Senescence is a fundamental biological process in plant life cycle, marking the final stage of leaf development and playing a critical role in nutrient recycling and survival under environmental challenges. During leaf senescence in Arabidopsis thaliana, various degradation processes are tightly regulated through chloroplast signaling, transcription factors, and autophagy. Among these, chloroplast, as a primary source of nutrient remobilization, must undergo degradation while maintaining their photosynthetic function. Thus, the regulation of chloroplast degradation through autophagy is essential to ensure proper senescence progression. Here, PHYTOCHROME INTERACTING FACTOR (PIF) 4 and PIF5 (PIF4/PIF5), positive regulators of leaf senescence, were shown to directly activate autophagy-related genes, as demonstrated through chromatin immunoprecipitation and dual-luciferase assays. In pif4pif5 mutants, reductions in autophagy flux and chloroplast degradation were observed, accompanied by a concomitant decrease in seed size. The partial recovery of chloroplast degradation and seed size reduction in proUBQ5:ATG8a-GFP transgenic lines crossed with pif4pif5 further supports the role of PIF4/PIF5 in modulating autophagy and chloroplast breakdown for nutrient remobilization during leaf senescence. Additionally, reduced PIF4 protein levels in the loss-of-function mutant of GENOME UPCOUPLED 1 (GUN1) suggest a regulatory mechanism aligning chloroplast degradation with developmental cues during leaf senescence. GUN1, a master regulator of retrograde signaling, was identified as a positive regulator of leaf senescence through its role in stabilizing PIF4 protein abundance. RNA-seq analysis of mature-green leaves from wild-type (Col-0) and gun1 identified approximately 700 PhD/NB 201946008 differentially expressed genes (DEGs), indicating that GUN1 affects nuclear gene expression before onset of visible leaf senescence phenotypes. Mechanistically, GUN1 interacts with HEMERA (HMR), a dual-localized protein with functions in both chloroplast and nucleus, to stabilize nuclear HMR, which subsequently regulates PIF4. In the hmr-22 mutant, where HMR’s transcription activation domain (TAD) is mutated, GUN1 was unable to bind HMR, underscoring the importance of TAD for the interaction. This study reveals that PIF4/PIF5 play a critical role in modulating autophagy and chloroplast breakdown for nutrient remobilization during leaf senescence, and further demonstrates that this autophagy regulatory pathway in senescent leaves is controlled by nuclear HMR which stabilized by GUN1, linking chloroplast signals to nuclear transcription networks. These findings shed light on how retrograde signaling coordinates autophagy and leaf senescence in response to developmental cues, facilitating nutrient remobilization and integrating chloroplast and nuclear functions during leaf senescence.|노화는 식물 생애 주기에서 필수적인 생물학적 과정으로, 잎 발달의 마지막 단계를 나타내며 영양소 재분배와 환경 스트레스에 대한 생존에 중요한 역할을 합니다. Arabidopsis thaliana의 잎 노화 동안 다양한 분해 과정은 엽록체 신호전달, 전사인자, 그리고 자가포식과 같은 메커니즘을 통해 정교하게 조절됩니다. 이 중, 영양소 재분배의 주요 원천으로 기능하는 엽록체는 분해 과정이 진행되면서도 광합성 기능을 유지해야 하므로, 자가포식을 통한 엽록체 분해의 조절은 적절한 노화 진행을 위해 필수적입니다. 본 연구에서는 잎 노화의 양성 조절자로 알려진 PHYTOCHROME INTERACTING FACTOR(PIF) 4와 PIF5(PIF4/PIF5)가 자가포식 관련 유전자(ATG 유전자)를 직접 활성화한다는 것을 크로마틴 면역침강 및 듀얼-루시페레이스 분석을 통해 밝혔습니다. pif4pif5 이중 돌연변이체에서는 자가포식 흐름 및 엽록체 분해가 감소하였으며, 이와 동시에 종자 크기도 감소하는 현상이 관찰되었습니다. 또한, pif4pif5 돌연변이체와 proUBQ5:ATG8a-GFP 형질전환체를 교배한 라인에서 엽록체 분해 및 종자 크기 감소가 부분적으로 회복된 결과는, PIF4/PIF5가 잎 노화 동안 자가포식 및 엽록체 분해를 조절하여 영양소 재활용에 기여한다는 점을 뒷받침합니다. 추가적으로, GENOME UNCOPLED 1 (GUN1) 기능 상실 돌연변이체에서 PIF4 단백질 수준이 감소하는 결과는, GUN1이 엽록체 분해와 발달 신호를 조정하는 조절 메커니즘의 일부임을 시사합니다. GUN1은 레트로그레이드 신호전달의 주요 조절자로서 PIF4 단백질의 안정성을 유지함으로써 잎 노화를 촉진하는 양성 조절자로 밝혀졌습니다. Col-0 및 gun1 젊은 잎에 대해 RNA-seq 분석을 수행한 결과 약 700개의 차등 발현 유전자(DEGs)가 식별되었으며, 이는 가시적인 잎 노화가 시작되기 전 GUN1이 핵 유전자 발현에 영향을 미친다는 것을 보여줍니다. 기전적으로, GUN1은 엽록체와 핵에 이중으로 위치하는 HEMERA(HMR)와 상호작용하여 핵 내 HMR 안정성을 유지하며, 이를 통해 PIF4를 조절합니다. 특히, HMR의 전사 활성화 도메인(TAD)이 변이된 hmr-22 돌연변이체에서는 GUN1이 HMR에 결합하지 못하는 결과가 관찰되었으며, 이는 GUN1-HMR 상호작용에서 TAD의 중요성을 강조합니다. 본 연구는 PIF4/PIF5가 잎 노화 동안 자가포식 및 엽록체 분해를 조절하여 영양소 재분배에 중요한 역할을 한다는 점을 밝혔으며, GUN1에 의해 안정화된 핵 내 HMR이 이 자가포식 조절 경로를 조정한다는 것을 보여줍니다. 이와 같이, 본 연구는 잎 노화 동안 엽록체 신호가 자가포식 및 핵 전사 네트워크와 어떻게 조화롭게 작용하는지를 규명하며, 발달 신호에 따른 영양소 재분배 및 엽록체-핵 기능 통합의 메커니즘을 통한 종자 발달에 대한 새로운 관점을 제시합니다.
