Hormonal regulation of drought stress responses and tolerance in Brassica napus L.

Doctoral Dissertation

Hormonal regulation of drought

stress responses and tolerance in

Brassica napus L.

Department of Animal Science and Bioindustry

Graduate School, Chonnam National University

La Van Hien

February 2020

I

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................... IV

LIST OF FIGURES ........................................................................................... V

ABBREVIATIONS ....................................................................................... VIII

ABSTRACT ..................................................................................................... 1

1. GENERAL INTRODUCTION ...................................................................... 3

1.1. Brassica species and drought stress ......................................................... 3

1.2. ROS is a primary stress signal for metabolism and transduction signaling 3

1.2.1. ROS generation and metabolism ...................................................................... 3

1.2.2. ROS role in transmitting signal......................................................................... 4

1.3. Proline is an elicitor in plants response to drought stress .......................... 5

1.3.1. Proline accumulation in stress response.......................................................... 5

1.3.2. Proline metabolism essential for stress response and tolerance .................. 6

1.4. Redox balance: A tools of plant defense against drought stress ................. 7

1.5. Plant responses to drought stress: A matter of hormones regulation .......... 8

1.5.1. Abscisic acid pathway........................................................................................ 8

1.5.2. Salicylic acid pathway........................................................................................ 9

1.5.3. Antagonism between abscisic acid and salicylic acid pathways.................. 9

1.6. Carbon and nitrogen metabolism in stress tolerance ............................... 10

1.6.1. Sucrose as a component of carbon source ..................................................... 10

1.6.2. Proline integrates nitrogen assimilation pathway ....................................... 11

Objectives ..................................................................................................... 12

2. MATERIALS AND METHODS .................................................................. 15

2.1. Plant culture ......................................................................................... 15

2.2. The parameters analysis ......................................................................... 15

2.2.1. Leaf water potential, osmotic potential, photopigment measurement........15

2.2.2. Collection of phloem exudates and xylem sap..........................................16

2.2.3. Determination of O2

-

, H2O2, and lipid peroxidation ..................................16

2.2.4. ROS localization in situ............................................................................16

2.2.5. Measurements of cytosolic Ca2+ concentration .........................................17

2.2.6. Measurements of antioxidant enzyme activities .......................................17

2.2.7. Measurement of proline and Δ1-pyrroline-5-carboxylate...........................17

2.2.8. Sugars and starch analysis ......................................................................18

II

2.2.9. Sucrose phosphate synthase, cell wall invertase activity assays.................18

2.2.10. Determination of nitrate assimilation enzymes activity...........................19

2.2.11. Hormone analysis.................................................................................20

2.2.12. Glutathione and pyridine nucleotide assays ...........................................21

2.2.13. RNA extraction and quantitative PCR....................................................22

2.2.14. Statistical analysis .................................................................................22

CHAPTER 1:.................................................................................................. 23

SALICYLIC ACID ALLEVIATES DROUGHT STRESS RESPONSES IN

CHINESE CABBAGE..................................................................................... 23

Abstract ........................................................................................................ 23

1.1. Introduction ......................................................................................... 24

1.2. Experiment design ................................................................................ 25

1.3. Results ................................................................................................. 26

1.4. Discussion ........................................................................................... 31

CHAPTER 2:.................................................................................................. 36

SALICYLIC ACID INVOLVES IN REDOX CONTROL BY MODULATING

PROLINE METABOLISM UNDER DROUGHT STRESS ................................ 36

Abstract ........................................................................................................ 36

2.1. Introduction ......................................................................................... 37

2.2. Experiment design ................................................................................ 38

2.3. Results ................................................................................................. 39

2.4. Discussion ............................................................................................ 49

CHAPTER 3:.................................................................................................. 54

SALICYLIC ACID INVOLVES IN DROUGHT TOLERANCE BY MODULATING

CARBOHYDRATE METABOLISM ................................................................ 54

Abstract ........................................................................................................ 54

3.1. Introduction ......................................................................................... 55

3.2. Experiment design ................................................................................ 56

3.3. Results ................................................................................................. 57

3.4. Discussion ............................................................................................ 65

CHAPTER 4:.................................................................................................. 71

INTERPLAY BETWEEN PHYTOHORMONE AND HYDROGEN PEROXIDE IN

NITROGEN ASSIMILATION AND PROLINE SYNTHESIS UNDER DROUGHT

STRESS CONDITION .................................................................................... 71

Abstract ........................................................................................................ 71

4.1. Introduction ......................................................................................... 72

III

4.2. Experiment design ................................................................................ 73

4.3. Results ................................................................................................. 74

4.4. Discussion ............................................................................................ 83

GENERAL CONCLUSION ............................................................................ 94

REFERENCES ............................................................................................... 95

LIST OF PUBLICATIONS............................................................................ 114

I. Papers published in scientific journals ...................................................... 114

II. Papers submitted in peer-reviewed scientific journals ............................... 115

III. Papers in preparation ............................................................................. 115

KOREAN ABSTRACT ................................................................................. 117

IV

LIST OF TABLES

Table 1. List of the primers used in this study.

