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 TABLE OF CONTENTS LIST OF TABLES IV LIST OF FIGURES V ABBREVIATIONS VIII ABSTRACT 1 GENERAL INTRODUCTION 1.1 Brassica species and drought stress 1.2 ROS is a primary stress signal for metabolism and transduction signaling 1.2.1 ROS generation and metabolism 1.2.2 ROS role in transmitting signal 1.3 Proline is an elicitor in plants response to drought stress 1.3.1 Proline accumulation in stress response 1.3.2 Proline metabolism essential for stress response and tolerance 1.4 Redox balance: A tools of plant defense against drought stress 1.5 Plant responses to drought stress: A matter of hormones regulation 1.5.1 Abscisic acid pathway 1.5.2 Salicylic acid pathway 1.5.3 Antagonism between abscisic acid and salicylic acid pathways 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 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 I 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 II 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 III LIST OF TABLES Table List of the primers used in this study CHAPTER 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 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 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 IV LIST OF FIGURES GENERAL INTRODUCTION Figure The main compartment of H2O2 production in photosynthetic cell Figure Model for proline synthesis pathway in plants CHAPTER Figure 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 droughtstressed 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 Figure 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 V 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 days of SA pretreatment) Figure 2.8 A proposed model for salicylic acid-mediated ROS, proline synthesis, and redox modulation under drought CHAPTER 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 (BAM1, A) and α-amylase (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 VI Figure 3.7 Heatmap analysis on the treatment effect and correlations among the variables measured at day 15 (after 10 days of drought, including days of SA pretreatment) CHAPTER Figure 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 VII ABBREVIATIONS ABA AMY3 APR3 Abscisic acid α-amylase Adenosine phosphosulfate reductase APX Ascorbate peroxidase ASC Ascorbate AsA Ascorbic acid AREB2 ABA-responsive element biding BAM1 β-amylase BSO Buthionine sulfoximine CAT Catalase CDPKs Calcium-dependent protein kinase CIPKs Calcineurin B-like interacting protein kinase CBL Calcineurin B-like CHK5 CHASE receptor kinase CHLG Chlorophyll synthase CK Cytokinin Cu/ZnCopper/Zinc superoxide SOD dismutase 2Cys-PRX 2-cys-peroxiredoxins CWINV Cell wall invertase GO GOGAT GPX Glycolate oxidase Glutamate synthease Glutathione peroxidase GR GRX GRXC9 GS Glutathione reductase Glutaredoxin CC-type glutaredoxin Glutamine synthease GshA GSH GSSG GST Glutamate-cysteine ligase Glutathione Glutathione disulphide Glutathione S-transferase G6PDH Glucose 6-phosphate dehydrogenase Hydroxyl radical Hydrogen peroxide Hexokinase 1-related gene Indole-acetic acid Isochorismate synthase DAB Diaminobenzidine MDHAR DCPIP DHAR Dichlorophenolindophenol Dehydroascorbate reductase MYC2 Mn-SOD γ-ECS GA γ-glutamylcysteine synthetase Gibberellic acid NBT NCED3 GA3 GDH GID1 Glu GLRs Gibberellin Glutamate dehydrogenase GA Insensitive DWARF Glutamate Glutamate receptor-like NO3NO2NH4+ NR NRTs VIII HOH2O2 HXK1 IAA ICS1 JA MAPKs Jasmonic acid Mitogen-activated protein kinase Monodehydroascorbate reductase MYC2 transcription factor Manganese superoxide dismutase Nitroblue tetrazolium 9-sis-epoxycarotenoid dioxygenase Nitrate Nitrite Ammonium Nitrate 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Hauser F, Jeon BW, Bader JS, Kwak JM, Schroeder JI, McKay JK, Assmann SM, 2016 Molecular and systems approaches towards drought-tolerant canola crops New Phytol 210, 1169–1189 113 LIST OF PUBLICATIONS I Papers published in scientific journals Van Hien