Genetic Mechanisms of Environmental Signal Integration and Developmental Regulation through Transcription Factors in Plants : 식물의 전사인자를 통한 환경 신호와 발달 조절 유전적 메커니즘 연구

다국어 초록 (Multilingual Abstract)

Secondary cell wall (SCW) formation in plants is a vital physiological process that provides mechanical support, enables efficient water transport, and enhances pathogen resistance. This process is intricately regulated by a hierarchy of transcription factors. Among them, NAC-domain transcription factors NST1 and NST2 are known as master regulators that control the expression of genes involved in SCW biosynthesis. Identifying the upstream regulators of these key NAC genes is essential for completing the transcriptional network that governs cell wall development. In this study, we demonstrate that the GATA family transcription factor AtGATA5 directly up-regulates NST1 and NST2 expressions and functions as an upstream regulator of the NST1 and NST2-centered transcriptional network.
Previous studies have reported that AtGATA5 plays a role in plant growth by responding to environmental cues such as light, suggesting that GATA transcription factors are closely associated with light signaling and plant morphogenesis. Our study expands this understanding by revealing a novel role for AtGATA5 in the structural development of plant stems through its involvement in SCW formation. Notably, AtGATA5 was found to bind to the NST1 and NST2 promoter region, thereby directly promoting its transcriptional activation. This finding adds a new upstream regulatory layer to the SCW transcriptional hierarchy.
These results propose a new gene regulatory model centered on the AtGATA5-NST1 and AtGATA5-NST2 axis and provide molecular evidence that a GATA transcription factor can function upstream of NAC-domain regulators. This expands the functional scope of GATA transcription factors from environmental signal response to tissue-specific developmental regulation. Our findings deepen the understanding of the hierarchical organization of gene regulatory networks related to SCW formation and suggest a potential molecular link between structural development and environmental response. This study may serve as a foundation for future research on SCW reprogramming and wood tissue development under varying environmental conditions.

국문 초록 (Abstract)

