Hormonal regulation of drought stress responses and tolerance in brassica napus

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 dro

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

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 ROSlocalization 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

2.2.9 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

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

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

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

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

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 H2O2conditions

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 H 2 O 2 treatments

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

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

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

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

increased sucrose content with the highest expression of ABA-dependent sucrose

signaling genes SnRK2.2 and AREB2 The highest sucrose accumulation due to

induce sucrose phosphate synthase (SPS) activity and starch degradation

enzymes-related BAM1 and AMY3 expression conferred by SA-pretreated plants

These results in considerable a mechanism shifting from ABA to SA role in sucrose accumulation adjusted osmotic potential or alleviated leaf senescence

To insight into hormones regulate proline accumulation in integration with nitrogen metabolism that deserves further characterization in chapter 4 Drought

trigger ABA inhibited ABI1 expression and upregulated CIPK23 and CBL9 in a

cytosolic Ca2+-dependent manner rather than in 0.1 mM SA- and/or 0.1 mM H2O2

treatments These complex signals negative effects on NO3- transport in xylem-phloem systemic and NO3- assimilation, in a part, to increase NO3-

accumulation Treatment with SA, H2O2, and drought limited NH4+ accumulation by activation of the NH4+ assimilation conferred by alternative glutamine synthase (GS)/glutamine oxoglutarate aminotransferase (GOGAT) cycle by glutamate dehydrogenase (GDH) activity The additional proline accumulation under drought stress accompanied to glutamate synthesis in the highest GDH activity, which involved in H2O2 and CBL9 signaling in the ABA regulatory pathway

Taken together, the results reveal that phytohormones involved in the modulation of carbohydrate and nitrogen metabolism in relation to phenotypic plant response to drought stress Among those hormones, antagonistic interaction between ABA and SA exhibited a more vital role to activate ROS, sucrose, proline

and redox state in terms of drought tolerance in Brassica napus

1 GENERAL INTRODUCTION

1.1 Brassica species and drought stress

Brassica species is a vegetable group provides the oil and protein in food for animal

and human Among, Brassica napus is one of the important agro-economic crops that

also source material to take oil and alternative fuel in industry bioenergy Oil rapeseed has much rich of polyunsaturated fatty acids, is considered for the healthy ingredient In animal feeds, canola oil as well a product because of it high protein content approximate 50% Like other field crops, oilseed rape is particularly assailable to environmental stress (Zhu et al., 2016; Elferjani and Soolanayakanahally, 2018)

Drought is one of the environmental stresses that a negative effect the growth and development, and the productivity of crops (Sinaki et al., 2007) In the case of canola, generally, drought stress directly effected on the flowering and grain filling stages, results in a severe reduction in both seed yield and seed quality (Sinaki et al.,

2007; Elferjani and Soolanayakanahally, 2018) The Brassica has been known the

vegetables are soft, succulent, and consist of more than 85% water Therefore, water deficit as well as excessive water significantly influences the yield and quality of this crop In decade recent year, through modern manipulation engineering allows to create the crops against stress and understand the defense mechanism that regulates

at the molecular level will be necessary for stress tolerance in Brassica napus (Zhang

et al., 2015; Zhu et al., 2016; Shokri-Gharelo and Noparvar, 2018)

1.2 ROS is a primary stress signal for metabolism and transduction signaling 1.2.1 ROS generation and metabolism

It is well reviewed that reactive oxygen species (ROS) are reactive forms of oxygen molecule including the hydroxyl radical (HO-), superoxide (O2-), hydrogen peroxide (H2O2) and singlet oxygen (1O2) ROS generated in plasma membrane NADPH oxidase (in plants called Respiratory Burst Oxidase Homologues [RBOHs], or peroxidases and oxidases in the apoplast (Wrzaczek et al., 2013; Waszczak et al., 2018) It has been known the cell organelles consist of the chloroplast, mitochondria,

and peroxisomes are major sources of intracellular ROS production in plant cells through photosynthesis, respiration, and photorespiration (Figure 1.1)

Figure 1.1 The main compartment of H2O2 production in photosynthetic cell GO, glycolate oxidase 3PGA, 3-phosphoglycerate POX, peroxidase RuBisCO, ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP, ribulose 1,5-bisphosphate SOD, superoxide dismutase XO, xanthine oxidase (from Mhamdi et al., 2010b)

In plants, ROS as a maker in plants acclimated to stresses through a change of internal or external metabolism In chloroplastic ROS most important in signal transduction event that mediates plant-environment interaction Hydrogen peroxide formation in chloroplastic and outcome to change of antioxidant system can be provided signals from environmental and metabolic changes to nucleus in

chloroplast retrograde signaling (Estavillo et al., 2013; Noctor and Foyer, 2016;

Waszczak et al., 2018)

1.2.2 ROS role in transmitting signal

It has been widely reviewed that H2O2 serves as a signaling molecule, enabling H2O2

to be a second messenger (Rejeb et al., 2014; Sies, 2017; Winterbourn, 2017) In ROS role in signal transduction implies that coordinated function of regulation networks

to maintain ROS in a delicate balancing between generations and scavenging of ROS, and to regulate ROS response and subsequent downstream process (Mittler, 2004) Numerous studies showed that the ROS generation contributed to an increase of antioxidant enzymes activity in abiotic stresses in different plant species (Lee et al., 2008; Mhamdi et al., 2010b) Intrinsic to this regulation is ROS production and

signaling that integrated with the action of calcium (Ca2+) signal, mitogen-activated protein kinase (MAPKs), and hormones in stresses response (Xia et al., 2015) Indeed, an increase of cytosolic Ca2+ was first active an upstream signal that controls ROS production via the modification of NADPH oxidase by calcium-dependent protein kinase (CDPKs) (Gao et al., 2013; Dubiella et al., 2013) Recent studies proposed Ca2+ can be acted in downstream ROS signaling (Wrzaczek

et al., 2013) Interaction of ROS and Ca2+ signal also activate antioxidant defense (Hu

et al., 2007) and mitogen-activated protein kinase (MAPKs) (Foyer and Noctor, 2005)

are involved in hormone production and signaling (Seyfferth and Tsuda, 2014;

Prodhan et al., 2018) Under stressed-plants, results in the activation of both ROS (H2O2 mainly) and phytohormone signaling acclimation stress (Xia et al., 2015; Choudhury et al., 2017; Hieno et al., 2019)

1.3 Proline is an elicitor in plants response to drought stress

1.3.1 Proline accumulation in stress response

Proline is an amino acid widely accumulated in abiotic stress response including drought (Lee et al., 2013; An et al., 2013), salt (Székely et al., 2008; Rejeb et al., 2015), oxidative (Yang et al., 2009) and pathogen stresses (Qamar et al., 2015) Proline has been known as a compatible osmolyte to improve drought stress tolerance With the role for an osmoprotectant, stabilizing membrane and detoxifying tissues of excess

