番茄SlWRKY45转录因子在响应低温和干旱胁迫中的功能(长英文摘要)

doi:10.11983/CBB24101

植物学报 ›› 2025, Vol. 60 ›› Issue (2): 186-203.DOI: 10.11983/CBB24101 cstr: 32102.14.CBB24101

番茄SlWRKY45转录因子在响应低温和干旱胁迫中的功能(长英文摘要)

樊蓓¹, 任敏¹, 王延峰¹^,², 党峰峰¹^,², 陈国梁¹^,², 程国亭¹^,², 杨金雨¹, 孙会茹¹^,²^,^*()

¹延安大学生命科学学院, 延安 716000
²延安大学生命科学学院, 陕西省黄土高原资源植物研究与利用省市共建重点实验室, 延安 716000

收稿日期:2024-07-08 接受日期:2024-10-14 出版日期:2025-03-10 发布日期:2024-10-16
通讯作者: 孙会茹
基金资助:
延安大学博士科研启动项目(YDBK2019-42);陕西省自然科学基础研究计划(2022JQ-159);陕西省大学生创新创业训练计划(S202310719073)

Functions of SlWRKY45 in Response to Low-temperature and Drought Stress in Tomato

Bei Fan¹, Min Ren¹, Yanfeng Wang¹^,², Fengfeng Dang¹^,², Guoliang Chen¹^,², Guoting Cheng¹^,², Jinyu Yang¹, Huiru Sun¹^,²^,^*()

¹College of Life Sciences, Yan’an University, Yan’an 716000, China
²Shaanxi Key Laboratory of Research and Utilization of Resource Plants on the Loess Plateau, College of Life Sciences, Yan’an University, Yan’an 716000, China

Received:2024-07-08 Accepted:2024-10-14 Online:2025-03-10 Published:2024-10-16
Contact: Huiru Sun

1. 附录.pdf(435KB)

摘要/Abstract

摘要： 番茄(Solanum lycopersicum)在生长发育过程中常受到低温和干旱等多种非生物胁迫的影响。WRKY转录因子参与调控植物多种非生物胁迫响应过程, 而SlWRKY45在番茄非生物胁迫中的功能尚不清楚。基因表达分析发现, 低温、干旱和ABA处理均可显著诱导SlWRKY45的表达; 过表达SlWRKY45可提高番茄对干旱和低温的耐受性; 在干旱和低温处理下, 过表达株系的光合指标、抗氧化酶活性和脯氨酸(Pro)含量显著高于野生型(WT), 活性氧(ROS)和丙二醛(MDA)含量显著低于WT。转录组数据分析显示, SlWRKY45主要通过调控抗氧化酶活性和胁迫响应途径介导番茄对低温胁迫的响应。双荧光素酶报告基因检测发现, SlWRKY45可直接激活SlPOD1的表达。酵母双杂交(Y2H)和双分子荧光互补(BiFC)试验结果表明, SlWRKY45与SlWRKY46存在相互作用。综上表明, SlWRKY45可能通过直接调控抗氧化酶途径增强转基因番茄的抗逆性, 为番茄的遗传改良提供了重要的候选基因资源。

关键词: 番茄, SlWRKY45, 干旱胁迫, 低温胁迫, 抗氧化酶

Abstract: INTRODUCTION Tomato (Solanum lycopersicum), a significant warm-season and water-dependent vegetable crop, is extensively cultivated worldwide. Whether grown in open fields or protected environments, tomatoes frequently encounter various environmental stresses, including drought and low temperatures, which significantly impact their yield and quality. Transcription factors play a pivotal role in plant stress responses by modulating the expression of specific target genes, thereby transmitting perceived stress signals downstream. WRKY transcription factors in tomatoes are known to regulate responses to multiple abiotic stresses. However, the specific role of the tomato SlWRKY45 in abiotic stress responses remains unclear. RATIONALEStudies have demonstrated that WRKY transcription factors play a crucial regulatory role in plant responses to abiotic stress. As an important economic vegetable crop, tomato is susceptible to various environmental stresses during its growth and development. By genetically overexpressing SlWRKY45 in tomato and investigating its function under low-temperature and drought stress conditions, the findings can provide a theoretical foundation for understanding the complex regulatory mechanisms of WRKY transcription factors. Additionally, this research offers valuable candidate genes for breeding stress-resistant tomato varieties. RESULTSExpression analysis revealed that low-temperature, drought, and abscisic acid (ABA) treatments significantly induced the expression of SlWRKY45. Overexpression of SlWRKY45 enhanced the resistance of tomato plants to drought and low-temperature stresses. Under drought and low-temperature conditions, the photosynthetic indices, antioxidant enzyme activities, and proline (Pro) contents in SlWRKY45 overexpression lines were significantly higher than those in wild-type (WT) plants. Conversely, the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) levels in SlWRKY45-OE plants was significantly lower than in WT plants under the same stress conditions. Transcriptome data analysis indicated that SlWRKY45 regulates tomato's response to low-temperature stress primarily by influencing antioxidant enzyme activities and stress response pathways. Dual-luciferase assays demonstrated that SlWRKY45 could directly activate the expression of SlPOD1. Furthermore, the interaction between SlWRKY45 and SlWRKY46 was confirmed through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. CONCLUSIONOur findings demonstrate that SlWRKY45 positively regulates drought resistance and low-temperature tolerance in tomato. Additionally, SlWRKY45 can interact with SlWRKY46 and directly activate the expression of SlPOD1. These results offer valuable insights for further research into the regulatory mechanisms underlying abiotic stress responses and provide potential gene resources for genetic improvement through molecular breeding. Phenotypes of SlWRKY45-overexpressing and wild-type plants under drought and low-temperature treatments

Key words: tomato, SlWRKY45, drought stress, low-temperature stress, antioxidase

中图分类号:

S641.2

樊蓓, 任敏, 王延峰, 党峰峰, 陈国梁, 程国亭, 杨金雨, 孙会茹. 番茄SlWRKY45转录因子在响应低温和干旱胁迫中的功能(长英文摘要). 植物学报, 2025, 60(2): 186-203.

