ROLES OF AUXIN RESPONSE FACTOR TRANSCRIPTION FACTOR (GmARF) IN SOYBEAN AND STRIGOLACTONE IN ARABIDOPSIS IN RESPONSE TO DROUGHT AND SALT STRESSES

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIET NAM ACADEMY OF AGRICULTURAL SCIENCES HA VAN CHIEN ROLES OF AUXIN RESPONSE FACTOR TRANSCRIPTION FA

MINISTRY OF EDUCATION

AND TRAINING

MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIET NAM ACADEMY OF AGRICULTURAL SCIENCES

HA VAN CHIEN

ROLES OF AUXIN RESPONSE FACTOR TRANSCRIPTION FACTOR (GmARF) IN SOYBEAN AND

STRIGOLACTONE IN ARABIDOPSIS IN RESPONSE

TO DROUGHT AND SALT STRESSES

Major: Biotecnology Code: 62 42 02 01

SUMMARY OF THE DOCTORAL THESIS

HA NOI - 2016

The doctoral thesis was completed in:

VIETNAM ACADEMY OF AGRICULTURAL SCIENCE

Supervisors:

1 Assoc Prof Dr Nguyen Van Dong

2 Dr Tran Phan Lam Son

at hours, day month year

The thesis can be referred to at:

1 Vietnam National Library

2 Library of Vietnam Academy of Agricultural Science

3 Library of Agriculture Genetics Institute

INTRODUCTION

1 Imperativeness of the thesis

The rapidly increasing of the world population has made food security one of the most important global issues, including Vietnam In addition, the food productivity as well as the sustainable agriculture development is also burdened by climate change and environmental stresses (such as drought, flooding, unpredictable epidemics, soil erosion and environment pollutants ) Understanding the stress responses in plants is necessary to mitigate the problems via creating stress-tolerant crop cultivars It has been demontrated that transcription factors and phytohormones (such as Abscisic acid (ABA), auxin, cytokinins (CK), strigolactones (SLs)) play important roles in gene expression regulation and physiological activities in plant Therefore, our research is aimed to identify and characterize candidate genes, which can be used to engineer stress-tolerant transgenic crops, through 2 approaches First, we address our self to study gene expression regulation mediating by transcription factors, namely auxin response factor transcription factors family (ARF); second, we concentrate on discovery of candidate genes involved in hormone metabolism and signaling in plant stress responses To achieve this goal, in

this thesis, we conduct the experiments on model plant - Arabidopsis thaliana and an economically important crop – soybean (Glycine max) at the same time The thesis entiled:

“Roles of auxin response factor transcription factor (GmARF) in soybean and strigolactone in

Arabidopsis in response to drought and salt stresses”

3.2 Roles of strigolactone in response to drought and salt stress in Arabidopsis

4 Scientific and practical significane

4.1 Scientific significance

Our study is the first publication that provides the scientific data about the function of ARF TFs coding genes in soybean, as well as the essential role of SLs involved in environmental stress responses, especially drought condition Our results are considered as the reliable references for education and research

4.2 Practical significance

- This research allowed us to identify genetic components that contribute not only to improve drought tolerance of soybean, but also for in-depth functional analysis that ultimately leads

to the development of soybean cultivars with improved tolerance to drought

- This research also provided a promising approach to reduce the negative impact of abiotic stresses on crop productivity based on the modulation of SL content/response

5 The novelty of the thesis

- Characterization and functional analysis of GmARF genes under drought conditions

- Our results classified some tissue-specific of GmARFs which are able to apply for genetic

engineering to develop the drought tolerant cultivars

- Our results also provided the roles of strigolactones in response to drought and high salinity in plant

- Our results opened a promising application approach to enhance the drought/salt tolerance

by strigolactone

6 Structure of the thesis

The main contents of the thesis are presented in 107 pages, including 28 figures and 8 tables

185 literature references were used to cite for this thesis, including 9 in Vietnamese and 176 in English and 8 webpages

