------------------IRLWTRALRPVQWYPGHIMKTEKELREQLKLMDVVIEVRDARIPLST-THP---KMDAWLG-NRKRILVLNREDMISNDDRNDWARYFAKQG--------------------------------I 176 DGP4 LGLVDDEDTKTEG------------------ETIEDLPKVVNVRDNSERAFYKELVKVIELSDVILEVLDARDPLGTRCTDMERMVMQAGP-NKHLVLLLNKIDLVPREAAEKWLMYLREEFP-------------------------AVAFKCS 204 DGP5 YEALVKKAAESQDAFEEKNGAGPSGEGGEEEDGFRDLVRHTMFEKGQSKRIWGELYKVIDSSDVIVQVIDARDPQGTRCHHLEKTLKEHHK-HKHMILLLNKCDLVPAWATKGWLRVLSKEY--------------------------------P 276 DGP6 VEKLDANEKQAFLTWRRK-----------LASLEEN-EKLVLTPFEKNLDIWRQLWRVLERSDLIVMVVDARDPLFYRCPDLEAYAQEID-EHKKTMLLVNKADLLPSYVREKWAEYFSRNNILFVFWSAKAATATLEGKPLKEQWRAPDTTQKT 260 DGP7 VEELDANEKQAFLNWRRM-----------LVSLEEN-EKLVLTPFEKNLDIWRQLWRVLERSDLIVMVVDARDPLFYRCPDLEAYAQEID-EHKKIMLLVNKADLLPTDVREKWAEYFRLNNILFVFWSAIAATATLEGKVLKEQWRQPDNLQKT 260 DAR G4____
sin2-1 S>F SIN2 DCIAINAHSRSSVMKLLDLVELKLKEV-IAR---------------EPTLLVMVVGVPNVGKSALINSIHQIAAARFPVQERLKRATVGPLPGVTQDIAGFKIAH-RPSIYVLDSPGVLVPSIPDIETGLKLALSGSVKDSVVGEERIAQYFLAI 241 DGP2 LSYAVNSHNKDCVKQLLNFLQSQVRELHKAG-------------HSGHTTTMMLLGIPNVGKSALSNSLHHIGRISAAEKGKLKHTTVSSQPGDTKDIMSLKIGS-HPNVYVLDTPGIFPPNLYDAEICAKLALTGAIPDDIVGELKLARLFLTI 245HsDAR1 NVIFTNCVKDENVKQIIPMVTELIGRSHRYHR------------KENLEYCIMVIGVPNVGKSSLINSLRRQHLRKG------KATRVGGEPGITRAVMSKIQVSERPLMFLLDTPGVLAPRIESVETGLKLALCGTVLDHLVGEETMADYLLYT 264 DGP3 KVIFTNGKLGMGAMKLGRLAKSLAGDVNGKRREKG---------LLPRSVRAGIIGYPNVGKSSLINRLLK-----------RKICAAAPRPGVTREMK---WVKLGKDLDLLDSPGMLPMRIDDQAAAIKLAICDDIGEKAYDFTDVAGILVQM 308 DGP4 TQEQRSNLGWKSSKASKPSNMLQTSDCLGADTLIKLLKNYSRSHELKKSITVGIIGLPNVGKSSLINSLKR-----------AHVVNVGATPGLTRSLQ---EVHLDKNVKLLDCPGVVMLKSSG-NDASIALRNCKRIEKLDDPVSPVKEILK- 343 DGP5 TLAFHASVNKSFGKGSLLSVLRQFARLKSDK----------------QAISVGFVGYPNVGKSSVINTLRT-----------KNVCKVAPIPGETKVWQ---YITLTKRIFLIDCPGVVYQS--RDTETDIVLKGVVRVTNLE---DASEHIGEV 396 DGP6 DNPAVKVYGRDDLLDRLKLEALEIVKMRKSRGVSATS-----TESHCEQVVVGFVGYPNVGKSSTINALVG-----------QKRTGVTSTPGKTKHFQTLIISED---LMLCDCPGLVFPSFSSSRYEMVASGVLPIDRMTEHLEAIKVVAELV 396 DGP7 DDPDIMIYGRDELLSRLQFEAQEIVKVRNSRAASVSSQ-SWTGEYQRDQAVVGFVGYPNVGKSSTINALVG-----------QKRTGVTSTPGKTKHFQTLIISDE---LMLCDCPGLVFPSFSSSRYEMIASGVLPIDRMTEHREAIQVVADKV 400 G1 G2 G3__
