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Functional characterisation of UPOX1, a
mitochondrial stress responsive protein in
Arabidopsis thaliana.
Vindya Uggalla, BSc (Hons)
This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia
School of Chemistry and Biochemistry
ARC Centre of Excellence for Plant Energy Biology
2017
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THESIS DECLARATION
I, Vindya Uggalla, certify that:
This thesis has been substantially accomplished during enrolment in the degree.
This thesis does not contain material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution.
No part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree.
This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text.
The work(s) are not in any way a violation or infringement of any copyright, trademark, patent, or other rights whatsoever of any person.
This thesis contains published work and/or work prepared for publication, some of which has been co-authored.
Signature:
Date: 21/12/2016
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ABSTRACT
Plant growth, health and productivity are greatly affected by stress. In order for plants
to survive and thrive they must respond and adapt to these stresses at molecular,
cellular, physiological and biochemical levels. Understanding plant mitochondria is
critical for elucidating plant stress mechanisms as they are both an important
production site and target for reactive oxygen species. Although many years have
been spent on plant research there still remains a critical gap in our scientific
knowledge in regards to mitochondrial plant stress mechanisms. The post genomic
era has significantly altered the way in which research can be conducted and there
are now many new possibilities for investigating these previously unknown
mitochondrial plant stress mechanisms. UPOX1 has been identified as a protein that
is putatively targeted to the mitochondria and has been labelled as a highly stress
responsive hallmark of oxidative stress in Arabidopsis thaliana (Gadjev et al., 2006).
To uncover more details on this protein and to characterise UPOX1, a number of
different approaches were employed, including transcriptomic, proteomic and
bioinformatics studies. The profiles and kinetics of transcript induction were
determined for UPOX1 and associated genes including paralogs, putatively co-
expressed and co-regulated genes. It was found that the kinetics of UPOX1 induction
were unique, as they were significantly different from other UPOX homologs.
Therefore, this suggests the existence of independent regulatory mechanisms
between these homologs.
By investigating several putative cis-acting regulatory elements in the UPOX1
promoter, it was found that some of the stress-responsive pathways tested
overlapped for the induction of UPOX1 and other co-expressed genes. Furthermore,
through the analysis of defense signalling mutants, detailed insights were gained into
the intricate mechanisms underlying gene expression under stress conditions.
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Transgenic plants with reduced or increased expression of UPOX1 were
characterised during normal and stress conditions. Finally, various proteomic
analyses were conducted to better understand the location of UPOX1. These
techniques included western blots, GFP tagging, in vitro uptake assays along with
alkaline extractions, PK digestions and BN-PAGE. These techniques confirmed that
UPOX1 is located in the mitochondrial intermembrane space and may also be
associated with the outer mitochondrial membrane.
Overall this research provides new detailed insights into this previously
uncharacterised marker gene of oxidative stress at both a transcriptomic and
proteomic level.
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TABLE OF CONTENTS
FUNCTIONAL CHARACTERIZATION OF UPOX1, A MITOCHONDRIAL STRESS
RESPONSIVE PROTEIN IN ARABIDOPSIS THALIANA. ........................................................ 1
ABBREVIATIONS .................................................................................................................... 14
CHAPTER 1 .............................................................................................................................. 17
INTRODUCTION ....................................................................................................................... 17
1.1 RESEARCH IN THE POST GENOMIC ERA ..................................................................... 18
1.2 PLANT STRESS RESPONSE AND MANAGEMENT ....................................................... 20
1.3 TRANSCRIPTIONAL REGULATION ................................................................................. 22
1.4 ENDOSYMBIOTIC ORIGIN OF ORGANELLES ................................................................ 24
1.5 ORGANELLE BIOGENESIS .............................................................................................. 26
1.6 MITOCHONDRIAL STRUCTURE ...................................................................................... 30
1.7 MITOCHONDRIAL FUNCTION .......................................................................................... 31
1.7.1 The tricarboxylic acid cycle .................................................... 33
1.7.2 The electron transport chain .................................................. 34
1.8 PLANT MITOCHONDRIA ................................................................................................... 35
1.8.1 A functional Alternative Plant Respiratory Pathway ................... 36
1.8.2. Import of organelle proteins in eukaryotic cells ....................... 37
1.9 MITOCHONDRIAL STRESS RESPONSE ......................................................................... 38
1.10 CO-ORDINATED EXPRESSION OF NUCLEAR AND ORGANELLAR GENES ............ 40
1.10.1 Anterograde regulation ........................................................ 41
1.10.2 Retrograde regulation ......................................................... 42
1.10.3 Mitochondrial Retrograde Regulation ..................................... 42
1.10.4 Organelle communications between mitochondria and chloroplasts
.................................................................................................. 44
1.11 PROJECT AIMS AND APPROACHES ............................................................................ 45
CHAPTER 2 .............................................................................................................................. 51
MATERIALS AND METHODS ................................................................................................. 51
2.1 GENERAL CHEMICALS .................................................................................................... 52
2.2 ARABIDOPSIS GROWTH CONDITIONS .......................................................................... 52
2.2.1 Arabidopsis suspension cell culture ......................................... 52
2.2.2 Arabidopsis water culture ...................................................... 52
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2.2.3 Arabidopsis plant material ..................................................... 52
2.2.4 Chemical treatment of Arabidopsis cell culture, water culture and
plant material ............................................................................... 53
2.2.5 UV treatment of Arabidopsis water culture ............................... 53
2.3 BACTERIAL TECHNIQUES ............................................................................................... 54
2.3.1 Bacterial cell strains .............................................................. 54
2.3.2 Transformation into JM109 and DH5 cells .............................. 54
2.3.3 Transformation into XL-Blue Super competent cells .................. 55
2.3.4 Transformation into XL-Gold Ultra competent cells .................... 55
2.4 NUCLEIC ACID MANIPULATION ...................................................................................... 55
2.4.1 Genomic DNA preparation ..................................................... 55
2.4.2 Total RNA isolation ............................................................... 56
2.4.3 Preparation of cDNA .............................................................. 57
2.4.4 Agarose gel electrophoresis ................................................... 58
2.4.5 Amplification of DNA by polymerase chain reaction ................... 58
2.4.6 TOPO-TA cloning into pCR2.1 ................................................. 58
2.4.7 Site Directed mutagenesis ..................................................... 59
2.4.8 Dpn1 Digestion .................................................................... 59
2.4.9 Restriction digests ................................................................ 60
2.4.10 Calf Intestinal Alkaline Phosphatase treatment ....................... 60
2.4.11 DNA ligation ....................................................................... 61
2.4.12 Plasmid Minipreps ............................................................... 61
2.4.13 Plasmid Midipreps ............................................................... 61
2.4.14 Plasmid Maxipreps .............................................................. 62
2.5 GENERATION OF ARABIDOPSIS MUTANT LINES ........................................................ 62
2.5.1 Preparation of Artificial microRNA constructs ............................ 63
2.5.2 Preparation of Agrobacterium tumefaciens electro-competent cells
.................................................................................................. 63
2.5.3 Transformation of electro-competent Agrobacterium ................. 63
2.5.4 Floral dipping ....................................................................... 64
2.5.5 Preparation of mutant lines .................................................... 64
2.6 TRANSIENT TRANSFORMATION OF ARABIDOPSIS CELL CULTURE AND LEAVES 65
2.6.1 Preparation of Arabidopsis suspension cells .............................. 65
2.6.2 Preparation of Arabidopsis leaves ........................................... 65
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2.6.3 DNA precipitation onto gold microcarriers ................................ 65
2.6.4 Transformation of tissue ........................................................ 66
2.6.5 Reporter gene assays for luciferase and β-glucuronidase ........... 66
2.6.6 Normalisation of LUC and MUG activities ................................. 67
2.7 QUANTITATIVE RT-PCR ................................................................................................... 67
2.7.1 DNA standards ..................................................................... 67
2.7.2 Primer design and optimization .............................................. 67
2.7.3 qRT-PCR using the Roche Light Cycler® 480 ............................. 68
2.7.4 qRT-PCR Analysis ................................................................. 68
2.7.5 qRT-PCR Cross reactivity assays ............................................. 69
2.8 ORGANELLE AND PROTEIN ISOLATION METHODS .................................................... 69
2.8.1 Mitochondrial Isolation .......................................................... 69
2.8.2 Chloroplast Isolation ............................................................. 71
2.8.3 Protein translation ................................................................ 71
2.9 IN VIVO AND IN VITRO ASSAYS ...................................................................................... 72
2.9.1 In vivo GFP Assays ............................................................... 72
2.9.2 In vitro import of precursor proteins into mitochondria .............. 73
2.9.3 In vitro import of precursor proteins into pea chloroplasts ......... 74
2.9.4 Mitoplast preparation following mitochondrial in vitro imports .... 74
2.9.5 Alkaline extractions .............................................................. 75
2.9.6 Proteinase K titrations ........................................................... 76
2.9.7 SDS-PAGE ........................................................................... 76
2.10 WESTERN BLOTTING ..................................................................................................... 77
2.10.1 Blotting with nitrocellulose membranes.................................. 77
2.10.2 Blotting with PVDF membranes ............................................. 78
2.10.3 Immuno-detection .............................................................. 78
2.11.1 Preparation of BN PAGE gels ................................................ 79
2.11.2 BN PAGE sample preparation ................................................ 80
2.11.3 Wet Transfer of BN PAGE gels .............................................. 80
CHAPTER 3 .............................................................................................................................. 82
EXPRESSION AND REGULATION OF UPOX1 ...................................................................... 82
3.1 INTRODUCTION ................................................................................................................. 83
3.2 AIMS AND STRATEGIES .................................................................................................. 84
3.3 RESULTS ........................................................................................................................... 85
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3.3.1 Comparisons between the UPOX1 family ................................. 85
3.3.2 Construction of a phylogenetic tree ......................................... 86
3.3.3 Establishment and optimisation of qRT-PCR assays ................... 86
3.3.4 Quantitative RT-PCR assays for UPOX1, UPOX2 and UPOX3 in
suspension cell culture .................................................................. 87
3.3.5 Transcript kinetics of nuclear genes encoding mitochondrial
proteins ....................................................................................... 89
3.3.6 Transcript analysis in response to UV stress ............................. 90
3.3.7 In silico response of UPOX1, UPOX2 and UPOX3 to a wide range of
stimuli and mutations .................................................................... 90
3.3.8 Mitochondrial genes induced under stress in signalling mutant
backgrounds ................................................................................ 91
3.3.9 GUS histochemical staining .................................................... 94
3.3.10 Identification of putative sequence elements in the upstream
region of UPOX1 ........................................................................... 94
3.3.11 Functional analysis of the B and I elements in the upstream
region of UPOX1 ........................................................................... 95
3.4 DISCUSSION ...................................................................................................................... 97
CHAPTER 4 ............................................................................................................................ 113
PROTEIN FUNCTION AND LOCALISATION ........................................................................ 113
4.1 INTRODUCTION ............................................................................................................... 114
4.2 AIMS AND STRATEGIES ................................................................................................ 115
4.3 RESULTS ......................................................................................................................... 116
4.3.1 Generation of UPOX1 under-expressing (knockdown) and over-
expression lines ........................................................................... 116
4.3.2 In vivo GFP ......................................................................... 118
4.3.3 In vitro mitochondrial imports ............................................... 119
4.3.4 In vitro chloroplast import .................................................... 120
4.3.5 Alkaline extractions ............................................................. 120
4.3.6 Proteinase K titrations into mitochondria and mitoplasts ........... 121
4.3.7 Blue Native PAGE ................................................................ 121
4.4 DISCUSSION .................................................................................................................... 122
CHAPTER 5 ............................................................................................................................ 133
GENERAL DISCUSSION ....................................................................................................... 133
5.1 SUMMARY OF AIMS AND APPROACHES .................................................................... 134
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5.2 SUMMARY OF RESULTS ................................................................................................ 134
5.3 FUNCTIONAL IMPLICATIONS OF THE MITOCHONDRIAL STRESS RESPONSE ..... 136
5.4 RETROGRADE REGULATION OF NUCLEAR GENES ENCODING MITOCHONDRIAL
PROTEINS DURING STRESS AND THE MITOCHONDRIAL DYSFUNCTION STIMULON 139
REFERENCES ........................................................................................................................ 143
APPENDIX 1 ........................................................................................................................... 157
APPENDIX 2 ........................................................................................................................... 159
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ACKNOWLEDGEMENTS
Coordinating Supervisor: Dr Olivier Van Aken
Co-supervisor: Professor James Whelan
Co-supervisor: Dr Monika Murcha
All experimental work and the initial thesis draft was carried out in the ARC Centre of
Excellence for Plant Energy Biology and the School of Chemistry and Biochemistry at
the University of Western Australia in Professor James Whelan’s lab.
This thesis would not have been possible without the help and guidance of several
individuals who contributed to the completion of this study.
I would like to thank my supervisor Prof Jim Whelan and for all his expertise, guidance
and for challenging me to do the best possible research throughout my PhD. My
sincere gratitude must go to Dr Olivier Van Aken for his encouragement, advice and
friendship throughout these years and supporting my thesis until the completion.
Thanks also to Dr Monika Murcha for her supervision of the proteomic studies and the
members of the Whelan lab for all the good times.
To my whole family, you are my whole world, you have made this life journey both
exciting and bearable. I know I always have you to count on and I’m so very grateful.
To Mali, Kasun’s Ammi, Thathi, Nisal and Sahan thank you for never giving up on me
and encouraging me to the finish line.
I especially want to thank my Ammi and Appachi, you have worked so hard and
sacrificed so much to give us every opportunity in life. You have always provided
unconditional love and care. I love you so much, I am so grateful for your unwavering
support and for all that you have given me. I would not have achieved anything in life
without your love and guidance.
To Kasun, my best friend, my soul-mate and my husband. There are no words to
convey how much I love you and how grateful I am to you for all that you have done to
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support me through these years. The past several years have not been easy
academically and you have been my pillar of strength throughout, without you I could
never have achieved this. Thank you.
I dedicate this thesis to my parents and Kasun.
“When life is good do not take it for granted as it will pass. Be mindful, be
compassionate and nurture the circumstances that find you in this good time so it will
last longer. When life falls apart always remember that this too will pass. Life will have
its unexpected turns.”
-- Ajahn Brahm
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AUTHORSHIP DECLARATION: CO-AUTHORED PUBLICATIONS
This thesis contains work that has been published.
Details of the work: Giraud E, Van Aken O, Uggalla V, Whelan J (2012) Redox Regulation of Mitochondrial
function in Plants; Plant, Cell & Environment 35(2): 271-280.
Location in thesis: Chapter 1&3
Student contribution to work: 10 %
Details of the work:
Van Aken O, Zhang B, Carrie C, Uggalla V, Paynter E, Giraud E and Whelan J (2009)
Defining the Mitochondrial Stress Response in Arabidopsis thaliana; Molecular Plant 2(6):
1310-1324.
Location in thesis: Chapter 4
Student contribution to work: 10 %
Details of the work: Ho LHM, Giraud E, Uggalla V, Lister R, Clifton R, Glen A, Thirkettle-Watts D, Van Aken O,
Whelan J (2008) Identification of regulatory pathways controlling gene expression of stress
responsive mitochondrial proteins in Arabidopsis; Plant Physiology 147(4): 1858-1873.
Location in thesis: Chapter 3
Student contribution to work: 10 %
Student signature:Date: 21/12/2016
13
I, Dr Olivier Van Aken certify that the student statements regarding their contribution to each of the works listed above are correct Coordinating supervisor signature: Date: 21-12-2016
Abbreviations
ABA abscisic acid
ABI4 abscisic acid insensitive 4
acetyl CoA acetyl coenzyme A
AOX alternative oxidase
At Arabidopsis thaliana
ATP adenosine tri-phosphate
BN-PAGE blue native polyacrylamide gel electrophoresis
CaMV cauliflower mosaic virus
CARE cis acting regulatory element
cDNA complementary deoxyribonucleic acid
CoA coenzyme A
Col-0 Columbia wild type
◦C degrees Celsius
DNA deoxyribonucleic acid
E.coli Escherichia coli
ETC electron transport chain
FAD flavin adenine dinucleotide
fmol femtomole
GC/MS Gas chromatography-mass spectroscopy
GUS β - glucuronidase
h hour
H2O2 hydrogen peroxide
HPLC high performance liquid chromatography
HR hypersensitive response
IM inner mitochondrial membrane
JA jasmonic acid
kb kilobase
kDa kilodalton
kPa kilopascal
L litre
LB Luria-Bertani
LUC luciferase
µg microgram
15
M molar
mA milliampere
MAPK mitogen activated protein kinases
Mb mega bases
MDS Mitochondrial Dysfunction Stimulon
mg milligram
min minutes
mL millilitre
mM millimolar
MnSOD Mn superoxide dismutase
mRNA messenger ribonucleic acid
MRR mitochondrial retrograde regulation
NAD(P)H nicotinamide adenine dinucleotide phosphate
NAD+ nicotinamide adenine dinucleotide (oxidised)
NADH nicotinamide adenine dinucleotide (reduced)
NADP-MDH NADP-Malate dehydrogenase
ng nanogram
NGEMP nuclear genes encoding mitochondrial proteins
OGDC 2-oxoglutarate dehydrogenase
OM outer mitochondrial membrane
PAGE polyacrylamide gel electrophoresis
PCD programmed cell death
PCR polymerase chain reaction
PD pyruvate dehydrogenase
PPR pentatricopeptide repeat
PVDF polyvinylidene fluoride
qRT-PCR quantitative real time polymerase chain reaction
redox reduction/oxidation
ROS reactive oxygen species
rpm revolutions per minute
s seconds
SA Salicylic acid
SAP shrimp alkaline phosphatase
SDS Sodium dodecyl sulphate
SDW sterile distilled water
TAE tris, acetic acid and EDTA
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TBE tris, boric acid and EDTA
TCA tricarboxylic acid cycle
T-DNA transfer DNA
TIC translocases of the inner chloroplast envelope
TIM translocase of the inner mitochondrial membrane
TOC translocases of the outer chloroplast envelope
TOM translocases of the outer mitochondrial membrane
UPOX upregulated by oxidative stress
UQ ubiquinone
VDAC voltage dependent anion channel
[v/v] volume per volume
[w/v] weight per volume
Chapter 1
Introduction
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1.1 Research in the Post Genomic Era
The complete sequencing of genomes has significantly altered the manner in which
research is conducted. In 1977 the bacteriophage φ X174 became the first
DNA-based genome to be completely sequenced (Sanger et al., 1977). The genomes
of more than 180 different organisms have been sequenced to date, with
representatives from all 5 biological kingdoms. In the year 2000 the genome for
Arabidopsis thaliana was completed making it the first plant genome to be sequenced
(The Arabidopsis Genome Initiative, 2000). Arabidopsis belongs to the Brassicaceae
family and over time it has become the pre-eminent plant model system. The reasons
as to why this model plant is extensively used in the field of molecular biology include
its relatively short life cycle of 6 weeks, small genome size of 125 Mb (Mega bases),
high progeny yield of up to 10 000 seeds per plant and the availability of more than 30
000 gene knockout lines. Consequently, there are large databases with information
pertaining to its growth and development (Lister et al., 2009). These factors in
combination with the high quality genome information have allowed for great progress
in both forward and reverse genetic research. The technological advances in the fields
of transcriptomics, proteomics and metabolomics that have arisen in the post genomic
era have now enabled research to be conducted at an unprecedented scale. It is
possible to gain significant insight into the complex physiological processes by
analysis at any of these 'omic' levels, and combinatorial approaches can be in
particular very powerful.
Transcriptomics is the study of the abundance of all transcripts in an organism. The
transcriptome is dynamic, as it is the sum of synthesis and degradation of thousands
of mRNAs in a cell (Meyuhas and Perry, 1979; Wilhelm et al., 2008). These changes
can be due to changes in the organism’s development or in response to external
stimuli (Holtorf et al., 2002). Microarrays investigating gene expression are a popular
method used to study whole genome transcriptomes. The basic principle behind
19
microarrays (e.g. Affymetrix GeneChip microarrays) involves the hybridisation of
fluorescently labelled cDNA (complementary deoxyribonucleic acid) to complementary
cDNA probes immobilised on small glass platforms or chips. The probes are designed
using genome sequence information and their position on the chip is catalogued. The
fluorescently labelled targets comprising cRNA or cDNA from individually prepared
samples can originate from for instance mutant lines, responses to external stimuli or
particular developmental stage (Ruan et al., 1998). The quantitative fluorescence
image can be monitored using a high resolution laser scanner from which the
transcript abundance can be inferred (Holtorf et al., 2002). A major limitation in the
use of microarrays is the requirement for well-annotated transcript information to
design the relevant probes. The development of high-throughput next generation
sequencing technologies allows for relatively rapid sequencing of the entire
transcriptome, circumventing the need for a priori genome information, and such
‘deep sequencing’ approaches are now more widespread and cost-effective (Wang et
al., 2012).
Proteomics is the study of the entire complement of proteins within an organism, cell
or tissue type (Chen and Harmon, 2006). Research using proteomic methods
significantly differ from that of transcriptomics. Transcriptomics can be performed
using a very small amount of mRNA sample by amplification of the starting material
via reverse transcription and PCR amplification. The main limitation to proteomics is
the large amount of starting material required to perform proteomic analysis. Another
limitation to proteomic studies is the efficiency with which proteins can be extracted
from tissues. However, over the last decade this area of research has been improving
at an increasing rate. These methods can evaluate small fragments of peptides using
tandem mass spectrometry (MS/MS) and several methods that involve affinity based
pull down assays using intact proteins to identify interactions such as Tandem affinity
purification coupled to MS/MS (ref). Protein complexes can be separated in their
20
native configurations using Blue Native PAGE analysis (BN-PAGE) (Cannon and
Webb-Robertson, 2006).
Metabolomics is potentially a very powerful ‘omic’ technique. Unlike transcriptomics
and proteomics this method of research doesn’t require a fully sequenced genome,
however, it does require a comprehensive database on which to cross check
molecular profiles against previously identified compounds. Until these databases
become sufficiently comprehensive, many questions pertaining to the metabolic
activities of organisms will remain unanswered (Fukushima et al., 2014). This method
involves extracting chemical constituents from cells and then identifying these
extracted fractions using techniques such as gas chromatography-mass spectrometry
(GC/MS) and high performance liquid chromatography (HPLC) (Roessner et al., 2001;
Holtorf et al., 2002).
To maintain the pace of research and to elucidate more in-depth mechanisms of
stress response and organelle biogenesis it is now required that multiple ‘omic’ levels
be studied in collaboration with each other.
1.2 Plant Stress Response and Management
All organisms are exposed to stress and have developed responses and adaptations
in order to survive. Plant stress has been defined as the conditions in which the
environment adversely affects plant growth and performance (Millar et al., 2001).
Stress can involve a constraint or a highly unpredictable fluctuation in the regular
metabolic patterns that usually cause injury and disease (Gaspar et al., 2002).
There are two types of stresses that may affect plants, abiotic and biotic. Abiotic
stress is caused by environmental factors such as drought, extreme temperatures,
soil, mineral deficiencies, salinity, chilling, hypoxia and high winds (Bartels and
Dinakar, 2013; Benikhlef et al., 2013; Jin et al., 2013; Mustroph et al., 2014;
Nakashima et al., 2014; Fernandes et al., 2016). Biotic stress is caused by pathogenic
21
organisms and includes viral, bacterial and fungal infections (Bahar et al., 2016; Misra
et al., 2016). To manage stress effectively the plant must firstly recognise the stress
imposed on it, activate stress response pathways and then respond to the stress
effectively.
Recognition of abiotic and biotic stresses affecting plants is required at an early stage
in order for plants to be resilient and resistant to stresses. A hypersensitive response
is commonly exhibited by plants in defense against pathogen invasion. This response
involves the formation of localised regions of cell death in order to limit cell transfer of
the pathogen (Heath, 2000).
The understanding of abiotic and biotic stress response in plants is an important and
challenging topic. The availability of the complete genome sequence of Arabidopsis
thaliana has facilitated access to physiological and molecular biological analysis.
Stress tolerance mechanisms include transcriptional changes in genes, in particular
stress responsive genes. The products of such genes can be grouped into two
categories; products that can directly protect plants from stresses and products that
produce a stress response by altering gene expression and/or signal transduction
pathways (Shinozaki et al., 2003; Teige et al., 2004). The first group includes
detoxification enzymes and osmo-protectants, both of which can directly protect plants
on a cellular level. Products from the second group include transcription factors and
mitogen activated protein kinases (MAPK).
A single stress condition can activate numerous signalling pathways, these pathways
may interact with one another and form intertwined response networks. Plants have
evolved complicated and sophisticated systems in response to complex stresses that
can affect growth, development and altered responses to endogenous and
environmental cues (De Caluwé et al., 2016). Salicylic acid (SA), abscisic acid (ABA),
jasmonic acid (JA) and ethylene are all hormones involved in plant stress signalling
pathways. SA accumulates following viral, fungal and bacterial pathogen infections
22
(White, 1979; Sticher et al., 1997; Huang et al., 2003) and results in the induction of
pathogen-related (PR) genes. SA has been shown to be involved in systemic acquired
resistance (SAR), in which a pathogen attack in one part of a plant can result in
pathogen resistance in non-infected areas (Vlot et al., 2009). Osmotic stress activates
the biosynthesis of ABA through the regulation of several biosynthetic proteins in the
plastid resulting in the accumulation of ABA within the cell (Skriver and Mundy, 1990).
ABA is produced in the roots of the plant under low water conditions and then
transported to the leaves where it reduces water loss e.g. by stimulating stomatal
closure (Schachtman and Goodger 2008). Wounding and specific fungal elicitors
increase the biosynthesis of JA and thus activate expression of proteins involved in
plant defense (Koo et al., 2009). The majority of the regulation following hormonal
stimuli occurs at the level of gene expression. Gene expression is a broad term,
defined as transcription and translation of a gene to produce a protein. There are
many possible levels of regulation i.e. transcriptional, post-transcriptional, translational
and post-translational, which together will lead to controlled levels of mRNA and
protein abundance
(Van Aken et al., 2016).
1.3 Transcriptional Regulation
In eukaryotes, transcriptional regulation primarily occurs at the level of transcriptional
initiation. This is facilitated by cis acting regulatory element (CAREs) within the
promoter areas of genes. The CAREs are recognised and bound by transcription
factors that recruit basal transcriptional machinery or chromatin remodelling
complexes (Gentry and Hennig, 2014; Han et al., 2015). These transcription factors
usually contain several domains including a DNA binding domain, oligomerisation site,
transcriptional regulation domain and nuclear localisation signal. Highly conserved
amino acids (that tend to be basic) determine the specificity of interactions within the
23
DNA binding site. The oligomerisation site is the site at which homo or hetero
oligomers are formed. The transcriptional regulation domain is the site of repression
or activation depending on the transcription factor thus allowing for inhibition or
excitation of a gene (Yanagisawa and Sheen, 1998; Liu et al., 1999). At each of these
sites there is the possibility of further specificity depending on the organism and the
complexity of its transcriptional regulation mechanisms.