N (Kim et al., 2004; Lee et al., 2013) In plants, proline accumulation in stresses is controlled by the balance of synthesis and catabolism Under water-stress, proline synthesis is activated and its degradation repressed, while re-watering triggered the opposite regulation (Szabados and Savoure, 2010; Krasensky and Jonak, 2012; Bhaskara et al., 2015)

Figure 1.2 Model for proline synthesis pathway in plants There are two ways

activates from ornithine d-aminotransferase (OAT) and glutamate (Glu)

Proline is synthesized by glutamate and ornithine pathway (Figure 1.2), but mainly from glutamate during abiotic stress (Székely et al., 2008) In both ways occurs in cytosol and chloroplast compartment, with 1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine d-aminotransferase (OAT) are two key enzymes involved in proline biosynthesis Another, proline catabolism occurs in mitochondria via the sequential action of proline dehydrogenase or proline oxidase (PDH) and P5C dehydrogenase (P5CDH), which converts P5C to glutamate (Szabados and Savoure, 2010; Kishor and Sreenivasulu, 2014) In addition, proline transport has been contributed to proline accumulation in stress condition For example, proline highly accumulated in the phloem sap under water-stressed plants (Girousse et al., 1996; Lee et al., 2009)

1.3.2 Proline metabolism essential for stress response and tolerance

Several studies reflect that proline predominance in the mechanism resistance other than osmotic adjustments, such as specific morphological and physiological modification (Deuschle et al., 2004; Chen et al., 2011; Lee et al., 2013) Indeed, the excessive amount of proline by exogenous application or endogenous over-production showed that the exhibition retarded for cell death (Deuschle et al., 2004) Available evidence indicates that high proline intracellular induces cell death

by increasing ROS production (Chen et al., 2011) The generation of ROS might be

derived from P5C–proline cycle or proline degradation (Hellmann et al., 2000; Rejeb

et al., 2014) In PDH or P5CDH overexpressing in Arabidopsis mutants, the

hypersensitive response would be increasing the chance of electrons generated from proline oxidation to glutamate, resulting in an elevated rate of electron transfer to

O2 leading to ROS production (Rejeb et al., 2014) Thus, proline degradation is an

important regulator of cellular ROS homeostasis and could influence the endogenous signal (Szabados and Savoure´, 2010) Several reviews have been emphasized that the assumption that proline accumulation in stressed plants has signaling molecule function in relation to metabolic regulatory pathways (Szabados

& Savoure´, 2010; Liang et al., 2013; Rejeb et al., 2014) An interaction between proline and ROS, antioxidant, redox balance and hormonal were observed in the response to abiotic stress (Chen et al., 2011; Sharma et al., 2011) For example, proline metabolism integrated with the alteration of ROS and ABA signaling pathway to trigger stress tolerance (Verslues et al., 2007; Székely et al., 2008) In

Arabidopsis mutant and transgenic plants, Deuschle et al (2004) reported that seem

to be altered SA level or SA signaling might be related to oxidative stress-induced

by proline toxicity

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

The stimulation of ROS often attached to highly activated scavenging system related antioxidants Glutathione (GSH) and ascorbic acid (AsA) are the major nonenzymatic antioxidants present in a plant cell, which maintains the intracellular redox homeostasis Of these, GSH is a thiol with the role in stabilizing enzymes of the Calvin cycle, and keeping AsA in the reduced form in chloroplast (Noctor et al., 2012) Further, maintenance of GSH in high GSH/GSSG ratios is critical for a redox balance due to AsA generated in AsA-GSH cycle (Mhamdi et al., 2010a; Pyngrope et al., 2013), involvement of the best described rout for H2O2 metabolism in oxidative stress (Foyer and Noctor, 2003; Noctor et al., 2012) Indeed, it can join in the ascorbate-glutathione cycle in which H2O2 reduction is ultimately linked to NADPH oxidation via ascorbate and glutathione pools Alternately APX activity could be coupled to NADPH oxidation independently of GSH via MDHAR activity While GSH can chemically reduce DHA by an enzymatic link between AsA and GSH pool

is catalyzed by DHAR (Noctor et al., 2012) Overexpression of this enzyme underlines the importance of GSH-dependent ascorbate pools in physiological processes like the regulation stress tolerance (Mhamdi et al., 2010b) The ascorbate-glutathione (AsA-GSH) cycle plays an important role in detoxification of

H2O2 and protects from oxidative stress

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

Plants exposed to various stresses result in the activation of both H2O2 and phytohormone signaling (Xia et al., 2015; Choudhury et al., 2017) In water stress, abscisic acid (ABA) is a central hormone signaling with regulation of plants response, and also participates in the complex interactions with or the other hormones including jasmonic acid (JA), salicylic acid (SA), cytokinin (CK),

gibberellic acid (GA), idole-3- acetic acid (IAA) (Yang et al., 2003; Yasuda et al., 2008;

Zhao et al., 2014; Muñoz-Espinoza et al., 2015; Huang et al., 2018; Wang et al., 2018)

1.5.1 Abscisic acid pathway

ABA has been well-known stress phytohormone, which is rapidly accumulated and induces the stress-responsive genes expression and the activation of plant’s cellular physiological adaptation to drought stress (Weiner et al., 2010; Fujita et al., 2011; Muñoz-Espinoza et al., 2015) Under stressed-plants subsequently induces ABA-dependent responses ABA is perceived by the pyrabactin resistance 1 (PYR1) and PYR1-like (PYL)/regulatory component of ABA receptor (RCAR) proteins (hereafter referred to as PYLs) (Ma et al., 2009; Park et al., 2009) PYLs are able to respond to ABA and inhibit clade A PP2Cs in an ABA-dependent or –enhanced manner, resulting in the activation of sucrose non-fermenting 1-related protein kinase 2 s (SnRK2s) (Fujii et al., 2009; Zhao et al., 2014; Yoshida et al., 2015) ABA-activated SnRK2s regulate the expression of ABA-responsive genes through phosphorylation of transcription factors, such as ABA-responsive element-binding factors (ABFs) (Furihata et al., 2006) and phosphorylate other substrates related to many processes In the ABA signaling pathway, SNF1-related kinase 2s (SnRK2s) are central regulators that mediate ABA-responsive transcription factors and genes

to activate ABA-mediated physiological processes (Yoshida et al., 2002; Fujita et al.,

2009; Umezawa, 2009; Kulik et al., 2011; Yoshida et al., 2015) In unstressed plants, the target of rapamycin (TOR) kinase phosphorylates PYLs to prevent activation of stress responses (Wang et al., 2018)

1.5.2 Salicylic acid pathway

SA plays an important role in the regulation of plant developmental and response to biotic and abiotic stress (Hayat et al., 2010; Miura et al., 2014) This reviewed elucidated that SA regulates the basal resistance to abiotic stresses and as a central key signal in regulating disease resistance (Seyfferth and Tsuda et al., 2014; Herrera-Vásquez et al., 2015; Islam et al., 2017) In several Arabidopsis mutants showed that endogenous SA accumulation and induction of SA-mediated disease resistance and water-deficit tolerance (Miura et al., 2013) In this case, SA involved

in the transduction signal system through SA-inducible genes nonexpressor pathogenesis related gene 1 (NPR1) and pathogenesis-related (PR) proteins (Mou et al., 2003) NPR1 is the central regulator in SA signaling which stimulates NPR1 interaction with TGA2 and TGA3, enhance its binding to TGA and WRKY boxes, forming a trans-activating complex for RNA polymerase II a large set of PR gene activation (Herrera-Vásquez et al., 2015) SA-induced drought tolerance mediated

induction of PR genes Members of PR genes such as PR1, -2, and -5 genes are

widely used as marker genes for SA-mediated drought tolerance (Liu et al., 2013).

