Molecular studies of abiotic stress and abscisic acid mediated stress response pathway in barley

366 Pages·2015·4.54 MB·English

by  Atul Kamboj

Molecular studies of abiotic stress and abscisic acid mediated
stress response pathway in barley

Atul Kamboj
Master of Science (Biotechnology)

This thesis is presented for the degree of Doctor of Philosophy
September 2014

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Abstract
Salt and drought stresses are the two most important environmental stresses which limit
plant growth and development. Salinity is currently affecting about 6 % of world’s total
land area and increased salinity may result in 50% loss of arable land by 2050. In
Australia alone, about 5 % is currently affected by salinity and another 17 million ha
(33 % of agricultural land) may be salinised or at risk by 2050. Drought stress also
impedes production of crops. Australian crop yields may drop up to 30% by 2050 as a
result of reduced rainfall. Thus, the major aim of the current study was to analyse the
global expression of barley genes to acute salinity and drought stress.

In this work, the utility of deep cDNA sequencing (mRNA-seq) for barley transcriptome
analysis is demonstrated as an alternative to microarrays despite the lack of a whole
genome reference sequence [barley whole genome sequence has only recently been
described; International Barley Genome Sequencing Consortium (IBSC)]. mRNA-seq
was performed on acutely salt, drought and exogenous ABA stressed and unstressed
leaf material of barley (Hordeum vulgare L. cv. Hindmarsh). The data yielded more
than 100 million sequence tags which aligned to more than 20,000 transcripts in the
IBSC database. Approximately 90% of the genes differentially expressed under salinity
or drought were also differentially expressed in exogenous ABA stress. Genes such as
chlorophyll a/b binding protein and MYB transcription factors were among the most
differentially regulated during salt and drought stresses. Analysis of gene ontology
showed “binding” as major molecular function in all three stress conditions. These
differentially regulated genes can be the candidate tolerance genes that need to be
further examined using reverse genetic approaches.

The phytohormone abscisic acid (ABA) is crucial for plant’s adaptive response to
salinity and drought stresses. ABA accumulates in plant cells to protect vegetative
tissues and regulates developmental events. The major components of ABA mediated
abiotic stress tolerance pathway include Pyrabactin resistance family
(PYR/PYL/RCAR; soluble ABA receptors), protein phosphatase 2C subfamily A
(PP2CA; negative regulators of ABA signalling cascade in the absence of ABA), SNF1
related protein kinase 2 (SnRK2; positive regulator of the cascade) and ABA transport
related ABC transporters (ABCG25 and ABCG40). ABA signalling pathway is well
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established in Arabidopsis, but is poorly defined in the major cereal crops, wheat and
barley. Thus, the present study aimed to identify and characterise barley ABA mediated
abiotic stress signalling related genes. Reciprocal BLAST search (RBH), an automated
reciprocal BLAST pipeline tool was developed enabling the rapid identification of
specific gene families of interest in related species, streamlining the collection of
homologs prior to downstream molecular evolutionary analysis. The expressed barley
ABA signalling related genes were initially identified using mRNA-seq and NCBI
Unigene database, followed by further searches in the IBSC database. The identified
sequences were analysed for certain functional motifs and key residues in the putative
proteins, which resulted in 13 PP2CAs, 9 PP2CDs (PP2C subfamily D), 10
PYR/PYL/RCARs, 5 SnRK2 and 4 ABC transporters. The amino acid involved in the
binding of PYR/PYL/RCAR and PP2C were strictly conserved among all sequences in
rice and barley. The consensus for the characteristic motifs of these genes was
generated for rice and barley. Of the above identified genes, only five barley PP2CAs,
three PYR/PYL/RCARs, two SnRK2s (subfamily II and III) and one each of ABCG25
and ABCG40 showed significant differential expression, while the rest were only
marginally affected. Putative PP2CAs, SnRK2s subfamily II, ABCG25 and ABCG40
were found to be up-regulated under all three abiotic stresses studied, whereas PP2CDs,
PYR/PYL/RCARs and SnRK2s subfamily III were down-regulated.