A co-ordinated and dynamic response to plant stress is required as the mechanism of
plant stress response is highly complex. Most genes are induced in a stimulus specific
manner, however stress responsive genes tend to display complex overlapping
patterns of response to some common biotic and abiotic stress conditions (Chen et
al., 2002; Weltmeier et al., 2009). The signalling pathways within Arabidopsis plants
following cold, drought and high salinity conditions showed a higher degree of overlap
than the signalling pathways following drought and heat stress conditions (Chow et
al., 2014). An overlap between different stress conditions is also evident in
transcriptomic profiling studies as shown by Gadjev et al., 2006; Gupta et al., 2016.
The large networks of transcription factors are the key to the plant genomes’ ability to
modulate its response to fluctuating environmental conditions. It was estimated that
the Arabidopsis genome encodes greater than 2000 transcription factors, however
less than 100 have available experimentally confirmed regulatory interactions (Barah
et al., 2015). Stress inducible transcription factors include the WRKY family, which is
a transcription factor family unique to plants involved in biotic, abiotic stresses, as well
as plant development (Rushton et al., 2010). Another example are the ethylene-
responsive element binding factor (ERF) family which is one of the third largest
transcription factor gene families in Arabidopsis (Singh et al., 2002; Shinozaki et al.,
2003). These transcription factors have been shown to work independently and
co-operatively to coordinate appropriate stress responses (Shinozaki et al., 2003).
24
Eukaryotic transcriptional regulation tends to be combinatorial and involves
interactions between several transcription factors that bind to the same or different
CAREs (Elkon et al., 2003). Studies have shown that transcription factors are over
represented in large genomes. This also correlates with an organism’s ability to adapt
and inhabit environmental conditions that are prone to change. Therefore it can be
concluded that having a larger genome and combinatorial transcriptional regulation
mechanisms is advantageous for the survival of multicellular organisms (Elkon et al.,
2003; Wray et al., 2003).
1.4 Endosymbiotic Origin of Organelles
A defining characteristic of eukaryotic cells is the presence of compartmentalised
specialised organelles. The formation of eukaryotic cells is hypothesised to have
occurred as a result of endosymbiotic events (Hedges et al., 2001). The
endosymbiotic origin was first hypothesised in the late 19th century however it was
largely ignored. It was only after the presence of overwhelming evidence for
endosymbiosis that the theory became widely accepted. The discovery of organellar
deoxyribonucleic acid (DNA) was a key element for the theory of endosymbiotic origin
of organelles. This is because the DNA within the mitochondrial and plastids differs
from that of the DNA within the nucleus, and it resembles the circular shaped DNA
found in bacteria. Further evidence for the endosymbiotic origin of organelles includes
the process of formation of mitochondria and plastids, as it involves a similar process
to binary fission. The proteins of bacteria and organelles both use N-formylmethionine
as an initiating amino acid and mitochondria have enzymes and transport systems
similar to that of bacteria. The ribosomes are similar to the 70S ribosomes found in
bacteria. Cyanobacteria have similar structures to the thylakoids and chlorophylls of
the chloroplasts and mitochondria and plastids are of similar size to bacteria. Finally
most of the genes needed by mitochondria and plastids are produced within the
25
nucleus and this is consistent with the theory that following endosymbiosis there is an
increased dependence of the eukaryotic host (Gray, 1989; van der Giezen et al.,
2005; de Duve, 2007; Poole and Penny, 2007).
This wide array of evidence also suggests that there were two symbiotic events in the
history of the eukaryotic cell; one pre-mitochondrial and a later event after
mitochondria were already assimilated (Hedges et al., 2001). The first endosymbiotic
event is estimated to have occurred 2 billion years ago between an anaerobic pro-
eukaryote that was dependent on glycolysis for energy production, and an α-
proteobacteria probably related to Rickettsia (Emelyanov, 2001). It is hypothesised
that at the time of this endosymbiotic event the atmospheric oxygen levels were
relatively low and the endosymbiont produced (Adenosine Tri-Phosphate) ATP and
hydrogen (Martin and Müller, 1998). The theory of endosymbiosis is also used to
explain the tendency of eukaryotic genes to display an archaeal or bacterial ancestry.
For example genes involved in processes such as transcription and translation tend to
be closely related to archaeal genes. However genes involved in cellular metabolic
processes such as lipid biosynthesis tend to be closely related to eubacterial
(Villanueva et al., 2016).
Approximately 1.5 billion years ago the second endosymbiotic event is hypothesised
to have taken place. The endosymbiotic event was believed to have occurred
between a eukaryote possessing mitochondria and a cyanobacterial ancestor that
was capable of photosynthesis. This resulted in the formation of plastids, leading to a
distinct lineage of eukaryotes, the forebears of modern plants (Palenik, 2002).
There still remains debate over the driving force behind the endosymbiotic events.
Current theories focus on the functions of the endosymbiont: hydrogen production,
and methane consumption are proposed by (Kim et al., 2014; Paranjape and
Hallenbeck, 2015). Additionally, the hypothesis of ‘ox-tox’ is also suggested by
26
(Stefano and Kream, 2016) that centres around the increasing oxygen levels and the
adaptations required for survival under these changing conditions.
1.5 Organelle Biogenesis
The transition from endosymbiont to a eukaryotic cell containing organelles resulted in
the formation of very diverse but distinct sets of enzymes, whose
compartmentalisation has allowed for a level of specialisation unachievable in
prokaryotes (Aitchison et al., 1992). The active proliferation and differentiation of
these organelles, that resulted in an increase in organelle number and mass, was
termed organelle biogenesis (Nisoli et al., 2004). An important event following
endosymbiosis was the vast genome reduction of the endosymbiont that took place
within the newly formed eukaryotic cells. The magnitude of the organelle genome
reduction can be fully comprehended when comparing the number of protein encoding
genes between -proteobacteria, modern day cyanobacteria and mitochondria. An -
proteobacterial genome can contain up to 9000 protein coding genes (Ettema and
Andersson, 2009), a cyanobacterium contains greater than 2000 protein coding genes
(Fujisawa et al., 2014) and in contrast, modern day mitochondria in P. falciparum and
R. americana respectively contain only 3 and 67 protein encoding genes respectively
(Gray et al., 1999).
Many organelle genes have been functionally transferred to the nucleus or have been
replaced by nuclear genes of similar function (Adams et al., 2002; Bergthorsson et al.,
2003). A clear example of this is demonstrated by the fact that there are over 2000
gene products forming the mitochondrial proteome however only 57 are encoded by
the mitochondrial genome in Arabidopsis thaliana (Unseld et al., 1997; Sato et al.,
1999). Despite the extensive reductions, the genetic function of organelle genomes
27
still remains well conserved. This vast reduction in the organelle genome necessitated
the evolution of mechanisms enabling organelle biogenesis. This included metabolite
exchange pathways, protein targeting and translocation systems (Dyall et al., 2004).
Considering the majority of proteins within mitochondria and chloroplasts require
importation from nuclear encoded cytosolicly synthesised precursors, organelles
require multi-subunit translocons and protein import machinery to facilitate this
process. In excess of 2000 proteins are imported into each organelle and although
there are similarities between the basics of the import apparatus of each organelle
they remain very specialised (Kleffmann et al., 2004; Millar, 2007). Examples of their
similarities include the presence of multi subunit translocons required to facilitate
protein import into the organelles. There are translocases of the outer chloroplast
envelope and a translocases of the outer mitochondria membrane (TOC and TOM)
respectively and there are also a translocases of the inner chloroplast envelope and
mitochondrial membrane (TIC and TIM) (Kleffmann et al., 2004; Millar, 2007). The
TOC and TOM complexes both have central components that precursor-proteins
dock to in order to mediate protein transfer, these are both cation selective channels
and the initial sorting of proteins occurs here. However the mode of translocation of
proteins differ between the two organelles; in mitochondria the passage of pre-
proteins is driven by their increasing affinity for the translocation subunits and within
chloroplasts GTP-binding and hydrolysis by the receptors are required for
translocation(Schleiff and Becker, 2010). Generally proteins are imported into
mitochondria and chloroplasts post translationally and several protein machineries
mediate this transport. Both the TOM and TOC complexes consist of receptors as
TOM20, TOM70, TOM22, TOC34, TOC64 and TOC159, furthermore there are pore
forming proteins such as TOM40 and TOC75. In addition there are complexes
involved in the assembly of additional TOM and TOC complexes, in the stabilisation of
28
these complexes and also the transfer of precursor proteins between complexes. In
plants there are isoforms of these TOMs and TOCs (Schleiff and Becker, 2010).
Within the chloroplasts there is a Toc-Tic supercomplex and Tic110, Tic22 and Tic20
are three inner membrane proteins that are associated with this supercomplex. They
assist in forming functional import sites on this supercomplex. To date it appears that
the assembly of a functional inner membrane translocon appears to be mediated by
associations with the Toc complex as there is no evidence to state that a stable
complex consisting only of Tic components exists (Soll and Schleiff, 2004). Similarly
to the Tic complexes there are several Tim complexes. These facilitate the
translocation of proteins across the inner membrane of the mitochondria. The main
Tim complexes include Tim23, Tim22 with the main subunits Tim17 and Tim 23. The
exact roles differ somewhat between these complexes as Tim23 facilitates the
translocation of proteins targeted to the matrix whereas Tim22 facilitates the
integration of carrier pre-proteins into the inner membrane of mitochondria. However
in general all the Tim complexes do allow for import of proteins into mitochondria
(Sirrenberg et al., 1996; Paschen et al., 2000). These similarities between Tic and
Tims and Tocs and Toms give rise to the concept that these organelles have co-
evolved, possibly explaining the economy of function via protein dual targeting (Carrie
et al., 2009).
There is debate over the method of biogenesis of mitochondria within the cell. It has
been hypothesised new mitochondria are derived via the fission of existing organelles
and alternatively it has also been hypothesised that the method of biogenesis of
mitochondria involves maturation of precursor organelles termed pro-mitochondria.
The pro mitochondria are more of a rest and storage state for instance during seed
production or spore formation etc. that needs to be resurrected when the seeds are
imbibed (Law et al., 2014). To date there is significant evidence for both models of
biogenesis.
29
Evidence for the first model involving fission is supported by the fact that mitochondria
and plastids both contain remnants of their ancestral genomes. These remnants are
essential components of these organelles and therefore these organelles are
incapable of arising in cells de novo. This contrasts against other organelles such as
the nuclei, peroxisomes and endoplasmic reticulum that have been observed to arise
de novo in cells (Dimitrov et al., 2013).
The complex machinery involved in the cell division of mitochondria and chloroplasts
include both host derived proteins and orthologs from their associated bacterial
ancestors(Yang et al., 2008). There are distinctions between the protein complexes
involved in both mitochondrial and chloroplastic fission however they function in the
same manner by a synchronized constriction of the outer and inner membranes in
order to produce two daughter cells (Osteryoung and Nunnari, 2003). There have
been significant studies examining mitochondrial populations in yeast and in
mammalian systems. These studies have accumulated further evidence to support the
growth and division model of mitochondrial biogenesis (Nisoli et al., 2004). Generally,
mitochondrial fission has been shown to coincide with mitosis with mitochondrial mass
increasing from the beginning of the S-phase until M-phase and results in each
daughter cell receiving a roughly equal complement of mitochondria(Sänger et al.,
2000). Although this is what is known to generally occur it has also been observed in
mammalian muscles that mitochondrial proliferation occurred in response to an
increase in physical activity (Brunk, 1981).
The second model for mitochondrial biogenesis centres on pro-mitochondria. There is
still no conclusive analysis outlining the origin of pro-mitochondria however it is
believed that they are caused by the degeneration of mature mitochondria (Day et al.,
2004). These pro-mitochondria are thought to be simple unstructured organelles
(Plattner et al., 1970). The evidence for this model was observed in yeast cells when
they transitioned from anaerobic to aerobic growth and also during seed germination
30
(Rosenfeld et al., 2004; Howell et al., 2006). In both these situations it appears that
the maturation model is applicable for cells undergoing a fundamental phase-
transition.
It has been shown under electron microscopy that during the phase transition energy
intensive period of seed germination the presence of poorly differentiated
mitochondria lacking cristae and matrix are evident. These pro mitochondria were
observed to have only a fraction of the usual protein complement and although they
possessed the protein machinery for import they were not capable of importing
proteins (Howell et al., 2006, 2007). Continuing observations taken 30 min following
the imbibition showed significantly increased rates of protein import. Thus it has been
proposed that during these fundamental phase changes such as during the
germination process, pro-mitochondria mature into metabolically active mitochondria,
which subsequently give rise to more mitochondria via growth and division (Howell et
al., 2006, 2007).
1.6 Mitochondrial Structure
Mitochondria are bioenergetic organelles, they display variations in form, however
generally vary in size between 1 µm to 2 µm in diameter (Siedow and Umbach, 1995).
Mitochondria contain four separate compartments, the outer membrane, the inner
membrane, the inter-membrane space and the mitochondrial matrix as shown in
Figure 1.1.The outer and inner membranes enclose a dense matrix incorporating
enzymes and multiple copies of a genome that encode a small number of inner
membrane proteins and the RNAs required for their translation (Frey and Mannella,
2000). The majority of mitochondrial proteins are nuclear encoded and imported from
the cytosol. The outer mitochondrial membrane (OM) is impermeable to large proteins
greater than 10kDa yet permeable to solutes, whilst the inner mitochondrial
membrane (IM) is osmotically active and impermeable to most solutes. A vital protein
31
required for import is Voltage Dependent Anion Chanel (VDAC) also known as Porin
and allows free passage to small molecules including NAD(P)H (Frey et al., 2002).
The IM contains most of the components required for the electron transport chain
(ETC) in addition to several other mitochondrial transporters. The shape of the
mitochondrial IM changes in response to metabolic and osmotic conditions. Oxidative
phosphorylation requires the rapid diffusion of ions and substrates across the IM.
Therefore the morphology of the cristae may regulate the rate of movement of ions
and substrates through the IM (Frey and Mannella, 2000). The intermembrane space
contains cytochrome c also essential for the ETC. The mitochondrial matrix is the
location where most of the tricarboxylic acid cycle (TCA) takes place, in addition to
various other metabolic processes such as amino acid metabolism and glycine
oxidation (Bowsher and Tobin, 2001; Rasmusson et al., 2004). Under normal
conditions the matrix is not electron dense and the matrix is expanded, however the
intra cristae spaces can become dilated and the matrix can become electron dense
and therefore this is part of a feedback mechanism that allows mitochondria to
respond to environmental perturbations (Frey and Mannella, 2000).
1.7 Mitochondrial Function
Mitochondria are multifunctional and semi-autonomous organelles. They carry out a
variety of functions, of which their primary roles are the oxidation of organic acids via
the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of
electrons from NADH to oxygen via the electron transport chain.
Mitochondria function in several different ways however the most prominent of them is
its role to produce ATP (by the phosphorylation of ADP) through respiration. In
addition to the TCA cycle mitochondria play a crucial role in plant defense
mechanisms. The hypersensitive response (HR) and programmed cell death (PCD)
32
are both influenced by mitochondrial function (Robson and Vanlerberghe, 2002;
Vacca et al., 2004).
There is evidence supporting a leading role for mitochondria in PCD involving
components of the mETC, FoF1 ATP synthase, cardiolipins, and ATPase AtOM66
(Zhang et al., 2014; Van Aken and Van Breusegem, 2015). Furthermore, it is
hypothesised that both the mitochondria and chloroplast both play an important role
during plant PCD. This is thought to occur by changes in redox levels and energy
states or metabolic equilibriums producing large quantities of ROS or the release of
pro-death factors which are yet to be fully understood. In addition to the mitochondria
other organelles that contribute to PCD include the vacuole (e.g., via vacuolar
processing enzymes and autophagy) and the ER (Van Aken and Van Breusegem,
2015).
VDAC is a large protein located on the outer mitochondrial membrane is known to be
an important component involved in PCD and HR. In Arabidopsis, proteins known as
hairpins that are produced in nature by certain bacterial plant pathogens were shown
to inactivate mitochondria in suspension cells. These cells were shown to have altered
mitochondrial functions. These were recognized by the enhanced generation of
mitochondrial reactive oxygen species and nitric oxide. In addition it was also evident
that there was a decrease of the mitochondrial membrane potential and of ATP
production, and the induced-expression of alternative oxidase gene and small heat
shock protein genes (Krause and Durner, 2004; Kusano et al., 2009). Further
evidence of this includes the fact that alternative oxidase (AOX) is known to be a
critical component involved in the HR salicylic hydroxamic acid-sensitive pathway
which is important in viral resistance (Ordog et al., 2002). The overexpression of AOX
in transgenic tobacco (Nicotiana tabacum) plants resulted in smaller hypersensitive
response lesions; this suggests a strong link is present between mitochondrial
function and programmed cell death. Furthermore VDAC a large protein located on
33
the outer mitochondrial membrane is also known to be an important component
involved in PCD and HR.
Mitochondria are also involved in a dynamic relationship with other cellular organelles
such as chloroplasts and peroxisomes to facilitate the efficient functioning of cells.
This can be evidenced during photorespiration, within photosynthetic cells a major
demand on mitochondria is the oxidation of glycine to serine generated during
photorespiration (Raghavendra and Padmasree, 2003; Balmer et al., 2004; Wong et
al., 2004). Furthermore mitochondria are able to sense and responds to
environmental changes and variations in metabolic demands of the other organelles,
for example mitochondrial and chloroplastic reductant export is suggested to co-exist
with peroxisomal and mitochondrial reductant import (Foyer and Noctor, 2005).
Mitochondria also play crucial roles in a number of essential biosynthetic processes
such as nitrogen fixation, the biosynthesis of amino acids and vitamin cofactors
(Mackenzie and McIntosh, 1999). For example plant mitochondria have been shown
to be the site of fatty acid synthesis and enzymes involved in the synthesis of cystein,
glycine and proline have been identified in mitochondria (Taylor et al., 2004).
1.7.1 The tricarboxylic acid cycle
The TCA cycle is the second stage of cellular respiration. Prior to the TCA cycle
glucose can be metabolised to pyruvate anaerobically as shown in Figure 1.2 A, this
however only synthesises a fraction of the ATP available from glucose. The aerobic
processing of glucose begins with the complete oxidation of glucose derivatives to
carbon dioxide. This oxidation takes place in the TCA cycle that is comprised of a
series of reactions also known as the citric acid cycle or the Krebs cycle. This
metabolic process occurs in most plants, animals, fungi and many bacteria. In all
organisms except bacteria the TCA cycle is carried out predominantly in the matrix of
the mitochondria. The larger molecules such as glucose, other sugars, fatty acids and
34
amino acids enter the TCA cycle after conversion to the break-down product acetyl
coenzyme A (acetyl CoA). The TCA cycle has 8 main steps as shown in Figure 1.2 B.
The first step is initiated by the reaction of acetyl CoA and oxaloacetate, this forms
citrate and produces the release of coenzyme A (CoA-SH). Following this, a
succession of reactions occur, citrate is rearranged to form isocitrate as shown in step
2 of Figure 1.2 B; isocitrate then loses a molecule of carbon dioxide and undergoes
oxidation to form alpha-ketoglutarate in step 3; alpha-ketoglutarate loses a molecule
of carbon dioxide and is oxidized to form succinyl CoA in step 4; in step 5 succinyl
CoA is enzymatically converted to succinate; succinate is then oxidized to fumarate in
step 6; in step 7 fumarate is hydrated to produce malate; and finally in step 8 malate is
oxidized to oxaloacetate. Each complete turn of the cycle results in the regeneration
of oxaloacetate and the formation of two molecules of carbon dioxide(Martin and
Vagelos, 1962).
Energy is produced and relayed through the many Redox (reduction/oxidation)
reactions and following several steps of the TCA cycle. One molecule of ATP is
produced following step 5 however a large proportion of energy is captured by
nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) that
are later converted to ATP. For each cycle three molecules of NAD+ are reduced to
NADH and one molecule of FAD is reduced to FADH2. The energy within these
molecules are then transfered to the electron transport chain, a pathway that is part
of the third stage of cellular respiration. The electron transport chain in turn releases
energy so that it can be converted to ATP through the process of oxidative
phosphorylation(Martin and Vagelos, 1962; Carrari et al., 2003; Pollard, 2003).
1.7.2 The electron transport chain
The ETC comprises several multi-subunit complexes and the redox energy from
NADH and FADH2 is transferred to oxygen via several steps as shown in Figure 1.1.
35
Complex I is a NADH-ubiquinone oxidoreductase, it catalyses the oxidation of NADH
and reduction of ubiquinone (UQ) a mobile redox carrier. Complex I comprise over 30
subunits in Arabidopsis however only 9 of them are encoded by the mitochondrial
genome. Complex II in Arabidopsis is composed of 8 nuclear encoded subunits and is
responsible for catalysing the transfer of electrons from succinate to UQ via covalently
bound FAD (Eubel et al., 2003). Complex III is also known as the bc1 complex, it is a
UQ H2-ferrocytochrome c oxidoreductase. Complex III then catalyses the transfer of
electrons from UQ to cytochrome c. This process involves the Rieske protein an (iron
sulfur center) and cytochrome b. Complex IV catalyses the oxidation of cytochrome c
and the reduction of O2 to water. This process is efficient however a small percentage
of electrons may prematurely reduce oxygen, this forms reactive oxygen species such
as superoxide. Superoxides can cause oxidative stress and damage. The transfer of
electrons through these complexes is coupled to the translocation of protons from the
matrix into the intermembrane space. As the proton gradient increases a strong
electrochemical gradient is formed across the inner membrane. This proton gradient
drives the synthesis of ATP from ADP and inorganic phosphate at Complex V which is
also refered to as ATP synthase, this process is called chemiosmosis (Mitchell and
Moyle, 1967).
1.8 Plant Mitochondria
Over the last 50-60 years researchers have been investigating the mitochondria of
plants and we now know there is a remarkable variation in the functional abilities of
this organelle relative to their mammalian and other non-photosynthetic counterparts.
In order for plants to survive as sessile organisms, plant mitochondria have evolved
unique strategies to enhance their survival capabilities. Mitochondrial genomes
encode a very small proportion of the genetic information required for their function;
36
the majority of the information is nuclear derived. This is hypothesised to have been a
result of nuclear-mitochondrial co-evolution within the plant kingdom.
1.8.1 A functional Alternative Plant Respiratory Pathway
As a unique advantage for survival, plants, some protists, fungi and algae possess a
(second) alternative respiration pathway as shown in Figure 1.1.These alternative
pathways bypass energy conservation by circumventing the utilization of the
electrochemical proton gradient. Components of this pathway include a type II
NAD(P)H dehydrogenases (NDHs), which bypasses proton-pumping complex I or
allows oxidation of cytoplasmic NAD(P)H; the alternative oxidase (AOX), which
bypasses proton -pumping complexes III and IV; and uncoupling proteins (UCP), that
bypass the ATP synthase by directly dissipating the proton gradient (Escobar et al.,
2004; Rasmusson et al., 2004).
The terminal oxidase in the alternative pathway is AOX. AOX is a nuclear encoded
mitochondrial protein of approximately 34 kDa. AOX is bound to the membrane bilayer
of mitochondria; it contains a coupled bi-nuclear iron centre with histidines and four
glutamate residues. It is a member of the di-iron carboxylate group that is
distinguishable by the presence of six conserved amino acids proposed to represent
iron-binding residues(Berthold et al., 2000).
The alternative respiratory pathway allows for a greater level of flexibility for regulating
the redox state of the mitochondrial and cytoplasmic matrix NAD(P)H pools as neither
of the AOX or NDHs contribute to proton pumping or ATP synthesis and bypass
adenylate control (Rasmusson et al., 2004). The level of ubiquinone can be altered by
the action of these alternative respiratory proteins consequently the level of reactive
oxygen species (ROS) can also be modified. There have been further observations
that show the enzymes of the alternative respiratory pathway play key roles in plant
stress response (Moore et al., 2002). In addition, findings also show that these
37
alternative respiratory proteins can also alter cellular redox, energy levels and can
minimise the production of ROS (van Lis and Atteia, 2004). Therefore mitochondria in
plants are both a target of oxidative damage and agents involved in stress response
and detoxification (Van Aken et al., 2009).
1.8.2. Import of organelle proteins in eukaryotic cells
The mitochondrial import apparatus is essential for the functioning of all eukaryotic
cells as the majority of more than 1000 proteins within the mitochondria are nuclear-
encoded and imported (Murcha et al., 2014). The process of discrimination and
sorting of nuclear encoded mitochondrial targeted proteins falls on the import
mechanisms. Although the Arabidopsis genome was sequenced many years ago
(AGI, 2000) the components and mechanisms involved in Arabidopsis protein import
continue to be studied in further detail. While the yeast model system doesn’t reflect
the complexity of higher plants there are several components of the protein import
machinery that have been conserved between these species. As the import
mechanisms in yeast have been studied more intensively this assists in the study of
the components involved in the import machinery within Arabidopsis.
There are a variety of mitochondrial import pathways that operate in eukaryotic
organisms. First, the general import pathway imports mitochondrial proteins with N-
terminal targeting signals. This pathways uses the translocase of the outer membrane
(TOM) and the translocases of the inner membrane 17:23 (TIM17:23). For these
proteins the primary signal defines the matrix location with secondary signals sorting
proteins into the inner membrane or intermembrane space (IMS) (Murcha et al.,
2014).
The mitochondrial carrier import pathway involves the import of inner membrane
proteins that contain multiple transmembrane regions. This pathway varies from the
38
general pathway as it utilises the TOM complex, a variety of small TIM proteins in the
intermembrane space and a translocase of the inner membrane 22 (TIM 22). Most of
these proteins’ targeting signals are located in the mature region of the protein rather
than the N-terminal region (Duncan et al., 2013).
The import of IMS proteins involves another protein import pathway. The signals on
these proteins are also in the mature protein and this pathway involves twin cysteine
residues and utilizes the TOM complex and the mitochondrial IMS assembly pathway
(MIA) machinery. The fourth import pathway involves the import of β-barrel proteins
into the outer membrane. This pathway uses a TOM and a sorting and assembly
machinery (SAM). In addition to these two outer membrane complexes small TIM
proteins from the IMS are also involved to import these β-barrel proteins (Murcha et
al., 2014). There are several additional specialised protein import pathways that are
known to exist for specific protein groups/proteins however to date these are not fully
understood (Bauer et al., 2015).