La, Bok-Rye Lee, Md Tabibul Islam, Sang-Hyun Park, Hyo Lee, Dong-Won Bae, Tae-Hwan Kim (2019) Antagonistic shifting from abscisic acid- to salicylic acid-mediated sucrose accumulation contributes to drought tolerance in Brassica napus Environmental and Experimental Botany 162: 38–47 Van Hien La, Bok-Rye Lee, Md Tabibul Islam, Sang-Hyun Park, Ha-il Jung, Dong-Won Bae, Tae-Hwan Kim (2019) Characterization of salicylic acid-mediated modulation of the drought stress responses: Reactive oxygen species, proline, and redox state in Brassica napus, Environmental and Experimental Botany Environmental and Experimental Botany 157: 1–10 Van Hien La, Bok-Rye Lee, Qian Zhang, Sang-Hyun Park, Md Tabibul Islam, Tae-Hwan Kim (2019) Salicylic acid ameliorates stress tolerance by regulating redox status and proline metabolism under drought stress in Brassica rapa Horticulture Environment and Biotechnology 60: 31–40 Hyo Lee, Bok-Rye Lee, Md Tabibul Islam, Van Hien La, Sang-Hyun Park, Tae-Hwan Kim (2020) Cultivar variation in hormones- and sugars-response reveals that abscisic acid-responsive sucrose phloem loading at the early regenerative stage is a significant determinant of seed yield in field grown Brassica napus Environmental and Experimental Botany 169: 103917 (published online) Md Tabibul Islam, Bok-Rye Lee, Sang-Hyun Park, Van Hien La, Woo-Jin Jung, Dong-Won Bae, Tae-Hwan Kim (2019) Hormonal regulation in soluble and cell-wall bound phenolic accumulation in two cultivars of Brassica napus contrasting susceptibility to Xanthomonas campestris pv campestris Plant Science 285: 132-140 Md Tabibul Islam, Bok-Rye Lee, Van Hien La, Hyo Lee, Woo-Jin Jung, Dong-Won Bae, Tae-Hwan Kim (2019) p-coumaric acid induces jasmonic acid-mediated phenolic accumulation and resistance to black rot disease in Brassica napus Plant Physiology and Biochemistry 106: 270-275 Md Tabibul Islam, Bok-Rye Lee, Protiva Rani Das, Van Hien La, Ha-il Jung, Tae-Hwan Kim (2018) Characterization of p-coumaric acid-induced soluble and cell wall-bound phenolic metabolites in relation to disease resistance to Xanthomonas campestris pv campestris in Chinese cabbage Plant Physiology and Biochemistry 125: 172-177 Md Tabibul Islam, Bok-Rye Lee, Sang-Hyun Park, Van Hien La, Dong-Won Bae, Tae-Hwan Kim (2017) Cultivar variation in hormonal Balance is a significant determinant of disease susceptibility to Xanthomonas campestris pv campestris in Brassica napus Frontiers in Plant Science 8: 2121 114 II Papers submitted in peer-reviewed scientific journals Van Hien La, Bok-Rye Lee, Md Tabibul Islam, Sang-Hyun Park, Dong-Won Bae, Tae-Hwan Kim Comparative hormonal regulatory pathway of the drought responses in relation to glutamate-mediated proline metabolism in Brassica napus (Re-submitting to Plants (Basel)) Md Al Mamun, Md Tabibul Islam, Bok-Rye Lee, Van Hien La, Dong-Won Bae, Tae-Hwan Kim Genotypic variation in Resistance genes-mediated calcium signaling involves in the hormonal signaling in relation to effector-triggered immunity or disease susceptibility in the black rot - Brassica napus pathosystem (Re-submitting to Plants (Basel)) III Papers in preparation Van Hien La, Bok-Rye Lee, Md Tabibul Islam, Dong-Won Bae, Tae-Hwan Kim Turnover of glutathione-integrated cysteine accumulation involves the modulation of salicylic acid and abscisic acid in the accumulation of hydrogen peroxide under water-stress Van Hien La, Bok-Rye Lee, Md Tabibul Islam, Md Al Mamun, Dong-Won Bae, Tae-Hwan Kim Nitrate and ammonium assimilation-integrated proline accumulation is associated with H2O2 interplay hormonal of the drought stress responses in Brassica napus L Md Tabibul Islam, Bok-Rye Lee, Van Hien La, Dong-Won Bae, Woo-Jin Jung, Tae-Hwan Kim Label-free quantitative proteomics analysis in susceptible and resistant Brassica napus cultivars infected with Xanthomonas campestris pv campestris Md Tabibul Islam, Bok-Rye Lee, Woo-Jin Jung, Van Hien La, Dong-Won Bae, Tae-Hwan Kim Evidence of salicylic