1.5.3 Antagonism between abscisic acid and salicylic acid pathways

Plants perceived stress signals often change in level of different hormones Among, ABA and SA are known to play important roles in plant responses to stress Several studies with the mutant plants indicated an enhanced basal resistance to abiotic stress mediated ABA (Muñoz-Espinoza et al., 2015), which suggests that ABA can interact with SA in the physiological responses to stress conditions (de Torres Zabala et al., 2009) Antagonistic hormonal interaction is involved in regulating defense responses (Anderson et al., 2004; Martínez-Medina et al., 2017) In a network metabolic interactions refer to hormone crosstalk that was observed in both abiotic and biotic stress (Berensa et al., 2019) The cost of ABA and SA interplay in plant defense well documented In the SA-ABA antagonism, NPR1 a component of

systemic acquired resistance (SAR) signaling downstream of SA, likely plays a

critical role in modulating the SA-mediated suppression of ABA signaling or vice

versa (Yasuda et al., 2008; Muñoz-Espinoza et al., 2015) In addition, studies on

tomato plants adaptation osmotic stress depend on SA concentration and also highlighted the difficulties in experimentally recreating the fine-balance and timing for endogenous levels of hormone For instance, Mur et al (2006) indicated that the low concentration reveals hormone synergistically, whereas a high concentration of one hormone antagonized the other one Despite this well-documented reciprocal inhibition, the relationship between SA and ABA is not always antagonistic Interestingly, SA treatments lead to an increase of ABA and proline in the barley leaves (Bandurska and Stroinski´, 2005; Wang et al., 2018) However, the relationship between SA and ABA signals in water deficit remains unknown

1.6 Carbon and nitrogen metabolism in stress tolerance

1.6.1 Sucrose as a component of carbon source

Sugars, especially sucrose are the main sugar for plant development, as a signaling

entity in the regulation of stress tolerance (Zheng et al., 2010; Ma et al., 2017; Sakr et

al., 2018) An accumulation of sugar is derived by hexose biosynthesis through hexokinase (HXK) phosphorylation activity (Moore et al., 2003; Ruan, 2014), sucrose synthesis catalyzed by sucrose phosphate synthase (Baxter et al., 2003), which either

by starch degradation and sugar transport (Durand et al., 2018) The best-known sucrose is a signaling molecule in phloem loading (Gong et al., 2015), which might control photo-assimilated partitioning between source-sink (Ruan, 2014) In the downstream of sucrose sensing, calcium and calcium-dependent protein kinase (CDPKs) supported transmit sucrose signal (Toyota et al., 2018), interact with bZIPs transcription factor (Sakr et al., 2018) The complex interplay of SnRK1/2 with the clade of PP2C phosphatase has been demonstrated in the ABA signaling pathway, among protein kinase encompass common downstream targets for metabolism signaling It is noteworthy that at least part of the gene responses related to the SnRK1 pathway might be independent of HXK1 signaling All these findings reveal that the question of sucrose sensors and sucrose signaling in the potential hormone

regulation are still open, it suggests that other factor such as sucrose synthease and sucrose –degradading enzyme) (Nguyen et al., 2015) or BZR1-BAM (a transcription factor contacting a non-catalytic-amylase (BAM)-like domain (Soyk et al., 2014) might be part of this mechanism

1.6.2 Proline integrates nitrogen assimilation pathway

It has been established as an accumulation of sugars and proline in abiotic stress tolerance Increase the evidence reveals that proline as a source of nitrogen and carbon metabolism (Kim et al., 2004; Verslues and Sharma, 2010) From proline synthesis pathway, glutamate was considered the substrate used for this process There is evidence that it can be important in supplying glutamate for proline synthesis (Kan et al., 2017) Indeed, the studies in amino acid labeling during stress suggest glutamate as a main precursor of proline (Szekely et al., 2008; Sharma and Verslues, 2010), which also mediated ammonium assimilation (Kim et al., 2004; Lee

et al., 2009, 2013) Glutamate availability is ultimately controlled by nitrogen assimilation, primarily through the action of glutamine synthetase (GS), which use glutamate and ammonium to produce glutamine Glutamate is then generated by action of glutamate synthetase (GOGAT) For example, wheat seeding in nitrogen starvation significantly decreased of NR, GS and GOGAT at both the transcript levels and enzyme activity (Balotf et al., 2016) Another possible route of generating glutamate in mitochondria is glutamate dehydrogenase (GDH), which can either directly produce glutamate from ammonium Furthermore, nitrogen assimilation or transport is attributed to proline accumulation involved in hormone signaling under water deficit still obscures

Based on the knowledge, the present context with a view to understading the hormones network involves in metabolite change during plant water-stressed response It is important to decipher their crosstalk with ROS, sucrose, proline and redox status underlines the emerging mechanism in term of drought stress tolerance