1.9 Mitochondrial Stress Response
As plants are continuously challenged by abiotic and biotic stresses it is vital that they
have mechanisms to counteract the damage caused by these external stresses. To
deal with stress, plants have several phyto-hormone-based pathways that signal
stress responses that crucially allow for stress tolerance (Bostock, 2005). These
stress responses include modification of the transcriptome and various post-
transcriptional changes (Koornneef and Pieterse, 2008). Mitochondria are a target of
oxidative stress and in wheat leaves they have been shown to be the main target of
oxidative stress (Bartoli et al., 2004). As targets of oxidative damage, processes within
plant mitochondria can be severely inhibited (Taylor et al., 2004). Key modulators of
the stress response such as salicylic acid and nitric oxide inhibit respiration in plants
39
(Millar et al., 2001). Furthermore various stresses are known to inhibit protein import
into mitochondria that in turn will affect several mitochondrial processes (Taylor et al.,
2003; Van Aken et al., 2009). Reactive oxygen species are most often a product of
stress. These products have been shown to cause oxidative damage to lipids,
proteins and nucleic acids (Gadjev et al., 2006). In mitochondria the main oxidative
damage targets appear to be Fe-S and lipoic acid containing enzymes, such as those
found in the respiratory chain components and the TCA cycle components
respectively (Taylor et al., 2004). Oxidative damage within the mitochondria can result
in the direct inhibition of Complex I and aconitase (Zhang et al., 1990; Verniquet et al.,
1991). The modification of lipoic acid residues has been shown to inhibit mitochondrial
pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) (Millar
and Leaver, 2000).
A possible reason why mitochondria are one of the main targets of oxidative damage
could be because they are capable of preventing the excessive build-up of ROS. If the
production of ROS was to be left unmonitored the likelihood of ROS induced cell
death is extremely high (Sun et al., 2002). There are known mechanisms within
mitochondria that suppress excessive ROS production. These include the Mn
superoxide dismutase (MnSOD) process that involves the conversion of superoxide
radicals to hydrogen peroxide and molecular oxygen. It has also been shown that the
resultant hydrogen peroxide can be further neutralised by mitochondrial ascorbate
peroxidase and gluthathione peroxidase (Chew et al., 2003; Navrot et al., 2006)
Another mechanism that prevents the excessive build-up of ROS includes the activity
of alternative oxidase and NAD(P)H dehydrogenases (NDs). Both these substances in
collaboration suppress ROS production by preventing the over-reduction of the ETC.
It has been found that a lack of AOX leads to a higher sensitivity to a combined stress
of drought and high light in Arabidopsis (Giraud et al., 2008) paired with an increase in
40
ROS formation (Vanlerberghe, 2013). These are clear demonstrations of the
importance of mitochondria in stress response and stress adaptation.
Mitochondria are not only a major target of oxidative stress they are also a major
source of cellular ROS. Complex I and Complex III of the electron transport chain are
involved in ubiquinone-ubiquinol inter-conversions, an intermediary of these reactions
is ubisemiquinone (Maxwell et al., 2002). It is thought that this is a major source of the
electrons that reduce molecular oxygen (O2) to O2- , H2O2 and the hydroxyl ionOH- is
the most damaging of all ROS species (Maxwell et al., 1999). ROS are in most cases
damaging to cells and toxic, however in some cases they can act as signals in stress
signalling cascades, second messengers during growth, movement and as a result of
hormone action (Suzuki et al., 2012). Thus, generation of ROS species in plant
mitochondria is an important mechanism by which the internal redox state can be
communicated to the nucleus and other cellular compartments.
1.10 Co-ordinated Expression of Nuclear and
Organellar Genes
The endosymbiotic theory on the origin of organelles as shown in Section 1.3
described the theory behind gene transfer. In Arabidopsis, only 33 proteins are
encoded in the mitochondria and only 128 proteins are encoded in the chloroplast,
and thus the majority of organellar proteins are encoded in the nucleus (Unseld et al.,
1997; Sato et al., 1999). In order for plants to function, a co-ordinated system between
the nucleus and organelles must occur. Therefore, nuclear encoded proteins are
transcribed in the nucleus, translated in the cytosol and then imported into their
corresponding organelle (McFadden, 1999; Van Aken et al., 2009). This multistep
pathway is controlled by anterograde mechanisms. In order for organelles to
communicate functional changes to the nucleus retrograde regulation is required. This
41
form of communication begins at the organelle and ends at the nucleus (Rhoads and
Subbaiah, 2007).
1.10.1 Anterograde regulation
Anterograde regulation has been described as “a forward flow” of information and has
also been described as a “top down” pathway of gene expression. There are many
questions regarding anterograde regulation and these include, “what are the
regulators of nuclear encoded organellar genes” and “how is this process regulated”.
From the complexity of these questions it is easy to understand that several levels of
anterograde regulation are required for the efficient functioning of these cells;
including the ability to change the level of transcripts of nuclear encoded mitochondrial
proteins (Kleffmann et al., 2004), control of transcript initiation, editing, processing and
maturation. Furthermore anterograde regulation controls almost entirely the
expression of the mitochondrial genome including DNA replication, transcription,
transcript processing and translation (Ng et al., 2014). The pentatricopeptide repeat
(PPR) family is the largest family encoding RNA-binding proteins in land plants and
they play an important role in anterograde regulation and gene expression. In
Arabidopsis there are approximately 450 family members and about 100 are predicted
to be targeted to organelle (Kotera et al., 2005). Other levels of anterograde regulation
include; the post translational import into the organelles, for chloroplasts the Tic and
Toc complexes are used and the Tim and Tom complexes are used for mitochondrial
import. This level of anterograde regulation is more complex for chloroplasts as the
import of proteins into the chloroplast is also dependent on the redox state of the
organelle, therefore further mechanisms that sense the redox state and respond to it
are involved (Küchler et al., 2002; Lister et al., 2004; Soll and Schleiff, 2004). Further
post translational actions require regulation from the nucleus and nuclear encoded
42
assembly factors, this often involves the assembly of multi protein complexes within
the mitochondria such as complex 1 (Shaughnessy et al., 2014).
1.10.2 Retrograde regulation
The mechanisms of retrograde regulation have evolved to communicate the functional
and developmental state of organelles to the nucleus, for this reason retrograde
regulation has been often referred to as the “bottom-up” pathway. The reason for this
form of communication may be as a result of alterations in organelle function from
multiple signals, stresses or mutations such as the presence of ROS or signalling
molecules or the availability of metabolites (Rhoads and Subbaiah, 2007). The
importance of this mechanism cannot be understated as it is crucial for resource
allocation under an altered organellar state (Woodson and Chory, 2008). An
interesting example of retrograde regulation can be seen in GUN mutants. The GUN1
mutant was mapped to a ~93 kilo base region of chromosome 2 and GUN1 alleles all
showed mutations in At2g31400 (Koussevitzky et al., 2007). Assays have shown that
GUN1 mutants were involved in tetrapyrrole biosynthesis (Kleine and Leister, 2016)
and the effects of Lincomycin on LHCB1.2 expression were suppressed in these
mutants (Gray et al., 2003). It is hypothesised that GUN1 mutants act in both
pathways and as a pentatricopeptide repeat-small MutS-related protein is presumably
involved in organelle gene expression (assuming that Norflurazon and Lincomycin
trigger different types of signalling) (Koussevitzky et al., 2007). Genetic evidence
suggests that GUN1 integrates signals derived from perturbations in the redox state,
chloroplast gene expression, and tetrapyrrole biosynthesis in seedlings. However, the
molecular mechanism by which GUN1 integrates retrograde signaling in the
chloroplast is unclear (Koussevitzky et al., 2007; Slonim and Yanai, 2009).
1.10.3 Mitochondrial Retrograde Regulation
43
Mitochondrial retrograde regulation (MRR) allows modulation of nuclear gene
expression in response to alterations in the mitochondrial state (Rhoads and
Subbaiah, 2007). This form of regulation is of critical importance for the smooth
functioning of organisms as mitochondrial dysfunction and the subsequent MRR is
thought to be a contributing factor in diseases such as Alzheimer’s, Parkinson’s,
Huntington’s, bipolar disorder, schizophrenia, Type II diabetes, cancer and the aging
process (Ben-Shachar and Laifenfeld, 2004; Butow and Avadhani, 2004; Beal, 2005;
Iwamoto et al., 2005; Kwong et al., 2006; Parihar and Brewer, 2007). MRR was first
demonstrated in yeast (Saccharomyces cerevisiae). The first target gene to be
identified of the retrograde pathway was shown in yeast to be CIT2, which encodes a
peroxisomal isoform of citrate synthase (Liu and Butow, 2006) and following genome
wide transcriptomic studies it has been shown that retrograde signalling is operational
in response to many signals in all eukaryotic organisms studied (Giraud et al., 2008;
Schwarzländer et al., 2011; Giraud et al., 2012; Quirós et al., 2016). AOX is the pre-
eminent model used to study MRR in plants (Rhoads and Subbaiah, 2007;
Vanlerberghe, 2013). AOX catalyses cyanide insensitive oxygen consumption and
oxidises its substrate without the generation of a proton motive force. At a
transcriptional, protein and activity level it is induced by a variety of abiotic and biotic
stresses, including photo-oxidative stress, temperature stress, nutrient deficiency,
water deficiency and oxygen deficiency (Vanlerberghe and McIntosh, 1997; Millar et
al., 2011; Ng et al., 2014). Several genes were shown to cluster with the expression
profile of AOX1a in response to perturbations tested in (Ng et al., 2014) including
Hsp60, sHSP23.5, sHSP23.6, MGE1, NDA2, NDB2 and UCP5, transporters and
several small auxin-up RNA (SAUR)-like proteins.
A prominent group of genes have been identified as all containing a cis-acting
regulatory element in their promoters to arbitrate the MRR mediated induction by
NAM, ATAF1/2 and CUC2 (NAC) transcription factors (De Clercq et al., 2013).
44
Several genes belonging to this group were clustered with AOX1a (Ng et al., 2014).
This group has been named the Mitochondrial Dysfunction Stimulon (MDS) and
UPOX1 is a member along with MGE1 (At5g55200), sHsp23.5 (At5g51440) and
AtOM66 (At3g50930) (Giraud et al., 2009; Van Aken et al., 2009; De Clercq et al.,
2013; Van Aken et al., 2013; Ng et al., 2014).
1.10.4 Organelle communications between mitochondria
and chloroplasts
Mitochondria and chloroplasts have an interdependent requirement on each other for
the survival of a eukaryotic cell. They are linked metabolically, because the
mitochondria oxidise the carbon that is fixed by the chloroplast during photosynthesis
and the chloroplast requires the ATP produced from mitochondrial dark respiration
(Leister, 2005). Although no known signalling molecules have been identified there
are several molecules implicated in playing a role in this organellar cross talk. ROS,
ascorbate and nitric oxide are all thought to play some role in the cross talk between
these organelles. In addition to these it is known that mitochondria produce redox
equivalents similar to that of the chloroplast, particularly the protectants against photo-
inhibition (Raghavendra and Padmasree, 2003; Lindemann et al., 2004). There are
also mutant lines that show indications of cross talk between these organelles. In
Arabidopsis AOX1a deficient mutants exhibited a high light and drought sensitive
phenotype with increased non-photochemical quenching and ROS levels. These
mitochondrial mutants also exhibited a decrease in transcript abundance for
photosynthetic and photo-respiratory transcripts. This is good evidence for organellar
cross talk, however, it still remains inconclusive, as these observations could have
been caused by indirect mechanisms (Giraud et al., 2008).
Recently, (Yoshida and Noguchi, 2011) have shown that under excess light reductant
are exported from the chloroplasts to the cytosol. It is hypothesised that this
45
communication occurs via NADP-Malate dehydrogenase (NADP-MDH)
and Malate-OAA shuttles and the mitochondrial non-phosphorylating pathways may
facilitate the dissipation of this excess reductant in the cell. Observations of organellar
communications were also observed in other species including maize, barley and
tobacco. The NCS6 maize mutant has a dysfunctional cytochrome oxidase subunit 2,
this consequently results in a dysfunction of the mitochondrial ETC and it has been
observed that this mitochondrial mutation corresponds with a decrease in
chloroplastic and nuclear encoded subunits of Photosystem I (Jiao et al., 2005). The
albostrians mutants in barley lack chloroplastic activity and it has been shown that this
corresponds with an increased mitochondrial gene copy number and an increase in
mitochondrial transcripts (Hedtke et al., 1999). Finally another mutant in tobacco, the
CMSII mutant that lacks the NAD7 subunit of complex I within the mitochondria has
been linked with a reduced rate of photosynthesis in dark to light transitions (Sabar et
al., 2000; Dutilleul et al., 2003).
1.11 Project Aims and Approaches
The best characterised processes in mitochondria are the production of ATP in the
process of oxidative phosphorylation and the TCA cycle. However mitochondria are
also critically important in plant defense in stress situations. Over the years many
mitochondrially located proteins have been identified as being induced by stress such
as AOX and NDB2, however the vast majority of mitochondrial proteins are yet to be
fully characterised.
This project aims to investigate the expression patterns and function of UPOX1 (Up
regulated due to OXidative stress). Prior to the start of this PhD, UPOX1 had not been
studied in depth. The first publications that used UPOX1 were studies by Clifton et al.,
2005, these experiments demonstrated that UPOX1 responded to a broad range of
abiotic stress treatments in cell cultures. Following this, the gene was named UPOX1
46
and described as one of the “hallmarks of oxidative stress” within the model plant
Arabidopsis thaliana (Gadjev et al., 2006). Apart from these transcriptomic studies no
in-depth transcriptomic or proteomic studies have been performed.
This project takes a multi-disciplinary approach to examining the induction of UPOX1
at a transcript and proteomic level with the eventual aim of elucidating the function of
UPOX1. The approaches taken include:
Transcript expression profiling-
This will involve investigations into the kinetics of UPOX1 induction with the aim of
identifying pathways that regulate stress induced gene expression. These assays will
be compared to that of known stress responsive genes such as AOX1a and other
mitochondrial proteins such as external NAD(P)H-dehydrogenase NDB2 and AtOM66.
In addition to this, transcript analysis will be conducted on defense signalling mutants
to further allude to the pathways that regulate stress induced gene expression.
Furthermore promoter regions will be investigated to determine the functional CAREs
that play a role in the response to stress treatments such as with H2O2, rotenone,
salicylic acid and combinations of these.
Proteomics -
Various proteomic methods will be employed including in vivo, in vitro localisation
methods and gel based isolation methods to further understand the proteomic profile
of UPOX1. Furthermore, transgenic lines that are over-expressers and under-
expressers for UPOX1 will be used to assist in determining the location of UPOX1
with the use of an antibody raised against UPOX1.
Global Transcriptomics –
In order to gain insights into the changes occurring within the transcriptome,
microarrays will be performed on the UPOX1 under-expressed and over-expressed
lines in comparison with Col-0 wild-type lines. Experiments will be conducted under
47
stress conditions and normal conditions. These microarrays may provide us with
more insight on the interactions and function of UPOX1 and associated transcripts.
48
49
50
Chapter 2. Materials and Methods
51
Chapter 2
MATERIALS AND METHODS
Chapter 2. Materials and Methods
52
2.1 General Chemicals
All reagents were molecular biology grade, analytical grade or equivalent, purchased
from Sigma-Aldrich (Sydney, Australia), Amersham Biosciences (Sydney, Australia)
Merck (Melbourne, Australia), Becton Dickinson (Melbourne, Australia), Bio-Rad
(Sydney, Australia), Univar APS Finechem (Melbourne, Australia) and AppliChem
BioChemica (Melbourne, Australia). Composition of solutions and media are listed in
Appendix 1. Water used for nucleic acid manipulations and water soluble solutions
was obtained from the Milli-Q Plus Ultrapure system (Millipore, Sydney, Australia) and
sterilized by autoclaving prior to use (denoted as SDW).
2.2 Arabidopsis Growth Conditions
2.2.1 Arabidopsis suspension cell culture
Arabidopsis thaliana suspension cell cultures (ecotype Landsburg erecta) (Sweetlove
et al., 2002) were cultivated in 100 mL cell culture media (Appendix 1) in 250 mL
flasks. Cell cultures were grown under long day light cycles of 16 h light
(~ 100 μE -2 s-1) and 8 h dark, rotating constantly at 120 revolutions per minute (rpm)
at 22°C. Cells were sub cultured at 7 day intervals with a 1:6 (v/v) dilution factor.
2.2.2 Arabidopsis water culture
Arabidopsis water cultures were cultivated in 80 mL water culture media (Appendix 1)
in 200 mL water culture containers. Cultures were grown under medium day light
cycles 12 h light (~ 100 μE -2 s-1) and 12 h dark, rotating constantly at 105 rpm at
22 °C.
2.2.3 Arabidopsis plant material
Arabidopsis plants were grown from seeds either in (4:1) soil mix : vermiculite or on
Arabidopsis solid media (Appendix 1) containing 50 µg/mL Cefotaxime® antifungal
Chapter 2. Materials and Methods
53
agent. Seeds were sterilized in a laminar flow hood using aseptic technique prior to
plating on solid media. Seeds were incubated in 1 mL sterilization solution (Appendix
1) for 5 min; this solution was removed and incubated in 1 mL 70% (v/v) ethanol. The
70% ethanol solution was removed and the seeds were washed in 100% ethanol
before being dried in the laminar flow. Seeds were resuspended in 200 μL of sterile
distilled water (SDW) and transferred to solid media. To synchronize germination,
seeds plated on solid media and in soil mix were stratified at 4 °C in the dark for 48 h,
and then grown at 22°C under long day conditions of 16 h light (~ 100 μE -2 s-1) and 8
h dark.
2.2.4 Chemical treatment of Arabidopsis cell culture,
water culture and plant material
Arabidopsis cell cultures were treated four days after subculturing. The following table
shows how the treatments were prepared and applied. The following treatments were
applied according to (Clifton et al., 2005).
Treatment Solvent Final
Concentration
Quantity Applied
Antimycin A ethanol 5 μM 12μLof 50μM
Glucose H2O 3 % (w/v) 1 mL of 2.17 M
H2O2 30% (v/v) 10 mM 130 μL of 8.82 M
Rotenone ethanol 40 μM 480 μL of 10 mM
Salicylic acid ethanol 100 μM 120 μL of 100 mM
All compounds were obtained from Sigma (Castle Hill, Australia).
2.2.5 UV treatment of Arabidopsis water culture
Chapter 2. Materials and Methods
54
Arabidopsis water cultures were treated with UV stress by exposing them to UV-C at
254 nm using cross linker (UVP 34-0042-01). Following irradiation, plants were
incubated under the same conditions used for routine growth unless stated otherwise
2.3 Bacterial Techniques
2.3.1 Bacterial cell strains
JM109:
recA1supE44endA1hsdR17gyrA96relA1thi(lac-proAB)F’[traD36proAB+,lacIqlacZM
15]
DH5:
supE44lacU169(80dlacZM15)hsdR17recA1endA1gyrA96thi-1relA1
XL1-blue:
supE44hsdR17recA1endgyrA46thirelA1lacF’[proAB+lacIqlacZ∆M15Tn10(tert)
XL10-Gold
Tetr∆(mrcA)183∆(mcrCB-hsdSMR-mrr)173endA1supE44thi-
1recA1gyrA96relA1lacHte[F’proAB+,lacIqZ∆M15Tn10(tetr)AmyCamr]
2.3.2 Transformation into JM109 and DH5 cells
Chemically competent cells (laboratory competent JM109 or DH5) were thawed on
ice, mixed gently and 50 μL were transferred to pre-chilled 15 mL round bottom tubes.
Up to 5 μL of DNA, containing up to 50 ng of plasmid DNA was added to the cells.
The transformations were incubated on ice for 10 min, heat shocked for 45 sec in a
water bath at 42°C and incubated on ice for a further 2 min. 500μL of LB media (for
JM109 cells) or 200 μL SOC media (Appendix 1) (for DH5) were added to the cells
and the transformations were incubated at 37°C for 1 hr, shaking at 220 rpm. The
transformations were then divided into 50 μL and 450 μL; these volumes were plated
Chapter 2. Materials and Methods
55
onto LB plates with the appropriate antibiotics (Kanamycin or Ampicillin) and
incubated overnight at 37°C.
2.3.3 Transformation into XL-Blue Super competent cells
Prior to transformation 4 units (U) of Dpn1 restriction enzyme was applied to the
polymerase chain reaction (PCR) reaction. 200 ng of the Dpn1 digested DNA was
added to 25μL of XL- Blue super competent cells and incubated for 30 min on ice in a
pre-chilled 15 mL round bottom tube. Cells were then heat shocked for 45 s at 42 °C
and incubated on ice for 2 min. Five hundred μL of pre-warmed NZY+ broth was
added to the transformations and they were incubated for 1 h at 37°C, shaking at 220
rpm. 50μL and 450 μL were plated onto separate LB plates with the appropriate
antibiotics (Kanamycin or Ampicillin) and incubated overnight at 37 °C.
2.3.4 Transformation into XL-Gold Ultra competent cells
Chemically competent cells were thawed on ice and 25 μL of cells were transferred to
pre-chilled 15 mL round bottom tubes. One μL of 50% (v/v) β-mercaptoethanol, were
added to the cells and incubated for 10 min on ice. Two to five μL of Dpn1 digested
DNA was added to the cells and mixed gently. The cells were heat shocked for 30 s at
42°C and incubated on ice for a further 2 min. 500μL of pre-warmed NZY+ broth was
added to the transformations and they were incubated for 1 h at 37°C, shaking at 220
rpm. Fifty μL and 450 μL were plated onto separate LB plates with the appropriate
antibiotics (Kanamycin or Ampicillin) and incubated overnight at 37 °C.
2.4 Nucleic Acid Manipulation
2.4.1 Genomic DNA preparation
Genomic DNA was isolated from Arabidopsis tissue using DNAeasy® Plant Mini Kit
(QIAGEN, Clifton Hill, Australia) as per manufacturer’s instructions.Plant samples
Chapter 2. Materials and Methods
56
were mechanically disrupted and chemically lysed, during the lysis step RNA was
removed by RNase digestion. Following the removal of cell debris the samples were
filtered by centrifugation through QIAshredder spin columns. Binding buffers and
ethanol were washed through the spin columns to promote the binding of DNA to the
membrane and remove contaminants. The precipitating proteins, polysaccharides and
lysate were then centrifuged through further spins. The DNA selectively bound to the
silica-gel membrane and contaminants passed through. Two further wash steps
ensured highly purified DNA. The DNA was finally eluted in 100 μL of SDW and
stored at -20°C.
2.4.2 Total RNA isolation
Total RNA was isolated using the RNeasy Plant Mini Kit (QIAGEN, Clifton Hill,
Australia). For each sample, approximately 100 mg of Arabidopsis plant tissue was
ground under liquid nitrogen with a mortar and pestle. These samples were then lysed
and homogenized in a highly denaturing guanidine-thiocyanate–containing buffer, this
inactivates RNases. Ethanol was added to the samples to promote the binding of RNA
to the RNease mini spin columns. The RNA bound to the membranes selectively
whilst contaminants were washed through. All reactions were treated with RNase-free
DNaseI (QIAGEN, Clifton Hill, Australia) and DNAfree™ (Ambion, Austin, USA)
according to manufacturer’s instructions to ensure the complete removal of any DNA
contamination. Ethanol precipitations were performed for all RNA samples extracted
from MS plates due to the high sugar content. RNA samples were eluted in80 μL of
RNase-free SDW and further diluted with 120μL of SDW. 20 μL of sodium acetate
was added with 440 μL of 100% (v/v) ethanol to the RNA. This reaction was kept at -
20°C for at least 2 h and centrifuged at 20 000 g for 25 min at 4°C. The supernatant
was removed and the remaining pellet was washed in 1 mL of 70% (v/v) ethanol,
centrifuged at 20 000 g for 20 min at 4°C and dried in a speedy-vac for 10 min. The
Chapter 2. Materials and Methods
57
pellet was resuspended in 40 μL of RNA-free SDW. RNA intended for qRT-PCR and
microarrays required stringent quality and quantity assessment. This was assessed
on 1.2 % (w/v) agarose gels and was based on the integrity of the 18S and 28S rRNA
bands. Quantity checks were also analysed using a Nanodrop ND-1000
Spectrophotometer (Biolab, Victoria, Australia) and further quality analysis was
performed using a Bioanalyzer 1200 (Agilent, California, USA).The A260/A280ratio
obtained using the Nanodrop and the Bioanalyser gave an indication of the purity of
the nucleic acid, typically ratios between 1.8 and 2.1 were considered pure.
2.4.3 Preparation of cDNA
Reverse transcription was performed to produce cDNA from total RNA. Using the
iScript cDNA synthesis kit (Bio-Rad, Sydney, Australia) 1 μg of total RNA was used
and cDNA was produced according to manufacturer’s instructions. To produce cDNA
an enzyme was used that contained a modified MMLV-reverse transcriptase with
RNase H+ activity. Also with the addition of SDW a five times transcription mix was
added to the reactions that contained RNase inhibitor, a blend of oligo(dT), random
hexamer primers and dNTPs. The reactions were incubated at 25 °C for 5 min, 42 °C
for 30 min, 85 °C for 5 min and held at 4 °C.
For qRT-PCR assays a negative control was always performed, these reactions
contained all the components described above except the reverse transcriptase
enzyme.
The cDNA samples were purified prior to quantitative PCR analyses, using the
QIAquick® PCR Purification Kit (Qiagen, Clifton Hill, Australia) as per manufacturer’s
instructions. A binding buffer was applied to the cDNA samples, this is a high-salt
buffer, DNA binds selectively to the silica membrane within the spin column. The wash
buffers wash all primers, nucleotides, enzymes and other impurities through the
column leaving the bound DNA samples attached to the membrane. Finally the cDNA
Chapter 2. Materials and Methods
58
samples were eluted in 30 µL of low salt buffer and stored at -20 °C. All samples to
be used in qRT-PCR were then diluted 1:10 in SDW.
2.4.4 Agarose gel electrophoresis
Agarose gels electrophoresis was used to analyse PCR products and restriction
digests by DNA separation. Nucleic acids were separated using 1% (w/v) agarose
(Roche, Castle Hill, Australia) and gels were made in 1X TAE buffer (Appendix 1)
containing 1 µg/mL ethidium bromide. After the Agarose gels solidified the samples
were mixed with loading buffer (Appendix 1) and electrophoresis took place in tanks
containing 1X TAE buffer under 70 Volts (V). Following electrophoresis, samples were
visualised under UV light using a gel doc system (Bio-Rad, Sydney, Australia).
