Differential Regulation of NO Homeostasis by AtGSNOR1 and AtPGB1 Coordinates Immunity and Submergence Tolerance in Arabidopsis thaliana

다국어 초록 (Multilingual Abstract)

Nitric oxide (NO) and ethylene are gaseous signaling molecules that regulate plant growth, development, and stress adaptation, forming a complex network of responses through interactions with reactive oxygen species (ROS). In this study, we used variants of phytoglobin 1 (AtPGB1), a scavenger of free NO, S-nitrosoglutathione reductase (AtGSNOR1), a key regulator of protein S-nitrosylation, to elucidate the response mechanisms under oxidative, pathogenic, and submergence stresses. In silico analysis of co-expression networks and regulatory enrichment resulted in the finding that AtPGB1 is related to ethylene-associated hypoxia signaling and broader stress responses. eFP browsers supported the result with a dynamic transcriptional response of AtPGB1 than AtGSNOR1 under hypoxia and Pseudomonas syringae pv. Tomato DC3000 infection. Furthermore, analysis of reactive oxygen/ NO stress at the germination stage revealed that the AtGSNOR1 influenced the regulation of oxidative stress, while AtPGB1 played a key role in alleviating NO stress. These results indicate that AtGSNOR1 and AtPGB1 contribute to distinct modes of NO regulation under stress. In PstDC3000 WT and avrB infection experiments, AtGSNOR1 deletion attenuated pattern-induced immunity (PTI) and effector-induced immunity (ETI), whereas AtPGB1 deletion induced excessive cell death and lipid peroxidation. Analysis of defense gene expression also revealed that AtGSNOR1 deletion induced suppression of defense responses, that AtPGB1 deletion increased NO and upregulated AtPR genes, and systemic acquired resistance (SAR). However, the amplification of defense signaling induced by deletion of AtPGB1 induced defense excess, leading to impaired oxidative balance. In submergence experiments, atgsnor1-1(OE) and 35S::AtACS11 showed high survival and recovery capacity, while atgsnor1-3(KO), atpgb1(KO), and atacs1(KO) showed susceptibility. Recovery differences were associated with changes in S-nitrosylated proteins, ethylene synthesis, and AtPGB1 expression, which were closely linked. Notably, 35S::AtACS11 strongly induced Ethylene-AtPGB1-ERF-VII changes, promoting fermentation-related gene expression and carbohydrate metabolic switching. In contrast, atgsnor1-1 promoted anaerobic energy metabolism with low ethylene production and no strong induction of AtPGB1 and ERFVII transcription factors, which differs from the pattern observed in the wild type. Collectively, we propose that AtGSNOR1 and AtPGB1 act as complementary but non-redundant regulators of NO homeostasis and are key axes of red-hormone signaling that determine immunity and submergence tolerance in Arabidopsis through integrated regulation with the ethylene pathway