Table 1 List of the primers used in this study

BrF-box Bra006317 F: 5′- GAGATAAGTCGCTTCCTACCG -3´ R: 5′- TGTTCCCATTGCCCTGTG -3´ BrP5CSA XM-009143589 F: 5′- GGTCATGCTGATGGAATCTG -3´ R: 5′- GCATTACAGGCTGCTGGATA -3´ BrP5CSB XM-009118014 F: 5´- TGGACAGAGCAGTCTCATGG -3´ R: 5′- AACCCTCATTTTCAGCATCG -3´ BrPDH EU186335 F: 5′- TCAGCGATCTTGTTCAATGC -3´ R: 5′- ATTGTGTCGTGGACTGGTGA -3´ BnActin AF111812 F: 5´- GATTCCGTTGCCCTGAAGTA -3´ R: 5´- GCGACCACCTTGATCTTCAT -3´ BnABI1 MF284702.1 F: 5´- GACTCCAGAGCCGTTCTTTG -3´ R: 5´- TCTTGACATGGCGAGAACAC -3´ BnAREB2 HE616526.1 F: 5´- AGATTGCTGCCAAAGATGCT -3´ R: 5´- CACCTCTTATCCCAGGACCA -3´ BnAMY3 XM_013846160.2 F: 5´- GGTTACCTCCACCGACAGAA -3´ R: 5´- GTTCAGACGCCCTCCAAATA -3´ BnAMT1.1 AF188744.1 F: 5´- CGTCTTGCTAGGCCCTAATG -3´ R: 5´- ATAGCGAAGCCAAGTTGCAT -3´ BnBAM1 XM_013852497.2 F: 5´- GAAGGTGGGGCTAAAGGTTC -3´ R: 5´- GCACGCATGAAATCAGAGAA -3´ BnCu/ZnSOD AY970822 F: 5´- TGCTAATCGTCATGCTGGAG -3´ R: 5´- CTCCCTTTCCAAGGTCATCA -3´ BnCAT1 JN163870 F: 5´- GATCCTGCGGATGAGGATAA -3´ R: 5´- AAGCAGCTTGTCATCCGAGT -3´ BnCHLG XM_013788949.1 F: 5´- CTACGAACTCGTCAC CAAAG -3´ R: 5´- AGGTCCAAACCAATGATTCT -3´ BnCPK5 JX122911.1 F: 5´- TGGAAGCGTGTCATTCTCTG -3´ R: 5´- TATAACACCAGCGGTCCACA -3´ BnCaM4 EU487185 F: 5´- CCTTGCTCCTGTCAAGGAAG -3´ R: 5´- ATTGGCTGTTGTGTCAACCA -3´ BnCBL9 NM_001315757.1 F: 5´- GACATGGACTGCACTGGCTA -3´ R: 5´- ACGAAGTCGCTCCATTCTGT -3´ BnCIPK11 XM_013893362.2 F: 5´- ACGTATCGGTGCCCTAGATG -3´ R: 5´- AACCAACCTCGTCGTGAAAC -3´ BnCIPK23 XM_013807024.2 F: 5´- AGATGAGCGTTGCGAAATCT -3´

R: 5´- CCTCGCGAACTTGACCTTAG -3´ BnCHK5 KF621033.1 F: 5´- GTTGCAGACCGGAAGAGAAG -3´

R: 5´- GCATGAACGGTGTGAAAATG- 3´ BnGRXC9 XM013875950 F: 5´- GTAACCCCAGCGGTTCTTGA -3´

R: 5´- ACCACAAAGCTCCAACATCC -3´ BnGLR1.3 XR_002655614.1 F: 5´- TGCTGGTGATAACCGTCAAG -3´

F: 5´- GAACCCACTCCACTGCTGTT -3´

BnHXK1 XM_013797259.1 F: 5´- TTCTCCGGATTTGAAGGTTG -3´

R: 5´- GTCTTTCGGTGCGTCTCTTC -3´ BnICS1 XM_013845172.1 F: 5´- TCAATCCCAGAACGAGATCC -3´

R: 5´- GACAGAAACCTTCGGATGGA -3´ BnMAPK6 XM_013888918.1 F: 5´-CCACACAAAACCGAAAGTCT-3´

R: 5´-CCTTTGCCAATAGGCATGAT-3´

All primers were designed directly from sequences in the public database, F: Forward; R: Reverse

BnMYC2 XM013880351 F: 5´- ACCAAACGTCTCGAAAATGG -3´ R: 5´- TGTCAACGAGCAAGAGGATG -3´ BnMnSOD EU487185 F: 5´- CCTTGCTCCTGTCAAGGAAG -3´ R: 5´- ATTGGCTGTTGTGTCAACCA -3´

BnMYB2.1 XM013787637 F: 5´- AAGAAGCTTCCACGTCCAGA -3´ R: 5´- TCTATGCCATTGCCAACGTA -3´ BnNRT1.5 EV220114 F: 5´- CAATCTACTTGATCGCATTG -3´ R: 5´- CCTGTAGGCTTGAAGTTTCG -3´

BjNRT1.7 KT119583.1 F: 5´- CGTCCGGTAGTTTCTCCAAA -3´ R: 5´- CCCAGTCTCCAAGCGTTC -3´

BnNAC55 NM_001315826.1 F: 5´- CGGGTTTAACCGAACAGAAA -3´ R: 5´- TGTTGCTGCGTCTTATCGTC -3´

BnNADPH

oxidase XM_013847449.1 F: 5´- CACCTCTCCCTCTTTCTGT -3´ R: 5´- CGTTGGGGTTTTGTCGCTAT -3´

BnNCED3 HQ260434 F: 5´- GGAGTGCTTCTGCTTCCATC -3´ R: 5´- TTCGAGGTTGACTTGCTCCT -3´

BnNPR1 EF613226 F: 5´- TGAGAACATTGCCAAGCAAG -3´ R: 5´- CAACAGCAAAATGGAGAGCA -3´ BnOXI1 XM_013843315.2 F: 5´- GCCACCAACTACCACAGGAT -3´ R: 5´- CCCAAGCAATGACAAAACCT -3´ BnPR1 XM_013826324 F: 5´- GAGTAGCGCCGACTTTTCTG -3´ R: 5´- TTTGCCACATCCAATTCTCA -3´

BnPR2 X69887.1 F: 5´- GAAGCTTTGAGGATGGCTTG -3´ R: 5´- TTCCAGGGTTAGTCGTGGTC -3´

BnPDF1.2 AY884023.1 F: 5´- TGTTTTTGCTGCTTTTGGTG -3´ R: 5´- TCGAATGCACTGATTCTTGC -3´

AtProT1 X95737.1 F: 5´- ACATGTTTAACGGCCCACTC -3´ R: 5´- GCCGACATAAGCGTGCTAAC -3´

BnSAG12 XM_013821610.2 F: 5´- AGAGAATACCAAACCAAACCGAA -3´ R: 5´- GCAACTCCCAAAATCTCAGGG -3´ BnSnRK2.2 LK937699.1 F: 5´- TGAAGATGAGGCTCGGTTCT -3´ R: 5´- TGCCATCATATTCCTGACGA -3´

BnSUT1 XM_013855840.2 F: 5´- GATCCTGCGGATGAGGATAA -3´ R: 5´- AAGCAGCTTGTCATCCGAGT -3´

BnSUT2 XM_022708891.1 F: 5´- TGAGAACATTGCCAAGCAAG -3´

R: 5´- CAACAGCAAAATGGAGAGCA -3´ BnSUT4 XM_022699588.1 F: 5´- AAGAAGCTTCCACGTCCAGA -3´

R: 5´- CGCGCATATACGTACATACTCACAA -3´ BnWRKY28 NM_001315647.1 F: 5´- GCCAGAGGAAACGAGAGTTG -3´

R: 5´- TATTTTTGAAGGCCTTTTGG -3´

2 MATERIALS AND METHODS

2.1 Plant culture

Chinese cabbage (Brassica campestris var pekinensis) was grown in 2-L pots that

contained a mixture of soil and perlite (70:30, v/v) Plants were cultivated in a glasshouse with day: night mean temperature of 25:20 °C, a relative humidity of 50-70%, and 16-h-day/8-h-night Natural light was supplemented with 200 μmol m-2

s-1 at the canopy height for 16 h day-1 Complete nutrient solutions containing the macroelements and microelements were continuously supplied until plants are ready to harvest (Lee et al., 2013)