2.4.5 Amplification of DNA by polymerase chain reaction
The Expand High Fidelity Polymerase Chain Reaction kit along with the dNTP kit
(Roche, Castle Hill, Australia) was used under manufacturer’s instructions to perform
all polymerase chain reactions (PCR). Primers were diluted to 20 ρmol/μL (Sigma-
Aldrich, Sydney, Australia; Invitrogen, Sydney, Australia) prior to PCR. Amplifications
were performed under the following parameters: 94°C for 2 min followed by 35 cycles
of 94°C for 30 s, 58°C for 2 min and 72°C for 5 min. Small adjustments were made to
the annealing temperature depending on the melting points of individual primers.
Following PCR, reactions were held at 4°C prior to purification. PCR products were
purified using the QIAquick PCR purification kit (QIAGEN, Clifton Hill, Australia) as
described in Section 2.4.3.
2.4.6 TOPO-TA cloning into pCR2.1
Following PCR purification TOPO-TA cloning kits (Invitrogen, Sydney, Australia) were
used to clone PCR products into the pCR2.1 vectors according to manufacturer’s
instructions. 4μL of PCR product was incubated with 1 μL of salt solution (consisting
Chapter 2. Materials and Methods
59
of 1.2 M NaCl and 0.06 M MgCl2) and 1μLof 10 ng/μL of vector for 10 min at room
temperature. 2μL of this reaction was transformed into the TOP10F’ cells supplied
with the TOPO-TA kit. The cells were then heat shocked for 30 s at 42°C and
incubated for 2 min on ice. 250μL of SOC media was added to the transformations
and incubated for 1 h at 37°C, shaking at 220 rpm. 40μL of 40 mg/mL X-gal (5-bromo-
4-chloro-3-indolyl-β-D-galactopyranoside) and 40 μL of 40 mg/mL IPTG (isopropyl-1-
thio-β-D-galactopyranoside) were added to LB plates containing the appropriate
antibiotics (Kanamycin or Ampicillin) prior to the transformations being plated.
2.4.7 Site Directed mutagenesis
Site directed mutagenesis (SDM) was performed using the QuickChange II or the
QuickChange XL II site directed mutagenesis kits (Stratagene, California, USA).
Generally QuickChange XL II kits were used for mutations that included more than 3
point mutations or for reactions that had been previously unsuccessful using the
QuickChange II kit. Mutagenesis was then performed according to the manufacturer’s
instructions as follows. Mutagenesis reactions used supercoiled double stranded DNA
(dsDNA) vectors, primers, dNTP mix, a 10 times reaction buffer, SDW and PfuTurbo
DNA polymerase. Primers were purchased from Invitrogen (Sydney, Australia) and
were designed with the desired mutation in both the forward and reverse orientations.
Cycling parameters were as follows: 95 °C for 1 min followed by 18 cycles of 95 °C
for 50 s, 55 °C for 50 s, 68 °C for 1 min/kb of plasmid followed by 68 °C for 7 min.
Cycling parameters were similar between with QuickChange II and the QuickChange
XL II, the major difference was the QuickChange II kit did not contain the final cycle of
68 °C for 7 min, all reactions were held at 4 °C.
2.4.8 Dpn1 Digestion
Chapter 2. Materials and Methods
60
Mutagenesis reactions were digested using 4U of Dpn1 endonuclease and incubated
at 37 °C for 1.5 hr. Dpn1 endonuclease removes parental, unmutated strands by
specifically digesting methylated and hemi-methylated DNA leaving only daughter
DNA containing the desired mutation. Following Dpn1 digestion SDM reactions were
transformed using XL-Blue Super competent cells (for QuickChange II reactions) or
XL-Gold Ultra competent cells for (QuickChange XL II reactions) according to Section
2.3.3 and Section 2.3.4.
2.4.9 Restriction digests
Restriction digests for sub-cloning purposes required ~1000 ng of purified insert DNA
and ~1000 ng of vector DNA. Digestions were set up using the appropriate restriction
buffers and enzymes depending on what the restriction site(s) were (Invitrogen,
Sydney, Australia). The digest reactions were made up to 30 μL and incubated at the
appropriate temperatures (most commonly 37 °C) for 2 hr. The restriction digests
were then incubated at 65 °C for 15 min to denature the enzymes. Following
inactivation of enzymes, the insert DNA along with 1 μL of vector DNA were analysed
using 1% (w/v) agarose gel electrophoresis using a DNA ladder containing known size
fragments (Invitrogen, Sydney, Australia).
2.4.10 Calf Intestinal Alkaline Phosphatase treatment
For sub-cloning, vector digests were treated with Calf Intestinal Alkaline Phosphatase
(CIAP) (Invitrogen, Sydney, Australia). CIAP prevents re-ligation of linearized vector
DNA by removing 5'-phosphate groups prior to 5'-end labelling of nucleic acids. Blunt
ended vector DNA digests were incubated at 37 °C for 1 hr, 5’ overhang vector
digests were incubated at 37 °C for 30 min. Following CIAP treatment any remaining
CIAP enzyme was inactivated at 65 °C for 15 min.
Chapter 2. Materials and Methods
61
2.4.11 DNA ligation
Following the PCR purification of the insert DNA and the CIAP treatment of the vector
DNA both samples were analysed using a Nanodrop to determine their DNA
concentrations. Calculations were performed to in order to obtain a 3:1 insert to vector
molar ratio in the ligation reaction. The formula for working out the correct molar ratio
is as follows: ((ng vector) x (kb size of insert)) / ((kb size of vector) x (molar ratio of
(insert/vector)) = (ng insert). T4 ligase buffer was added to a final concentration of 1 x
and 1 U of T4 ligase was added (Roche, Castle Hill, Australia). The reactionswere
incubated at 14 °C overnight prior to transformation with chemically competent cells.
2.4.12 Plasmid Minipreps
Single E.coli bacterial colonies containing a plasmid were used to inoculate 2 mL of
LB media containing appropriate antibiotics. The cultures were grown for 16 h at
37 °C with shaking at 1400 rpm. Cells were centrifuged for 1min at 20 800 g, the
supernatant was removed and the plasmid DNA was isolated from the cell pellet using
the QIAGEN Plasmid Mini kit (QIAGEN, Clifton Hill, Australia) according to
manufacturer’s instructions. The process by which QIAGEN Plasmid Mini kits purify
DNA involved the use of spin columns made with anion-exchange resin. Following
the lysis of the bacterial pellets the alkaline lysate was applied onto the anion-
exchange resin tip, under centrifugation and low salt conditions the lysis was cleared
leaving the DNA bound to the membrane. RNA proteins, dyes, and low-molecular-
weight impurities were then removed by the addition of a medium-salt wash under
centrifugation and plasmid DNA was eluted in a high salt buffer. The DNA was
desalted and concentrated following isopropanol precipitation and collected by
centrifugation to be finally eluted in 20 μL of SDW and stored at -20 °C.
2.4.13 Plasmid Midipreps
Chapter 2. Materials and Methods
62
Single E.coli bacterial colonies containing a plasmid were used to inoculate 50 mL of
LB media containing appropriate antibiotics. The cultures were grown for 16 h at
37 °C with shaking at 220 rpm. Cells were collected by centrifugation for 15 min at
3000 g. The plasmids were then purified using the QIAGEN Plasmid Midi kit
(QIAGEN, Clifton Hill, Australia) as per manufacturer’s instructions. The principles by
which the QIAGEN Plasmid Midi kit are based on involve the use of a anion-exchange
resin column and selective binding under varying salt and pH conditions that operate
under gravity flow, they are the same principles as that described for QIAGEN
Plasmid Mini kit and the process occurs as described in Section 2.4.13. The key
differences involve the use of larger liquid quantities for the required binding, washing
and elution buffers. The DNA was eluted in 100 μL of SDW and stored at -20 °C.
2.4.14 Plasmid Maxipreps
Single E.coli bacterial colonies containing a plasmid were used to inoculate 150 mL of
LB media containing appropriate antibiotics. The cultures were grown for 16 h at 37
°C with shaking at 220 rpm. Cells were collected by centrifugation for 30 min at
3000 g. The plasmids were then purified using the QIAGEN Plasmid Maxi kit
(QIAGEN, Clifton Hill, Australia) as per manufacturer’s instructions. The principles by
which the QIAGEN Plasmid Maxi kit are based on involve the use of a anion-
exchange resin column and selective binding under varying salt and pH conditions
that operate under gravity flow, they are the same principles as that described for
QIAGEN Plasmid Mini and Midi kits and the process occurs as described in Section
2.4.13. The key differences involve the use of larger liquid quantities for the required
binding, washing and elution buffers. The DNA was eluted in 1 ml of SDW and stored
at -20 °C.
2.5 Generation of Arabidopsis mutant lines
Chapter 2. Materials and Methods
63
2.5.1 Preparation of Artificial microRNA constructs
For Arabidopsis stable transformations the experimental procedure was carried out
according to the Web MicroRNA Designer 2.0 (Schwab et al., 2006). The artificial
microRNAs (AmiRNAs) were generated by overlapping PCRs and the MIR319a
precursor was used as the plasmid to engineer the AmiRNA. The AmiRNA fragment
was sub-cloned from the MIR319a precursor into the pRS300 plasmid and
consequently sub-cloned into the pCambia1301 plasmid for agrobacterium mediated
transformation according to Section 2.4.9 to Section 2.4.12.
2.5.2 Preparation of Agrobacterium tumefaciens electro-
competent cells
Two hundred μL of Agrobacterium stock was inoculated with 10 mL LB and
Rifampicin to a final concentration of 50 μg/mL. This culture was grown over night at
30 °C shaking at 220 rpm. One hundred μL of overnight culture was added to 900 μL
of LB with Rifampicin and the amount of starting culture required to inoculate 100 mL
of LB with Rifampicin was calculated. The 100 mL culture was grown over night at
30 °C shaking at 220 rpm until the optical density was between 0.4 and 0.6. This
culture was centrifuged at 4 °C for 10 min at 10 000 rpm and the pellet was
resuspended in 10mLice cold1 mM HEPES buffer. The centrifugation was repeated
and the pellet was resuspended in 10mLice cold of 1 mM HEPES, 10 % (w/v)
glycerol. The centrifugation was repeated, the resultant pellet was resuspended in
10mL of 1 mM HEPES 10 % (w/v) glycerol and the suspensions were pooled. The
solutions were centrifuged at 4°C for 5 min at 12 200 g and the pellet was
resuspended in 200 μL of 1 mM HEPES, 10 % glycerol. The samples were aliquoted
into 100 μL samples and snap frozen at -80°C.
2.5.3 Transformation of electro-competent
Agrobacterium
Chapter 2. Materials and Methods
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One hundred μL of Agrobacterium competent cells were thawed on ice and 2 μL of
plasmid (200-400ng) was added, the reaction was chilled on ice for 2 min. Then
transferred to a cuvette and 500 μL of LB was pipetted and immediately pulsed using
the Gene Pulser (Bio-Rad, Sydney, Australia). The cells were shocked with 2.5 kV,
with a capacitance of 25 μF and a resistance of 400 Ω. Immediately following
electroporation, 500 μL of LB was added and the reaction was transferred to a 15 mL
round bottom tube and incubated for 2 h at 30 °C shaking at 220 rmp. 50 μL and
200 μL of culture were plated onto LB plates containing Rifampicin in conjunction with
other appropriate antibiotics (Kanamycin or Ampicillin).
2.5.4 Floral dipping
Based on the methods detailed in (Clough and Bent, 1998) Arabidopsis plants were
grown until they started to bolt and seven days prior to transformation the bolts were
clipped. The agrobacterium was inoculated in 5 mL of LB with required antibiotics and
grown for 2 days at 28 °C shaking at 200 rpm. The small culture was transferred to
500 mL of LB media with required antibiotics and grown overnight at 28 °C, shaking at
200 rpm until the optical density reached 0.6 ~ 0.8. The agrobacterium was then
centrifuged for 20 min at 2500 g and resuspended in 500 mL of 5 % (w/v) sucrose,
0.03% (v/v) silwet L-77. The above soil parts of the Arabidopsis plants were dipped in
the agrobacterium for 10 s with gentle agitation. The remaining solution was pipetted
onto the leaves. The plants were covered for one to two nights to maintain high
humidity levels. The plants were grown as usual and the dry seeds were harvested
and the transformants were selected using hygromycin selection on MS agar plates.
2.5.5 Preparation of mutant lines
The full length coding sequence of UPOX1 was cloned into the p35S overexpression
vector pK7WG2 and homozygous transgenic plant lines were created by Dr Van Aken
Chapter 2. Materials and Methods
65
and Dr Inge De Clercq within Prof. Frank Van Breusegem's lab (VIB Ugent, Belgium),
this line was provided for this collaboration. This line was also confirmed for
homozygosity within the Whelan lab by myself prior to qRT-PCR analysis.
Promoter:GFP-GUS lines were generated by isolating DNA from Col-0 plants with
DNeasy plant kits (Qiagen) according to the manufacturer's instructions. Following
amplification of the 1500 bp genomic region upstream of the start codon with Platinum
Taq High Fidelity DNA polymerase (Invitrogen), the PCR product was cloned into
pDONR221 (Thermofisher, Melbourne Australia) and cloned by recombination to
pBGWFS7 (VIB-Ghent University, Ghent, Belgium) (Karimi et al., 2002), generating a
transcriptional GFP-GUS fusion. These constructs were then transformed into
Arabidopsis Col-0 by Agrobacterium tumefaciens-mediated floral dipping.
2.6 Transient transformation of Arabidopsis
cell culture and leaves
2.6.1 Preparation of Arabidopsis suspension cells
Arabidopsis cell culture was harvested at 4 days and stress treatments were applied
in the manner described in Section 2.2.4 for 1 hr. Cells were aseptically syringed onto
filter paper and left for 2 hours on solid osmotic media (Appendix 1) prior to biolistic
transformation.
2.6.2 Preparation of Arabidopsis leaves
Four week old rosette leaves were excised and stress treatments were applied in the
manner described in Section 2.2.4 for 3 hr. Leaves were placed on filter papers which
were then placed on solid osmotic media prior to biolistic transformation.
2.6.3 DNA precipitation onto gold microcarriers
Chapter 2. Materials and Methods
66
To a 1.5 ml tube 30 mg of 1 µm gold particles was aliquoted, this was washed in 1 ml
of 70% ethanol and following the removal of the ethanol the gold particles were
resuspended in 500 µl of 50% glycerol. The gold particles were aliquoted into 50 µl
lots. To tube, 5 µl of DNA (at 1 µg/µl), 50 µl 2.5 M CaCl2 and 20 µl of 100 mM
Spermidine was added prior to biolistic transformation. After vortexing and
precipitation, the gold beads were washed with ethanol.
2.6.4 Transformation of tissue
Using the PDS-1000 (Bio-Rad, Sydney, Australia) transformations of each construct
were performed in triplicate according to manufacturer’s instructions. A hepta adapter
was used and all transformations were performed under a vacuum pressure of 27
inches/Hg and a helium pressure of 1300 psi. The rupture disks (Bio-Rad, Sydney,
Australia) were rated at 1100 psi. Following transformation of cell culture and leaves
they were placed under light at 22 °C for 24 hr.
2.6.5 Reporter gene assays for luciferase and
β-glucuronidase
Tissue was ground under liquid nitrogen using stainless steel ball bearings and the
Mixer Mill 301 (Retsch, Haan, Germany). The tissue was extracted according to the
manufacturer’s instructions using the lysis buffer supplied with the Luciferase Assay
System (Roche, Sydney, Australia).The Polarstar Optima (BMG Lab technologies,
Offenburg, Germany) was used to perform Luciferase (LUC) and 4-methylumbelliferyl
β-D-glucuronide dehydrate (MUG) activity assays. Activities for LUC were measured
at 4 s after the addition of substrate. MUG activities were assayed by measuring the
accumulation of MUG fluorescent product over 1 h with measurements at 1 min
intervals using a fluorimetric assay described by (Jefferson et al., 1987). All samples
were excited at 355 nm and emission was measured at 460 nm.
Chapter 2. Materials and Methods
67
2.6.6 Normalisation of LUC and MUG activities
An estimation of the transformation efficiency was provided by dividing the LUC
activities by the highest LUC value for the set. All MUG values were consequently
divided by this value to give a normalized MUG value. Standard errors were then
calculated by dividing the standard deviation by the square root of the number of
transformations performed for each construct.
2.7 Quantitative RT-PCR
2.7.1 DNA standards
Gene specific DNA standards are required in order to enable the measurement of
absolute cDNA abundance for quantitative real time polymerase chain reaction
(qRT-PCR). The gene of interest was cloned using cDNA as a template and specific
forward and reverse cloning primers. The DNA standards were then amplified via
PCR. The PCR products were separated by agarose gel electrophoresis and purified.
The amount of DNA was quantitated using the PicoGreen® dsDNA quantitation kit
(Molecular Probes, Eugene, USA) according to manufacturer’s instructions. DNA
standards were kept at 0.1 fmol/μL and diluted to 0.01 fmol/μL as a working stock.
2.7.2 Primer design and optimization
Primers for qRT-PCR were all designed to be intron spanning with an amplicon size of
approximately 200 bp in size. Gene specificity tests were conducted to ensure no
cross amplification for genes within a family, the primers used for qRT-PCR are listed
in Appendix 2.
The maximum efficiency for the qRT-PCR reactions were individually tested by using
serial dilutions of the standard DNA template, (by varying the Mg2+ concentration and
the primer concentrations). Following qRT-PCR runs on the Light Cycler® 480 (Roche,
Chapter 2. Materials and Methods
68
Castle Hill, Australia) melt curve analysis was performed to determine if any primer-
dimer and/or non-specific amplification had occurred.
The absence of genomic DNA contamination was confirmed by assaying all no
reverse transcriptase reactions (no-RT). If any no-RT samples showed specific
amplification all samples were discarded and the process was repeated from the
beginning with new RNA.
2.7.3 qRT-PCR using the Roche Light Cycler® 480
Serial dilutions were prepared from the DNA standards, these ranged from
0.01 fmol/μL to 0.00000001 fmol/μL. Five μL reactions were assayed with varying
primer concentrations from 0.3 to 0.9 μM, to these reactions 2.5 μL of Roche SYBR
Green and 1 ul of cDNA was added. Prior to the commencement of all qRT-PCR runs
all plates were mixed gently and spun down at 1000 g for 1 minute.
More specific primer hybridization was provided by the pre-incubation step performed
at 95 °C for 10 min. The remainder of the qRT-PCR assayed consisted of the
following parameters: amplification for 45 cycles; 95 °C for 10 s, 60 °C for 10 s and
72°C for 10 s with a single data acquisition point. Melt curve consisted of the following
parameters: 95 °C for 10 s, 65 °C for 1.01 min, 95 °C continuous with 5 data
acquisitions per cycle and cooling at 40 °C for 10 s. The results were analysed using
the software on the Light Cycler® 480.
2.7.4 qRT-PCR Analysis
Prior to analysing the qRT-PCR results melt curve analysis was performed, the
principle behind melt curve analysis involves the denaturation of base-base hydrogen
bonds. The energy required for this break is dependent on the length, GC content and
Chapter 2. Materials and Methods
69
complementarity of a sequence. Therefore any non-specific products or primer
dimmers amplified during a qRT-PCR run can be detected by SYBR green.
The data analysis software calculated a threshold cycle for each sample, this
represents the cycle at which the fluorescence signal in the sample first increased
significantly above the background level. From the threshold cycles (Ct) of the
standard DNA dilution series a standard curve was generated between 0.01 fmol/μL
and 0.00000001 fmol/μL. This was then used to determine the starting concentration
of transcript in each cDNA sample based on its threshold cycle.
2.7.5 qRT-PCR Cross reactivity assays
Performed on the Roche Light Cycler, melt peaks confirmed if the primers were
specific for the target transcript. This tool plots temperature against fluorescence
units. The fluorescence increases as the temperature increases until a point at which
the dsDNA denatures to form ssDNA which involves unbinding the SYBR® Green dye
and losing fluorescence. This data is represented by plotting temperature against the
negative derivative of the relative fluorescence units. A single peak at a specific
temperature represents the specific product of uniform size. Any non-specific product,
such as primer dimmer could be visualized as a substantially smaller peak occurring
at a lower temperature. This method allowed products of different size to be
differentiated with 1 °C accuracy or 1 bp accuracy.
2.8 Organelle and protein isolation methods
2.8.1 Mitochondrial Isolation
All mitochondrial isolation steps were carried out at 4 °C and centrifugation was
performed with the use of the Beckman J-26XD and J301 centrifuges with JA-20
Chapter 2. Materials and Methods
70
rotors. All media, centrifuge tubes and apparatus were pre-chilled at 4 °C prior to use.
The procedure for mitochondrial isolation was based on the methods described by
(Day et al., 1985) as follows.
Mitochondrial isolations from water cultures and whole tissues involved the same
buffers and centrifugations spins, for isolation of mitochondria from cell cultures there
were slight variations in the buffers used and the centrifugation speeds, the principles
were however the same. Four week old plants grown on soil and 10-14 day old water
cultures were used for all mitochondrial isolations. Grinding and wash buffers were
made (Appendix 1) in advance and were kept at 4°C. On the morning of the
mitochondrial isolation 1.06 g (per 300ml media) of sodium ascorbate and 0.74 g (per
300ml media) of L-cysteine were added to the grinding media to stabilize the
mitochondria during the grinding process. The plant material was homogenized by
hand using mortar and pestles and filtered through 4 layers of miracloth, this process
was repeated twice. The filtered homogenate was centrifuged under 2 450 g for 5 min
to remove the cell debris and nuclei. The supernatant was transferred to new tubes
containing 1 times wash buffer and centrifuged at 17 400 g for 20 min to pellet the
mitochondria and peroxisomes. The pellets were resuspended in ~2 mL of 1 times
wash buffer and the previous low and high speed centrifugation steps were repeated
to further purify the crude mitochondrial fraction. Following this, the crude
mitochondrial pellets were layered over 0-40% (w/v) PVP gradients (Appendix 1).
These gradients were centrifuged for 40 min at 40 000 g. The PVP served to narrow
the distribution of mitochondria within the gradients, mitochondrial bands visibly form a
yellow/grey band low in the tube. The layers above were aspirated off and the
mitochondrial bands were distributed in 1 times wash buffer to remove the percoll and
other impurities. Following two high speed spins at 31 000 g for 15 min the final
mitochondrial pellet was isolated.
Chapter 2. Materials and Methods
71
Mitochondria following this isolation method was sufficiently pure to perform in vitro
imports however if mitochondria required further purification for downstream
application such as proteinase K titrations a second gradient was performed prior the
final high speed spins at 31 000 g.
2.8.2 Chloroplast Isolation
All pea leaf chloroplast isolation steps were carried out at 4 °C and centrifugation was
performed with the use of the Beckman J-26XD and J-301 centrifuges with JA-20
rotors. All media, centrifuge tubes and apparatus were pre-chilled at 4 °C prior to use.
The procedure for chloroplast isolation was based on (Bruce et al., 1994) as follows.
Prior to a chloroplast isolation the day/light cycle at 26 °C was set so as chloroplasts
were isolated at the end of a dark period, this prevents the accumulation of starch.
The peas harvested ranged in age between 7 and 10 days and the percoll gradients
(Appendix 1) were prepared in advance. The plant tissue was homogenized in two
times grinding buffer using a standard blender with 5-6 short bursts. The
homogenized material was then filtered through one layer of miracloth and centrifuged
at 2000 g for 5 min to pellet the crude chloroplasts. The resuspended pellet was then
pipetted over two gradients and centrifuged at 12 100 g. Chloroplasts form a light
green band low in the tube, this fraction was then washed and centrifuged at 2000 g
for 4 min. The resultant pellet was then assayed for its chlorophyll concentration prior
to use in in vitro imports.
2.8.3 Protein translation
Radiolabelled [35S]-methionine precursor proteins were translated using the TNT®
Coupled Transcription Translation Rabbit Reticulocyte system by Dr Murcha. As this
translation was weak it was enhanced by myself with two additional methionines at
the start and end of the protein (Stratagene, California, USA). This in vitro synthesis
Chapter 2. Materials and Methods
72
system provided a master mix containing reticulocyte lysate, RNA polymerase, amino
acids, buffer, RNase inhibitor and [35S]-methionine. Translations were all prepared on
ice, mixed with plasmid DNA, incubated for 2 h at 30 °C and frozen until use. Prior to
the use of translation in imports the translations were tested by running 2 μL on SDS-
PAGE gels (Section 2.9.7 SDS-PAGE) and exposing them on Fuji BAS TR2040
phosphor imaging plates.
2.9 In vivo and in vitro assays
2.9.1 In vivo GFP Assays
The GFP is composed of 238 amino acids, with a predicted mol mass of 26.9 kDa. It
absorbs blue light maximally at 395 nm with a minor peak at 470 nm and emits a
green light at 509 nm with a shoulder at 540 nm (Phillips, 2001).
The process involved in creating GFP fusions is relatively straight forward; this was
carried out via PCR and cloning strategies following the initial sub-cloning of UPOX1
into the pCR2.1 by Prof Ryan Lister (within the Whelan lab). The GFP fusions were
then subsequently sub-cloned by using the coding sequences (CDS) which were
amplified from Arabidopsis cDNA and cloning them into GFP fusion vectors, for
UPOX1, UPOX2 and UPOX3 both C-terminal (CDS-GFP) and N-terminal (GFP-CDS)
GFP fusions were constructed. The choice of the carboxy or amino terminals is
usually based on predicting which region of the proteins will tolerate the addition of
GFP and remain functional (Pédelacq et al., 2005). As this was a preliminary
examination of these proteins both terminals were used in individual constructs.
These fusion vectors were always co-transformed with RFP control constructs in order
Chapter 2. Materials and Methods
73
to define the sub cellular localisation of GFP with certainty. The RFP fusions used as
mitochondrial controls were AOX transit peptide red fluorescent proteins (AOX-RFP)
which were created by Dr Murcha and Dr Chris Carrie (Murcha et al., 2007; Carrie et
al., 2009).
These GFP and RFP fusions were transformed into Arabidopsis cell culture and onion
epidermal cells prior to visualisation microscopically. However akin to any type of
scientific technology there are limitations. Proteins with very short half-lives are
difficult to measure and proteins must be in enough quantity so as an adequate level
of detection is possible. Finally, experimental controls are performed. For example the
intensity and foci of fluorescence are monitored and checked to confirm that
fluorescence was due to localisation of a protein and not protein aggregation on the
microscope.
2.9.2 In vitro import of precursor proteins into
mitochondria
The procedures for in vitro imports were based on the methods described in (Whelan
et al., 1996). Import reactions used 180 μL of import master mix that was made up of:
2x import buffer, 1M MgCl2, 100 mM methionine, 100 mM ADP, 100mM ATP, 0.5 M
succinate, 0.5 M of fresh DTT, 0.5 M NADH and 100 mM GTP. Arabidopsis
mitochondrial imports were always carried out using freshly isolated mitochondria
from swirling liquid plant cultures. For a 4 lane import 100 μg of fresh mitochondria
were used with precursor protein (Whelan et al., 1996).