Epigenetic factors, such as histone modification and DNA methylation, play a
significant role in regulating gene transcription. The present study aimed to investigate
the distribution of one such modification, the tri-methylation of lysine 4 of histone H3
(H3K4me3) that is involved in gene activation, in barley during abiotic stress.
Chromatin immuno-precipitation and deep sequencing (ChIP-seq) identified 19,015 and
19,005 genomic regions exhibiting differential H3K4me3 under salt and drought
stresses, respectively. The ChIP-seq dataset was then searched for putative genes with a
priori annotations relating to ABA mediated signalling, which lead to identification of
three PP2CA, four PP2CD, two PYR/PYL/RCAR, three SnRK2 and two ABC
transporters type G. H3K4me3 was found at the 5’UTR, exon and intron regions of
PP2CAs, whereas H3K4me3 was found at exon, intron and 3’ regions of PP2CDs and
SnRK2s. For PYR/PYL/RCARs, H3K4me3 was found at 5’UTR, exon, intron and
3’UTR regions. On the other hand, H3K4me3 was restricted only to exon and intron
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regions of ABC transporters. Three PP2CAs, three PP2CDs, two PYR/PYL/RCARs,
three SnRK2s and one ABCG40 were found to be significantly de-methylated at H3K4
during abiotic stress, whereas one each of PP2CDs and ABCG25 were significantly tri-
methylated at H3K4.

This work also assessed the salt tolerance levels of sixteen barley varieties by key
physiological assays including relative water content, levels of ABA and Na+/K+ ratio.
The results showed a significant variation, ranging from highly salt tolerant to sensitive
varieties. Further, the differential expression of key genes in the ABA-mediated abiotic
stress tolerance pathway was analysed by quantitative real-time PCR of leaf RNA.
Importantly, an arbitrary ranking method was developed and applied to both
physiological and gene expression analysis. The tolerance ranking by gene expression
closely correlated that by physiological indices. Thus, expression analysis of the ABA
pathway can be used for rapid identification of potentially salt-tolerant barley varieties
before undertaking physiological studies.

The work provides insights into genome-wide effects of salinity and drought stresses
and is a new resource for the study of gene regulation in barley. The bioinformatics
workflow may be applicable to other non-model plants to establish their transcriptomes
and identify unique sequences. The in-depth expression and chromatin profiling of
genes involved in ABA mediated abiotic stress signalling make it possible to identify
candidates for future functional testing and development of tolerant plants through
genetic modification or breeding.

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Acknowledgements
Firstly, I would like to thank my principle supervisor, Prof. Mrinal Bhave for accepting
me into her research group. Her unwavering zeal for research has been a great source of
inspiration. I am also very indebted for all her support and guidance, the long hours
spent on problem-solving discussions and always read every line of everything that I
sent to her and gave her time selflessly. I benefitted immensely from your invaluable
wisdom and experience. Thank you for being my teacher. To my co-supervisor, Dr
Mark Ziemann, I am grateful for all your feed-backs, advice, kind words and constant
encouragement. Thank you for all the late sittings, guidance in performing mRNA-seq,
ChIP-seq and bioinformatics based analysis. To my co-supervisor, A/Prof Assam El-
Osta, I am grateful for allowing me to use facilities at his lab and support the work.

I am also grateful to Swinburne University for awarding me the SUPRA scholarship,
which allowed me to study without financial constraints. Thank You to Dr. Runyararo
Memory Hove, a fellow PhD student then for sharing the mRNA-seq I work with me.
Thank you to Soula Mougos, Ngan Nguyen, Chris Key, Dr Huimei Wu and Angela
McKellar for their advice, and skilled technical assistance. I would also like to thank
former and current PhD students, Dr Peter Gollan, Dr Abirami Ramalingam, Dr
Rebecca Alfred, Dr Saifone Chuaboonmee, Dr Shanthi Joseph, Ms. Azadeh Niknejad,
Ms. Gurvinder Kalra, Ms. Thi Hoang Yen Dang, Ms. Nadine Shagaghi, Mr. Avinash
Karpe, Ms. Snehal Jadhav and Mr. Rohan Shah for the support, advice and
encouragement.

To my, dearest mama, papa and Kanu (best parents and sister) thank you for all your
encouragement, patience and ever-lasting support to constantly excel. Thank you for
being outstandingly understanding throughout this journey. It really helped to keep me
going. It was just not possible without you guys.

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Publications arising from this work

Refereed journal articles
Ziemann M, Kamboj A, Hove R, Loveridge S, Bhave M (2013) Analysis of the barley
leaf transcriptome under salinity stress using mRNA-seq. Acta Physiologiae Plantarum
35: 1915-1924.

Ziemann M, Kamboj A, Bhave M (2013) OrthoRBH: A streamlined pipeline for mining
large gene family sequences in related species. Bioinformation 9: 267-269.