CHAPTER 1: OVERVIEW AND SCIENTIFIC BACKGROUND 1.1 Introduction

Soybean is one the world’s leading economic oilseed crops, providing the largest source of vegetable oil, proteins, macronutrients and minerals for human consumption and animal feed Unfortunately, the low productivity of soybean is mainly attributed to evironmental stresses, in cluding drought Plants, especially soybean, activate various mechanisms to adapt with drought stress In the last 20 years, many genes, including both regulatory and functional genes, have been

discovered in important crops, such as rice (Oryza sativa) and soybean (Glycine max), which are

involved in defense mechanisms and functioned in increasing drought tolerance However, the detail information and the relationship between the regulation of TFs and gene expression have not been elucidated yet Therefore, identification and characterization of TFs family in soybean is necessary to understand plant stress responses

On the other hand, high-salinity is also a typical stress that have influence to crop yield To elucidate plant responses mediated by the phytohormone – SLs signaling pathway to high-salinity

and drought stress conditions, we conduct the experiment on model plant – Arabidopsis thaliana Because Arabidopsis has many advantage characteristics (such as its short life-cycle, small size and

fully sequenced genome, easy to grow and transform, closely related to a major crop species) It has been shown that SLs play a typical role in regulation of many physiological processes in plant However, the involvement of SLs in plant drought and high-salinity stress responses has not been revealed yet So that to identify and study the relationship between SLs and drought and high-salinity stress responses is essential to compliment to the biological knowledge as well as the potential application in sustainable agriculture and cultivars improvement

In short, to evaluate the good candidates for genetic engineering, in this study, we will focus on the auxin response factor transcription factor family in soybean (GmARF) and the pivotal role of

SL in abiotic stress response in plant

1.2 The mechanisms of plant responses to environmental stresses

Plants are always exposed to environmental stresses, such as drought, salt, cold, light

or humidity, however, they are lack of movement ability, so that they have to respond and adapt to stresses to survive In response to environmental stresses, plants must activate

plenty of complexity pathways and mechanisms (Manavalan et al.,, 2009) In term of

phisiological, plants attempt to close the stomata, reduce respiration and photosynthesis frequency, water volume in tissues and plant growth, induce the root development to enhance the water-absorbance ability (Tran and Mochida, 2010) In term of molecular mechanisms, there are many genes that encoded for the stress responsive protein (Dorothea

stress signal perception to stress-responsive gene expression, various transcription factors (TFs) and

their DNA binding sites, the so-called cis-acting elements, act as molecular switches for

stress-responsive gene expression, enabling plants adapt better to the adverse stressor Futhermore, phytohormone also play a typical role in plant abiotic stress responses and contribute to the adaptative mechanisms

1.3 Introduction of the auxin response factor transcription factor family in plant

1.3.1 Concept and classification of transcription factor

be classified into one TFs family

1.3.2 Structure and Function of transcription factor

The structure of TFs contain several specific domains including DNA-binding domain

(DBD), trans-activating domain (TAD), and signal sensing domain (SSD)

Firsly, the basic function of TFs is the involvement in regulation of gene expression (Weinzierl, 1999) Then, the appearance of TFs can verify the specificity of the transcription from DNA to RNA as well as control the cell development (Lobe, 1992) One of the crucial role

of TFs is the participation in biotic and abiotic stress responses (Fujita et al.,, 2005; He et al.,, 2005; Hu et al.,, 2006; Yamaguchi-Shinozaki and Shinozaki, 2006; Fang et al.,, 2008;

Nakashima et al.,, 2009; Cutler et al.,, 2010; Fujita et al.,, 2011)

1.3.3 The research situation of transcription factor in response to enviroinmental stresses 1.3.4 The auxin response factor transcription factor

The phytohormone auxin has been known to regulate various aspects of plant growth and

development (Kieffer et al., 2010; de Jong et al., 2011; Lau et al., 2011;Ha et al., 2012) Numerous genetic and biochemical studies in Arabidopsis have provided evidence that transcriptional

regulation of auxin response genes are regulated by two large TF families, the auxin response factor (ARF) and the auxin/indole acetic acid (Aux/IAA) families.(Guilfoyle and Hagen 2007)