Protein imports were commenced by incubating the two reactions, one reaction with
the addition of Valinomycin (Val) and the other without at 25 °C for 20 min shaking at
350 rpm. The reactions were stopped by placing them on ice for 3 min. The reactions
containing no Val and Val were again divided into two. Proteinase K (PK) was added
Chapter 2. Materials and Methods
74
to one of the reactions with Val and one reaction without Val. All reactions were
incubated for a further 30 min on ice and 1μL of 100mM PMSF was added to the
reactions containing PK in order to inactivate the PK (Whelan et al., 1996). All
mitochondrial reactions were centrifuged for 3 min at 4°C at max speed to pellet the
mitochondria. The pellets were resuspended in 30 μL of 2x sample buffer, boiled for 3
min and run on a 16 % (v/v) SDS-PAGE gel (12 % (v/v) if the proteins were larger
than 20 kDa). The gels were coomassie stained for 2 hr, destained for 2hr, dried at 80
°C for 2h and exposed to a BAS TR2040 plate for 2 nights. The exposed plates were
read with the Personal Molecular Imager (Bio-Rad, Sydney, Australia).
2.9.3 In vitro import of precursor proteins into pea
chloroplasts
An import master mix was made prior to import commencement this contained: 1 x
import buffer, 100 mM MgCl2, 100 mM ATP, 100 mM Methionine, 1 M potassium
acetate and 1 M NaHCO3. Imports were always carried out using freshly isolated
chloroplasts, 50 μL of 500 μg chlorophyll/mL chloroplasts were added to each reaction
with 10 μL of precursor protein. The reactions were incubated for 25 min at 25 °C with
gentle agitation. The reaction was divided into two separate reactions, to one reaction,
1.5 μL of 5 mM CaCl2 and 2 mg/mL thermolysin was added. This reaction was
incubated on ice for 30 min and following incubation 2 μL of 0.5 M EDTA was added
to inactivate the thermolysin. Both reactions were centrifuged for 2 min at 830 g at 4
°C, the pellets were resuspended in 2 x sample buffer, boiled for 3 min and run on a
SDS-PAGE gel (Section 2.9.7 SDS-PAGE).
2.9.4 Mitoplast preparation following mitochondrial
in vitro imports
Following in vitro imports, outer mitochondrial membranes were ruptured via osmotic
swelling (Whelan et al., 1996). This produced mitoplasts that allowed the inner
Chapter 2. Materials and Methods
75
membrane and inter membrane space to become accessible to PK treatment.
Following in vitro imports mitochondria were pelleted by centrifugation at 20 000 g for
10 min at 4 °C. The supernatant was removed and the pellets were resuspended in
10 μL SEH buffer (250 mM sucrose, 1 mM EDTA, 10 mM Hepes-KOH, pH 7.4). To
this reaction 155 μL of 20 mM Hepes-KOH, pH 7.4 was added and left on ice for 15
min. The iso-osmotic conditions were restored by adding 25 μL of 2 M sucrose, to
release the inter membrane space proteins 10 μL of 3 M KCL was added. The
mitoplast samples were then divided into two, to which 50 μg of PK was added to one
reaction; both reactions were left on ice for 30 min. Following incubation 2 mM PMSF
was added to inhibit the PK activity. All mitoplast reactions were pelleted by
centrifugation at 20 000 g for 10 min at 4 °C, the pellets were resuspended in 2 x
sample buffer, boiled for 3 min and run on a 16 % (w/v) gel (12 % (w/v) if the proteins
were larger than 20 kDa) (Whelan et al., 1996). As with the in vitro import the gels
were coomassie stained, dried and exposed to a BAS TR2040 plate for 48 hand these
plates were read with the Personal Molecular Imager (Bio-Rad, Sydney, Australia).
2.9.5 Alkaline extractions
Alkaline extractions were performed following mitochondrial in vitro imports in order to
determine if proteins were membrane associated or if they were soluble (Terziyska et
al., 2005). Imports were carried out as described in Section 2.9.1 and the pellets
produced were resuspended in 0.1 M Na2CO3. Following incubation on ice for 10 min
the samples were centrifuged for 30 min at 20 000 g at 4 °C to separate the soluble
fraction in the supernatant from the insoluble fraction in the pellet. The soluble fraction
was treated with 10 % (w/v) TCA, heated to 65 °C to inactivate the protease,
incubated on ice for 10 min and centrifuged for 10 min at 20 000 g at 4 °C. The
soluble proteins contained in the pellet following this spin were resuspended in 1 x
Tris-glycine based running buffer and centrifuged for 10 min at 20 000 g at 4 °C. The
Chapter 2. Materials and Methods
76
insoluble fraction and the soluble fraction were both resuspended in 2 x sample buffer
and loaded onto an SDS-PAGE gel (Section 2.9.5).
2.9.6 Proteinase K titrations
Imports were carried out as described in Section 2.9.1 with increasing amounts of
proteinase K added. Six lanes of mitochondria and six lanes of mitoplasts had 0 μg,
2.4 μg, 4.8 μg, 9.6 μg, 19.2 μg and 38.4 μg of PK added. These samples were then
incubated on ice for 30 and following incubation, 1 μL of 100mM PMSF was added to
the reactions containing PK in order to inactivate the PK. All samples were centrifuged
for 3 min at 4 °C at max speed to pellet the mitochondria and mitoplasts. The pellets
were resuspended in 30 μL of 2x sample buffer, boiled for 3 min and run on a 16 %
(w/v) gel (12 % (w/v) if the proteins were larger than 20 kDa). The gels were
coomassie stained, dried and exposed to a BAS TR2040 plate for 48 h. The exposed
plates were read with the Personal Molecular Imager (Bio-Rad, Sydney, Australia).
2.9.7 SDS-PAGE
Polyacrylamide gels were used to run protein samples following import, alkaline
extractions and PK titrations (Lister et al., 2001). These were assembled using the
Bio-Rad PROTEAN IITM system (Bio-Rad, Sydney, Australia).Gels were typically
made of a 16 % (w/v) acrylamide separating component and a 4 % (w/v) acrylamide
stacking component. The separating gel was made with 375 mM Tris-HCL pH 8.8, 16
% (w/v) acrylamide and 0.1 % (w/v) SDS. This was degassed for 5 min prior to the
addition of 0.05 % (w/v) ammonium persulphate (AMPS) and 0.05 % (v/v) TEMED. To
resolve larger proteins on SDS gels 12 % (w/v) acrylamide was used in the separating
component. After the separating gel was set the stacking gel was prepared by adding
125 mM Tris-HCl pH 6.8, 4 % (w/v) acrylamide and 0.1 % (w/v) SDS. Similarly this
Chapter 2. Materials and Methods
77
solution was degassed for 5 min prior to the addition of 0.05 % (w/v) AMPS and 0.05
% (v/v) TEMED.
Gels were run in a discontinuous Tris-glycine buffer system consisting of; 25 mM Tris-
HCL pH 8.3, 200 mM glycine and1 % (w/v) SDS. The gel was run for 10 h at 10 mA
per gel limited to 200 V. Most gels were stained with coomassie stain, that consisted
of 0.25 %(w/v) Coomassie Brilliant blue R250, 40 % (w/v) ethanol and 10 % (v/v)
glacial acetic acid for 3 h. Gels that were not stained were used for Western blotting.
Following the staining, gels were destained in 30 % (v/v) ethanol, 12 % (v/v) glacial
acetic acid and 2% (v/v) glycerol for 2 h. Gels that contained radiolabelled proteins
were dried under a vacuum drier for 2 h at 80 °C using the Gel dryer 583 (Bio-Rad,
Sydney, Australia). The dried gels were carefully wrapped in cling wrap, exposed to a
BAS TR2040 plate for 2 nights in a film cassette and then the plate was read using
the Personal Molecular Imager (Bio-Rad, Sydney, Australia).
2.10 Western Blotting
2.10.1 Blotting with nitrocellulose membranes
Following the separation of proteins on Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), the gels were transferred to 0.45 μm supported hybond
C+ nitrocellulose membrane (Amersham, Sydney, Australia) (Jaeger and Wigge,
2007). The gels were equilibrated in ponceau (Appendix 1) for 1 h on a rocker whilst
10 sheets of Whatman® filter paper and 1 membrane were cut to the exact
measurements of the gel. The filter paper and membrane were both equilibrated in
transfer buffer for 10 min prior to transfer. Five sheets of Whatman® filter paper were
placed on the Phamacia (Amersham Pharmacia Biotech, Sydney, Australia) semi-dry
blotting apparatus, followed by the membrane, the gel, the remaining 5 sheets of filter
Chapter 2. Materials and Methods
78
paper and the cathode lid of the blotting apparatus. Gels were transferred for 1 h at
0.8 mA per cm2. Following transfer the membrane was quickly stained with
Ponceau-S stain (Appendix 1) for less than 1 min to confirm the locations of the
molecular weight markers and consistent transfer throughout the gel. The membrane
was destained in TBS-Tween (Appendix 1) and incubated in 1 % (v/v) blocking
solution from the Chemiluminescence Blotting substrate kit (BM, Sydney, Australia)
overnight at 4°C (Jaeger and Wigge, 2007).
2.10.2 Blotting with PVDF membranes
For proteins smaller than 20 kDa in size Immobilon-PSQ membranes were used. Gels
and Whatman® filter paper were equilibrated in Polyvinylidene fluoride (PVDF) transfer
buffer (Appendix 1) for 10 min whilst the membranes were washed in methanol for 15
min until they became opaque, the membranes were then rinsed in milli-Q water and
equilibrated in PVDF transfer buffer for 10 min. The filter papers, membranes and gels
were assembled and transferred on the Phamacia semi-dry blotter as described in
Section 2.10.1 and blocked overnight in 1 % (v/v) blocking solution prior to immune-
detection.
2.10.3 Immuno-detection
The UPOX1 antibody was expressed and purified by Dr Murcha in the Whelan lab and
provided for use in this thesis. This was produced using aa 1-137 of UPOX1, this
protein was then expressed in pdest15 fused to a GST tag. The GST tag was
removed using a Precision Protease leaving a 14 kDa protein for inoculation. The
protein was confirmed by mass spectrometry prior to injection into rabbit at 4 x 0.25
mg. The immuno detection experiments were blocked overnight, following this the
membranes were washed with TBS-Tween and incubated with primary antibody
diluted in 20 mL of TBS-Tween for 1 h with rocking. Following incubation with primary
Chapter 2. Materials and Methods
79
antibody the membranes were rinsed twice for 5 min and once for 15 min with TBS-
Tween. The membranes were incubated for 1 h in a 1 in 10 000 dilution of secondary
antibody (either anti-mouse or anti-rabbit) with rocking and then rinsed twice for 5 min
and once for 15 min with TBS-Tween.
Detection solution was made up using the substrates in the Chemiluminescence kit 30
min prior to use and equilibrated at room temperature. The 3 mL detection mixture of
1 % (v/v) substrate A in substrate B was poured onto the membrane, a plastic sheet
was placed underneath and above the membrane and all air bubbles were removed
prior to detection (Lister et al., 2007). Visualisation of the membrane was carried our
using the Image Quant RT ECL (GE Amersham Bioscience, Sydney, Australia)
according to manufacturer’s instructions.
2.11 BN PAGE
2.11.1 Preparation of BN PAGE gels
Prior to the preparation of Blue Native PAGE (BN-PAGE) gels, all solutions, casting
devices and pumps were chilled for at least 2 h at 4 °C. To prevent leakage of the 1.5
mm thick protein gel two pieces of Whatman® filter paper were placed under and 1
sheet of parafilm was placed above the rubber seal at the bottom of the glass plates.
Once the tubing is placed around the pump the injection needle is injected through the
bottom of the glass plates through the rubber seal. A pre-chilled light solution
(Appendix 1) was pumped through into the gel and once the level of the light solution
and heavy solutions (Appendix 1) were equal the valve for the heavy solution was
opened creating an increasing gradient higher in the gel. After the gel (in liquid form)
was fully injected between the two glass plates it was moved to room temperature to
speed the polymerization process which took approximately 1.5 h. After
Chapter 2. Materials and Methods
80
polymerization of the separating gel a 4 % (v/v) casting gel was poured on top with a
10 lane comb placed in it.
2.11.2 BN PAGE sample preparation
Digitonin was used to solubilise the mitochondrial samples for analysis by blue native
PAGE. Mitochondrial samples were resuspended in 5 % (w/v) digitonin extraction
buffer and incubated on ice for 20 min, 250 μg of protein was dissolved in 25 μL of
digitonin buffer with 1.25 mg digitonin. The samples were then centrifuged at 15 000 g
for 20 min at 4 °C and the supernatant containing the mitochondrial proteins were
again resuspended in 5 % (w/v) Serva Blue G prior to loading onto a blue native
PAGE gel.
To support the transfer of the protein into the gel a tension of 100 V was selected for
45 min with a variable current between 6 to 8 mA. For the separation of the proteins a
current of 15 mA was selected for 6 h with a limit to 500 V. After the gels completed
running they were washed with milliQ water and prepared for wet transfer.
2.11.3 Wet Transfer of BN PAGE gels
Prior to wet transfer the gel was soaked in transfer buffer for 30 min and PVDF
membrane cut to the exact dimensions of the gel were soaked in transfer buffer for
5 min along with two sheets of filter paper cut to the size of the sponges. The electro
blotting apparatus was assembled in transfer buffer by placing 1 sheet of filter paper
on top of the cathode cassette, followed by the gel, the membrane and the second
filter paper. All bubbles were rolled out with every layer and the cassette was placed
in the tank and electro blotted for 11 h at 50 mA. After transfer blue die was visible on
the membrane this indicated a good transfer, this was washed with methanol for 3
min. After marking the location of the complexes the membrane was immuno-detected
according to Section 2.10.3.
Chapter 2. Materials and Methods
81
2.12 Construction of a phylogenetic tree
Two phylogenetic trees of UPOX were created by using ClustalW2 and PANTHER
(Protein ANalysis THrough Evolutionary Relationships). ClustalW2 uses the
Neighbour-Joining method (Saitou and Nei, 1987). This algorithm functions by
clustering sequences by minimizing the sum of branch lengths. An output format that
ClustalW2 produces is the input format required by the tree-displaying program
FigTree v1.4.3. The PANTHER database was used for the second phylogenetic tree.
This resource is used for the classification of protein sequences by evolutionary
history, and by function. The database consists of protein-coding genes from 104
organisms that are classified by evolutionary relationships, by structured
representations of protein function including the Gene Ontology and biological
pathways. PANTHER phylogenetic trees show the evolutionary events including
speciation, gene duplication and horizontal gene transfer. Using these phylogenetic
trees orthologs, paralogs and xenologs are predicted. A hidden Markov model (HMM)
is constructed for each family and subfamily (Mi et al., 2016).
Chapter 3 Expression and regulation of UPOX1
82
Chapter 3
Expression and regulation of UPOX1
Chapter 3 Expression and regulation of UPOX1
83
3.1 Introduction
UPOX1 is a 104 amino acids long and is known as a highly stress-responsive gene
that was identified as one of the hallmarks of oxidative stress (Gadjev et al., 2006)
within the model plant Arabidopsis thaliana. To understand this gene in more depth
orthologs will be first examined. Two nuclear encoded genes have been identified as
exhibiting high levels of amino acids sequence identity to At2g21640 (UPOX1). These
genes have been named UPOX2 (At4g39235) and UPOX3 (At3g05570) (Uggalla,
2006). Gene duplication is a major evolutionary mechanism leading to the emergence
of new functions (Gu et al., 2004) and these genes may be a result of gene
duplication. In plant genomes the existence of multi-gene families are common and
one reason for maintaining these genes is that they have become functionally
specialized (Lynch and Conery, 2000). Genes within a family are known to exhibit
diverse transcriptional responses to oxidative stress (Dong and Adams, 2011). A
study by (Kim et al., 2005) identified that from 4222 duplicated gene pairs 22%
contained at least one member that was responsive to oxidative stress but from this
22% only 3% contained gene pairs that were both responsive to oxidative stress.
Possible orthologs of UPOX1 have been identified using BLAST (Uggalla, 2006).
Orthologs by definition (orthos meaning ’right’) are the same genes in a different
species that have evolved from a common ancestral gene by speciation, therefore
they should retain the same function. Paralogs on the other hand do not have any
functional connotation and the sequence similarity is the result of a gene duplication
that occurred after the speciation, in-paralogs are within a species whereas
out-paralogs are not (Fitch, 1970).
In this study we have analysed and compared how UPOX1 has diverged from UPOX2
and UPOX3 by examining their transcript expression patterns by performing qRT-PCR
and by using Genevestigator. In addition to these homologs, several other
Chapter 3 Expression and regulation of UPOX1
84
mitochondrial genes that are induced by treatments that perturb mitochondrial function
were also examined in this study.
Microarray analysis suggests that UPOX1 may be co-expressed with AOX1a and
NDB2 (Clifton et al., 2005), however, there is no data that shows the transcript profiles
of these genes in response to stress treatments over a time period. Therefore qRT-
PCR analysis was also conducted in order to observe the alterations in transcript
abundance during a time course for AOX1a, NDB2, AtOM66, UBC (AT5G25760) and
PR1 in Col-0 plants and in suspension cell cultures.
To further examine possible pathways that regulate mitochondrial stress response at
a transcriptional level the transcript abundances of UPOX1 and various other nuclear
encoded mitochondrial proteins known to be stress responsive were analysed in
defense signalling mutants and Col-0 plants.
Complex regulatory networks are formed from the interactions of different transcription
factors influencing the expression of genes in response to various internal and
external stimuli (Elkon et al., 2003). In addition to the cis-acting elements present
upstream of the transcriptional start site, it has been documented that 5’ introns can
also influence the level of expression at many levels including initiation of
transcription, mRNA metabolism, mRNA editing and translation. In order to obtain a
comprehensive understanding of the function of UPOX1 an investigation into the
induction of UPOX1 at a molecular level is required. Therefore, the upstream region of
UPOX1 was studied in detail and a set of potential regulatory elements were
functionally characterised as UPOX1 has a 5' intron 477 bp upstream of the
translational start site.
3.2 Aims and Strategies
Chapter 3 Expression and regulation of UPOX1
85
The specific aim of the research described in this chapter is to develop a
comprehensive understanding of the transcript expression pattern and regulation of
UPOX1.
This will be conducted by studying;
- The transcript expression kinetics of UPOX1 under various stress conditions
- The transcript expression profile in various mutants
- Promoter- GUS histochemical staining of plants
- The promoter region of UPOX1 via biolistic transformation using bioinformatic
tools
3.3 Results
3.3.1 Comparisons between the UPOX1 family
As UPOX2 and UPOX3 display high levels of sequence identity to UPOX1, the protein
and DNA coding sequences for these genes were investigated. Nucleic acid and
protein sequences were obtained from TAIR (Swarbreck et al., 2007) and multiple
sequence alignments were performed using ClustalW2 (Larkin et al., 2007). This
analysis revealed that only the N terminal region differentiates UPOX1 from UPOX2
and UPOX3 as the C terminal regions are highly conserved amongst all three genes.
Figure 3.1 A shows the entire 104 amino acid length of UPOX1. UPOX1 appears to
have additional amino acids at the N terminal region and it is thought that this is a
characteristic of mitochondrial targeted sequences (Glaser et al., 1998). Their
deduced amino acid sequences show 76% sequence identity between UPOX2 and
UPOX3 and 52% sequence identity when UPOX2 and UPOX3 are compared with
UPOX1 (Figure 3.1 B). The coding sequences of UPOX2 and UPOX3 have 80%
sequence identity between them and they both have 75% sequence identity with
UPOX1 (Figure 3.1 C).
Chapter 3 Expression and regulation of UPOX1
86
3.3.2 Construction of a phylogenetic tree
Orthologs of UPOX1 were identified using two independent applications. The first
phylogenetic tree (Figure 3.2 A) was generated by ClustalW2 and displayed using
FigTree v1.4.3 following the use of Basic Local Alignment Search Tool (BLAST). The
second phylogenetic tree (Figure 3.2 B) was generated using PANTER (Protein
ANalysis THrough Evolutionary Relationships) databases (Mi et al., 2016).. Analysis
of the phylogenetic tree created through the PANTHER database reveals more
orthologous relationships than that using ClustalW. Analysis of this second tree
reveals that there are seven orthologs of UPOX1 in Oryza sativa, one in Sorghum
bicolor, one in Brachypodium distachyon, five in Glycine max, three orthologs in
Populus trichocarpa, two in Solanum lycopersicum and one orthologs in
Physcomitrella patens. It is also apparent that UPOX2 and UPOX3 may be related
closer to each other than they are to UPOX1. Interestingly upon further investigations
using SUBA3 a subcellular localisation tool for Arabidopsis (Tanz et al., 2012; Hooper
et al., 2014) the predicted in silico subcellular localisation of UPOX2 and UPOX3 were
found to be in both the nucleus and cytosol whereas UPOX1 had mixed predictions
with 2 out of 12 predictors suggesting mitochondrial localisation. There were further in
silico localisations available for the orthologs in Oryza sativa and these were localised
to multiple subcellular localisation including the chloroplast, cytosol and golgi.
3.3.3 Establishment and optimisation of qRT-PCR assays
The full length coding sequences for UPOX1, UPOX2 and UPOX3 were amplified and
qRT-PCR primers (Appendix 2) were designed for each gene as per Section 2.7. The
oligonucleotides were 16 to 20 base pairs (bp) in length with GC contents ranging
from 47% to 62% and melting temperatures ranging from 50 °C to 70 °C. The
sequence identity between UPOX1, UPOX2 and UPOX3 was high and therefore the
primers were designed in the regions that displayed lower sequence identity in order
to obtain gene specific primers sets.
Chapter 3 Expression and regulation of UPOX1
87
Quantitative RT-PCR standards were generated for UPOX1, UPOX2 and UPOX3.
The full length expected coding sequence was used and the sizes for UPOX1,
UPOX2 and UPOX3 were 315 bp, 261 bp and 273 bp respectively. The standards
were quantitated using PicoGreen® and diluted to produce a master stock at 1 mol/
L. Serial dilutions of this master stock were used as a standard in all qRT-PCR
assays. The qRT-PCR reactions were optimised for linearity and specificity. Linearity
is essential for all qRT-PCR assays as this determines the accuracy with which
samples of unknown concentration are determined. A four point standard curve was
utilised for all the assays conducted and qRT-PCR efficiencies of greater than 95%
were maintained for all assays performed (Giraud et al., 2008).
Cross reactivity assays were also performed using the Roche Light Cycler® software
and melt peak charts according to Section 2.7.5 which confirmed that the primers
were specific for the target transcript.
3.3.4 Quantitative RT-PCR assays for UPOX1, UPOX2 and
UPOX3 in suspension cell culture
Transcript levels of UPOX genes were monitored in Arabidopsis cell culture lines
treated with a variety of chemicals. A range of chemical treatments were selected for
their broad impact on cellular function or for their ability to inhibit a specific
mitochondrial or cellular process. The cell cultures used for the qRT-PCR assays
were isolated from three biological replicates, from each of these, two technical
replicates were isolated. From 120 ml flasks grown under the conditions in Section
2.2.1 a 5 mL aliquot was removed at the 0 h time point prior to the addition of any
chemicals. A range of chemical treatments were selected for their broad impact on
cellular function or for their ability to inhibit a specific mitochondrial or cellular process.
Following the addition of stress treatments 5 ml samples were taken, at 3 h, 12 h
and 24 h after the stress treatment was applied. These samples were then vacuum
filtered and snap frozen. From this, the RNA was isolated and cDNA was synthesized
Chapter 3 Expression and regulation of UPOX1
88
according to (Clifton et al., 2005). Following this, qRT-PCR assays (using the primers
in Appendix 2) were conducted and the results were normalized to ubiquitin (UBC) as
a housekeeping gene (Clifton et al., 2005; Czechowski et al., 2005). The stress
treatments used included citrate, hydrogen peroxide (H2O2), rotenone and salicylic
acid. Citrate is a TCA metabolite and is known to disturb metabolic homeostasis
(Vanlerberghe et al., 2002). H2O2 is a ROS signal and an agent for cellular damage
(Maxwell et al., 2002). Rotenone inhibits complex I and thus affects the functioning on
the mitochondria (Li et al., 2003). Lastly, salicylic acid is an un-coupler of respiration
and is a common defense signalling hormone .
The qRT-PCR assays in Figure 3.3 demonstrate that the transcript abundance of
UPOX1, UPOX2 and UPOX3 increase with these treatments. However, there were
differences in the magnitude and kinetics of their response. In the case of UPOX1 the
response to all treatments except salicylic acid peaked 24 h after stress treatment;
salicylic acid produced a peak at the 12 h time point with transcript abundance
increasing approximately 25-fold. At the 24 h time point there were significant
increases in the transcript abundance in response to H2O2, rotenone and salicylic
acid. The changes in transcript abundance corresponded to-5 fold, 20-fold and 5-fold
increase respectively. The increase in transcript abundance for UPOX2 and UPOX3
were of a significantly lesser magnitude than that of UPOX1. However there was still a
significant increase in transcript abundance in response to salicylic acid for UPOX2.
The response peaked at the 3 h time point with transcript abundance increasing 7-
fold, this was maintained at the 12 h time point and at the 24 h time point a slight
reduction in transcript abundance was measured that corresponded to a 6-fold
increase. The transcript abundance of UPOX3 in response to citrate, H2O2, rotenone
and salicylic acid did not increase above 3.5-fold and the only significant changes
observed were in response to citrate. This change in transcript abundance showed a
Chapter 3 Expression and regulation of UPOX1
89
3-fold increase at the 12 h time point and peaked with a 3.5 fold increase at the 24 h
time point.
3.3.5 Transcript kinetics of nuclear genes encoding
mitochondrial proteins
The results shown in Figure 3.4 were produced using the same suspension cell
cultures used to produce the results for Figure 3.3. This permits the expression
kinetics of UPOX1, UPOX2 and UPOX3 to be compared to the results obtained in
Figure 3.4. Following micro array analysis it was observed that the expression of
AOX1a was co-expressed with NDB2 and UPOX1 (Figure 3.6). To examine the
alterations in transcript abundance of several nuclear genes encoding mitochondrial
proteins in response to stress treatments that inhibit specific mitochondrial or cellular
processes qRT-PCR analysis was conducted; the genes tested were AOX1a, NDB2,
AtOM66, UBC and PR1. UBC (AT5G25760) was used as a housekeeping gene as it’s
known to be unresponsive to a wide range of treatments and PR1 is a pathogen
related protein 1a and is a positive control for a response to SA (Clifton et al., 2005;
Czechowski et al., 2005).