| sin2-2 | SIN2 LNIRGTPLHWKYLVEGINEGPHADCIDKPSYNLKDLRHQRTKQPDSSALHYVGDMISEVQRSLYITLSEFDGDTEDENDLECLIEQQFEVLQKALKIPHK-ASEARLMVSKKFLTLFRTGRLGPFILDDVPETETDHPNSKRVVVL--------- 386 DGP2 LNSSHEYKKWAKLCKSQDLTESLSDESSKS--------DAKHKRQYATDHTQDFIVYDVRRVLYETISAFDGNLEDEISMGNLIETQFAALRSVLRVPEEASEFADLRVASKILNLYRTGRLGHYTLEHVSALAKSYTYL--------------- 377HsDAR1 LNKHQRFGYVQHYG--------------------------------------------------LGS-ACDNVERVLKSVAVKLGKTQKVKVLTGTGNVNVIQPNYPAAARDFLQTFRRGLLGSVMLDLDVLRGHPPAETLP------------- 355 DGP3 LARIPEVG-----AKALYNR------------------------------------------------------------YKIQLEGNCGKKFVKTLGLNLFGGDSHQAAFRILTDFRKGKFGYVSLERPPL----------------------- 375 DGP4 --LCPKDMLVTLYKIPS---------------------------------------------------------------FEAVDDFLYKVATVRGKLKKGGLVDIDAAARIVLHDWNEGKIPYYTMPPK---RDQGGHAESKIVTELAKDFNID 430 DGP5 LRRVKKEHLQRAYKIKD---------------------------------------------------------------WEDDHDFLLQLCKSSGKLLKGGEPDLMTGAKMILHDWQRGRIPFFVPPPKLDNVASESEVIVPGIDKEAIADNSQ 488 DGP6 PRHAIEDVYNISLPKPKSYEPQS-------------------------------------------------------RPPLASELLRTYCLSRGYVAS-SGLPDETRAARQILKDYIEGKLPHFAMPPE--ITRDDENETADDTLGAETREGS- 492 DGP7 PRRVIESVYNISLPKPKTYERQS-------------------------------------------------------RPPHAAELLKSYCASRGYVAS-SGLPDETKAARLILKDYIGGKLPHYAMPPG--MPQADEPDIEDTQELEDILEGS- 496
LXG__
DGP4 EVYSGESSFIGSLKTVNEFNPVIIPSNGPLNFDETMIEDESKTQTEEEAEHESDDDESMGGEEEEEAGKTKEKSETGRQNVKLYAAESMLNTKKQKAEKKKRKKAKKAGADEEDLMDGDYDFKVDYRKNKDGEDEEFQIDAKIPMAGLLPEE 582 DGP5 AAAALKAIAGIMSTQQQKDVPVQRDFYDEKDLKDDKKAKESTETDAENGTDAEEDEDAVSEDGVESDSDADEDAVSENDEEDESDSAE---------------------------------------------------------------- 576 DGP6 QTEKKGEEAPSLGLDQVLDDLSSFDLANGLVSSKTKQHKKSHRKQ----------------------------------------------------------------------------------------------------------- 537 DGP7 ESDDSAVGDETENEQVPGIDDVLDDLSSFDLANGLKSSKKVTAKKQTASHKQHKKPQRKKDRTWRVQNTEDGDGMPSVKVFQKPANTGPLTMR----------------------------------------------------------- 589
Clade1
Clade4
Clade2
Clade3
A
Figure 4
BSIN2 139-VMVVGVPNVGKSALI-153 169-TVGPLPGVTQD-180EngA1 13-VALVGRPNVGKSTLF-28 38-LVADFPGLTRD-49EngA2 213-LAIVGRPNVGKSTLT-228 238-VVYDMPGTTRD-249ThdF 219-VVIAGRPNAGKSSLL-234 242-IVTDIAGTTRD-253Era 11-FAIVGRPNVGKSTLL-26 35-ITSRKAQTTRH-46Ras 6-LVVVGAGGVGKSALT-21 27-IFVDEYDPTIE-38
DAR
G1 G2
SIN2 48-DLVIEVRDARIP-60EngA1 83-DVVLFMVDARAG-95
C
D