In Arabidopsis, there are 23 ARFs most of which contain a conserved N-terminal DNA-binding

domain (DBD), a variable middle transcriptional regulatory region (MR) and a carboxy-terminal

dimerization domain (CTD).(Perez-Rodriguez et al., 2010; Zhang et al., 2011) The DBD of ARFs

specifically binds to the conserved auxin response element (AuxRE, TGTCTC) in promoter regions of primary or early auxin-responsive genes The structure of the TRR of each ARF determines whether the ARF acts as an activator or repressor Activation domain (AD) of ARFs is usually enriched in glutamine (Q), serine (S) and leucine (L), while repression domain (RD) is enriched in either S, L and proline (P); S,

L and/or glycine (G) or S The ARF CTD is modular with amino acid sequence related to domains III and

IV in Aux/IAA proteins, making it function as a dimerization domain among the ARF CTDs or with several Aux/IAA proteins (Guilfoyle and Hagen 2007)

In Arabidopsis, mutations in the paralogous AtARF01 and AtARF02 resulted in delayed leaf senescence and floral organ abscission (Ellis et al.,, 2005; Lim et al.,, 2010) Similarly, AtARF07 and AtARF19 were shown to play a positive role in regulation of lateral root development (Fukaki et

al.,, 2006) Given the importance of ARF TFs in diverse biological and physiological processes, and

their potential applications for the development of improved stress-tolerant transgenic crop plants, the ARF TF families have been identified and characterized in a number of crop species, such as

maize (Zea mays) (Xing, Pudake et al., 2011; Wang, Deng et al., 2012), rice (Oryza sativa) (Jain and Khurana 2009; Song, Wang et al., 2009; Shen, Wang et al., 2010), sorghum (Sorghum bicolor) (Wang, Bai et al., 2010), tomato (Solanum lycopersicum) (Wu, Wang et al., 2011), Chinese cabbage (Brassica rapa) (Mun, Yu et al., 2012) and Citrus sinensis (Li et al.,, 2015)

1.3.5 Transcription factor in soybean

There are 61 transcription factor families in soybean containing 5035 TFs However, 857 TF genes have not study in characterization, functional analysis and their roles in soybean plant Some

TF families were determined the roles of them in response to environmental stresses, including

GmNACs (Le et al.,, 2011), GmNFYAs (Ni et al.,, 2013), GmWRKYs (Lou et al.,, 2013)

1.4 Introduction of strigolactone

1.4.1 Concept and classification of strigolactone

Strigolactones (SLs), a small class of carotenoid-derived compounds, were first characterized over 45 years ago as seed germination stimulants in root parasitic plants, such as

Striga, Orobanche and Phelipanche species (Xie and Yoneyama 2010; Ruyter-Spira, Al-Babili et al., 2013) SL was later reported as a root-derived signal that can enhance symbiosis between plants

and arbuscular mycorrhizal fungi (AMF) possibly through its ability to induce AMF hyphal

branching (Akiyama, Matsuzaki et al., 2005) More recently, SL was reported to play an important

role in the suppression of shoot branching by inhibiting the outgrowth of axillary buds (Umehara,

(+)-5-Deoxystrigol is thought to be the precursor of other strigolactones (Matusova, Rani et al., 2005)

1.4.3 Biosynthesis of strigolactone

Strigolactone, a small class of carotenoid-derived compounds, were found in many plant

species In Arabidopsis, MAX3 and MAX4 encode CCD7 (carotenoid cleavage dioxygenase 7) and

CCD8, respectively, which catalyze sequential carotenoid cleavage reactions to produce an

apo-carotenone called carlactone, a proposed SL precursor (Alder, Jamil et al., 2012) MAX1 is a

cytochrome P450 monooxygenase that is presumably involved in a catalytic step downstream of

MAX3 and MAX4 (Ruyter-Spira, Al-Babili et al., 2013)

1.4.4 Signaling of strigolactone

Strigolactone is transported and percepted by the specific system Two components of the

sitrolactone signaling are α/β-fold hydrolase, D14(Arite, Iwata et al., 2007; Arite, Umehara et al., 2009; Hamiaux, Drummond et al., 2012; Waters, Nelson et al., 2012) and F-box protein,