The qRT-PCR analysis demonstrated the transcript abundance for AOX1a in
response to H2O2, rotenone and SA all peaked at the 3 h time point. The transcript
abundance increased greater than 6-fold and at the consequent 12 h and 24 h time
points the transcript abundance of AOX1a in response to all the treatments decreased
whilst maintaining a higher abundance than the levels observed at the 0 h time point.
The transcript profile of NDB2 most resembles that of AOX1a, however, there were
distinct differences. The transcript abundance of NDB2 peaked at the 12 h time point,
an approximate 4-fold increase was observed in response to H2O2 and SA whilst an 8-
fold induction was observed in response to rotenone treatment. NDB2 was the only
gene to significantly increase in transcript abundance in response to citrate with a
Chapter 3 Expression and regulation of UPOX1
90
relatively modest 2-fold increase observed at the 24 h time point. The transcript
profiles of AOX1a and NDB2 were similar in the aspect that the transcript abundance
of both genes decreased at the 24 h time point. The expression profile of AtOM66
differed greatly as its response to SA produced the greatest increase in transcript
abundance at all time points after the 0 h time point. At the 12 h time point a 30-fold
increase was observed and at the 24 h time point a 25-fold increase was observed in
response to SA. The transcript abundance of AtOM66 in response to citrate and H2O2
treatments produced inductions below 5-fold at all 4 time points whilst the response to
rotenone was slightly higher than 5 fold for the 12h and 24 h time points. The
transcript abundance of PR1 was as expected with only SA producing a significant
change in transcript abundance with a greater than 140-fold induction observed at the
24 h time point.
3.3.6 Transcript analysis in response to UV stress
To further investigate the response of UPOX1 and AOX1a in Col-0 plants to
environmental stress qRT-PCR analysis was conducted in response to UV stress
(Figure 3.5). Arabidopsis water cultures were monitored 6 h following 5 min treatment
of UV-C according to Section 2.2.5. For each sample, total RNA was isolated from
three pools of tissue, each consisting of three independent pots. The qRT-PCR
analysis demonstrated the transcript abundance for UPOX1 and AOX1a increased
significantly following UV stress.
3.3.7 In silico response of UPOX1, UPOX2 and UPOX3 to
a wide range of stimuli and mutations
Within the Genevestigator database the Response Viewer tool was used to extract the
relative expression data as fold changes for UPOX1, UPOX2, UPOX3, AOX1a, NDB2,
AtOM66, PR1 and UBC (Hruz et al., 2008). This was viewed as a heat map in
response to stimuli and mutations, the 50 most up-regulated and down-regulated
Chapter 3 Expression and regulation of UPOX1
91
stimuli and mutations can be seen in Figure 3.6. Interestingly among the top 50 stimuli
that up regulate the expression of UPOX1, 17 were chemical treatments, 14 were
stress treatments, 10 were light treatments, 4 were biotic treatments, 3 were nutrients,
1 was a hormone and the 1 remaining was a cell sorting and protoplasting stimulus.
The stimuli that produced UPOX1 to be most down regulated were represented by 12
stress treatments, 12 nutrients, 9 chemicals, 7 hormones, 3 light treatments, 2
temperature treatments, 2 biotic treatments, 1 elicitor, 1 photoperiodic shift and 1
programmed cell death stimulus. It is also clear that the expression of UPOX2 and
UPOX3 to these stimuli did not result in a change in transcript abundance to the
degree shown by UPOX1. From the top 50 stimuli that up regulate UPOX1, 46 (92 %)
of these stimuli also up regulated AOX1a. In addition to this, 70% of these 50 stimuli
also caused an up regulation of NDB2 and AtOM66. Furthermore from the top 50
stimuli that caused a down regulation in UPOX1 greater than 50 % of these stimuli
also produced a down regulation of AOX1a.
3.3.8 Mitochondrial genes induced under stress in
signalling mutant backgrounds
By observing the expression patterns of UPOX1, UPOX2, UPOX3, AOX1a, NDB2,
AtOM66, PR1 and UBC in various mutants it can assist in characterizing the pathways
that regulate their expression (Figure 3.6). There were significant changes in the
expression of UPOX1 in several mutant lines. UPOX1 was most up-regulated in the
flu.2 mutants and this mutant is a negative regulator of impaired chlorophyll
biosynthesis and results in ROS production when shifted from dark to light
(Meskauskiene et al., 2001). The mutant in which UPOX1 was second most up-
regulated was the ang4 (Berná et al., 1999; Robles and Micol, 2001) mutant, this
mutant increased cell cycle duration in young leaves. The third mutant that UPOX1
was most up-regulated in was the csn5 mutant (Dohmann et al., 2005), these mutants
Chapter 3 Expression and regulation of UPOX1
92
have a characteristic phenotype of photo-morphogenic growth in the dark and
post-germination growth arrest. The flu and csn mutants comprise the top 7 mutants
that most upregulate UPOX1. This provides a clue as to the pathways that UPOX1
could be functioning in as it may be related to these mutants. Furthermore of the top
50 mutants that caused UPOX1 to be up regulated, the top 16 mutants that most up
regulated UPOX1 also produced an up regulation of AOX1a, NDB2 and AtOM66. An
additional 15 of the 50 mutants also produced an up regulation in the transcript levels
of AOX1a, NDB2 and AtOM66.
To investigate the signalling pathways that regulate UPOX1, UPOX1 expression
levels were assessed in several additional mutant lines compared to Col-0 plants as
shown in Figure 3.7. The transcript abundance of several genes encoding
mitochondrial proteins was also assessed in a variety of mutant lines. The genes
tested alongside UPOX1 included a highly stress responsive alternative oxidase
AOX1a (Yu et al., 2001), AtOM66 that encodes a protein that is present in a homo-
multimeric protein complex on the outer mitochondrial membrane and plays a role in
cell death and amplifying salicylic acid signalling (Zhang et al., 2014), a gene that
encodes an external NAD(P)H dehydrogenase NDB2 that follows a similar pattern of
transcript expression to AOX1a (Clifton et al., 2005), a pathogen related protein that
was used as a positive control for salicylic acid (SA) PR1a (Shah, 2003) and a house
keeping gene that is unresponsive to a wide range of treatments UBC (Czechowski et
al., 2005). The qRT-PCR for AOX1a and NDB2 were conducted in a collaboration
within the Whelan lab by Dr Giraud and Dr Ho. The mutant lines chosen were all
affected in phytohormone signal transduction pathways. NahG mutant plants break
down SA by expressing bacterial salicylate hydroxylase (Lawton et al., 1995; Bowling
et al., 1997), pad4 is a phytoalexin deficient mutant that is essential for the SA
dependent defense pathways (Glazebrook and Ausubel, 1994; Glazebrook et al.,
2003), eds1 is an enhanced disease susceptible mutant (Falk et al., 1999) and npr1
Chapter 3 Expression and regulation of UPOX1
93
mutants are non-expressers of the gene PR1 which is required for the expression of
defense genes (Cao et al., 1994; Shah, 2003). Pad 4 mutants act upstream of SA
whereas eds1 mutants and npr1 mutants act downstream of SA. The other mutant
lines chosen were the jar1 mutant line that are associated with insect and
necrotrophic defense and is deficient in the jasmonic acid signalling pathway
(Staswick et al., 1992), the abi3 line that interacts with the abscisic acid (ABA)
responsive transcription factors by encoding a viviparous like transcription factor
(Giraudat et al., 1992) and the etr1 mutant line that confers ethylene insensitivity in
Arabidopsis (Chang et al., 1993).
In NahG plants relative to Col-0 plants it was observed that the transcript abundance
of AtOM66 and PR1 had reduced by approximately 100-fold, whereas there was only
a 2.5 fold reduction of AOX1a and 5-fold reduction in transcript abundance of NDB2
and UPOX1 (Figure 3.7). In the pad4 and eds1 mutant lines the transcript abundance
of AOX1a differed greatly to that of NDB2 and UPOX1, there was a greater than 2-fold
increase in AOX1a transcript abundance whilst the transcript abundance of NDB2,
UPOX1 and AtOM66 resulted an approximate 5-fold reduction in the pad4 plants. In
the eds1 line there was a slight increase in the transcript abundance of UPOX1, a 5-
fold decrease in AtOM66 and the transcript abundance of NDB2 remained
unchanged. The expression of PR1 in the pad4, eds1, npr1 and jar1 plants was
similar to that expressed in the NahG plants, the transcript abundance was only just
detectable and it was approximately 100-fold reduced relative to Col-0 plants, the only
significant increase in abundance of PR1 was observed in the abi3 plants. The
expression pattern of AtOM66 clearly differs from that of AOX1a, NDB2 and UPOX1
in the npr1, jar1, abi3 and etr1 plants. From its induction pattern AtOM66 is consistent
with a role for SA signal induction as its transcript abundance decreases in NahG,
pad4, eds1 plants. However it distinguishes itself from PR1 by showing that it is not
dependent on NPR1. The expression patterns of AOX1a, NDB2 and UPOX1 were
Chapter 3 Expression and regulation of UPOX1
94
similar in the npr1, jar1, abi3 and etr1 lines, the transcript abundance of all these
genes increased greater than 2-fold (Ho et al., 2008).
3.3.9 GUS histochemical staining
GUS histochemical analysis is another method to confirm and localise the expression
of genes of interest. The upstream 1.5 kb region of the UPOX1 promoter was fused to
β-glucuronidase and transformed into Arabidopsis according to the methods detailed
in Skirycz et al., (2010). This line was obtained in collaboration with Dr Van Aken and
Inge De Clercq in the lab of Prof. Van Breusegem (VIB Ugent, Belgium). The GUS
activities in the whole root tip, vascular tissue, the siliques and mature flowers were
assayed by myself. Figure 3.8 shows 6 images and the blue regions indicate the
degree to which the GUS has stained the plant material. The root tip, the base ends of
the silique and the mature flowers reveal strong GUS staining and the localisation of
this signifies the localisation of UPOX1 expression in Arabidopsis plants. Overall, the
UPOX1 promoter appears to be active in a variety of tissues throughout the plant.
3.3.10 Identification of putative sequence elements in
the upstream region of UPOX1
To obtain a better insight in how the stress-responsive expression of UPOX1 is
regulated, the UPOX1 promoter was analysed and searched for the presence of
regulatory elements. In total 14 putative regulatory cis-sequence elements were
identified in the upstream region of UPOX1 using five prediction programs; Plant Care
(Rombauts et al., 1999), PLACE signal scan (Higo et al., 1999), AthaMap (Steffens et
al., 2004), Athena (O'Connor et al., 2005), and AGRIS (Palaniswamy et al., 2006).
Of these 14 elements, there was 1 MYB motif, 1 I and B overlapping elements which
were found to be functional in AOX1a (Clifton et al., 2005), there were 2 WRKY
elements both in the forward orientation, these elements have been found to be
Chapter 3 Expression and regulation of UPOX1
95
involved in stress and defense signalling (Eulgem and Somssich, 2007). Also present
were 2 RAV1 elements, the RAV1 elements in pepper have been identified as having
a role in biotic and abiotic stress resistance (Sohn et al., 2006) and there were 6
elements that are known as stress responsive elements, these elements are highly
over represented in stress responsive genes (Figure 3.9). Furthermore a
CTTGGCCACG motif was identified that is known to be overrepresented in genes
responding to mitochondrial dysfunction and is bound by NAC-type transcription
factors (Van Aken and Whelan, 2012; De Clercq et al., 2013; Ng et al., 2014).
.
3.3.11 Functional analysis of the B and I elements in the
upstream region of UPOX1
The B and I element had been previously identified to be functional in AOX1a and
NDB2 genes (Ho et al., 2008). These genes are co-expressed with UPOX1 and
therefore the functionality of these elements were investigated in UPOX1 (Figure
3.10). In order to test sequence elements for functionality, the 1100 bp upstream
region of UPOX1 was cloned into the pLUS vector (Ho et al., 2008). The pLUS vector
was created so promoters could be analysed via biolistic transformation. The
promoter of interest was fused to the glucuronidase reporter gene, and a
35S:luciferase gene was used for normalisation as transformation control.
Biolistic transformations were performed in suspension cell cultures that were
maintained as described in Section 2.2.1 and 4 week old Arabidopsis Col-0 leaves
maintained as described in Section 2.2.3. The I and B sequence elements were
mutated from a wild type promoter via site directed mutagenesis as described in
Section 2.4.7. Following the individual deletion of the I and B elements a construct
was also created that had both the I and B elements mutated. The effect of the
deletion of the sequence elements on the promoter’s ability to drive GUS expression
under stress treatments was also tested in both suspension cell cultures and Col-0
Chapter 3 Expression and regulation of UPOX1
96
leaves. The stress treatments used were H2O2 and rotenone. The GUS activity of the
wild type promoter under no stress treatment was set to 100% and the activity for the
deletions under mock (water/ethanol) treatments and stress treatments were
expressed relative to this.
(Rosner, 2006). The t-test assuming unequal variance was used to determine the
statistical difference in endogenous levels of GUS activity observed between the wild
type promoter and the mutated promoter within a treatment and also the statistical
difference in GUS activity between mock treated samples versus treated sample
within a promoter construct, in all situations the significance level was set at p ≤ 0.05.
In untreated suspension cell cultures the deletion of the I element from the upstream
region of UPOX1 resulted in a loss of GUS activity by ~40%. The deletion of the B
and the I+B combination elements resulted in a loss of GUS activity by ~50%. For the
wild type region, the mutated I element region and the mutated B element region the
addition of H2O2 produced an approximate doubling in GUS activity. Following the
deletion of the I+B element the increase observed following the addition of H2O2 was
eliminated. No significant responses to rotenone treatment were observed for the wild
type region or the mutated regions.
In untreated 4 week old Arabidopsis leaves the deletion of the I element and the I+B
element from the upstream region of UPOX1 resulted in a loss of GUS activity by
~75% under control conditions. The deletion of the B element resulted in a loss of
GUS activity by ~50%. For the wild type region, the addition of H2O2 produced an
increase in GUS activity by ~ 70%, however, following the deletion of the I, B and I+B
elements this induction following the addition of H2O2 was eliminated. There was no
significant response to rotenone treatment observed in the wild type region or the
mutated B element region, however, following the addition of rotenone to the mutated
I and I+B element regions there was an ~50% reduction in GUS activity.
Chapter 3 Expression and regulation of UPOX1
97
From the results obtained from the suspension cell assays it appears that all three
elements, I, B and I+B are positive regulators, as they show a decrease in GUS
activity when deleted. They appear to be positive regulators under untreated
conditions and following the treatment of H2O2. The result obtained from the 4 week
old Arabidopsis leaves concurs with the results obtained in the suspension cell assays
apart from one aspect. The assays in the 4 week old Arabidopsis leaves show that the
addition of rotenone in the mutated I and I+B element regions also appears to be a
negative regulator.
3.4 Discussion
A comprehensive study on the transcript expression kinetics of UPOX1 and its
homologs, UPOX2 and UPOX3, was conducted. This study incorporated qRT-PCR
analysis, promoter activity and microarray data analysis. This study established that
the expression profiles of these homologs differed considerably. This unique pattern
of transcript expression could suggest that the regulatory mechanisms controlling the
expression of each of these genes are largely independent and unrelated. This
conclusion correlates with previously conducted research which has established that
expression diversity is a means by which genes are evolutionarily retained (Yanhui et
al., 2006; Dong and Adams, 2011).
Overall the qRT-PCR assays in Figure 3.3 showed that UPOX1 is significantly more
stress responsive than its homologs UPOX2 or UPOX3. Furthermore, this analysis
revealed the kinetics of its stress response. The expression levels for UPOX1 peaked
at the 24 h time point. This indicates that the stress treatments used (citrate, H2O2,
rotenone and SA) likely cause a secondary stress response in UPOX1.
Chapter 3 Expression and regulation of UPOX1
98
Genevestigator analysis illustrated that the expression profile of UPOX1 in response
to various stress treatments and in various mutant lines differs from the expression
profiles of UPOX2 or UPOX3. This evidence correlates with the hypothesis that genes
within a family specialize and exhibit differing expression profiles.
Investigations into the transcript abundances of UPOX1 compared to that of AOX1a,
NDB2 and AtOM66 in response to external stimuli and in mutant backgrounds found a
high degree of correlation between UPOX1, AOX1a, NDB2 and AtOM66 and in
particular between UPOX1 and AOX1a. This suggests that there may be an overlap in
the pathways responsible for the induction of these genes.
Further qRT-PCR assays allowed the transcript abundance of UPOX1 to be
compared to AOX1a, NDB2, AtOM66 and PR1. The pattern of response of AOX1a
and NDB2 revealed that they did not co-ordinate with UPOX1. This was important as
the transcript abundance changes of AOX1a has been previously characterised as
important in the typical mitochondrial stress response pathway (Rhoads et al., 2006;
Suzuki et al., 2012). The difference in the transcript responses indicates involvement
in a potentially uncharacterised signal transduction pathway(s) regulating gene
expression for proteins that function in mitochondria.
An alternative approach was taken to further characterise the pathways that regulate
the expression of mitochondrial targeted stress responsive proteins by observing their
transcript abundance in defense signalling mutants. It was found that there were
distinctive pathways that regulate mitochondrial stress response at a transcriptional
level for UPOX1, AOX1a, NDB2 and AtOM66. This was first evident in the NahG
plants: UPOX1, AOX1a and NDB2 transcript abundance were reduced by
approximately 5-fold, however, AtOM66 expression was reduced greater than 100-
fold. This reveals that there were two distinct modes of action of SA on these
transcripts. AtOM66 was highly responsive to the application of SA and indicates a
Chapter 3 Expression and regulation of UPOX1
99
direct effect on transcription initiation. Conversely from the analysis of the promoters
and transcripts in this study it is proposed that SA acts to increase transcript
abundance for AOX1a, NDB2, and UPOX via a posttranscriptional mechanism. There
is further support for this hypothesis as a similar response was observed from
UPOX1, AOX1a and NDB2 in the etr1, abi3, jar1 and npr1 mutant lines, again
differing from the expression of AtOM66. Finally it was clear that the transcript
abundance of only AOX1a increased in the pad4 and eds1 mutant plants, this
indicates a separate regulatory pathway is involved for AOX1a and the induction of
AOX1a is SA independent in these lines. However from this study alone it is
impossible to conclude the exact effects of these mutants on altering transcript
abundances and it must be acknowledged that other factors could be acting in
conjunction with the effects of these mutant lines as the interactions between various
phytohormone signalling pathways are very complex (Erb et al., 2012).
To determine if UPOX1 was not only co-expressed with AOX1a but also co-regulated,
two elements previously found to be functional in AOX1a were assayed for
functionality within the upstream region of UPOX1. Elements I and B largely overlap
with a previously identified potential binding site for the transcription factor Abscisic
acid insensitive 4 and were tested independently and together. It was found that in all
three cases they acted as positive regulators. In addition the response of the UPOX1
promoter to H2O2 was consistent with it being defined as a marker of oxidative stress
(Gadjev et al., 2006). However as UPOX1 promoter activity was largely unresponsive
to rotenone and this differed considerably to the promoter activity of AOX1a (Ho et al.,
2008) it must be inferred that there are differences present in the nature of the signals.
In recent publications it has been found that the palindromic overlapped region of
element I+B has been called the NAC binding site (NACBS) and this has been found
to bind both ANAC017 and ANAC013 (Van Aken and Whelan, 2012; De Clercq et al.,
2013; Ng et al., 2014).
Chapter 3 Expression and regulation of UPOX1
100
In conclusion the use of transcript analysis, defense signalling mutants and promoter
activity allows for a thorough study of the intricate mechanisms underlying gene
expression under stress conditions. Through this research and following from what is
presented in (Ho et al., 2008) it can be concluded that there were 3 general pathways
that mediate mitochondrial stress response at the transcriptional level. The first is a
SA dependent pathway represented by AtOM66, therefore this implies that only
AtOM66 is regulated at a transcriptional level whereas UPOX1, AOX1a and NDB2 are
regulated at a post-transcriptional level. The second is illustrated by the lack of
response by UPOX1 to rotenone which differs significantly to AOX1a. This implies that
a set of different transcriptional factors are involved in the targeting of CARES by
H2O2 and rotenone. The third pathway is revealed by AOX1a as its expression differs
from UPOX1 and NDB2, this suggests a different pathway involved via EDS1 and
PAD4 to regulate only AOX1a. From the transcript analysis performed as part of this
study and further investigations of the promoter elements (Ho et al., 2008) it can also
be concluded that SA acts to increase transcript abundance for UPOX1, AOX1a and
NDB2 via a post-transcriptional mechanism, in contrast to AtOM66 which is under
direct transcriptional control. It can also be concluded that although H2O2 and
rotenone can both act transcriptionally and their CAREs overlap there is a distinction
in the response mechanism of UPOX1 compared to the other genes tested. The fact
that UPOX1 shows a peak in response to rotenone at the 24 h time point is the
evidence for the different transcription factors being involved, this is diagrammatically
represented in Figure 3.11.
Chapter 3 Expression and regulation of UPOX1
101
At4g39235 ---------METQTNQS---PKPAM--TSCRKKVKDD-ATFFEDVKDHIDDFIHASMDEH 45
At3g05570 ---------METQKTQAENPPKPAT--SSCRKKTKDD-ANFLEDVKDHIDDFINASMDDH 48
At2g21640 MASQTNQTVVDTKKIETENPPKPQVPASSCRKRVKDDNATFFANLKDHMDEFIHASMDEH 60
::*:. :: *** :****:.*** *.*: ::***:*:**:****:*
At4g39235 KSCFQKTIKKMFG---LSKAVAEKQAVEAKG-VESQLPLQTTVSE 86
At3g05570 KNCFNKTIKKMFG---LSKAVADKQQSEAKGGVESYLPLQTTVSD 90
At2g21640 KTCFKNTMDKIFGSFSKAEAVAEKQ-IEAKEVVEIHSPLQTAVTK 104
*.**::*:.*:** ::***:** *** ** ****:*:.
UPOX1 UPOX2 UPOX3
UPOX3 75 80 100
UPOX2 75 100
UPOX1 100
UPOX1 UPOX2 UPOX3
UPOX3 52 76 100
UPOX2 55 100
UPOX1 100
A)
B) C)
UPOX 1
UPOX 2
UPOX 3
UPOX 1
UPOX 2
UPOX 3
Figure 3.1 Comparisons of UPOX1, UPOX2 and UPOX3 sequences.
A) A ClustalW2 multiple sequence alignment of UPOX1, UPOX2 and UPOX3 protein
coding sequences. B) Percentage sequence identity between protein sequences.
C) Percentage sequence identity between nucleic acid sequences.
The colours represent the physicochemical propeties of the protein residues. (small
and small hydrophobic residues are depicted in red, acidic residues are blue, basic
residues are magenta, hydroxyl/ sulfhydryl/ amine are in green and other unusal
amino/ imino acids are in grey). An * (asterisk) indicates positions which have a
single, fully conserved residue. A : (colon) indicates conservation between groups of
strongly similar properties - scoring > 0.5 in the Gonnet PAM 250 matrix. A . (period)
indicates conservation between groups of weakly similar properties - scoring =< 0.5
in the Gonnet PAM 250 matrix.
Chapter 3 Expression and regulation of UPOX1
102
A)
Fig
ure
3.2
A)
UP
OX
1o
rth
olo
gu
es
.(A
)P
hylo
gene
tic
tre
ein
ferr
ed
from
asim
ultane
ous
com
pa
riso
nofU
PO
X1
ort
holo
gues
based
on
se
quen
ce
sim
ilari
ty.T
he
tree
was
con
str
ucte
dusin
gC
lusta
lW2
an
dF
igT
ree
v1.4
.3.
PO
PT
R_
000
5s22720
Pop
ulu
str
ich
ocarp
a
PO
PT
R_
000
4s16280
Po
pu
lus
tric
ho
carp
a
OS
03
G0
74555
0O
ryza
sativa
UPOX3At3g05570
Ara
bid
opsis
Thalia
na
UPOX2(A
t4g3
92
35
)A
rabid
op
sis
Tha
liana
PH
YP
AD
RA
FT
_1
9838
Physcom
itre
llapate
ns
UPOX1(At2g21640)
Ara
bid
op
sis
Th
alia
na
Os03g53390
Ory
za
Sativa
Os03
0724
600
Ory
za
Sa
tiva
0.4
5
0.3
30
.24
5
0.2
50.0
73
0.0
41
0.0
99
0.1
34
0.1
52
0.0
73
0.0
67
0.0
15
0.0
21
0.0
41
0.0
43
Chapter 3 Expression and regulation of UPOX1
103
Embryophyta
Magnoliophyta
Poaceae
Poaceae
Poaceae
BEP_clade
AN16
BEP_clade
Os03g53390 Oryza sativa
Os030724600 Oryza sativa
OS03G0745550 Oryza sativa
Os03g51470 Oryza sativa
Sb01g009650 Sorghum bicolor
BEP_clade
AN13
OJ1499_D04.6 Oryza sativa
BRADI1G10250 Brachypodium distachyon
OsJ_26398 Oryza sativa
Os08g0204632 Oryza sativa
Sb01g009660 Sorghum bicolor
Poaceae
Pentapetalae
rosidsfabids
AN47GLYMA11G07305 Glycine max
GLYMA04G16401 Glycine max
GLYMA01G38260 Glycine max
GLYMA06G46580 Glycine max
GLYMA02G18260 Glycine max
AN53
POPTR_0004s16280 Populus trichocarpa
POPTR_0005s22720 Populus trichocarpa
POPTR_0002s05810 Populus trichocarpa
AN42
AT3G05570 Arabidopsis thaliana
At2g21640 Arabidopsis thaliana
AT4G39235 Arabidopsis thaliana
VIT_03s0063g02630 Vitis vinifera
AN37
Solyc02g085260.1 Solanum lycopersicum
Solyc01g109680.2 Solanum lycopersicum
PHYPADRAFT_19838 Physcomitrella patens
Figure 3.2 B) UPOX1 orthologs. (A) Phylogenetic tree inferred from a simultaneous
comparison of UPOX1 orthologs based on sequence similarity. The tree was
constructed by PANTHER. PANTHER defines a controlled vocabulary for protein anno-
tation (the PANTHER/X ontology), as well as a method for classifying new sequences
(scoring against the PANTHER/LIB library of Hidden Markov Models).
Chapter 3 Expression and regulation of UPOX1
104
0
5
10
15
20
25
30
UPOX1
Rela
tive
tra
nsc
rip
ta
bu
nd
an
ce
* *
*
*
**
Time (hours)
0
1
2
3
4
5
6
7
8
903 12 24
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 3 12 24
0
3 12 240
UPOX2
UPOX3
* **
Figure 3.3 Transcript abundance of UPOX 1, UPOX 2 and UPOX 3.