Brassica napus (cv Capitol) seeds were sown in bed soil in a tray Plants with the

4-leaf stage were transferred to 2-L pots containing a mixture of soil and perlite (70:30, w/w) and grown in a greenhouse A complete nutrient solution was continuously supplied to plants (Lee et al., 2015) Natural light was supplemented

by metal halide lamps, which generated c 200 μmol photons m-2s-1 at the canopy height for 16 h per day

2.2 The parameters analysis

2.2.1 Leaf water potential, osmotic potential, photopigment measurement

Leaf water potential was evaluated immediately as the petiole xylem pressure potential using a pressure chamber (PMS Instrument Co., Corvallis, OR USA) To detection of osmotic potential, fresh leaves were frozen in liquid nitrogen and then

allowed them to thaw, followed by centrifugation at 13000 g for 15 min The

collected sap was used for measuring osmolality by using a vapor pressure osmometer (Wescor 5100; Wescor Inc., Logan, UT), as previously described by Lee

et al (2013) The osmotic potential (Ψw) of leaves was measured according to Bajji et

al (2001), using the formula: osmotic potential (Ψw) = - c (mosmol/kg) × 2.58 × 10-3 For total chlorophyll and carotenoid content, fresh leaves were immersed in 10 mL

of 99% dimethyl sulfoxide (Richardson et al., 2002) After 48 h, the absorbance of the supernatants was read at 480, 510, 645 and 663 nm by using a microplate reader (Synergy H1 Hybrid Reader; Biotek, Korea)

2.2.2 Collection of phloem exudates and xylem sap

Phloem exudates were collected using the facilitated diffusion method by using ethylenediaminetetraacetic acid (EDTA), according to Lee et al (2009b) The forth fully extended leaf was cut and immediately immersed in 20 mM EDTA solution (pH 7.0) for 5 min, and then the EDTA solution was discarded The leaf was rinsed, transferred to a new tube containing 5 mM EDTA solution, and kept for 6 h in a growth chamber with 95% relative humidity under dark condition The xylem sap was collected from the cut stems under high pressure by using an exhausted syringe The phloem exudates and xylem sap were stored at -20 C for further analysis

2.2.3 Determination of O 2- , H 2 O 2 , and lipid peroxidation

The detection of O2- was made by hydroxylamine oxidation (Lee et al., 2009a) About 200 mg sample of leaf was homogenized in 50 mM potassium phosphate

buffer (pH 7.8) and centrifuged at 12, 000 g for 10 min at 4°C A mixture of 0.5 mL of

enzyme extract and 1 mL of 10 mM NH2OH-HCl was incubated for 60 min at 25°C,

and then reacted with 1 mL of 17 mM p-aminobenzene sulfonic acid and 7 mM (N-(1-Naphthyl) ethylenediamine) at 25°C for 20 min The absorbance was

determined at 530 nm and calculated from a standard curve prepared by NaNO2 For H2O2 determination, leaves were extracted with phosphate buffer and reacted with titanium chloride The absorbance was immediately read at 410 nm and calculated using the coefficient of absorbance, 0.28 µM-1 cm-1 (Lee et al., 2009a) The malondialdehyde (MDA) content were measured according to Lee et al (2009a)

2.2.4 ROSlocalization in situ

For O2- visualization, leaf discs were immersed in 0.1% solution of nitroblue tetrazolium (NBT) in K-phosphate buffer (pH 6.4), containing 10 mM Na-azide Visualization of H2O2, leaf disc were immersed in 1% solution of 3,3’-diaminobenzindine (DAB) in Tris-HCl buffer (pH 6.5) Then were vacuum-infiltrated for 60 min, and illuminated until the appearance of dark spots, which are characteristic of the blue formazan precipitate for O2•− or brown spots from DAB and H2O2 reaction Detection O2- and H2O2 used a light microscope (Leica

2.2.5 Measurements of cytosolic Ca 2+ concentration

Cytosolic Ca2+ levels were estimated using aequorin luminometry detection (Tanaka

et al 2010) with some modification Briefly, 200 mg leaves were extracted in reconstitution buffer (1 mM KCl, 1 mM CaCl2, and 10 mM MgCl2, with pH adjusted

to 5.7 using Tris-base) and centrifuged at 12,000 ×g for 10 min For each sample, the

resulting supernatant was collected 100 µl and incubated for 30 min with 1 µl of 0.1

mM coelenterazine-h in the 96-well plate Incubation was performed in the dark to facilitate binding between coelenterazine-h (Sigma) and aequorin After incubation,

an equal volume of 2 M CaCl2 (in 30% ethanol, v/v) was added to each sample to discharge the remaining aequorin Calcium concentration was determined by

luminescence, according to Knight et al (1996)

2.2.6 Measurements of antioxidant enzyme activities

The antioxidant enzymes were extracted with 100 mM KPO4- buffer (pH 7.5), as described by Lee et al (2013) The activity of superoxide dismutase (SOD; EC 1.15.1) was determined by measuring its ability to inhibit the photoreduction of NBT (Lee

et al., 2009a) One unit of enzyme activity was defined as the amount of enzyme required to inhibit 50% of the NBT photoreduction in comparison with tubes lacking the plant extract Catalase (CAT; EC 1.11.1.6) activity was assayed using the method

of Lee et al (2013) Degradation of H2O2 content was calculated using the coefficient,

ε = 36 mM-1 cm-1 One unit of enzyme activity was defined as the amount of enzyme that causes the degradation of 1 mM H2O2 per min

2.2.7 Measurement of proline and Δ 1 -pyrroline-5-carboxylate

Approximately 200 mg fresh leaves were homogenized in 3% sulfosalicylic acid and centrifuged at 13, 000  g for 20 min The supernatant was mixed with concentrated

acetic acid and acid ninhydrin reagent prepared by dissolving 1.25 g in 30 mL of 6

M H3PO4 and 20 mL of acetic acid The mixture was boiled for 1 h and then added to toluene The concentration of proline in the toluene fraction was determined by measuring the absorbance at 520 nm with a microplate reader (Synergy H1 Hybrid Reader, Biotek, Korea) Proline concentration was calculated with L-proline as the standard (Lee et al., 2013) P5C content was determined according to method

described by Mezl and Knox (1976) The supernatants were mixed with 10 mM of 2-aminobenzaldehyde dissolved in 40% ethanol Then, the mixture was incubated at

37 oC for 2 h to develop the yellow color The absorbance was measured at 440 nm and calculated by using an extinction coefficient 2.58 mM-1 cm-1