Kamboj A, Ziemann M, Bhave M (2014) Identification of salt tolerant barley varieties
by a consolidated physiological and molecular approach. Under review

Conference presentations
Kamboj A, Ziemann M, Bhave M (2011) Identification of key components of the
Abscisic acid signalling pathway in barley by next generation mRNA sequencing. 61th
Australian Cereal Chemistry conference, Coolangatta, New South Wales (4-8
September 2011) (Talk).

Kamboj A, Ziemann M, Bhave M (2012) Use of next generation sequencing to profile
the barley leaf transcriptome under salt stress. XXII International Congress on Sexual
Plant Reproduction, Melbourne (13-17 February 2012) (Poster).

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Declaration

I, Atul Kamboj, declare that the PhD thesis entitled ‘Molecular studies of abiotic stress
and abscisic acid mediated stress response pathway in barley’ is no more than 100,000
words in length, exclusive of tables, figures, appendices, references and footnotes. This
thesis contains no material that has been submitted previously, in whole or in part, for
the award of any other academic degree or diploma, and has not been previously
published by another person. Except where otherwise indicated, this thesis is my own
work.

Atul Kamboj
2014

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Abbreviations

Standard chemical symbols, genetic notations, gene names and SI units are used without
definition. Full gene names are given at the first mention
ABA abscisic acid
At Arabidopsis thaliana
BLAST Basic Local Alignment Search Tool
BDT Big Dye Terminator
bp base pair (s)
cDNA complimentary DNA
CDPK Ca2+-dependent protein kinase
CDS coding sequence
C-terminal carboxyl terminal
cv cultivar
DNA deoxyribonucleic acid
DNase deoxyribonuclease
dNTP deoxyribonucleotide
EDTA ethylenediaminetetraacetic acid
EST expressed sequence tag
gDNA genomic DNA
Hv Hordeum vulgare (barley)
IBSC International Barley Genome Sequencing Consortium
IPTG isopropyl β-D-1-thiogalactopyranoside
Kb kilobase pairs
LB Luria-Bertani media
mRNA messenger RNA
MW molecular weight
NCBI National Centre for Biotechnology Information
NGS next-generation sequencing
N-terminal amino terminal
ORF open reading frame
Os Oryza sativa (rice)
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PEG polyethylene glycol
PCR polymerase chain reaction
PM plasma membrane
PP2C Protein phosphatase 2C
PYR/PYL/RCAR Pyrabactin resistance/PYR like/ regulator component of ABA
receptor
RNA ribonucleic acid
RNase ribonuclease
Rpm revolutions per minute
RT room temperature
SNP single nucleotide polymorphism
SnRK2 Sucrose non fermenting 1 related kinase 2
Ta Triticum aestivum (common wheat)
TA transcript assembly
TAE tris acetate ethlenediaminetetraacetic acid buffer
TC tentative consensus sequence
TIGR The Institute for Genomic Research
TMH transmembrane helical domain
UV ultra-violet
X-gal 5-bromo-4-chloro-3-indolyl-b-D galactopyranoside

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Table of contents

Chapter1 General introduction and literature review
1.0 Introduction 2
1.1 Barley 2
1.1.1 Origin and evolution of cultivated barley 3
1.1.2 Importance of barley as crops 3
1.1.3 Current barley genome sequencing projects 4
1.1.4 World barley production 5
1.1.5 Barley production in Australia 5
1.1.6 Abiotic stresses limiting the growth of barley 5
1.2 Phytohormones 7
1.2.1 Abscisic acid 8
1.2.2 Discoveries in relation to abscisic acid 8
1.2.3 Biophysical and biochemical properties of abscisic acid 9
1.2.4 Regulation of ABA biosynthesis 11
1.2.5 ABA biosynthetic pathway 12
1.2.6 ABA catabolism 15
1.2.7 Self-regulation of ABA biosynthetic genes 17
1.3 ABA transporters for intercellular signalling 18
1.4 Regulatory proteins in abscisic acid signalling 20
1.5 ABA mediated regulatory components involved during plant 21
development
1.5.1 Flowering Time Control proteins A 21
1.6 ABA mediated regulatory components involved during abiotic 23
stresses
1.6.1 Membrane bound receptors 23
Soluble Receptors 25
1.6.2 The putative ABA Receptor 26
1.6.3 Pyrabactin resistance (PYR)/ PYR like (PYL)/ Regulatory 28
component of ABA receptor (RCAR)

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