QRT-PCR analysis of transcript abundance for UPOX1 homologous
genes over 24 h in response to the addition of citrate, H2O2, rotenone and
salicylic acid to Arabidopsis cell cultures. The amount of transcript prior
to the addition of compounds was set to 1 and changes expressed in a
relative manner. An asterisk indicates a significant difference
(P ≤ 0.05) relative to the 0 h timepoint for each individual treatment.
*
Citrate
H2O2
Rotenone
Salicylic Acid
Chapter 3 Expression and regulation of UPOX1
105
0
2
4
6
8
0 3 12 24
Citrate
H2O2
Rotenone
Salicylic Acid
0
2
4
6
8
10
0 3 12 24
0
510
15
20
2530
35
40
0 3 12 24
AOX1a NDB2
ATOM66
Rela
tiv
etr
an
sc
rip
tab
un
dan
ce
*
** *
* * *
**
*
* *
**
**
*
** *
*
*
* **
* *
*
* *
*
0
0.4
0.8
1.2
1.6
0 3 12 24
UBC
*
*
**
* * * **
*
*
0 3 12 240
20
40
60
80
100
120
140
0 3 12 24
PR1
Time (hours)
2.5
1.5
0.5
*
*
*
*
* *
*
**
*
*
Rela
tiv
etr
an
sc
rip
tab
un
dan
ce
Rela
tiv
etr
an
scri
pt
ab
un
dan
ce
Time (hours) Time (hours)
Time (hours)
Figure 3.4 Transcript abundance of AOX1a, NDB2, ATOM66, UBC and
PR1a. QRT-PCR analysis of transcript abundance for various genes over 24 h
in response to addition of citrate, H2O2, rotenone and salicylic acid in Arabidop-
sis cell cultures. The amount of transcript prior to the addition of compounds
was set to 1 and changes expressed in a relative manner. An asterisk indicates
a significant difference (P ≤ 0.05) relative to the 0 h timepoint for each individual
treatment and gene.
Time (hours)
Chapter 3 Expression and regulation of UPOX1
106
0
5
10
15
20
25
0h 1h 3h 6h
UPOX1
Aox1a
Rela
tive
tran
script
ab
und
an
ce
Figure 3.5 Transcript abundance of UPOX 1 and Aox1a. QRT-PCR
analysis of transcript abundance for UPOX1 and Aox1a in response to the
application of UV light stress at 280nm. An asterisk indicates a significant
difference relative to the 0 h timepoint for each gene, (P ≤ 0.05).
*
**
*
Time (h) after treatment
Chapter 3 Expression and regulation of UPOX1
107
Mutations
Figure 3.6 Relative transcript abundances of UPOX1, UPOX2, UPOX3, AOX1a,
NDB2, AtOM66, PR1 and UBC. The relative expression data for these genes as viewed
as a heat map in response to stimulus and mutations using Genevestigator. Displayed
are the top 50 (most upregulated and down regulated) stress treatments and mutations.
Red indicates an increase and green indicates a decrease in abundance.
Stimulus
UP
OX
1
UP
OX
2
UP
OX
3
AO
X1
a
ND
B2
AT
OM
66
PR
1
UB
C
UP
OX
1
UP
OX
2
UP
OX
3
AO
X1
a
ND
B2
AT
OM
66
PR
1
UB
C
Chapter 3 Expression and regulation of UPOX1
108
Figure 3.7 Expression of AOX1a, NDB2, UPOX, ATOM66, PR1a, and UBC in
defence signalling mutants. The relative transcript abundance was determined
by qRT-PCR in the defense signalling mutants etr1, abi3, pad4, npr1, jar1, and
eds1, and NahG and compared to Col-0 where expression was set to 100%.
Experiments conducted in triplicates.
ATOM66
UBC
PR1
UPOX1
NDB2
AOX1a
Chapter 3 Expression and regulation of UPOX1
109
Figure 3.8 Histochemical localisation of GUS activity in Arabidopsis
transformed with the full-length UPOX1 upstream region fused to the GUS
reporter gene. A) Inflorescence (flower stalk). B) Root tip. C) Emerging lateral
root primordium. D) Flower/pedicel. E) Seed Silique. F) Anthers at anthesis
A) B) C)
D) E) F)
Chapter 3 Expression and regulation of UPOX1
110
CT
TA
A
CA
AC
A
TTA
AG
GT
CA
AT
TG
AC
WR
KY
RA
V1
Str
ess
Resp
onsiv
e
ele
men
ts
AC
GT
GI
ele
men
t
CT
TA
A
AA
CA
AA
CM
YB
CA
GA
T
CG
TG
AT
Be
lem
ent
-400
AT
G-
20
0-
10
00
-8
00
CT
TA
AC
GT
GA
T
AC
GT
G
CT
TG
GC
CA
CG
-60
0
GT
CA
A
CT
TA
A
GT
CA
A
AA
CA
AA
C
CA
AC
A
CT
TA
A
CT
TA
A
CT
TA
A
CA
GA
T
UP
OX
1U
pstr
ea
mre
gio
n
Fig
ure
3.9
Su
mm
ary
of
the
pre
dic
ted
cis
-acti
ng
reg
ula
tory
ele
me
nts
inth
eU
PO
X1
pro
mo
ter
reg
ion
.P
uta
tive
cis
-acting
reg
ula
tory
ele
men
ts
were
identified
inth
eU
PO
X1
pro
mote
rre
gio
nu
sin
ga
va
rie
tyof
pre
dic
tion
meth
ods.
Nu
mberin
gis
fro
mth
etr
ansla
tio
na
lsta
rtsite.
The
ele
ments
pre
dic
ted
inclu
ded
the
MY
B,
WR
KY,
RA
V1
an
dstr
ess
resp
onsiv
ee
lem
ents
.E
lem
ents
Ban
dI
that
ha
ve
bee
np
revio
usly
iden
tified
tobe
active
in
the
AO
X1a
pro
mote
r,th
ese
ele
men
tsw
ere
als
oid
entified
tobe
inth
eU
PO
X1
pro
mo
ter.
Chapter 3 Expression and regulation of UPOX1
111
20
100
80
60
40
140
120
0
0
50
100
150
200
250
250
GU
SA
ctiv
ity
Mock
H2O2
Rotenone
**
*
*
*
*
*
***
0
50
100
150
200
250
Cell Culture
GU
SA
ctiv
ity
20
100
80
60
40
140
120
0
Leaves
Figure 3.10 Functional analysis of the B and I predicted motifs in the promoter of
UPOX1. Sequence elements identified in the promoter region of UPOX1 were tested for
functionality by deleting the elements and testing the ability of the mutated promoter to
drive GUS expression. Cell cultures and 4 week old Col-0 leaf tissue were used to for
biolistic transformation assays. GUS activity of the untreated wild-type promoter was
normalised to 100% with all other activities expressed as a percentage of this. A red
asterisk indicates a significant difference between the GUS activity of the wild-type
promoter and the mutated promoter within a treatment. A green asterisk indicates a
significant difference between the GUS activity of a mock-treated versus treated
samples within a promoter. Significance is calculated using the student's t test with a P
value ≤ 0.05.
35S
35S
**
*
*
*
*
*
*
UPOX1 wt
UPOX1I
UPOX1B
UPOX1I +
B
UPOX1 wt
UPOX1I
UPOX1B
UPOX1I +
B
Chapter 3 Expression and regulation of UPOX1
112
Nu
cle
us
H2O
2
Ro
t
AO
X1
a
ATO
M66
UP
OX
1
ND
B2
AO
X1
a
AtO
M66
UP
OX
1
ND
B2
Cyto
pla
sm
AA
AA
AA
AA
AA
AA
AA
AA
TS
S
TS
S
TS
S
TS
S+++
+
+-
+
ED
S1
PA
D4
S.A
Fig
ure
3.1
1A
su
mm
ary
of
the
sig
nallin
gp
ath
ways
that
reg
ula
testr
ess
ind
uced
tran
scri
pt
ab
un
dan
ce
for
UP
OX
1an
dco
-exp
ressed
gen
es
en
co
din
g
mit
och
on
dri
alp
rote
ins.T
hre
edis
tinctive
path
ways
regula
tem
itochondrials
tress
response
ata
transcriptionall
evel,
an
SA
-dependentpath
way
thatis
repre
-
sente
dby
AtO
M66.
The
second
path
way
repre
sents
aconverg
ence
poin
tfo
rsig
nals
genera
ted
by
H2O
2and
rote
none
that
act
via
the
sam
eC
AR
Es.
H2O
2
and
rote
none
act
at
atr
anscriptional
level.
There
are
diffe
rences
inth
isactions
betw
een
genes
nam
ely
rote
none
als
ohas
are
pre
ssiv
eeffect
on
specific
regio
ns
ofth
eA
OX
1a
pro
mote
r,how
ever
rote
none
has
no
directeffecton
the
pro
mote
rofU
PO
X.T
he
third
path
way
thatacts
via
ED
S1
and
PA
D4
and
regu-
late
sonly
AO
X1a.
The
AO
X1a
pro
mote
ris
str
ongly
repre
ssed
under
norm
alconditio
ns
and
isde-r
epre
ssed
follo
win
gtr
eatm
ent.
The
regula
tion
of
transcript
abundance
follo
win
gtr
eatm
ent
with
SA
isre
gula
ted
at
atr
anscriptionalle
velfo
rU
PO
X1,A
tOM
66
and
post
transcriptionally
for
AO
X1a
and
ND
B2.
SA
has
a
sm
all
butsig
nific
anteffecton
the
UP
OX
pro
mote
rand
this
may
be
indirectby
trig
gering
oxid
ative
str
ess.
Chapter 4 Protein Function and Localisation
113
Chapter 4
Protein Function and Localisation
Chapter 4 Protein Function and Localisation
114
4.1 Introduction
To determine the intra-organelle localisation of UPOX1 several in vivo and in vitro
methods were utilised, including GFP-localisation, import assays and BN-PAGE
analysis. To investigate this stress responsive gene at a functional level several
under-expressing lines and over-expressing lines were generated. To confirm these
lines, Western blots were performed with an antibody which was raised against
UPOX1, see Section 2.10.3. Furthermore, the UPOX1 over-expressing and
under-expressing lines were used in BN-PAGE gels in order to observe possible
proteins that may associate with UPOX1 in its native form.
The results from the in vitro assays were all visualised on either SDS-PAGE or
BN-PAGE gels. SDS-PAGE gels separate proteins according to their electrophoretic
mobility which is a function of the length of the polypeptide chain or molecular weight.
The detergent used is sodium dodecyl sulphate denatures proteins and provides a
negative charge to the protein that is proportional to its mass (Davis and Ornstein,
1959). Native PAGE separations are contrastingly run under non-denaturing
conditions with no detergents. Therefore complexes remain associated and folded as
they would otherwise in the cell. For BN-PAGE gels Coomassie Brilliant Blue dye is
used to provide a charge to the protein complexes during electrophoretic separation
(Schägger and von Jagow, 1991). Therefore, by performing all these assays it’s
possible to obtain a more thorough and in depth understanding of the intra-organelle
location of UPOX1.
Chapter 4 Protein Function and Localisation
115
4.2 Aims and Strategies
The specific aim of the research outlined in this chapter was to confirm and
characterise the function, location and interaction of UPOX1 within the plant cell.
This was conducted by performing;
- Generation and analysis UPOX1 T-DNA under-expressing lines, UPOX1
over-expressing line and Col-0 wild type
- In vivo GFP assays
- In vitro mitochondrial import assays
- In vitro chloroplast import assays
- Alkaline extractions
- Proteinase K titrations
- Blue Native PAGE assays to determine if UPOX1 is bound to a particular complex
Chapter 4 Protein Function and Localisation
116
4.3 Results
4.3.1 Generation of UPOX1 under-expressing
(knockdown) and over-expression lines
In order to characterise the function of UPOX1, multiple knock down-lines were
generated including an artificial microRNA (AmiRNA) line and an UPOX1
over-expressing line. Furthermore, several under-expressing lines were also sourced
from the Arabidopsis Biological Research Center (ABRC) including SALK_10761 and
SALK_125930. To determine if the site of the T-DNA insertions was within the exonic
coding region, the UPOX1 genomic regions were re-sequenced. DNA sequencing
confirmed that the T-DNA insertion was within the exonic region of UPOX1 for the
SALK_10761 line (Figure 4.1 A). The primers used are in Appendix 2. As the
SALK_125930 line did not contain its T-DNA insertion in the exonic regions it was not
used for further analysis. Homozygous stocks of the SALK_10761 line were grown in
soil mix pots according to Section 2.2.3, stratified for 3 days at 4ºC then grown under
long-day conditions (16 h light/8 h dark) at 22ºC. The plants were then confirmed to
be homozygous by PCR-based genotyping (primer sequences in Appendix 2), the
Sigma RedExtract PCR Kit Protocol and gel electrophoresis (according to the
methods outlined in Section 2.4.4) prior to being used for further transcriptomic and
proteomic analysis.
The amiRNA line was generated according to the methods outlined in Section 2.5 and
sequenced for confirmation. To determine the level of transcript downregulation
qRT-PCR analysis was conducted (Section 2.7). Following the qRT-PCR it was
concluded that the amiRNA line did not show a significant downregulation in the level
of UPOX1 transcript. Therefore, this line was not used for further analysis.
To examine the effect of increased levels of UPOX1 transcripts in plants, a
homozygous over-expressing line for UPOX1 was generated by Dr Olivier Van Aken
Chapter 4 Protein Function and Localisation
117
and Dr Inge De Clercq in the lab of Prof. Van Breusegem (VIB Ugent, Belgium) and
provided for use in this collaboration. This line was then consequently confirmed by
qRT-PCR for homozygosity prior to further analysis and use for this thesis (Section
2.7). Figure 4.1 B depicts the levels of UPOX1 in three lines including: the
SALK_10761 under-expressing line, the UPOX1 over-expressing line and Col-0 wild
type lines with UBC used as a housekeeping gene. From this qRT-PCR analysis it
was confirmed that the SALK_10761 under-expressing line showed a significant
decrease (approximately -90%) in UPOX1 transcript abundance relative to the Col-0
and the UPOX1 over-expressing line showed a significant increase (approximately
+400%) in UPOX1 transcript abundance relative to the Col-0.
The under-expressing, over-expressing and Col-0 lines were grown in water cultures
(according to the methods in Section 2.2.2) and purified mitochondria were purified as
described in Section 2.8.1. Mitochondrial proteins were separated on 16% SDS-PAGE
gels, transferred to PVDF membranes and probed with antibodies to UPOX1 and
Porin (generated by our laboratory). UPOX1 runs at an apparent molecular weight of
16 kDA on SDS-PAGE gels. Probing with the UPOX1 antibody resulted in a band that
was detected in mitochondria isolated from Col-0 plants but not from mitochondria
isolated from the UPOX1 under-expressing plants (Figure 4.2). A band with strong
intensity was detected in the over-expressing line with the same apparent molecular
mass as that detected in mitochondria isolated from Col-0 plants. Probing with the
Porin antibody control resulted in a cross-reacting band of uniform intensity in all three
mitochondrial samples. This position of this band corresponds to a molecular mass of
30 kDA. Thus the over-expressing and under-expressing lines were confirmed to
contain excess or no detectable quantities of UPOX1 protein respectively.
Under normal growth conditions (12 h light/12 h dark) at 22ºC, no phenotypic
differences existed between the Col-0 line, the under-expresser and over-expresser
lines as depicted in Figure 4.1 C. As UPOX1 is known to be a highly stress responsive
Chapter 4 Protein Function and Localisation
118
gene several stress assays were conducted under drought, salinity, heat and UV
exposure. To test these three lines under drought stress, plants were grown for 3
weeks and then water was withheld for 17 days following which the plants were re-
watered, this was then repeated. For salinity and heat stress phenotypic studies the
plants were treated according to the methods outlined in (Suter and Widmer, 2013)
and for UV stress 4 week old plants plants were treated with UV-C at 254 nm for 5
min. Additional stress treatments measuring root length and plant formation were also
conducted on MS-Agar plates according to the methods outlined in (Allu et al., 2014).
The left hand side agar plate shows the result of no UV-C stress and the agar plate on
the right hand side shows the effect of UV-C applied as mentioned above. There were
no phenotypic variations in either mutant line relative to the Col-0 line following these
stress treatments as can be seen in Figure 4.1 D or E.
4.3.2 In vivo GFP
In order to determine the organelle and intra-organelle localisation, chimeric proteins
were created by fusing GFP to the N and C-terminus of UPOX1 as described in
Section 2.9.1. These constructs were then biolistically transformed into Arabidopsis
suspension cell culture (Thirkettle-Watts et al., 2003; Lee and Whelan, 2004). As a
mitochondrial control, red fluorescent protein (RFP) was fused to the targeting signal
of soybean alternative oxidase (Aox-RFP) (Murcha et al., 2007) and the subsequent
fluorescence patterns were observed 48 h after transformation. The N terminal
UPOX1 GFP proteins displayed co-localisation with Aox-RFP following the merging of
their individual images, this indicated that UPOX1 was mitochondrially located (Figure
4.3A). Furthermore, the N-terminal UPOX1 GFP signal produced circular ring
structures (Figure 4.3 B), this can be indicative of proteins targeted to the outside of
the inner membrane or the outer mitochondrial membrane such as the outer
mitochondrially located protein Metaxin (Lister et al., 2007). N and C terminal GFP
Chapter 4 Protein Function and Localisation
119
constructs were also prepared and tested for UPOX2 and UPOX3. From the images
in Figure 4.3 it is clear that UPOX2 and UPOX3 are not localized to the mitochondria
in vivo as the two proteins do not overlap with the Aox-RFP control. Their appearance
is indicative of proteins localised to the cytosol.
4.3.3 In vitro mitochondrial imports
In vitro translation of the UPOX1 precursor protein generated a protein band on SDS-
PAGE gels with an apparent molecular mass of 16 kDa (Figure 4.4A, lane 1) with a
14 kDa band also present probably as a result of an alternative translation start. The
precursor protein was then incubated with freshly isolated mitochondria from 4 week
old Arabidopsis plants (lane 2). These conditions support in vitro import and the
protein was taken into the mitochondria. UPOX import is shown to be unaffected by
Valinomycin (Lane 4). Furthermore, upon rupture into mitoplasts (Lanes 6-9), UPOX
becomes exposed. This is evidence that the protein is not moving across the inner
membrane. This in vivo import is in correlation with the GFP results and suggests that
UPOX1 is not imported across the IMM and is localised to the OMM or IMS. As a
control, the TIM23-2 precursor protein was translated to generate a product with an
apparent molecular mass of 20 kDa. Also present was a 16kDa band that is a likely a
more protease resistant fragment of imported 23-2 (Figure 4.4B, lane 3). The addition
of valinomycin abolished import as evidenced by the fact that no protein was
protected by the addition of PK after the import assay (Figure 4.4B, lane 5). The outer
membrane was ruptured to confirm the protease protected protein was inserted into
the inner membrane. Following the rupture of the outer membrane and the addition of
PK a protease protected band with an apparent molecular mass of 16 kDa was
observed. This represented the portion of Tim23-2 that was present in the inner
membrane, protected from PK digestion. The addition of valinomycin to the import
Chapter 4 Protein Function and Localisation
120
assay followed by PK digestion of outer membrane ruptured mitochondria resulted in
no import being detected.
The import of AOX was also used a control. The AOX precursor protein was
translated to generate a product with an apparent molecular mass of 36 kDa. The
protein was imported and processed to produce a mature product of 32 kDa. The
mature protein was transported into the inner mitochondrial membrane as it was
confirmed to be PK resistant and sensitive to the addition of valinomycin (Figure
4.4C).
4.3.4 In vitro chloroplast import
In order to exclude the possibility that UPOX1 was also targeted to chloroplasts, i.e. a
dual targeted protein, in vitro import assays into chloroplasts were performed (Figure
4.5). The extensively studied small subunit of 1,5-ribulose bisphosphate
carboxylase/oxygenase (Rubisco SSU) was used as a control (Carrie et al., 2008).
Lane 1 for both UPOX1 and SSU show a precursor at 16 and 20 kDa respectively.
Following the incubation of the SSU precursor with chloroplasts a band was present at
20 kDa and a mature band was also present at 14 kDa. Following the addition of
thermolysin the mature band was present at 14 kDa showing that SSU was imported
into the chloroplast. Following the incubation of UPOX1 with chloroplast there appears
to be no band evident. Therefore it can be deduced from these experiments that
UPOX1 is not imported into chloroplasts and appears to be specifically imported into
the mitochondria.
4.3.5 Alkaline extractions
To determine if UPOX1 was located or associated in/with the mitochondrial
membrane or soluble fraction, alkaline extractions were performed according to
Section 2.9.5 (Figure 4.6). AOX was used as a control as it has been well established
that it is an inner membrane protein and therefore would be found in the insoluble
Chapter 4 Protein Function and Localisation
121
fraction (Lister et al., 2004). In vitro import of AOX and UPOX1 followed by alkaline
extraction resulted in AOX only being detected in the insoluble (membrane) fraction
while UPOX1 was detected in both the soluble and insoluble fractions (Figure 4.6).
This suggests that UPOX1 is associated with the outer membrane and aqueous
compartments or inter-membrane space of mitochondria.
4.3.6 Proteinase K titrations into mitochondria and
mitoplasts
In the previous import experiments, UPOX1 was partially resistant to PK treatment.
(As in Figure 4.4). In order to determine the efficiency of protease treatment, PK
titrations were carried out on mitochondria and mitoplasts. These were then immune
detected with several antibodies. The controls used included TOM20-2 which has
been previously localised to the outer mitochondrial membrane (Murcha et al., 1999;
Werhahn et al., 2001) AOX which has been previously localised to the inner
membrane and HSP 60 which is a matrix control protein (Millar et al., 2001) (Figure
4.7). Titrations ranging from 0 to 256 µg of PK were carried out per sample.
As expected, following PK titration the TOM20-2 protein depletes immediately; this is
to be expected as this protein is an outer mitochondrial protein and is immediately
sensitive to proteinase digestion. Protein bands are present for AOX and HSP60
following PK titrations on mitochondria and mitoplasts up to 128 µg of PK as expected
as they are located in the inner membrane and the matrix respectively. The amount of
UPOX1 diminishes with an increasing concentration of PK in mitochondria. Following
the rupture of the outer membrane of the mitochondria and the addition of PK no
protein bands are detectable. This suggests that UPOX1 may be located in or near to
the inner mitochondrial space. These results correlate with the in vitro import results
and the GFP analysis.
4.3.7 Blue Native PAGE
Chapter 4 Protein Function and Localisation
122
Blue Native Polyacrylamide gel electrophoresis (BN-PAGE) was conducted to
determine if UPOX1 was associated with a mitochondrial respiratory chain complex.
This experiment was conducted by performing BN-PAGE according to Section 2.11.2
BN PAGE sample preparation. BN-PAGE gels allow for the visualisation of the
mitochondrial multi-subunit respiratory chain complexes as they are prevented from
dissociation as no SDS is used (Swamy et al., 2006). The first panel shows the
Coomassie stained gel directly proceeding electrophoresis. The major respiratory
chain complexes and super complexes (CI+CIII, CI, CV, CIII, CIV and CII) are clearly
visible in Figure 4.8. For this analysis, mitochondria were isolated from Arabidopsis
Col-0 plants, the SALK_10761 UPOX1 under-expressing line and the UPOX1
over-expressing line.
These gels were then transferred onto PVDF membranes according to Section 2.11.3
Wet Transfer of BN PAGE gels and immune detected using several different
antibodies. For a complex I control NAD9 was used. BN-PAGE imports were carried
out to determine if UPOX1 was imported into any complexes. These gels were then
dried and exposed on Fuji BAS TR2040 plates. It was determined from these
experiments that UPOX1 was not imported into either complex I or the super complex
CI+CIII. Therefore the bands present for the UPOX1 western following BN-PAGE can
only be attributed to either minor associations without import or non-specific binding of
the antibody to these complexes. The import of UPOX1 and consequent BN-PAGE
for this result shows that there is a strong band very low in the BN-PAGE image. This
suggests that UPOX1 runs further into the gel than all the larger respiratory chain
complexes (without incorporation into the complexes).
4.4 Discussion
A number of approaches were undertaken to determine the sub-cellular localisation of
UPOX1. The mitochondrial localisation was confirmed using a number of methods,
Chapter 4 Protein Function and Localisation
123
namely western blot analysis, in vivo GFP tagging and in vitro uptake assays. The
intra-organellar localisation was investigated using alkaline extraction, PK digestions
and BN-PAGE. Several approaches are necessary to determine the location of
uncharacterised proteins as each of these techniques alone has its limitations and no
technique should be used alone. Only after several different approaches are taken
can a hypothesis be made about the organelle and intra-organelle location of any
protein of interest. In this case the results of all these approaches together correlate
with each other and suggests that UPOX1 is located in the mitochondrial
intermembrane space and may also be associated with the outer mitochondrial
membrane. Following phenotypic studies on the under-expressing and
over-expressing mutant lines in response to stress treatments no phenotypes were
detected. The lack of a phenotype could be explained by the compensation of
duplication genes (Gu et al., 2003) or by the mechanisms that allow stress networks
to buffer the effects of perturbations in related pathways and thereby allowing for
homeostasis to be maintained (Cutler and McCourt, 2005).
Chapter 4 Protein Function and Localisation
124
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Chapter 4 Protein Function and Localisation
125
16 kDa
Col-0UPOX1 over expresser
30 kDa
Figure 4.2 Western blot analysis of isolated mitochondria from 2 week
old water culture plants confirming the mutant under expressing and
over expressing lines. The mitchondria were isolcated from Col-0 water
cultures, UPOX1 under expressing line SALK_10761 water cultures and the
UPOX1 over expressing line water cultures. Mitochondrial proteins were
probed with an antibody raised against UPOX1, Porin was used as a control.
UPOX1 under expresser
Chapter 4 Protein Function and Localisation
126
A)
Chapter 4 Protein Function and Localisation
127
B)
Figure 4.3 B) In vivo sub cellular localisation of N’ UPOX1. GFP fusion
construct using full length coding sequences were transformed into Arabidopsis
cultured cells using biolistic transformation. This image is a zoomed up image
from Figue 4.3 A) showing the circular ring structures.
Ring Structures
UPOX1GFP
10µm
Chapter 4 Protein Function and Localisation
128
Mito - + + + + + + + +
PK - - + - + - + - +
Val - - - + + - - + +
MP - - - - - + + + +
Lane 1 2 3 4 5 6 7 8 9
UPOX1
TIM23-2
AOX
16 kDa
36 kDa
16 kDa
32 kDa
20 kDa
Figure 4.4 Import of radiolabelled UPOX1, TIM23-2 and AOX into mitochon-
dria. Lane 1, precursor protein only. Lane 2, precursor protein incubated with
mitochondria under conditions that support import. Lane 3, as lane 2 with 0.4
ug/ul proteinase K (PK) added. Lane 4, as lane 2 with 1 uM valinomycin (Val)
added. Lane 5, as lane 2 with PK and Val added. Lane 6 to 9 as lanes 2 to 5
respectively with ruptured outer mitochondrial membranes.