2.2.8 Sugars and starch analysis

Soluble sugar was extracted from 200 mg of fresh leaves by using 1 mL of 80% ethanol Sucrose content was determined according to the method of Van Handel (1968) For this, 100 µL of the supernatant was mixed with 100 µL of 30% KOH and boiled at 95 °C for 10 min After cooling, 3 mL of 0.15% anthrone reagent was added

to the mixture and incubated at 40 °C for 15 min Absorbance was recorded at 620

nm and calculated using sucrose as the standard Glucose content was determined using the method of Koehler (1952) The reaction mixture containing 100 µL of the supernatant and 0.2% anthrone reagent (w/v) was boiled at 100 C for 8 min After cooling, absorbance was measured at 625 nm and calculated using glucose as the standard Fructose content was measured using the method of Davis and Gander (1967) The supernatant was mixed with 1.5 mL of 12 N HCl and 0.05% resorcinol reagent and incubated at 77 C for 8 min Absorbance was recorded at 420 nm and calculated using fructose as the standard

Starch content was measured using the method of Baxter et al (2003) with some modification After sugar extraction, the pellet was dried, suspended with 1 mL of distilled water, and heated at 100 °C for 10 min The pH of the solution was adjusted

to 5.1 by adding 400 µL of 200 mM acetate buffer Starch was digested by adding 100

µL of reaction mixture containing 0.2 U of amyloglucosidase and 40 U of α-amylase

to the solution and then incubating at 37 °C for 24 h After the mixture was

centrifuged at 14000 g for 2 min, glucose was measured as mention above Starch

concentration was estimated as 0.9 × glucose concentration

2.2.9 Sucrose phosphate synthase, cell wall invertase activity assays

Sucrose phosphate synthase (SPS) activity was measured by quantifying the fructosyl moiety of sucrose by using the anthrone reagent (Baxter et al., 2003) About

200 mg of fresh leaves was homogenized with 1.5 mL of 100 mM K-PO buffer

solution (pH 7.0) containing 2 mM phenylmethylsulphonyl fluoride and 1 mM EDTA Samples (100 µL) were mixed with 50 µL of buffer (50 mM HEPES pH 7.5, 20

mM KCl, and 4 mM MgCl2) containing 12 mM uridine 5ʹ-diphosphoglucose (UDPGlc), 10 mM fructose-6-phosphote (Fru6P), and 40 mM of glucose-6-phosphate (Gluc6P) (1:4 ratio with Fru6P:Glu6P), and incubated at 25 C for 20 min The reaction was stopped by boiling at 95 C for 5 min, and the mixture was centrifuged

at 12000 g at 4 C for 5 min The absolute amounts of sucrose-6-phosphate obtained during incubations were calculated by measuring the amount of sucrose in the supernatant as mentioned above One unit of enzyme activity was defined as the amount of enzyme that causes the formation of 1 µg sucrose used per min

Cell wall invertase (CWIVN) activity was measured according to the method of French et al (2014) Fresh sample (100 mg) was homogenized in extraction buffer (50 mM Hepes-NaOH (pH 7.5) containing 8 mM MgCl2, 2 mM EDTA, 12.5%

glycerol, and 50 mM 2-mercaptoethanol (ratio 1: 5) and centrifuged at 12000 g for 10

min The pellets were collected and washed with water twice They were suspended with 1 mL of 100 mM acetate buffer (pH 4.8) containing 1 M NaCl and kept on ice with gentle agitation for 1 h The mixture was centrifuged, and 100 µL of the supernatant was collected and mixed with 100 mM sodium acetate buffer (pH 4.8) containing 20 mM sucrose for invertase activity assay The reaction mixture was incubated at 37 C for 1 h, and the reaction was stopped by boiling for 3 min The

resultant glucose from the reactions was determined as mentioned above One unit

of enzyme activity was defined as the amount of enzyme that causes the formation

of 1 µg glucose used per min

2.2.10 Determination of nitrate assimilation enzymes activity

The activity of nitrate reductase (NR) was measured as the rate of NO2- production according to a method previously described (Kaiser and Lewis, 1983) with some mofification About 200 mg fresh leaves were homogenized in 1 mL of 100 mM phosphate buffer (pH 7.8) containing 0.1 mM Na2EDTA, 0.1 mM PMSF, 1% PVP One hundred microliter was mixed with 50 µL 100 mM KNO3, 250 µL 100 mM phosphate buffers The reaction was initiated by addition 100 µL of 2 mM NADH After incubation at 28 C for 15 min, rapid addition of 0.5 mL of 1% sulfanilamide

(w/v) and 0.5 mL of 0.02% N-(1-napthyl) ethylendiamine dihydrochloride (NED) The reaction developed color at room temperature in 30 min and record absorbance

at 540 nm One unit of NR activity was defined as the amount of enzyme that the liberation of 1 µmol of NO2- per min

Glutamine synthase (GS) activity, 200 mg fresh leaves was extracted in 1 mL Tris-HCl buffer (pH 7.5) containing 10 mM Na2EDTA, 0.1 mM PMSF, and 1% PVP Reaction was initiated by addition of 100 µL of supernatant extracted to 400 µl assay mixture contained 50 mM glutamate, 10 mM ATP, 30 mM MgSO4, 20 mM

NH2OH-HCl and 80 mM Tris-HCl (pH 8.0) The reaction was incubated at 28 C for

15 min, the color reagent development by adding 1 mL of 2.5% (w/v) FeCl3 and 5% (w/v) trichloroacetic acid (TCA) in 1.5 mM HCl and absorbance at 540 nm One unit

of GS activity was expressed as 1 µmol glutamylhydroxamate produced per min Glutamine 2-oxoglutarate aminotransferase (GOGAT) activity, fresh leaves was homogenized in 1 mL of 80 mM Tris-HCl (pH 8.0) containing 100 mM KCl, 5 mM

Na2EDTA, 1 mM PMSF, 0.1% 2-mercaptoethanol, and 0.05% triton X-100 The assay mixture contained 5 mM 2-oxoglutarate, 1 mM EDTA, 100 mM KCl, 1 mM NADH and 100 µL enzyme extract The reaction was started by adding 20 mM L-glutamine with the decrease in absorbance at 340 nm for 5 min GOGAT activity was calculated using the NADH standard curve

Glutamate dehydrogenase (GDH) activity, using 200 mg fresh leaves was extracted in the same buffer analysis for GS activity GDH activity was determined

in 100 µL reaction mixture containing 100 mM Tris-HCl (pH=8.0), 20 mM alfa-ketoglutarate, 200 mM NH4C1, 10 mM CaCl2, 0.2 mM NADH and 90 µL H2O The reaction start by adding 10 µL enzyme extracted The absorbance at 340 nm was monitored for 5 min, 30 °C (kinetic used), and the activity of GDH was expressed as nmol NADH mg-1 Pro·min-1

2.2.11 Hormone analysis

Quantitative analysis of SA, ABA, and JA in leaf tissues was performed according to Pan et al (2010) Fifty milligram of fresh leaves in 2 mL tube was frozen in liquid nitrogen and ground using a Tissuelyser II (Qiagen) The ground sample was extracted with 500 µL of extraction solvent 2-propanol/HO/concentrated HCl