식물의 2차 세포벽 형성은 기계적 지지와 수분 수송, 병저항성 확보에 핵심적인 역할을 수행하는 생리적 과정으로, 다양한 전사인자들에 의해 정교하게 조절된다. 이 가운데 NAC 계열 전사인자인 NST1과 NST2는 이차 세포벽 생합성 유전자들의 발현을 조절하는 최상위 조절인자로 알려져 있으며, 이들의 발현을 상위에서 조절하는 전사인자들의 정체를 밝히는 일은 세포벽 형성과정의 유전자 네트워크를 완성하는 데 있어 중요한 과제로 남아 있다. 본 연구는 GATA family 전사인자인 AtGATA5가 NST1의 발현을 직접적으로 증가시키며, 이를 통해 NST1기반 전사 조절 네트워크의 상위 조절인자 역할을 수행한다는 사실을 밝혔다.
AtGATA5는 빛과 같은 환경 자극에 반응하여 식물 생장을 조절하는 기능이 선행 연구들을 통해 보고되어 왔으며, 이는 GATA 전사인자들이 광신호 전달계 및 식물체의 형태 형성과 밀접하게 연관되어 있다는 점을 시사한다. 본 연구에서는 AtGATA5가 이러한 기존의 역할을 넘어, 식물체의 구조적 발달 과정인 줄기 내 이차 세포벽 형성에 관여한다는 새로운 기능적 가능성을 제시하였다. 특히 AtGATA5는 NST1의 프로모터에 결합하여 직접적으로 전사 활성을 촉진하는 것으로 나타났으며, 이는 기존에 제안되어온 이차 세포벽 조절 네트워크에 새로운 상위 조절 계층을 추가하는 결과이다.
이와 같은 결과는 AtGATA5-NST1 축을 중심으로 하는 새로운 유전자 조절 모형을 제안하며, GATA 전사인자가 NAN-domain 전사인자의 상위에서 기능할 수 있음을 분자생물학 수준에서 입증한 사례로 해석된다. 이는 GATA 전사인자의 역할을 기존의 환경 신호 반응 조절에서 조직 특이적 발달 조절로 확장시키는 것으로, 이차 세포벽 형성과 관련한 유전자 네트워크의 계층 구조에 대한 이해를 높일 수 있을 것이다. 아울러 본 연구는 구조 형성과 환경 반응 간의 유기적 연결 가능성을 제시함으로써, 향후 환경 조건에 따른 목질 조직 발달 및 세포벽 재구성 전략 연구에도 기초자료를 제공할 수 있을 것으로 기대된다.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• 1. Environmental regulation of plant growth 1
• 2. Role of GATA transcription factors in plant development 5
• 3. Regulation of stem development in Arabidopsis thaliana 8
• 4. Secondary cell wall : Formation and applications 10
• 5. Objective of this study 13
• Ⅱ. Materials and Methods 14
• 1. Experimental materials 14
• 2. Development of a set of binary vectors for plant transformation 15
• 2.1. pTK-BMLC 15
• 2.2. pTrTK-BMLC 16
• 2.3. pTA7002-BMLC 17
• 3. Plant materials and growth conditions 18
• 4. Gene cloning 19
• 4.1. gDNA extraction 19
• 4.2. Total RNA extraction and DNaseⅠ treatment 19
• 4.3. First-Strand cDNA synthesis 20
• 4.4. Polymerase chain reaction (PCR) 20
• 4.5. DNA sequencing 21
• 4.6. Preparation of Escherichia coli competent cell 21
• 4.7. Preparation of Agrobacterium tumefaciens competent cell 21
• 4.8. Transformation of Arabidopsis thaliana 22
• 5. Identification of GATA5 transcription factor 24
• 5.1. Phylogenetic analysis 24
• 5.2. Subcellular localization 24
• 6. Generation of transgenic plants 25
• 6.1. Generation of GATA5 overexpression transgenic plants 25
• 6.2. Generation of GATA5 promoter transgenic plants 25
• 6.3. Generation of inducible expression transgenic plants 25
• 7. Knockout plants generated using the CRISPR/Cas9 system 27
• 7.1. Designing the common gRNA target sequence 27
• 7.2. Constructing the CRISPR/Cas9 binary vector 27
• 7.3. Generation of CRISPR/Cas9-mediated gene editing transgenic plants 27
• 7.4. Identification of the gata5 mutants and validation of CRISPR/Cas9-mediated target mutation 28
• 8. Morphological characterization of GATA5 transgenic plants 31
• 9. Gene expression patterns of GATA5 transgenic plants 31
• 9.1. Dexamethasone-inducible system and effect of dexamethasone treatment 31
• 9.2. Semi-quantitative PCR 33
• 9.3. Quantitative real-time PCR (qRT-PCR) 33
• 10. Validation and development of transcriptional activation analysis 36
• 10.1. Transient activity assay (TAA) 36
• 11. Statistical analysis 41
• Ⅲ. Results 42
• 1. Phylogenetic analysis of GATA transcription factors 42
• 2. Visualization of GATA5 transcription factor expression organization 44
• 3. Nuclear localization of AtGATA5 suggests its potential function as a transcription factor 46
• 4. Histological evidence of AtGATA5 function in SCW formation 48
• 5. Up-regulation of NAC-domain transcription factors in AtGATA5-OX transgenic plants 50
• 6. Positive regulation of NAC-domain transcription factors by AtGATA5 52
• 7. Gene expression analysis of NAC-domain transcription factors in the gata5 knockout mutant 54
• 8. Stem morphology analysis of AtGATA5 inducible transgenic plants 57
• 9. Inducible expression of AtGATA5 activates NST1 and NST2 in transgenic lines 59
• 10. Direct activation of NST1 and NST2 by GATA5 transcription factor 61
• 11. Transient complementation assay for functional validation of GATA5 in the gata5 mutant background 63
• 12. Characterization of direct GATA5 binding sites in NST1 and NST2 65
• 13. Identification of direct binding sites of GATA5 transcription factor on NST1 and NST2 67
• Ⅳ. Discussion 69
• Ⅴ. Reference 72