14 kDa
A)
B)
C)
Chapter 4 Protein Function and Localisation
129
Chapter 4 Protein Function and Localisation
130
AOX
Lane
UPOX1
1 2 3
Mitochondria +_ +
Figure 4.6 Alkaline extractions of AOX and UPOX1. Alkaline
extractions following in vitro imports assays. Lane 1, precursor
protein alone. Lane 2, soluble fraction. Lane 3, insoluble fraction
Chapter 4 Protein Function and Localisation
131
Tom20-2
AOX
HSP 60
0 16 32 64 128 256
Lane
PK 0 16 32 64 128
1 2 3 4 5 6 7 8 9 10 11
MP - - - - - - + + + + +
Figure 4.7 Western blot analysis of proteinase K titrations of TOM20-2, AOX, HSP
60 and UPOX1. Both mitochondria and mitoplast (MP) samples were treated with
increasing amounts of PK. Lane 1, mitochondria with no PK added, Lanes 2, 3, 4, 5
and 6 as lane 1 with the addition of 16, 32, 64, 128 and 256ug PK respectively. The outer
membrane was ruptured via osmotic shock for lanes 7 to 12. Lane 7 as lane 1 except
that mitochondria were osmotically shocked prior to PK digestion. Lanes 8, 9, 10 and 11
as lanes 2-5 except that mitochondria were osmotically shocked prior to PK digestion.
UPOX1
23 kDA
30 kDa
60 kDA
16 kDA
Chapter 4 Protein Function and Localisation
132
CV
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21
34
56
Chapter 5 General Discussion
133
Chapter 5
General Discussion
Chapter 5 General Discussion
134
5.1 Summary of Aims and Approaches
This study involved carrying out the most comprehensive and in-depth research on
UPOX1 to date. The aims of this project involved taking a multi-disciplinary approach
in order to characterise UPOX1, this has incorporated various transcriptomic,
proteomic and bioinformatic approaches. Specifically, the approaches taken included:
1) Investigations into the kinetics of UPOX1 induction with the aim of identifying
pathways that regulate stress induced gene expression. These assays were
compared and contrasted against other known stress responsive genes such
as AOX1a and other mitochondrial proteins such as external NAD(P)H-
dehydrogenase NDB2 and AtOM66.
2) Transcript analysis was conducted on defense signalling mutants to determine
the pathways that regulates this stress induced gene.
3) The promoter regions were examined to determine the functional CAREs that
play an active role in the response to stress treatments such as with H2O2,
rotenone, salicylic acid and combinations of these.
4) The co-expression and co-regulation of UPOX1 was examined.
5) Several proteomic methods were employed to determine the localisation of
UPOX1 within the plant cell and more specifically the mitochondria. These
approaches involved; conducting in vivo GFP analysis in conjunction with
antibody based in vitro isolation methods
6) The use of an over-expressing line and under-expressing line for UPOX1 for
functional analysis.
5.2 Summary of Results
UPOX1 to date in scientific journals is still often categorised as an ‘unknown stress
responsive gene’. The research conducted in this PhD has added valuable, new and
Chapter 5 General Discussion
135
in depth knowledge on this gene, its corresponding protein, how it is co-regulated and
where it is located within the cell.
Specifically it has been elucidated that the homologs of UPOX1, (UPOX2 and
UPOX3) differ considerably from the qRT-PCR transcript profiles of UPOX1.This
suggests that the regulatory mechanisms controlling the expression of each gene may
be largely unrelated and/or independent. In addition, qRT-PCR assays also showed
that UPOX1 was found to be significantly more stress responsive than its two
homologs. Further investigations focused on the kinetics of transcript induction and it
was established that UPOX1 following the treatment of a wide array of stress
treatments responded late as it peaked 24 h after stress treatment. From these
studies, it was concluded that UPOX1 was involved in a secondary stress response
pathway. The kinetics of induction were also tested for several other putatively co-
expressed genes and following these qRT-PCR assays it was confirmed that the
pattern of transcript induction for NDB2 and AOX1a differed from that of UPOX1.
Investigations were carried out to determine if UPOX1 was co-expressed or
co-regulated with any other genes. It was first confirmed that of all the genes tested
AOX1a was the gene that was most co-expressed with UPOX1. This suggested that
there is a possible overlap in the pathways responsible for the induction of these
genes. Following on from this, research was carried out to confirm whether UPOX1
and AOX1a were co-regulated. These experiments involved initially narrowing down
putative functional elements in the upstream regions and mutating these regions.
Biolistic transformation assays confirmed that UPOX1 and AOX1a share the same
functional motif in their upstream region and therefore these genes were confirmed as
being co-regulated.
In compiling the transcriptomic and biolistic transformation data three pathways that
mediate mitochondrial stress response were identified. The first was a SA dependent
pathway in which only AtOM66 is regulated at a transcriptional level while UPOX1,
Chapter 5 General Discussion
136
AOX1a and NDB2 are regulated at a post-transcriptional level. The second pathway is
the lack of response by UPOX1 to rotenone which differs significantly to AOX1a. The
third pathway is represented by AOX1a as its expression differs from UPOX1 and
NDB2, this suggests a different pathway involved via EDS1 and PAD4 to regulate only
AOX1a. From these investigations into the promoter elements in UPOX1, AOX1a and
NDB2 it was concluded that SA acts to increase transcript abundance of these via a
post-transcriptional mechanism in contrast to AtOM66 which is under direct
transcriptional control. Finally it was also found that H2O2 and rotenone can both act
transcriptionally and their CAREs overlap however different transcription factors are
thought to be involved for UPOX1, AOX1a and NDB2. Therefore transcript analysis,
defense signally mutants and promoter activity studies together have provided new
and detailed insights into the intricate mechanisms underlying the expression of these
genes under stress conditions.
By conducting western blot analysis, in vivo analysis, GFP tagging, in vitro uptake
assays along with alkaline extractions, PK digestions and BN-PAGE it was confirmed
that UPOX1 is located in the mitochondrial intermembrane space and may also be
associated with the outer mitochondrial membrane. Interestingly, UPOX1 seems to be
associated with both membrane and soluble fractions. It has been recently found by
ID Blue Native PAGE that UPOX1 may also be associated with the membrane arm of
Complex I (Senkler et al., 2016).
5.3 Functional implications of the
mitochondrial stress response
Mitochondria have several crucial roles in the functioning of plants; these include the
integration of carbon and nitrogen into the metabolism of plants, the biosynthesis of
pyrimidines, heme, vitamins, iron-sulphur centres. Additionally they are involved in
Chapter 5 General Discussion
137
signalling programmed cell death and the production of ATP during phosphorylation
and the TCA cycle. Several studies over the years have demonstrated how
mitochondria are the site of and target of a variety of stresses (Bartoli et al., 2004;
Giraud et al., 2008) yet there is still very little known about many of the smaller
proteins that are encoded in the nucleus, targeted to the mitochondria and are stress
responsive. This thesis has focused on one such protein UPOX1, a hallmark of
oxidative stress (Gadjev et al., 2006) and using a multitude of approaches many of the
mitochondrial stress pathways in which it is involved in have been deduced. A main
sub-topic in relation to mitochondrial stress response (MSR) has revolved around the
production of ROS species and the targeting of ROS as it is known to critically
damage proteins, nucleic acids and lipids (Fujita et al., 2006). Within our lab it has
been shown that the activity of AOX in conjunction with NAD(P)H dehydrogenases
enable the ETC to continue functioning by preventing over reduction of the
constituents of the ETC, furthermore, it has also been shown that the lack of AOX
produces a heightened sensitivity to combined drought and light stress in Arabidopsis
(Giraud et al., 2008; Van Aken et al., 2009).
AOX1a, the alternative respiratory pathway and the ETC have been studied
extensively in relation to MSR however it is now clear that MSR pathways are much
wider than just encompassing these components. For example the mitochondrial
substrate carrier protein family (MCF) are the strongest represented protein family in
MSR pathways. The nature of the MCFs by their broad stress responsiveness which
may be involved in Pi (At3g48850), ADP/ATP (At4g28390) and dicarboxylic acid
transport suggests that under stress conditions, mitochondria requires an increased
exchange of respiration and TCA cycle substrate (At4g27940 and At2g22500) (Hamel
et al., 2004; Palmieri et al., 2008).
In order to gain a further understanding of the functional implications of the
mitochondrial stress response 26 mitochondrial stress responsive genes, including
Chapter 5 General Discussion
138
UPOX1 were investigated within the Whelan lab and these genes were examined in
16 selected microarray datasets (Van Aken et al., 2009). The 16 selected microarrays
spanned various stress conditions including heat, UV, ozone, salt and osmotic stress.
Cluster analysis was conducted on this data as information about a gene’s function
can be deduced by identifying genes that share its expression pattern. Genes that are
co-expressed under a variety of different circumstances may work together in the cell,
they may be involved in a co-ordinated activity and may encode proteins that are part
of the same multi-protein machine. The cluster analysis showed that in all conditions
tested there was a predictable trend of up regulation for most genes although a few
treatments that are often considered to be physiologically comparable such as salt
and osmotic stress did not show a similar transcriptomic response. For example
UPOX1 and sHSP23.5 were observed to be strongly down regulated during osmotic
stress although up regulated during salt stress. Saying this however, most other cases
did show comparable treatments clustering together (Van Aken et al., 2009).
In terms of gene specific expression patterns, the clustering analysis revealed
correlations that reveal functional implications for UPOX1 and mitochondrial import
pathways. UPOX1 and Tim17-1 were shown to be highly co-expressed. Tim17-1 is
the only import component known to date to be altered by stress. Another interesting
note to mention is that UPOX1 along with most other mitochondrial proteins are
imported into the mitochondria and therefore it’s likely that the rate of mitochondrial
import changes during stress.
The clustering analysis also revealed that UPOX1 is also co-expressed with
sHSP23.6, sHSP23.5, AOX1a and NDB2 (Van Aken et al., 2009). As members of the
alternative respiratory pathway AOX1a and NDB2 can form a complete respiratory
chain, dissipating energy as heat whilst not contributing to ATP synthesis. The
co-expression of AOX1a, NDB2 with UPOX1 has been documented before in (Clifton
et al 2005) and the research conducted in this thesis shows the extent to which
Chapter 5 General Discussion
139
UPOX1 and AOX1a are also co-regulated as a result of CAREs within their promoter
regions. This implies that the observed expression patterns of MSR genes are at least
partially regulated by underlying co-regulation mechanism (Van Aken et al., 2009).
Additional studies have investigated the expression of known stress responsive genes
in mutant atphb3 lines on (Van Aken et al., 2010). Both AOX1a, a marker of
mitochondrial retrograde signalling in plants as well as UPOX1 were significantly
upregulated (UPOX1 was upregulated 40-fold). This further indicates that at least part
of the mitochondrial stress response can be triggered by retrograde signalling.
5.4 Retrograde regulation of nuclear genes
encoding mitochondrial proteins during
stress and the mitochondrial dysfunction
stimulon
Many molecular components of retrograde signalling pathways that regulate
expression of nuclear genes encoding mitochondrial proteins (NGEMP) are known to
interact with chloroplast, growth and stress signalling pathways in the cell at different
levels. Some components are involved in the transmission and execution of these
signals. This positions mitochondria as important hubs for signalling in the cell, not
only in direct signalling of mitochondrial function but also in sensing and/or integrating
a variety of other internal and external signals. This integrates growth with energy
metabolism and stress responses.
A review by Ng et al., 2014 analysed the expression characteristics of all NGEMPs in
response to chemical and genetic mitochondrial perturbations. The MRR signalling
pathways were assessed and from this meta-analysis at least two transcriptomic
footprints were apparent from the clustering: genes that are generally up-regulated
and genes that are generally down-regulated under most of the conditions. As
Chapter 5 General Discussion
140
predicted UPOX1 was upregulated over all five categories, including abiotic, biotic,
nutrient, chloroplast and mitochondrial perturbations. More specifically this included
UV, salt, water and oxygen availability, temperature and many pathogens (Ng et al.,
2014).
Some of these genes identified as being generally stress responsive were also part of
the so-called Mitochondrial Dysfunction Stimulon (MDS) (De Clercq et al., 2013) of
which UPOX1 is a member along with MGE1 (At5g55200), sHsp23.5 and AtOM66
(Van Aken et al., 2009). The genes belonging to the MDS share a common cis-
regulatory element in their promoters that have been named the mitochondrial
dysfunction motif (MDM). This arbitrates the MRR-mediated induction by NAM, ATAF
½ and CUC2 (NAC) transcription factors (De Clercq et al., 2013; Ng et al., 2014). Of
these, the transcription factors ANAC017, ANAC013, ANAC053 and ANAC078 both
belonging to the transcription factor class NAM target UPOX1 (De Clercq et al., 2013).
It has been suggested that there is an integration of multiple MRR pathways at the
transcriptional or more upstream level as the MDM seems to be sufficient for
responsiveness to various genetic mitochondrial perturbations such as the prohibitin
mutant atphb3 (Van Aken et al., 2007; Van Aken et al., 2016), pharmacological
perturbations such as rotenone, Antimycin A and H2O2. Furthermore, a W-box cis-
element is also present in the MDS promoters and the W-box binding WRKY
transcription factors regulate the expression of these genes under various conditions
including many mitochondrial perturbations and high-light (Van Aken and Whelan,
2012; Van Aken et al., 2013).
Further investigations within the lab have suggested that the MDS genes act as a
non-specific and primary defense layer in response to a multitude of stress conditions
by avoiding (through alternative respiration) and by alleviating the effects of oxidative
stress, which is a common factor during different adverse conditions (Giraud and Van
Aken, 2012). Genome wide transcription studies by Rhoads and Subbaiah, 2007;
Chapter 5 General Discussion
141
Schwarzlander et al., 2012 also concur with the hypothesis that multiple MRR
pathways are present. This hypothesis has been further re-enforced by the promoter
analysis in this thesis that have tested transcript abundances of four stress responsive
NGEMPs (AOX1a, AtOM66, NDB2, and UPOX1) in response to rotenone, H2O2, and
salicylic acid and have found that discrete regulatory mechanisms exist.
In the last few years more details on how transcription factors control their target
genes have been elucidated. For example; AtWRKY40 has been shown to limit the
induction of UPOX1, AOX1a, NDB2 and AtOM66 after high light and AA stress
treatments and AtWRKY63 has been shown to positively affect the expression of
these genes (Van Aken et al., 2013). Furthermore, the expression of UPOX1 along
with AOX1a and several other genes encoding mitochondrial and non-mitochondrial
proteins have been found to be positively regulated by a group of NAC transcription
factors. These NAC transcription factors are bound to the endoplasmic reticulum
membrane and are released following mitochondrial inhibition. This discovery helps to
explain how signals could be transmitted from mitochondria to the nucleus as the
location of the ER bound NAC transcription factors associated with actin filaments
facilitates a close interaction between mitochondria and the ER (Ng et al., 2014).
5.5 Future Directions
The understanding of the molecular components that control the expression of
NGEMPS’s has significantly progressed in the last decade. This thesis continues to
add to the knowledge base but there is much we still don’t understand as only a
portion of these regulators and their gene targets are known to date. Several more
studies incorporating various stress responsive genes, transgenic lines and promoter
analysis experiments are required to demonstration a comprehensive understanding
of this field.
Chapter 5 General Discussion
142
In summary, the data presented in this thesis comprises the most comprehensive,
integrated analysis of the highly stress responsive hallmark of oxidative stress UPOX1
performed to date. In this study, this previously un-characterised protein was found to
be located in the mitochondria, elucidated the transcript kinetics and identified various
pathways in which it is involved. To investigate and characterise the function of
UPOX1 further advanced proteomic and metabolomic approaches could be used in
conjunction with stress treatments and new transgenic lines. Other future approaches
could involve the identification of potential protein interaction partners of UPOX1 and
genome-wide expression studies of the UPOX loss- and gain-of-function lines under
specific stress conditions.
Chapter 5 General Discussion
143
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Appendix 1
Plant Growth Media
Cell Culture Media
1 sachet/L Murashige & Skoog Salt Mixture (N1145; Invitrogen, Australia), 30 g/L
sucrose, 500 µl 1 mg/ml Naphthalene acetic acid (K salt), 50 µl 1 mg/ml kinetin.
Liquid Culture Media
1 sachet/L Murashige & Skoog Salt Mixture (N1145; Invitrogen, Australia), 400 µl
Gamborg’s B5 vitamins (Sigma Aldrich, Australia), 0.32 g MES, 16 g sucrose. Adjust
pH to 5.7 using KOH, and autoclave to sterilise. Add 50 µg/ml cefotaxime sodium
antibiotic (Claforan®).
Solid Culture Media
1 sachet/L Gamborg’s B5 Basal Salt mixture (G5768; Sigma Aldrich, Australia), 1 ml/L
1000X Gamborg’s B5 vitamins, 0.5 g/l MES, 30 g/L sucrose. Adjust pH to 5.7 using
KOH, add 6 g/L agar and autoclave to sterilise. Prior to pouring media, add 50 µg/ml
cefotaxime sodium antibiotic (Claforan®).
Seed Sterilization Solution
After washing the seeds in 70% ethanol a solution of 5% bleech with 0.1% Tween 20
was applied to the seeds. This solution was washed with SDW prior to sowing.
Bacterial Growth Media
SOC Media
20 g/L tryptone peptone, 5 g/L yeast extract, 10 ml/L 1 M NaCl, 2.5 ml/L KCl.
Autoclave to sterilize. Add 5 ml/L 1 M MgCl2, 5 ml/L 1 M MgSO4, 10 ml/L filter
sterilized 2 M glucose prior to use.
Luria-Bertani (LB) media
10 g/L tryptone peptone, 5 g/L yeast extract, 10 g/L NaCl, adjust pH to 7.0 using 1 M
NaOH. Sterilize by autoclaving liquid. Add 15 g/L bacto agar prior to autoclaving to
Appendix 1
158
make solid media LB plates. Add appropriate antibiotics following cooling of liquids
such as Amicillin to a final concentration of 50 g/ml prior to pouring plates.
NZY+ media
10 g/L NZ amine (casein hydrolysate), 5 g/L yeast extract, 5 g/L NaCL. Sterilize by
autoclaving. Add the following prior to use; 12.5 ml/L 1 M MgCl2, 12.5 ml/L MgSo4 and
10 ml/L 1 M filter sterilized 2 M glucose.
Agarose gel electrophoresis solutions
TAE Buffer
48 g/L Tris, 11.8 ml/L glacial acetic acid, 10 mM EDTA. Dilute 1 in 10 in ddH2O for
use as 1 x TAE.
Loading Buffer
30% (v/v) glycerol, 0.05% (v/v) bromophenol blue.
5 x Running Buffer
15 g/L Tris base, 72 g/L glycine, 5 g/L SDS. Do not adjust pH but check it it lies
between 8.3-8.5. Dilute 1 in 5 with ddH2O for use as 1 x Running Buffer.
Tris-Tricine 1 x Cathode Buffer
12.11 g/L Tris base, 17.92 g/L tricine. 1 g/L SDS. Do not adjust pH
Tris-Tricine 5 x Anode Buffer
121.1 g/L Tris base. Adjust pH to 8.9 with concentrated HCL.
Coomassie stain solution
100 ml/L glacial acetic acid, 400 ml/L 100% ethanol, 1 g/L Coomassie Brilliant Blue R-
250. Vacuum filtered to remove undissolved grains.
Appendix 1
159
Coomassie destain solution
150 ml/L glacial acetic acid, 375 ml/L 100% ethanol, 25 ml/L glycerol.
SDS-PAGE solutions
Separating buffer
1.5 M Tris-HCL, pH8.8
Stacking buffer
0.5 M Tris-HCL, pH 6.8
Separating gel (12%)
Add 5.25 ml separating buffer, 8.4 ml 30% (w/v) acrylamide/bisacrylamide and 210 µl
10% (w/v) SDS to 7 ml ddH2O. Degas via vacuum manifold for 5 min and add 120 µl
10% (w/v) ammonium persulfate and 10.5 µl TEMED just prior to use.
Separating gel (14%)
Add 5.25 ml separating buffer, 9.8 ml 30% (w/v) acrylamide/bisacrylamide and 210 µl
10% (w/v) SDS to 5.6 ml ddH2O. Degas via vacuum manifold for 5 min and add 120 µl
10% (w/v) ammonium persulfate and 10.5 µl TEMED just prior to use.
Stacking gel (4%)
Add 2.5 ml stacking buffer, 1.3 ml 30% (w/v) acrylamide/bisacrylamide and 100 µl
10% (w/v) SDS to 6.1 ml ddH2O. Degas via vacuum manifold for 5 min and add 50 µl
10% (w/v) ammonium persulfate and 10 µl TEMED just prior to use.
2 x Sample buffer
25 ml 10% (w/v) SDS, 10 ml glycerol, made up to 50 ml with stacking buffer and
several grains of bromophenol blue added. Prior to use, add 20% (v/v) β-
mercaptoethanol.
Western Blotting Solutions
Appendix 1
160
Transfer Buffer
2.93 g/L glycine, 5.81 g/L Tris base,0.375 g/L SDS, 200 ml/L methanol. Vacuum
filtered
10 x Ponceau stain
20 g/L Ponceau S, 300 g/L trichloroacetic acid, 300 g/L sulfosalicylic acid. Dilute 1 in
10 with ddH2O for use as 1 x Ponceau stain.
10 x TBS
90 g/L NaCL, 12.11 g/L Tris. Adjust pH to 7.4 using HCL and sterilize by autoclaving.
1 x TBS- Tween
100 ml/L 10 x TBS, 1 ml/L Tween® 20.
Blue Native PAGE Solutions
5 x Cathode Buffer
44.8 g/L Tricine, 15.7 g/L Bis-Tris, 1g/L Coomassie 250G. Adjust pH to 7.0 with HCL
at 4ºC and dilute to 1x working solution.
6 x Anode Buffer
62.75 g/L Bis-Tris. Adjust pH to 7.0 with HCL at 4ºC and dilute to 1x working solution.
6x Blue Native Gel buffer
168.8 g/L Amino caprioc acid, 31.2 g/L Bis- Tris. Adjust pH to 7.0 with HCL at 4ºC and
dilute to 1x working solution.
Amino Caprioc Acid (ACA) 750mM
98.4 g/L ACA, 10.46 g/L Bis-Tris, 0.2 g/L EDTA. Adjust pH to 7.0 with HCL at 4ºC.
Appendix 1
161
5% Serva Blue G
0.984 g/L ACA, 0.5 g/L Coomassie 250G.
1 x Sample Buffer
750 µl/ml ACA 750, 150 µl/ml n-Dodecylmaltoside 10%, 150 µl/ml 5% Serva Blue G.
Arabidopsis Mitochondrial isolation media
Grinding media
0.45 M mannitol, 50 mM anhydrous sodium pyrophosphate (10.H2O), 0.5% (w/v) BSA,
0.5% (w/v) PVP-40, 2MM EDTA. Adjust pH to 8.0 using H3PO4. Add 20mM cysteine
just prior to use.
1 x Wash medium
0.3 M mannitol, 10mM TES, 0.2% (w/v) BSA. Adjust pH to 7.5 using HCL.
1st Gradient
0.6 M mannitol, 20mM TES, 0.2% (w/v) BSA. Adjust pH to 7.5 using HCL.
2nd Gradient
0.6 M sucrose, 20mM TES, 0.2% (w/v) BSA. Adjust pH to 7.5 using HCL.
40% (v/v) Gradient 1
40% Percoll® in 50% (v/v) gradient buffer 1 in ddH2O.
21% (v/v) Gradient 1
21% Percoll® in 50% (v/v) gradient buffer 1 in ddH2O.
16% (v/v) Gradient 1
16% Percoll® in 50% (v/v) gradient buffer 1 in ddH2O.
Appendix 1
162
Gradient
5 ml 40% (v/v) gradient 1, 20 ml 21% (v/v) gradient 1, 10 ml 16% (v/v) gradient 1.
In vitro import Solutions
In vitro transcription/translation reaction master mix (50 µl)
25 µl TNT® rabbit reticulocyte lysate, 4µl TNT® reaction buffer, 2 µl T7 or SP6 RNA
polymerase, 2 µl 1 mM amino acid mixture minus methionine, 2 µl RNasin®
ribonuclease inhibitor (40 units/µl; Roche, Australia), 1 µl [35S]-methionine (> 1000
Ci/mmol at 10 mCi/ml; Amersham Pharmacia Biotech, Australia)
2 x Mitochondrial import buffer
0.6 M sucrose, 100 mM KCL, 20 mM Mops, 10 mM KH2PO4, 0.2% (w/v) BSA. Adjust
pH to 7.5 with HCL.
Import Master Mix
1 ml of 2 x import buffer, 2 µl 1 M MgCl2, 20 µl of 100 mM methionine, 4 µl 100 mM
ADP, 15 µl 100 mM ATP, 20 µl 0.5 M succinate, 20 µl 0.5 M DTT. This solution was
made up with ddH2O to 2 ml prior to use.
159
Appendix 2
QRT-PCR Primers. UPOX1, UPOX2 and UPOX3, their chromosomal locus, the primer sets used, their melting temperatures and the optimum primer
concentration given in µM for the respective primer set
Name Accession # Sequence 5’-3’ G/C: A/T Melting
Temperature
Optimum Primer Concentration for
qRT-PCR
UPOX1 Fwd At2g21640 CCGAGAACCCGCCAAAACC 12:7 62 °C 0.9 M
UPOX1 Rev At2g21640 GCTTCTCTGCAACTGCCTC 11:8 60 °C 0.9 M
UPOX2 Fwd At4g39235 GAGACCCAGACAAATCAAAG 9:11 58 °C 0.7 M
UPOX2 Rev At4g39235 CTGCGACCGCCTTTGACAATCC 13:9 70 °C 0.7 M
UPOX3 Fwd At3g05570 GACGCAGAAAACTCAAG 8:9 50 °C 0.7 M
UPOX3 Rev At3g05570 CAGCCTTTGACAACCCAAAC 10:10 60 °C 0.7 M
159
SALK 010761 T-DNA screening primers.
Name Accession # Sequence 5’-3’ CG% Melting Temperature
SALK 010761 Fwd At2g21640 GCTTCGATTCAGTATCACGGG 52 57.4 °C
SALK 010761 Rev At2g21640 CTTAAGGGAGGAAGAGTCATGC 50 56.8 °C
160