CHAPTER 1

Table 1.1. Effects of salicylic acid pretreatment on shoot biomass and osmotic

potential in leaves of the control or salicylic acid-pretreated plants

under well-watered or drought-stressed conditions.

Table 1.2. Changes in the antioxidative system, including superoxide dismutase

(SOD), catalase (CAT), guaiacol peroxidase (GPOD), and ascorbate

peroxidase (APOD) in the leaves of Chinese cabbage in the control or

salicylic acid pretreated plants under well–watered or drought

stressed conditions.

CHAPTER 2

Table 2.1. Effects of salicylic acid (SA) pretreatment on hormonal status in

leaves of Brassica napus under well-watered or drought-stressed

conditions.

Table 2.2. Effects of salicylic acid (SA) pretreatment on redox status in leaves of

Brassica napus under well-watered or drought-stressed conditions.

CHAPTER 3

Table 3.1. Effects of salicylic acid (SA) pretreatment on leaf biomass (g-1 plant,

FW), leaf water potential (MPa), and chlorophyll content (mg g-1 FW)

in the leaves of Brassica napus under well-watered or drought-stressed

condition.

Table 3.2. Effects of salicylic acid (SA) pretreatment on soluble sugars (mg g-1

FW) and starch (mg g-1 FW) in the leaves of Brassica napus under

well-watered or droughtstressed condition.

V

LIST OF FIGURES

GENERAL INTRODUCTION

Figure 1. The main compartment of H2O2 production in photosynthetic cell.

Figure 2. Model for proline synthesis pathway in plants.

CHAPTER 1

Figure 1. Experimental design of salicylic acid treated to Brassica rapa plants

under non-drought and drought stress conditions.

Figure 1.1. Effects of salicylic acid pretreatment on morphological changes (A),

chlorophyll (B), and carotenoid (C) in leaves of the control or salicylic

acid pretreated plants under well-watered or drought-stressed

conditions.

Figure 1.2. Effects of salicylic acid pretreatment on O2

- (A), H2O2 (B), and MDA

concentrations (C), and O2

-

localization (D) in leaves of the control or

salicylic acid-pretreated plants under well-watered or drought￾stressed conditions.

Figure 1.3. Effect of salicylic acid pretreatment on GSH (A), GSSG (B), Ratio of

GSH/GSSG (C), NADPH (D), NADP+ (E) and ratio of

NADPH/NADP+ (F) in leaves of the control or salicylic acid-pretreated

plants under well-watered or drought-stressed conditions.

Figure 1.4. Effect of salicylic acid pretreatment on proline content (A) and relative

expression of P5CSA (B), P5CSB (C) and PDH (D) in leaves of the

control or salicylic acid-pretreated plants under well-watered or

drought-stressed conditions.

CHAPTER 2

Figure 2. Experimental design of salicylic acid treated to Brassica napus plants

under non-drought and drought stress conditions.

Figure 2.1. Effects of salicylic acid (SA) pretreatment on plants morphology (A)

and osmotic potential (B) in leaves of Brassica napus under

well-watered or drought stress conditions.

Figure 2.2. Effects of salicylic acid (SA) pretreatment on the expression of SA

regulated gene NPR1 (A), PR-1 (B), ABA synthesis-related gene

NCED3 (C), ABA-signaling gene MYC2 (D), and JA-signaling gene

PDF1.2 (E) in leaves of Brassica napus under well-watered or drought

stress conditions.

VI

Figure 2.3. Effects of salicylic acid (SA) pretreatment on ROS accumulation in

leaves of Brassica napus under well-watered or drought stress

conditions.

Figure 2.4. Effects of salicylic acid (SA) pretreatment on antioxidant enzymes

activities and their encoding genes expression in leaves of Brassica

napus under well-watered or drought stress conditions.

Figure 2.5. Effects of salicylic acid (SA) pretreatment on the expression of redox

regulating genes in leaves of Brassica napus under well-watered or

drought stress conditions.

Figure 2.6. Effects of salicylic acid (SA) pretreatment on proline content and the

expression of proline metabolism-related genes in leaves of Brassica

napus under well-watered or drought stress conditions.

