未折叠蛋白反应通过调控色氨酸代谢提高植物抗病性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0275

摘要/Abstract

摘要：

目的探究内质网胁迫（endoplasmic reticulum stress, ERS）引发的未折叠蛋白反应（unfolded protein response, UPR）与色氨酸代谢和植物抗病性的关系；明确UPR中的转录因子bZIP28和bZIP60在色氨酸代谢中的调控机制及对植物抗病性的影响。方法利用转录组测序和RT-qPCR分析内质网胁迫后，色氨酸代谢通路相关基因的表达；检测内质网胁迫对色氨酸代谢产物植保素-亚麻荠素（camalexin）和吲哚-3-乙酸（indole-3-acetic acid, IAA）合成的影响；检测病原菌侵染后bZIP28和bZIP60的表达及其过表达植株的抗病能力；通过酵母单杂交和双荧光素酶报告实验探究bZIP28/60对WRKY33的调控。结果 UPR使色氨酸代谢基因表达上调，植保素Camalexin和IAA的水平升高；病原菌感染上调bZIP28和bZIP60的表达，且bZIP28和bZIP60过表达植株的抗病性增强；bZIP28和bZIP60间接调控WRKY33的转录，且WRKY33启动子的-364- -566 bp区段为核心序列。结论内质网胁迫通过转录因子bZIP28和bZIP60调控WRKY33的表达，进而上调色氨酸代谢基因的表达，提高Camalexin和IAA的水平，抵御病原菌的感染，缓解内质网胁迫。

关键词: 内质网胁迫, 未折叠蛋白反应, bZIP28, bZIP60, WRKY33, 色氨酸代谢, 植保素, 吲哚乙酸

Abstract:

Objective To investigate the relationship between endoplasmic reticulum stress (ERS) induced unfolded protein response (UPR), tryptophan metabolism, and plant disease resistance, and to clarify the regulatory mechanisms of the transcription factors bZIP28 and bZIP60 in tryptophan metabolism and their impact on plant disease resistance. Method RNA-seq and qRT-PCR were used to analyze the expressions of genes involved in the tryptophan metabolism pathway under ER stress conditions. The synthesis of tryptophan-derived metabolites, camalexin and IAA, was measured under ER stress. The expressions of bZIP28 and bZIP60 after pathogenic infection and the disease resistance of plants overexpressing these genes were assessed. Yeast one-hybrid assays and dual-luciferase reporter assays were conducted to examine the regulation of WRKY33 by bZIP28/60. Result ER stress triggered the UPR, leading to the up-regulation of tryptophan metabolic genes and increased levels of camalexin and IAA. Pathogenic infection up-regulated the expressions of bZIP28 and bZIP60. Plants overexpressing bZIP28/60 demonstrated enhanced resistance to disease. bZIP28/60 indirectly regulated the expression of WRKY33, with the core regulatory sequence located within the -364 to -566 bp region of the WRKY33 promoter. Conclusion ER stress modulates the expression of WRKY33 through the transcription factors bZIP28 and bZIP60, resulting in elevated levels of camalexin and IAA, thereby enhancing resistance to pathogen infection and alleviating ER stress.

Key words: ER stress, unfolded protein response, bZIP28, bZIP60, WRKY33, tryptophan metabolism, camalexin, IAA

曹园园, 周舒浩, 张海荣, 崔晓娜. 未折叠蛋白反应通过调控色氨酸代谢提高植物抗病性[J]. 生物技术通报, 2025, 41(8): 155-164.

CAO Yuan-yuan, ZHOU Shu-hao, ZHANG Hai-rong, CUI Xiao-na. Unfolded Protein Response Enhances Plant Resistance to Disease by Regulating Tryptophan Metabolism[J]. Biotechnology Bulletin, 2025, 41(8): 155-164.