Table Of Contents
List of Contents Abstract i Preface iii List of Tables viii List of Figures ix I. Background Introduction 1 1.1 Plant Senescence 1 1.2 PIF4/PIF5 as Senescence Regulators 4 1.3 Autophagy 4 1.4 Nutrient Remobilization 8 1.5 HEMERA’s Role in the Nucleus and Chloroplasts 10 1.6 Retrograde Signaling and GUN1 13 Ⅱ. Phytochrome–interacting factors PIF4 and PIF5 directly regulate autophagy during leaf senescence in Arabidopsis 19 2.1 Introduction 19 2.2 Results 21 2.2.1 PIF4/PIF5 are activators of ATGs transcription via direct binding to their promoters. 21 2.2.2 PIF4/PIF5 control age-dependent upregulation of ATGs. 36 2.2.3 PIF4/PIF5 modulate autophagy activity by increasing the transcript levels of ATG genes during leaf senescence. 39 2.2.4 Overexpression of ATG8s accelerates age-dependent leaf senescence. 47 2.2.5 PIF4/PIF5 promote carbon and nitrogen deprivation-induced leaf senescence 55 2.2.6 ATG8a-OE suppresses the delayed-senescence phenotypes of pif4, pif5, and pif4pif5. 61 2.2.7 PIF4/PIF5 improve seed quality by increasing ATG8s during leaf senescence 66 2.3 Discussion 72 Ⅲ. Role of GUN1–HMR–PIF4 Module in Regulating Leaf Senescence Through Retrograde Signaling 76 3.1 Introduction 76 3.2 Results 80 3.2.1 GUN1 as a positive regulator of leaf senescence. 80 3.2.2 GUN1 influences nuclear gene expression before the onset of leaf senescence. 83 3.2.3 GUN1 interacts with HEMERA in chloroplast. 87
3.2.4 GUN1 and HMR interaction affects HMR protein levels in the nucleus. 97 3.2.5 GUN1 affects the accumulation of PIF4 through its interaction with HMR 103 3.2.6 GUN1 and PIF4 genetically interact to regulate leaf senescence. 112 3.2.7 GUN1 regulates autophagy gene via HMR-PIFs module. 116 3.3 Discussion 119 Ⅳ. Conclusion of this thesis 123 Ⅴ. Materials and Methods in this thesis 125 5.1 Plant materials 125 5.2 Natural leaf senescence assay 126 5.3 RNA Extraction and RT-qPCR Analysis 126 5.4 Protein Extraction and Western Blot Analysis 127 5.5 Chromatin Immunoprecipitation (ChIP) and ChIP–seq Analysis 127 5.6 Electrophoretic Mobility Shift Assay (EMSA) 128 5.7 Dual-Luciferase Assay 128 5.8 Observation of GFP-ATG8a-Labeled Autophagosomes 129 5.9 Autophagy Flux Assay 129 5.10 Nutrient Starvation Assays 129 5.11 Total Nitrogen Content Determination 129 5.12 Transcriptome analysis 130 5.13 Co-immunoprecipitation (Co-IP) Assays 130 5.14 Nucleus and Chloroplast Isolation 131 5.15 Structural Modeling Using AlphaFold3 132 5.16 Fluorescence resonance energy transfer (FRET) analysis coupled with immunofluorescence 132 5.17 Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) analysis 133 5.18 Statistical Analysis 134 5.19 Schematic diagrams 134 Table 2. Primer list 135 Appendix 138 Table 4. Gun1 interaction partners identified in mature green leaves 138 Reference 144 요약문 151