Figure 2.7. Heatmap analysis on treatment effect and correlations among the

variables measured at day 15 (after 10 days of drought including 5

days of SA pretreatment).

Figure 2.8. A proposed model for salicylic acid-mediated ROS, proline synthesis,

and redox modulation under drought.

CHAPTER 3

Figure 3.1. Effect of exogenous salicylic acid (SA) on chlorophyll synthase gene

(CHLG, A) and senescence-associated gene 12 (SAG12, B) expressions

in the leaves of control or SA-pretreated plants under well-watered or

drought-stressed condition.

Figure 3.2. Effect of exogenous salicylic acid (SA) on hormonal status and its

signaling related genes in the leaves of control or SA-pretreated plants

under well-watered or drought-stressed condition.

Figure 3.3. Effect of exogenous salicylic acid (SA) on the activities of sucrose

phosphate synthase (SPS, A) and cell wall invertase (CWINV, B) and

expression of hexokinase 1-related gene (HXK1, C) in the leaves of

control or SA-pretreated plants under well-watered or

drought-stressed condition.

Figure 3.4. Effect of exogenous salicylic acid (SA) on the expression of starch

degradation enzyme-related genes β-amylase 1 (BAM1, A) and α-amylase

3 (AMY3, B) in the leaves of control or SA-pretreated plants under

well-watered or drought-stressed condition.

Figure 3.5. Effect of exogenous salicylic acid (SA) on sucrose transportation in the

leaves of control or SA-pretreated plants under well-watered or

drought-stressed condition.

Figure 3.6. Effect of exogenous salicylic acid (SA) on osmotic potential (A) and

contribution of sucrose to osmotic potential (B) in the leaves of control

or SA-pretreated plants under well-watered or drought-stressed

condition.

VII

Figure 3.7. Heatmap analysis on the treatment effect and correlations among the

variables measured at day 15 (after 10 days of drought, including 5

days of SA pretreatment).

CHAPTER 4

Figure 3. Experimental design of salicylic acid, hydrogen peroxide, glutathione

and drought stress treated to Brassica napus plants.

Figure 4.1. Changes in plant morphology and redox state in the leaves of B.napus

exposed to salicylic acid or drought with or without H2O2 conditions.

Figure 4.2. Hormonal status change in the leaves of B.napus exposed to salicylic

acid or drought with or without H2O2 conditions.

Figure 4.3. Hormone defense-related gene expression in the leaves of B.napus

exposed to salicylic acid or drought with or without H2O2 conditions.

Figure 4.4. Nitrate and ammonium status and enzyme activity-related nitrogen

assimilatory pathway in the leaves of B.napus exposed to salicylic acid

or drought with or without H2O2 conditions.

Figure 4.5. Oxidative burst, Ca2+ content and its kinase sensors, and glutamate

receptor response in the leaves of B.napus exposed to salicylic acid or

drought with or without H2O2 conditions

Figure 4.6. Calcium sensors signaling, nitrate and ammonium transporters related

gene expression in the leaves of B.napus exposed to salicylic acid or

drought with or without H2O2 conditions.

Figure 4.7. Changes in proline metabolism in the leaves of B.napus exposed to

salicylic acid or drought with or without H2O2 conditions.

Figure 4.8. Proline transport in phloem, xylem and its accumulation in roots of

B.napus exposed to salicylic acid or drought with or without H2O2

conditions.

Figure 4.9. Pear correlations analysis among the variables in plants exposed to

salicylic acid or drought with or without H2O2 conditions.

Figure 4.10. Proposed model for assimilation and transport of nitrate and ammonium

modulated by ABA and/or SA regulation under salicylic acid or drought with

or without H2O2 treatments.