图/表 6

图1 色氨酸代谢基因在衣霉素处理后和lew1突变体中的表达A：RNA-seq热图，颜色代表log2倍数差异；B：色氨酸代谢通路图；C：5 μg/mL衣霉素处理幼苗，通过qPCR检测色氨酸代谢相关基因的相对表达量，误差标尺表示±标准差，不同字母表示差异显著P<.05，下同

Fig. 1 Expressions of tryptophan metabolism genes under TM treatment or in the lew1 mutantA: RNA-seq heatmap illustrates the gene expressions. The color gradient indicates the log2fold change. B: Tryptophan metabolism pathway. C: Seedlings were treated with 5 μg/mL TM, and the relative expressions of tryptophan metabolism genes were detected by qPCR. The data indicate the mean fold difference ± SD of three biological replicates. Statistically significant differences (P<0.05) are indicated by distinct letters using Student's t-test, the same below

图2 衣霉素处理的抗病分析及衣霉素处理和lew1突变体中生长素的应答A：Camalexin的含量：5 μg/mL衣霉素处理14 d的幼苗24 h，DMSO处理为对照，通过HPLC检测Camalexin的含量。B：灰霉菌感染表型：5 μg/mL衣霉素处理叶片，DMSO处理为对照，在叶片中央滴加B.cinerea孢子悬液，3 d后观察表型；wrky33突变体和WRKY33过表达植株（WRKY33OE）叶片作对照，标尺=5 mm。C：用Image J软件对图B叶片的菌斑大小进行测量。D：暗处理5 d后野生型和lew1突变体下胚轴的伸长表型。E：用Image J软件对图D中的下胚轴长度进行测量。F：对4 d的幼苗进行GUS染色，检测DR5：‍：‍GUS在野生型和lew1突变体中的表达。WT：野生型；TM：衣霉素；DMSO：二甲基亚砜。*表示差异显著，P<0.05；**表示差异极显著，P<0.01，下同

Fig. 2 Disease resistance treated with TM and auxin response after TM treatment and in the lew1 mutantA: Camalexin levels: 14-day-old seedlings were treated with 5 μg/mL TM for 24 h, with DMSO treatment as control, then the Camalexin contents were quantified using HPLC. B: Phenotypes after B.cinerea infection: Leaves were treated with 5 μg/mL TM, using DMSO treatment as control, followed by adding B. cinerea spore suspension onto the leaves, then the phenotypes were observed after 3 d; wrky33 mutant and WRKY33 over-expression plants were used as controls, bar = 5 mm. C: The lesion sizes on the leaves in figure B were measured using Image J. D: Elongation phenotype of hypocotyls of wild type and lew1 mutant after 5 d of dark treatment. E: The hypocotyl lengths in figure D were measured using Image J software.. F: GUS staining of 4-day-old seedlings was conducted to detect the response of DR5::GUS in WT and lew1 mutant. WT: Wild type; TM: tunicamycin; DMSO: dimethyl sulfoxide. * indicates significant differences (P<0.05); ** indicates significant differences (P<0.01), the same below

图3 链格孢菌和灰霉菌感染后bZIP28和bZIP60的表达A：灰霉菌感染不同时间后基因的表达。B：链格孢菌感染不同时间后基因的表达。将生长6 d的野生型幼苗转移到MS液体培养基中，24 h全光照继续培养6 d，用8×104孢子/mL的A.brassicicola或B.cinerea孢子悬液处理0、4、8、12和24 h，通过RT-qPCR检测bZIP28、bZIP60和WRKY33的相对表达量。hpi：感染后几小时