MAX2/D3/RMS4 (Dun, Hanan et al., 2009; Nelson, Scaffidi et al., 2011)

and arbuscular mycorrhizal fungi (AMF) possibly through its ability to induce AMF hyphal

branching (Akiyama, Matsuzaki et al., 2005) More recently, SL was reported to play an important

role in the suppression of shoot branching by inhibiting the outgrowth of axillary buds

(Gomez-Roldan, Fermas et al., 2008; Umehara, Hanada et al., 2008; Xie and Yoneyama 2010)

1.4.6 Potential application of strigolactone

Strigolactone is an important regulator for growth and development of plant Strigolactone and its functions could become a promising approach for developing the methods and new biotechnology for sustainable agriculture

CHAPTER 2: MATERIALS AND METHODS

2.1 MATERIALS, CHEMICALS AND MACHINES

2.1.1 Materials

The model plant cultivar - Williams 82 was used for study the Auxin-response factor transcription factor family in soybean

The max2-3 (SALK_092836), max2-4 (SALK_028336), 11 (SALK_023975),

max3-12 (SALK_015785), max4-7 (SALK_082552) and max4-8 (SALK_072750) mutants on Arabidopsis thaliana Columbia-0 genetic background (Col-0, wild-type, WT) were used in this

study These mutants are well-characterized by the previous study (Umehara, Hanada et al., 2008)

2.3 Methods

2.3.1 Plant growth, treatments and collection of tissues

2.3.1.1 Plant growth, treatments and collection of tissues for soybean

2.3.1.2 Plant growth, treatments and collection of tissues for Arabidopsis

2.3.2 Identification of the GmARF members and strigolactone-related genes in soybean

All predicted GmARF TFs in soybean were collected for manual analysis from various plant

TF databases, (Mochida, Yoshida et al., 2009; Mochida, Yoshida et al., 2010; Wang, Libault et al., 2010; Zhang, Jin et al., 2011) and only those GmARFs containing full open reading frames (ORFs),

as predicted by Glyma v1.1 (http://www.phytozome.net/soybean), were used for further analyses Genes with threshold of ≥ 90% nucleotide sequence identity were considered as duplicated genes

(Cheung, Estivill et al., 2003)

Strigolactone biosynthetic and signaling genes in soybean were predicted and classified by

BLAST method using the Arabidopsis homolog genes

2.3.3 Phylogenetic analysis

Sequence alignments of all identified ARFs from Arabidopsis and soybean were performed

with a gap open penalty of 10 and a gap extension penalty of 0.2 using ClustalW implemented on

MEGA 5 software (Thompson, Gibson et al., 1997; Tamura, Dudley et al., 2007) The alignments

were subsequently visualized using GeneDoc (http://www.nrbsc.org/gfx/genedoc/) as presented in Supplementary Fig S1 The sequence alignments were also used to construct the unrooted phylogenetic tree by the neighbor-joining method using MEGA 5 The confidence level of monophyletic groups was estimated using a bootstrap analysis of 10,000 replicates Only bootstrap values higher than 50% are displayed next to the branch nodes

2.3.4 Expression analyses of GmARF genes using microarray data and soybean Illumina

expression data

For tissue-specific expression analysis of GmARF genes, microarray-based expression data

for 68 types of tissues and organs housed in Genevestigator (https://www.genevestigator.com/)

were used.(Hruz, Laule et al., 2008) Illumina transcriptome sequencing data provided by Libault et

al.,(Libault, Farmer et al., 2010; Libault, Farmer et al., 2010) were also used to evaluate the

expression of GmARF genes in 8 tissues: nodules of 35-d-old soybean plants (harvested after

32 days of inoculation of the 3-d-old plants), 14-d-old shoot apical meristem (SAM), flowers (reproductive R2 stage), green pods (R6 stage), 18-d-old trifoliate leaves, roots (V2 stage), root tips and root hairs of 3-d-old seedlings

For expression analysis of GmARF genes in soybean leaves at V6 and R2 stages under

drought stress, which was imposed on the plants by withholding water from the pots until the

volumetric soil moisture content reduced to below 5%, microarray data recently published by Le et

al., was used.(Le, Nishiyama et al., 2012) At the V6 stage, soybean plants had six unrolled trifoliate

leaves and seven nodes, while at R2 full bloom stage, open flowers were found on any of the top two nodes on the main stem