VIII

ABBREVIATIONS

ABA Abscisic acid GO Glycolate oxidase

AMY3 α-amylase 3 GOGAT Glutamate synthease

APR3 Adenosine phosphosulfate

reductase 3

GPX Glutathione peroxidase

APX Ascorbate peroxidase GR Glutathione reductase

ASC Ascorbate GRX Glutaredoxin

AsA Ascorbic acid GRXC9 CC-type glutaredoxin 9

AREB2 ABA-responsive element

biding 2

GS Glutamine synthease

BAM1 β-amylase 1 GshA Glutamate-cysteine ligase

BSO Buthionine sulfoximine GSH Glutathione

CAT Catalase GSSG Glutathione disulphide

CDPKs Calcium-dependent protein

kinase

GST Glutathione S-transferase

CIPKs Calcineurin B-like interacting

protein kinase

G6PDH Glucose 6-phosphate

dehydrogenase

CBL Calcineurin B-like HO- Hydroxyl radical

CHK5 CHASE receptor kinase 5 H2O2 Hydrogen peroxide

CHLG Chlorophyll synthase HXK1 Hexokinase 1-related gene

CK Cytokinin IAA Indole-acetic acid

Cu/Zn￾SOD

Copper/Zinc superoxide

dismutase

ICS1 Isochorismate synthase 1

2Cys-PRX 2-cys-peroxiredoxins JA Jasmonic acid

CWINV Cell wall invertase MAPKs Mitogen-activated protein

kinase

DAB Diaminobenzidine MDHAR Monodehydroascorbate

reductase

DCPIP Dichlorophenolindophenol MYC2 MYC2 transcription factor

DHAR Dehydroascorbate reductase Mn-SOD Manganese superoxide

dismutase

γ-ECS γ-glutamylcysteine synthetase NBT Nitroblue tetrazolium

GA Gibberellic acid NCED3 9-sis-epoxycarotenoid

dioxygenase

GA3 Gibberellin 3 NO3

- Nitrate

GDH Glutamate dehydrogenase NO2

- Nitrite

GID1 GA Insensitive DWARF 1 NH4

+ Ammonium

Glu Glutamate NR Nitrate reductase

GLRs Glutamate receptor-like NRTs Nitrate transporters

IX

NRT NADPH-thioredoxin reductase RCAR Regulatory component of

ABA receptor

NPR1 Nonexpressor of

pathogenesis-related protein 1

ROS Reactive oxygen species

OAT Ornithine aminotransferase RuBisCO Ribulose 1,5-bisphosphate

carboxylase/oxygenase

PDF1.2 Plant defensin 1.2 RuBP Ribulose 1,5-bisphosphate

PDH Proline dehydrogenase SA Salicylic acid

PEPc Phosphoenolpyruvate

carboxylase

SAG12 Senescence-associated gene12

3PGA 3-phosphoglycerate SAT2.1 Serine acetyltransferase

POX Peroxidase SOD Superoxide dismutase

PMS Phenazine methosulfate SnRK2.2 Sucrose non-fermenting

related kinase 2

PR Pathogenesis-related gene SPS Sucrose phosphate synthase

ProT1 Proline transporter 1 SUT Sucrose transporters

PRX Peroxiredoxin 1O2 Oxygen singlet (1O2)

PP2Cs Clade A phosphatases type-2C O2

•- Superoxide radical

PYR1 Pyrabactin resistance 1 OXI1 Oxidative signal –inducible 1

P5C Pyrroline-5-carboxylic acid TGA1 TGACG sequence-specific

binding proteins 1

P5CS 1- pyrroline-5-carboxylate

synthetase

TRXh5 Thioredoxin-h5

P5CDH P5C dehydrogenase XO Xanthine oxidase

qPCR Quantative polymerase chain

rection

WRKY40 WRKY transcription factor 40

RBOHs Respiratory Burst Oxidase

Homologues

1

Hormonal regulation of drought stress responses and tolerance

in Brassica napus L.

La Van Hien

Department of Animal Science and Bioindustry

Graduate School, Chonnam National University

(Supervised by Professor Kim, Tae-Hwan)

ABSTRACT

This study aimed to characterize hormonal regulation of drought stress response

and tolerance, especially in the exert synergistic or antagonistic effects on various

biological processes. In chapters 1 to 3, the characterization of salicylic acid (SA) role

modulation of drought tolerance was accessed. Chapters 4, studies on the hormonal

regulation of nitrogen metabolism involved in proline accumulation, and as well as

the glutamate synthesis in drought stress tolerance.

In chapters 1 to 3, identifies the role of SA-mediated transcriptional regulation

in a possible interaction with other hormones, ROS, antioxidant, proline, sugars is

involved in drought tolerance, particularly for the redox modulation. Drought stress

induced ROS and proline accumulation, as well as the enhancement oxidized state

of redox. Pretreatment of 1.5 mM SA substantially ameliorated the negative effect of

drought to Chinese cabbage by further activation of ascorbate peroxidase activity

scavenged O2

-

, H2O2, and lipid peroxidation. According to proline and glutathione

were further accumulated by SA-pretreated plants under drought stress. The

detailed underlying mechanism interplay between SA, ROS and proline in redox

control was assessed in Brassica napus. Treatment with 0.5 mM SA scavenged

drought-induced O2

- accumulation, but not H2O2. SA-mediated NRP1 controlled

TRXh5 and GRXC9 redox signaling transcriptionnals response by which SA reset of

redox state and showing an increase in proline synthesis, with an antagonistic

depression of ABA- and/or JA-signaling. On another hand, drought induced mainly

hexose levels with depressed expression of hexokinase gene HXK1 and, in part, to