Fig. 3 Expression of bZIP28 and bZIP60 after Alternaria brassicicola and Botrytis cinerea infectionA: Gene expressions after Botrytis cinerea infection for different times. B: Gene expressions after Alternaria brassicicola infection for different times. 6-day-old WT seedlings were transferred into MS liquid medium and subsequently cultured under continuous light conditions for 6 d. The seedlings were then immersed in suspension containing 8×104 spores/mL of either A. brassicicola or B. cinerea for 0, 4, 8, 12, and 24 h. RT-qPCR analysis was performed to determine the relative expressions of bZIP28, bZIP60, and WRKY33. The data represent the mean fold difference ±SD of three biological replicates, Statistically significant differences (P<0.05) are indicated by distinct letters. Student's t-test was used to determine the P-values. hpi: Hours post injection

图4 bZIP28和bZIP60过表达对植物抗病性的影响A：bZIP28和bZIP60过表达植株感染灰霉菌的表型，标尺=2 mm，OE#4/#6和OE#3/#8表示不同的转基因株系。B：bZIP28和bZIP60在过表达植株中的表达。C：bZIP28和bZIP60过表达植株感染灰霉菌后的菌斑大小统计

Fig. 4 Effects of bZIP28 and bZIP60 overexpression on plant resistance to diseaseA:Phenotypes of ZIP28 and bZIP60 over-expressed plants infected with B. cinerea, bar = 2 mm, OE#4/#6 and OE#3/#8 indicate different transgenic lines. B: Expression of bZIP28 and bZIP60 in over-expressed plants, ** indicates significant difference. C: Statistical analysis of lesion size in bZIP28 and bZIP60 overexpressed plants after Botrytis cinerea infection

图5 双荧光素酶报告实验检测bZIP28和bZIP60对WRKY33的转录调控作用A：5 μg/mL衣霉素处理24 h幼苗，通过RT-qPCR检测基因的相对表达。B：双荧光素酶报告实验所构建的载体：Full表示WRKY33启动子全长，启动子被截短成a（-1--566 bp）和b（-566--1 724 bp）片段，启动子a段被截短成a1（-364--566 bp）和a2（-1--364 bp）片段。C：bZIP28或bZIP60表达载体与融合有WRKY33启动子全长的报告载体共转化拟南芥原生质体，检测荧光素酶活性比值，EV（空载体）为对照。D：bZIP28和bZIP60对WRKY33启动子a和b段的调控。E：bZIP28和bZIP60对WRKY33启动子a1和a2段的调控

Fig. 5 Transcription regulatory roles of bZIP28 and bZIP60 on WRKY33 detected using dual-luciferase reporter assayA: Seedlings were treated with 5 μg/mL TM for 24 h, and the relative expressions of genes were detected by RT-qPCR. B: Constructed vector used in the dual-luciferase reporter assay: Full indicates the full-length WRKY33 promoter, the promoter is truncated into segments “a” (-1 to -566 bp) and “b” (-566 to -1724bp); segment “a” in the promotor is further truncated into “a1” (-364 to -566 bp) and “a2” (-1 to -364 bp). C: bZIP28 or bZIP60 expression vector and constructs fused with full-length WRKY33 promoter reporter vector were co-transformed into Arabidopsis protoplasts to measure the ratio of luciferase enzyme activity, with EV (empty vector) as a control. D: Regulation of WRKY33 promoter segments “a” and “b” by bZIP28 and bZIP60. E: Regulation of WRKY33 promoter segments “a1” and “a2” by bZIP28 and bZIP60

图6 酵母单杂交检测bZIP28和bZIP60对WRKY33的转录调控作用将WRKY33启动子不同区段分别连接pHIS2，与pGADT7-bZIP28D或pGADT7-bZIP60S共转酵母Y187。空载体AD为阴性对照，共转pHIS2-53和AD-53为阳性对照

Fig. 6 Transcription regulatory roles of bZIP28 and bZIP60 on WRKY33 detected using yeast one-hybrid assayDifferent segments of the WRKY33 promoter were separately fused to pHIS2 and co-transformed into Y187 with either pGADT7-bZIP28D or pGADT7-bZIP60S. The empty vector AD served as a negative control, while co-transformation of pHIS2-53 and AD-53 served as a positive control

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