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1 What is nitric oxide 1
• 1.2 Physiological traits of NO 2
• 1.3 NO production pathways 4
• 1.4 NO scavenging pathways: PGB1 vs GSNOR1 5
• 1.5 PTM by NO: Snitrosylation vs Tyrosine nitration 6
• 1.6 ROS in plant signaling 6
• 1.7 Mechanisms of redox signaling 7
• 1.8 Crosstalk of NO and ROS 8
• 1.9 Synthesis and function of ethylene 10
• 1.10 NO-Ethylene Crosstalk 10
• Chapter 2. Integrative Characterization of AtPGB1 in Comparison with AtGSNOR1 12
• 2.1 Introduction 12
• 2.2 Materials and Methods 13
• 2.2.1 Computational analysis of AtPGB1 co-expression and regulatory networks 13
• 2.2.2 Plant materials 13
• 2.2.3 Seed sterilization and growth conditions 14
• 2.2.4 DNA extraction 15
• 2.2.5 Genotyping of T-DNA insertion mutant lines 15
• 2.2.6 Arabidopsis crossing and Generation of Double Mutants 18
• 2.2.7 PCR amplification of AtACS5 for cloning 18
• 2.2.8 TA cloning into pCR8/GW/TOPO® vector 19
• 2.2.9 Plasmid extraction 20
• 2.2.10 Gateway® LR recombination cloning 21
• 2.2.11 Agrobacterium transformation 23
• 2.3 Results 26
• 2.3.1 Network analysis of AtPGB1 26
• 2.3.2 Confirmation of homozygote T-DNA insertion knock-out mutants and 35S promoter overexpression mutants 31
• 2.3.3 Development of homozygous atgsnor1/atpgb1 double mutant lines 33
• 2.3.4 Phenotypic characterization of AtGSNOR1-, AtPGB1-, and AtACS-Related Mutants 34
• 2.3.5 MS media reactive oxygen and nitrogen species (ROS/RNS) stress assay 37
• 2.3.6 AtACS5 cloning through pCR8/GW/TOPO® and gateway LR reaction 43
• 2.4 Discussion 47
• 2.4.1 Comparison of functional context and developmental phenotype of AtGSNOR1 and AtPGB1 47
• 2.4.2 Functional differentiation of the ACS family and cloning of AtACS5 48
• 2.4.3 Differential responses under ROS, RNI, and NO inhibition conditions 49
• 2.4.4 Implications for pathogen infection and submergence tolerance 52
• Chapter 3. AtPGB1 Balances Nitric Oxide Homeostasis and Modulates Immune Response during PTI and ETI 53
• 3.1 Introduction 53
• 3.2 Materials and methods 54
• 3.2.1 Pathogen strains and culture preparation 54
• 3.2.2 Plant materials and experimental design 54
• 3.2.3 Bacterial growth assay (CFU measurement) 55
• 3.2.4 Histochemical staining assays 56
• 3.2.5 Oxidative stress marker assays 57
• 3.2.6 Protein extraction and quantification 58
• 3.2.7 Antioxidant enzyme activity assays 58
• 3.2.8 RNA extraction 60
• 3.2.9 cDNA synthesis and quantitative real-time PCR 60
• 3.2.10 Statistical analysis 62
• 3.3 Results 63
• 3.3.1 Resistance responses of AtGSNOR1 and AtPGB1 mutants to Pst DC3000 63
• 3.3.2 Hypersensitive response and cell death regulation in Pst DC3000 AvrB–induced ETI 65
• 3.3.3 DAB staining for ROS detection 67
• 3.3.4 H2O2 accumulation, lipid peroxidation, and antioxidant enzyme activities in response to Pst DC3000 WT and AvrB 68
• 3.3.5 Defense gene expression patterns in PTI and ETI 70
• 3.3.6 Relative expression of pathogen marker genes on distal leaves 72
• 3.3.7 Induction of protein S-nitrosylation in distal leaves 74
• 3.4 Discussion 75
• 3.4.1 Central role of AtGSNOR1 and fine-tuning by AtPGB1 in plant immunity 75
• 3.4.2 Modulation of S-nitrosylation and peroxynitrite under immunity 76
• 3.4.3 Local–systemic immune imbalance and SAR responses 77
• Chapter 4. Submergence Stress Responses Integrating NO and Ethylene Pathways 78
• 4.1 Introduction 78
• 4.2 Materials and methods 79
• 4.2.1 Plant materials and experimental design 79
• 4.2.2 Biomass measurement and recovery assessment 80
• 4.2.3 Detection of nitric oxide by DAF-FM DA staining 80
• 4.2.4 Nitrate reductase activity assay 81
• 4.2.5 Nitrate content determination 82
• 4.2.7 Total carbohydrate content 83
• 4.2.8 Total soluble sugar content 83
• 4.2.9 Quantification of S-nitrosylated protein 83
• 4.3 Results 87
• 4.3.1 Submergence tolerance and recovery of AtGSNOR1, AtPGB1, AtACS mutants 87
• 4.3.3 Interplay between S-nitrosylation and ethylene production under submergence. 93
• 4.3.4 Tyrosine nitration under submergence 95
• 4.3.5 Differential ROS accumulation and oxidative damage under submergence 96
• 4.3.6 Differential regulation of enzymatic antioxidants 98
• 4.3.7 Expression profiles of ERF-VII transcription factors under submergence 100
• 4.3.8 Carbohydrate content and fermentation-related gene expression 102
• 4.3.9 Phytohormone dynamics under submergence 104
• 4.4 Discussion 105
• 4.4.1 Integrative roles of NO-ethylene signaling in submergence response 105
• 4.4.2 ROS regulation and the contribution of ethylene signaling 105
• 4.3.5 Differential regulation of ERF-VII transcription factors 108
• 4.4.6 Unique nitrate and fermentative metabolic responses of atgsnor1-1 109
• 4.5 Conclusion 111