(2:1:0.002, v/v/v) containing d6-ABA, d6-SA and H2JA s an internal standard for ABA, SA and JA, respectively 24 h at 4oC Dichloromethane (1 mL) was added to the

supernatant and then centrifuged at 13 000 g for 5 min at 4oC The lower phase, which was taken into a clean screw-cap glass vial, was dried under nitrogen and resolved in pure methanol Complete dissolved extract ensured by vortexing and sonicating was transferred to a reduced volume liquid chromatography vial Hormones were analyzed by a reverse phase C18 Gemini high-performance liquid chromatography (HPLC) column for HPLC electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) analysis The chromatographic separation of hormones and its internal standard from the plant extracts was performed on an Agilent 1100 HPLC (Agilent Technologies), Waters C18 column (15092.1 mm, 5l m), and API3000 MSMRM (Applied Biosystems) using a binary solvent system comprising 0.1% formic acid in water (Solvent A) and 0.1% formic acid in methanol (Solvent B)

at a flow rate of 0.5 mL/min The solvent gradient was used as follows: 0 min (99.9%

A, held for 0.5 min), 1 min (80% A), 1.5 min (40% A, held for 0.5 min), 2.5 min (35%

A, held for 0.5 min), and 4 min (99.9% A, held for 1.8 min)

2.2.12 Glutathione and pyridine nucleotide assays

To extract glutathione, approximately 200 mg fresh leaves were homogenized in 5%

of 5-sulfosalicylic acid and centrifuged at 12,000  g for 10 min at 4°C Glutathione

content was determined by microplate assay using the GSH/GSSG Kit GT40 (Oxford Biomedical Research Inc)

Determination of oxidized and reduced pyridine nucleotide content was conducted as described by Queval and Noctor (2007) For the NAD(P)+ and NAD(P)H extraction, 200 mg fresh leaves were homogenized with 0.8 mL of 0.2 N HCl and 0.2 M NaOH, respectively One hundred microliters of extracts was heated

at 95oC for 1 min and stopped in an ice-bath For the NAD(P)+ assay, the supernatant was neutralized by 0.2 M NaOH to a final pH of 5-6, and NAD(P)H was neutralized by 0.2 N HCl to a final pH of 7-8 Forty microliters was added to the reaction mixture containing 0.1 M HEPES (pH 7.5) that consisted of 2 mM Na2EDTA, 1.2 mM dichlorophenolindophenol (DCPIP), 20 mM phenazine methosulfate (PMS), and 10 mM glucose-6-phosphate for NADPH/NADP+, and to the reaction mixture

replaced glucose-6-phosphate by 15 µL absolute ethanol for NADH/NAD+

measurement The reaction started by adding 2 µL glucose 6-phosphate dehydrogenase (G6PDH, 200U) for NADPH/NADP+ or 10 µL alcohol dehydrogenase (ADH, 2500U) for NADH/NAD+ measurement The content of NAD(P)+ and NAD(P)H were determined by the standard curve with contents 1-100 pmol

2.2.13 RNA extraction and quantitative PCR

Total RNA was isolated from 200 mg leaf tissues using an RNAiso Plus (Takara) The cDNA was synthesized using the GoScript Reverse Transcription System (Promega) Gene expression levels were quantified on a light cycle real-time PCR detection system with Bio-Rad with SYBR® Premix Ex TaqTM (Takara, DALIAN) The PCR reaction were initiated at 95 oC for 5 min, and then followed by 45 cycles of

95 oC for 30 s, 54 oC – 60 oC for 30 s (depend on target primers), 72 oC for 30 s, and final extension at 72 oC for 5 min The gene-specific primers used for the qRT-PCR application are given in supplementary data Table S1 The qPCR reaction was performed in duplicate for each of three independent samples The relative expression level of target genes was calculated from threshold values (Ct) using the actin gene as an internal control Quantification of the relative transcript levels used the 2-∆∆CT method (Livak and Schmittgen, 2001)

2.2.14 Statistical analysis

A completely randomized design was used with three replicates for four treatments and sampling date Analysis of variance (ANOVA) was applied to all data Duncan’s multiple range test was employed to compare the means of separate replicates All statistical tests were performed using SAS 9.1 (SAS Institute Inc.,

2002-2003) Differences at P < 0.05 were considered significant The analysis of

heatmap was conducted using MetaboAnalyst 3.0 (http://www.metaboanalyst.ca)

To investigate the role of salicylic acid (SA) in the tolerance mechanism with regard

to cellular redox control and proline metabolism Chinese cabbages were pretreated

or not with SA for 7 days, and then each group was grown under drought stressed

or well-watered conditions for 14 days Drought decreased osmotic potential, and chlorophyll and carotenoid content The negative effect of drought on these parameters was substantially ameliorated in SA-pretreated plants Drought increased O2-, H2O2, and malondialdehyde (MDA) content in non-SA pretreated plants relative to that of SA-pretreated plants Superoxide dismutase, catalase, guaiacol peroxidase, and ascorbate peroxidase were highly activated in drought-stressed plants, whereas these substances were further activated in SA-pretreated plants under drought stress Drought significantly decreased the GSH/GSSG ratio and the NADPH/NADP+ ratios, whereas these factors were recovered to the control levels by SA pretreatment SA pretreatment significantly increased proline content by up-regulating pyrroline-5-carboxylate synthase (P5CS) gene expression and down-regulating proline dehydrogenase (PDH) gene expression compared to that of non-SA pretreated plants, under drought conditions These results indicate that SA pretreatment improves drought stress tolerance by maintaining redox homeostasis and activating proline biosynthesis

1.1 Introduction

Salicylic acid (SA) is a known phytohormone related to phenolic compounds involved in the regulation of plant defenses against pathogens (He et al., 2005) SA also plays an important role in modulation of plant defense against various abiotic stresses (Hayat et al., 2008; Khan et al., 2013; 2014) SA promotes protective reactions involving photosynthesis, leading to increased CO2 assimilation, RuBisCO activity, stomatal conductance, and chlorophyll and carotenoid content (Rao et al., 1997; Habibi, 2012) SA application to water deficient plants reduces cell membrane damage in leaves, because it reduces cellular lipid peroxidation and H2O2

accumulation (Khan et al., 2014) In addition, reduction of H2O2 accumulation is associated with enhancing the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPOD), when plants were pretreated with SA (Saruhan et al., 2012) Besides, activities of glutathione (GSH) and ascorbate (AsA) cycle enzymes, ascorbate peroxidase (APOD) and glutathione reductase (GR), were also modulated with SA in plants to drought stress (Nazar et al., 2015) GSH and AsA are major non-enzymatic antioxidants and directly involved in detoxification of H2O2, and its high efficiency is responsible for the alleviation of oxidative stress under abiotic stress (Asada, 2006) GSH is also well recognized in enhancing stress tolerance by regulating redox molecules, because reduced GSH/oxidized glutathione (GSSG) couple indirectly interacts with the NADPH/NADP redox couple (Noctor et al., 2011; Nazar et al., 2015) In previous works, SA application significantly increased AsA and GSH contents and transcripts

of the genes encoding AsA-GSH cycle enzymes (Li et al., 2013) Although the role of

