Identification of transcription factors that regulate <i>ATG8</i> expression and autophagy in <i>Arabidopsis</i>

Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. In <i>Arabidopsis thaliana</i>, the core machinery of autophagy is well defined, but its transcriptional regulation is largely unknown. The ATG8 (autophagy-related 8) protein plays central roles in decorating autophagosomes and binding to specific cargo receptors to recruit cargo to autophagosomes. We propose that the transcriptional control of <i>ATG8</i> genes is important during the formation of autophagosomes and therefore contributes to survival during stress. Here, we describe a yeast one-hybrid (Y1H) screen for transcription factors (TFs) that regulate <i>ATG8</i> gene expression in <i>Arabidopsis</i>, using the promoters of 4 <i>ATG8</i> genes. We identified a total of 225 TFs from 35 families that bind these promoters. The TF-<i>ATG8</i> promoter interactions revealed a wide array of diverse TF families for different promoters, as well as enrichment for families of TFs that bound to specific fragments. These TFs are not only involved in plant developmental processes but also in the response to environmental stresses. TGA9 (TGACG (TGA) motif-binding protein 9)/AT1G08320 was confirmed as a positive regulator of autophagy. TGA9 overexpression activated autophagy under both control and stress conditions and transcriptionally up-regulated expression of <i>ATG8B, ATG8E</i> and additional <i>ATG</i> genes via binding to their promoters. Our results provide a comprehensive resource of TFs that regulate <i>ATG8</i> gene expression and lay a foundation for understanding the transcriptional regulation of plant autophagy. <b>Abbreviations:</b> ABRC: <i>Arabidopsis</i> biological resource center; AP2-EREBP: APETALA2/Ethylene-responsive element binding protein; ARF: auxin response factor; ATF4: activating transcription factor 4; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; DAP-seq: DNA affinity purification sequencing; FOXO: forkhead box O; GFP: green fluorescent protein; GO: gene ontologies; HB: homeobox; LD: long-day; LUC: firefly luciferase; MAP1LC3: microtubule associated protein 1 light chain 3; MDC: monodansylcadaverine; 3-MA: 3-methyladenine; OE: overexpressing; PCD: programmed cell death; qPCR: quantitative polymerase chain reaction; REN: renilla luciferase; RT: room temperature; SD: standard deviation; TF: transcription factor; TFEB: transcription factor EB; TGA: TGACG motif; TOR: target of rapamycin; TSS: transcription start site; WT: wild-type; Y1H: yeast one-hybrid.

自噬（autophagy）是真核生物中一类保守的分解代谢过程，可帮助细胞应对多种胁迫，同时对维持机体适合度至关重要。在拟南芥（*Arabidopsis thaliana*）中，自噬的核心机制已得到明确阐释，但其转录调控机制仍知之甚少。
ATG8（autophagy-related 8，自噬相关蛋白8）在自噬体膜修饰以及结合特定货物受体招募货物至自噬体的过程中发挥核心作用。我们提出，ATG8基因的转录调控在自噬体形成过程中至关重要，因此有助于胁迫条件下的细胞存活。
本研究利用4个ATG8基因的启动子，通过酵母单杂交（yeast one-hybrid, Y1H）技术筛选拟南芥中调控ATG8基因表达的转录因子（transcription factor, TF）。研究共鉴定得到35个家族的225种可结合上述启动子的转录因子。
TF与ATG8启动子的互作分析显示，不同启动子可结合多样化的TF家族，同时结合特定片段的TF家族存在富集现象。这些转录因子不仅参与植物生长发育过程，还参与环境胁迫应答。
我们验证了TGA9（TGACG（TGA）基序结合蛋白9，TGACG (TGA) motif-binding protein 9 / AT1G08320）作为自噬的正调控因子：TGA9过表达可在正常培养及胁迫条件下激活自噬，并通过结合ATG8B、ATG8E及其他ATG基因的启动子，在转录水平上调这些基因的表达。
本研究提供了调控ATG8基因表达的转录因子的综合资源，为解析植物自噬的转录调控机制奠定了基础。
**缩写说明：**
ABRC：拟南芥生物资源中心（Arabidopsis biological resource center）
AP2-EREBP：APETALA2/乙烯响应元件结合蛋白（APETALA2/Ethylene-responsive element binding protein）
ARF：生长素响应因子（auxin response factor）
ATF4：激活转录因子4（activating transcription factor 4）
ATG：自噬相关（autophagy-related）
ChIP：染色质免疫共沉淀（chromatin immunoprecipitation）
DAP-seq：DNA亲和纯化测序（DNA affinity purification sequencing）
FOXO：叉头框O转录因子（forkhead box O）
GFP：绿色荧光蛋白（green fluorescent protein）
GO：基因本体论（gene ontologies）
HB：同源异型盒（homeobox）
LD：长日照（long-day）
LUC：萤火虫荧光素酶（firefly luciferase）
MAP1LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）
MDC：单丹磺酰尸胺（monodansylcadaverine）
3-MA：3-甲基腺嘌呤（3-methyladenine）
OE：过表达（overexpressing）
PCD：程序性细胞死亡（programmed cell death）
qPCR：定量聚合酶链式反应（quantitative polymerase chain reaction）
REN：海肾荧光素酶（renilla luciferase）
RT：室温（room temperature）
SD：标准差（standard deviation）
TF：转录因子（transcription factor）
TFEB：转录因子EB（transcription factor EB）
TGA：TGACG基序（TGACG motif）
TOR：雷帕霉素靶蛋白（target of rapamycin）
TSS：转录起始位点（transcription start site）
WT：野生型（wild-type）
Y1H：酵母单杂交（yeast one-hybrid）

搜集汇总

背景与挑战

背景概述

该数据集通过酵母单杂交筛选，识别了拟南芥中调控ATG8表达和自噬的转录因子，包含225个转录因子来自35个家族，并确认TGA9为正调控因子。数据集提供了原始实验数据（如Excel文件），支持研究植物自噬的转录调控机制，适用于生物化学、植物生物学和遗传学等领域。