acid-induced phenolic metabolites and antifungal activity in Chinese cabbage (Brassica rapa var pekinensis) 115 ABSTRACT IN KOREAN 유채의 식물 호르몬에 의한 가뭄 스트레스 반응 및 저항성 조절 기전 La Van Hien 동물산업학과 전남대학교 대학원 (지도교수 김태환) 초록 본 연구는 가뭄스트레스 반응과 저항성에 대한 호르몬 조절을 시너지 효과 또는 길항 효과가 일어나는 다양한 생물학적 과정에서 특성화하기 위하여 수행되었다 장부터 장 까지는, 살리실산 (SA) 가뭄 저항성 조절에 대한 역할을 평가하였다 장에서는, 가뭄스트레스에 대한 저항성 기전에서 글루탐산의 합성 뿐만 아니라 프롤린의 축적을 포함한 질소 대사기전을 호르몬 반응으로 설명하였다 장부터 장까지는, SA 매개 전사적 조절의 역할을 가뭄 저항성, 특히 redox 조절에 포함된 다른 호르몬들, ROS, 항산화제, 프롤린, 당과의 가능한 상호관계를 특정화 함으로 밝혀냈다 가뭄스트레스는 ROS 와 프롤린 축적을 야기했을 뿐만 아니라 redox 의 산화 상태를 증가시켰다 1.5 mM SA 전처리는 배추에서 ascorbate peroxidase 의 활성이 증가하여 O2•-, H2O2 및 지질과산화가 제거됨으로써 가뭄스트레스에 대한 부정적 영향이 상당히 저감되었다 SA 전처리는 식물의 가뭄 스트레스 조건에서 프롤린과 글루타티온의 축적을 야기하였다 산화환원 반응 조절에서 SA, ROS 및 프롤린 상호작용의 상세한 근본적인 메커니즘은 유채에서 평가되었다.0.5mM SA 처리는 가뭄에 의해 유도된 O2•- 축적을 저감시켰으나, H2O2 에는 영향을 주지 못했다 SA 매개 NPR1 은 TRXh5 와 GRXC9 redox 신호 전사를 조절하였고, 이 신호에 의하여 SA 는 redox 상태를 재설정하고, 프롤린 합성 증가 및 ABA 및/또는 JA 신호의 길항적 감소를 보여주었다 반면에, 가뭄은 주로 hexokinase 유전자인 HXK1 발현 감소로 hexose 수준을 증가시켰고, 부분적으로 ABA-dependent sucrose signaling 유전자인 SnRK2.2 와 AREB2 의 높은 유전자 발현으로 sucrose 함량을 116 증가시켰다 SA 전처리한 식물에서 sucrose phosphate synthase (SPS) 의 활성과 전분 분해 효소와 관련된 BAM1 과 AMY3 유전자 발현 증가로 인해 sucrose 축적이 가장 높게 일어났다 이러한 결과들은 sucrose 축적 기전에서 ABA 로부터 SA 로의 상당한 매커니즘의 변화가 삼투압 조절 또는 잎의 노화를 완화시키는 것을 보여준다 장에서는 프롤린 축적에 의한 호르몬 조절과 질소 대사를 통합하여 특성화 하였다 가뭄에 의해 유도된 ABA 는 0.1 mM SA 와 0.1 mM H2O2 처리구 보다 ABI1 의 발현을 감소시켰고, 세포질 내 Ca2+ 의존적인 방식으로 CIPK23 과 CBL9 발현을 증가시켰다 이러한 복잡한 신호들은 물관부-체관부 시스템 내의 NO3이동과 NO3- 의 동화작용에 부정적인 영향을 미쳤으며, 부분적으로는 NO3- 의 축적을 증가시켰다 SA, H2O2 및 가뭄 처리는 GDH 활성에 의한 대체 GS/GOGAT cycle 에 의해 진행된 NH4+ 동화 작용 활성화로 NH4+의 축적을 제한하였다 가뭄 스트레스 조건에서 ABA 조절 기전의 H2O2 와 CBL9 신호와 관련된 가장 높은 GDH 활성에서 추가적인 프롤린 축적은 글루탐산 합성과 동반했다 이러한 결과들은 식물 호르몬들이 가뭄스트레스에 의한 식물의 반응과 관련된 탄소와 질소대사의 조절에 관여한다는 것을 보여준다 여러 호르몬들 중 ABA 와 SA 의 길항적 상호작용은 유채에서 가뭄 저항성 측면에서 ROS, 당, 프롤린 및 redox 상태를 활성화하는데 더욱 중요한 역할을 했다 117 ACKNOWLEDGMENTS The journey of this thesis has been an important turning point and an amazing invaluable experience in my life I thanks to all who contributed to the completion of this thesis First of all, I would like to give my sincere thanks to my supervisor, Professor Tae-Hwan Kim, for all of his academic guidance, kindly support, and encouragement during my doctoral study I would like to especially thank to the committee members for my thesis, Prof Sejong Oh, Prof Kil-Yong Kim, Prof Woo-Jin Jung, and Prof Sung-Hak Kim for their encouragement, excellent advices, mentorship, criticisms and insightful scientific comments which enabled me to make the necessary improvements of my thesis I express my very sincere thanks to Dr Bok-Rye Lee for her valuable comments on my research career and assistance for my experiments Profound thanks are due to all my labmates as Dr Zhang, Dr Tabibul Islam, Dr Sang-Hyun Park, Ms Wang Xinlei, Ms Hyo, and Mamun who had shared their outstanding methodological knowledge, laboratorial skills to help during my study in Korea I acknowledge my gratitude to all the members in Prof Oh and WCU Labs for the instrument support I am also thankful to Dr Dong-Won Bae for hormone analysis Finally, warmest thanks go to my family for their spiritual love and moral support I deeply thank to my father made me more confident, stronger and keep going the right way until now I am also grateful to my girlfriend Duong Thi Tham for her’s warm love and allways encourages me in the difficult situation, which motivated me to my best My family always proud of me and they are the most important people in my world and I dedicate this thesis to them Sincerely thanks all! Gwangju-South Korea, February 2020 La Van Hien 118 ... 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... ABA-responsive element biding BAM1 β-amylase BSO Buthionine sulfoximine CAT Catalase CDPKs Calcium-dependent protein kinase CIPKs Calcineurin B-like interacting protein kinase CBL Calcineurin B-like CHK5... 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;