SA in plant stress resistance has long been known, the physiological mechanism regarding this stress tolerance remains largely unknown

Proline accumulation occurs in many plants in response to environment stress, including drought (Lee et al., 2009; Kubala et al., 2015) It was considered as a compatible solute that maintains turgor pressure and protects cellular structure (Hare and Cress, 1997) In addition, proline accumulation enhances plant resistance against adverse environmental conditions by detoxifying ammonia accumulation and reactive oxygen species (ROS), and activating antioxidant enzymes such as CAT,

POD and SOD (Iqbal et al., 2014) Thus, many studies focused on modification of proline metabolism to enhance stress tolerance Overexpression of proline biosynthetic enzymes, pyrroline-5-carboxylate (P5C) synthase 1 (P5CS1) and P5C reductase (P5CR), results in higher proline content and lower H2O2 content, followed by a lower content of malondialdehyde (MDA), a marker of

membrane-lipid peroxidation (Zhu et al., 1998) Conversely, the p5cs1 mutants

reduced proline level and decreased stress resistance, increasing ROS damage under salt stress (Székely et al., 2008) Proline dehydrogenase (PDH) and P5C dehydrogenase (P5CDH) enzymes catalyze conversion of proline back to glutamine These enzymes activities are suppressed in photosynthetic tissue and improved resistance to salinity and freezing (Nanjo et al., 1999) Recently, a link between proline metabolism and cellular redox status has been observed in drought-stressed plants In proline biosynthetic process, NADPH is used as an electron donor, thereby generating NADP+ which is used as an electron acceptor in photosystem I (PSI) in thylakoid membrane resulting in decrease of singlet oxygen production (Sharma et al., 2011; Giberti et al., 2014) Therefore, proline synthesis is thought to help maintain a proper NADPH/NADP+ ratio Proline is also accumulated by SA, activating the enzymes of proline metabolism (Khan et al., 2013) However, how proline synthesis is regulated and interacts with SA on drought stress tolerance has not yet been fully documented

In the present study, we hypothesized that pretreatment with SA will contribute

to the maintenance of cellular redox homeostasis through the regulation of proline biosynthesis under drought stress conditions To test this hypothesis, plants were sprayed with SA for 7 days and then they were exposed to drought stress for 14 days ROS content, antioxidant enzymes activity, redox status (including GSH and GSSG, and pyridine nucleotides), proline biosynthesis and degradation were assessed

1.2 Experiment design

Approximately 10 weeks later, plants were treated without (control) or with salicylic acid (SA) For the SA treatment, 30 mL of 1.5 mM SA was sprayed to 6

plants, whereas same volume of water was sprayed to 6 plants for the control for 7 days After then, the half of control and SA-pretreated plants were exposed to drought stress Daily irrigation of 150 mL or 30 mL of nutrient solution per pot was applied to the well-watered (control) and drought stress treatment, respectively, for

14 days For drought-stress treatment, 30 mL of nutrient solution, containing same amount of nutrient as applied to the control pot, was administered (Figure 1) Afterwards, leaves were harvested and stored in a deep freezer (-80°C) for further analysis The change in plant morphology, biomass, stress symptom development and biochemicals were measured in SA or drought treatment condition

Figure 1 Experimental design of salicylic acid treated to Chinese cabbage plants

under non-drought and drought stress conditions

to that in the control (Table 1.1)

Table 1.1 Changes in the fresh weight of Brassica rapa in the control or salicylic acid

pretreated plants under well–watered or drought stressed conditions

Values are presented as means ± SE for n = 3 Values in a vertical column followed

by different letters are not significantly different (P > 0.05) according to Duncan’s

multiple range test

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 There are significant differences at P ≤ 0.05 according to Duncan’s multiple range test Values

are presented as means ± SE for n = 3

Chlorophyll and carotenoid contents in drought-stressed plants without SA pretreatment were significantly decreased by 22.7% and 21.8%, respectively, compared to that in the control (Figure 1.1B, C) In contrast, decrease rate was lower

in SA-pretreated plants (-9.2% compared to that of the control)

SA pretreatment suppressed oxidative stress induced by drought stress

O2- content in SA pretreated plants was increased by 56.7% regardless of drought stress, whereas it was further increased 170% in drought-stressed plants without SA pretreatment, compared to that of the control (Figure 1.2A) This result was

Treatments Fresh weight

(g -1 plant)

Dry weight (g -1 plant)

Osmotic potential (MPa)

consistent with accumulation of O2- in situ, indicated by deep brown spots (Figure

3d) Drought stress increased H2O2 content by 102.8% in non-SA pretreated plants and 74% in SA-pretreated plants compared to that of the control (Figure 1.2B) Similarly, content of MDA, a product of lipid peroxidation, was strongly increased

by 52.9% in drought-stressed plants without SA pretreatment, whereas an induction

of 31.6% occurred in SA-pretreated plants under drought stress conditions (Figure 1.2C)

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 There are significant differences at P ≤ 0.05 according to Duncan’s multiple range test Values

are presented as means ± SE for n = 3 Localization of O2- is indicated by black parts Histochemical staining was performed by the NBT method and the picture was taken under a microscope (40X)

To scavenge ROS (O2- and H2O2), activities of antioxidant enzymes such as SOD, CAT, GPOD, and APOD were significantly increased 160.9%, 71.8%, 90.6%, and 62.6%

in drought-stressed plants, respectively, compared to that of the control (Table 1.2) However, they were further increased by SA pretreatment, except for GPOD These results indicated that SA pretreatment proved effective in lowering oxidative stress under drought 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

U: unit Values are presented as means ± SE for n = 3 Values in a vertical column

followed by different letters are not significantly different (P > 0.05) according to

Duncan’s multiple range test n.s: Non-significant The asterisk indicates significant

difference compared with the control: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

SA pretreatment is involved in the regulation of redox balance under drought stress conditions

Drought stress significantly decreased GSH (reduced glutathione) content, but slightly increased GSSG (oxidized glutathione) content, compared to that of the control (Figure 1.3A, B) In contrast, SA pretreatment of drought-stressed plants significantly induced GSH and GSSG content by 65.3% and 70%, respectively, compared to that of the control The ratio of GSH to GSSG was remarkably reduced

by 69.7% in drought-stressed plants without SA pretreatment, whereas it was recovered to the control levels by SA pretreatment (Figure 1.3C) Similarly, NADPH content in drought stressed plants was decreased by 35% compared to control, whereas it was increased by 24.5% and 34.5% in SA pretreated plants under well-watered and drought stressed condition, respectively (Figure 1.3D) Compare

to control, NADP+ content was significantly increased over than 40% in all three treatments (Figure 1.3E) The ratio of NADPH to NADP+ was markedly reduced by 47.3% in drought-stressed plants, whereas its decrease rate was lower in SA

(U mg -1 protein)

CAT (U mg -1 protein)

GPOD (U mg -1 protein)

APOD (U mg -1 protein)