2.3.5 RNA isolation, DNaseI treatment and cDNA synthesis

2.3.6 Dehydration treatment and microarray analysis in Arabidopsis

WT and max2-3 plants (30 plants/each) were grown in soil as described previously (Nishiyama, Watanabe et al., 2011) and in the drought tolerance assay Aerial portions of 24-d-old plants were

detached and exposed to dehydration by placing them on paper towels on a lab bench At the indicated time points, RWC of treated samples was measured (n = 5) Rosette leaves of 3 independent WT and

SL-signaling max2-3 mutant plants treated for 0, 2, 4 and 6 h were then collected to make three

biological replicates for microarray and expression analyses Purification of total RNA from plant

samples and microarray analysis using the Arabidopsis Oligo 44K DNA microarray (Version 4.0, Agilent Technology) were performed as described in (Nishiyama, Le et al., 2012) The raw microarray

data and a detailed protocol were deposited in the Gene Expression Omnibus database (GSE48949)

MapMan (http://mapman.gabipd.org) and VirtualPlant (http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/)

were used to analyze the data In some cases, ABA and stress-responsive gene expression was analyzed

using Genevestigator (https://www.genevestigator.com) or the Arabidopsis eFP browser

(http://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpWeb.cgi)

2.3.7 Assessment of drought, salt, and osmotic Sstress tolerance

2.3.7.1 Drought stress tolerance assay in Arabidopsis

2.3.7.2 Salt stress tolerance assay

2.3.7.3 Germination assay for salt stress for Arabidopsis:

2.3.7.4 Root growth assay under salt and osmotic stress conditions in Arabidopsis

2.3.7.5 Stomatal closure assay and measurement of stomatal density in Arabidopsis

2.3.7.6 Assay for sensitivity to ABA in Arabidopsis

2.3.8 qRT-PCR and statistical analysis of the data

qRT-PCR reactions and data analyses were performed according to previously published

methods.(Le, Nishiyama et al., 2011) The 60s and polyubiquitin 10 (UBQ10) genes were used as reference genes in soybean and Arabidopsis The delta-CT method was used to calculate initial amount of target genes When appropriate, a Student’s t-test (one tail, unpaired, equal variance) was

used to determine the statistical significance of the differential expression patterns between tissues and/or between treatments Considering the biological significance of the differential expression in this study, we adopted a cutoff value of 3-fold for tissue-specific expression, and 2-fold (at least at one time point) when analyzing stress induction or repression The expression levels were designated as “tissue-specific”, “induced” or “repressed” only if such differences met the above

criteria and passed the Student’s t-test

CHAPTER 3: RESULTS AND DISCUSSIONS

3.1 Roles of auxin response factor transcription factor family under drought stress conditions

in soybean

3.1.1 Identification of the GmARF members in soybean

Currently, there are three databases, namely SoybeanTFDB (Mochida, Yoshida et al., 2009), SoyDB (Wang, Libault et al., 2010) and PlantTFDB (Zhang, Jin et al., 2011) provide access to the

TF repertoire of soybean, which was obtained by genome-wide analysis of the Glyma v1.0 model

We were able to identify 51 GmARFs with annotated full ORF, and only these full-length (FL) GmARF TFs were used for further analyses

3.1.2 Chromosomal distribution, structural and phylogenetic analyses of the GmARFs

Among 51 GmARF genes, we found 17 duplicates; each pair shares ≥ 90% nucleotide

sequence identity The GmARF functions were predicted by using the phylogenetic analysis between GmARFs and their Arabidopsis ARF counterparts (AtARFs) (Figure 3.2)

Figure 3.1 Chromosomal distribution of 51 soybean GmARF genes identified in this study

and structural analysis of the GmARF proteins (A) Chromosomal distribution of GmARF genes with indication of percentages of GmARFs located on each chromosome (B) Graphical representation for chromosomal localization of GmARF genes Greek numbers indicate chromosome numbers (C) Graphical representation for domain organization of GmARF proteins

A typical ARF contains a DNA-binding domain (DBD), which consists of a B3 subdomain and an auxin-response (ARF) subdomain, a middle region (MR) and a carboxy-terminal dimerization domain (CTD)