Bei Fan, Min Ren, Yanfeng Wang, Fengfeng Dang, Guoliang Chen, Guoting Cheng, Jinyu Yang, Huiru Sun. Functions of SlWRKY45 in Response to Low-temperature and Drought Stress in Tomato. Chinese Bulletin of Botany, 2025, 60(2): 186-203.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24101

https://www.chinbullbotany.com/CN/Y2025/V60/I2/186

图/表 7

图1 低温处理下SlWRKYs的表达热图(A)以及SlWRKY45在低温(B)、干旱(C)、脱落酸(ABA) (D)处理下和不同组织(E)中的表达 (B), (C), (D) SlWRKY45的相对表达量分别以4°C、ABA和PEG6000处理0小时为对照; (E) SlWRKY45的相对表达量以茎为对照。** 表示在P<0.01水平上差异显著。

Figure 1 Heat map of SlWRKYs under low-temperature treatment (A) and SlWRKY45 expression under 4°C (B), drought (C), and abscisic acid (ABA) (D) treatments and in different tissues (E) (B), (C), (D) The relative expression levels of SlWRKY45 at 0 h under treatments of 4°C, ABA and PEG6000 were utilized as controls, respectively; (E) The relative expression of SlWRKY45 in stems served as the control. ** represents significant differences at P<0.01.

图2 SlWRKY45的转录自激活 pGBKT7-53+pGADT7-T和pGBKT7空载质粒分别为阳性对照和阴性对照。

Figure 2 Transcriptional self-activation of SlWRKY45 pGBKT7-53+pGADT7-T and the empty vector pGBKT7 represent the positive control and negative control, respectively.

图3 干旱处理下SlWRKY45-OE转基因和WT番茄植株的表型(A)、光合指标(B)、DAB和NBT染色(C)及生理生化指标(D) (B), (D) **表示在P<0.01水平上差异显著。DAB: 二氨基联苯胺; NBT: 氮蓝四唑; WT: 野生型; SOD: 超氧化物歧化酶; POD: 过氧化物酶; MDA: 丙二醛; Pro: 脯氨酸. #1和#2为不同的转基因株系。Bars=1 cm

Figure 3 Phenotype (A), photosynthesis indicators (B), DAB, and NBT staining (C) and physiological indicators (D) of SlWRKY45-OE transgenic and WT tomato plants under drought treatment (B), (D) ** represent significant differences at P<0.01. DAB: Diaminobenzidine; NBT: Nitrotetrazolium blue chloride; WT: Wild type; SOD: Superoxide; POD: Peroxidase; MDA: Malondialdehyde; Pro: Proline. #1 and #2 are different transgenic lines. Bars=1 cm

图4 SlWRKY45-OE转基因和WT番茄植株在4°C处理下的表型(A)、光合指标(B)、DAB和NBT染色(C)及生理指标(D), (E) (B)-(E) *和**分别表示在P<0.05和P<0.01水平上差异显著。WT: 野生型。DAB、NBT、SOD、POD、MDA和Pro同图3。#1、#2为不同的转基因株系。Bars=1 cm

Figure 4 Phenotype (A), photosynthesis indicators (B), DAB, and NBT staining (C), and physiological indicators (D), (E) of SlWRKY45-OE transgenic and WT tomato plants under 4°C treatment (B)-(E) * and ** represent significant differences at P<0.05 and P<0.01, respectively. WT: Wild type. DAB, NBT, SOD, POD, MDA, and Pro are the same as shown in Figure 3. #1 and #2 are different transgenic lines. Bars=1 cm

图5 SlWRKY45-OE转基因番茄和WT在低温处理前后的RNA-seq分析 (A) 差异表达基因(DEGs)火山图(红色、蓝色和灰色分别代表上调、下调和无差异表达基因); (B) GO聚类(红色星号和线条标出的为显著富集的通路); (C) KEGG富集分析(红色星号和线条标出的为显著富集的通路); (D) 挑选的6个DEGs的表达热图; (E) 6个DEGs的相对表达量(以常温下WT为对照。**表示在P<0.01水平上差异显著。WT: 野生型。#1和#2为不同的转基因株系)

Figure 5 RNA-seq analysis of SlWRKY45-OE transgenic tomato and WT plants before and after low-temperature treatment (A) Volcano diagram of DEGs (red, blue and gray represent upregulated, downregulated and undifferentially expressed genes, respectively); (B) GO cluster (the red asterisks and lines indicate significantly enriched pathways); (C) KEGG enrichment (the red asterisks and lines indicate significantly enriched pathways); (D) The heatmap of 6 selected DEGs; (E) The relative expression of the 6 selected DEGs (the relative expression of WT under normal temperature was used as control. ** represent significant differences at P<0.01. WT: Wild type. #1 and #2 are different transgenic lines)

图6 双荧光素酶报告基因检测分析SlWRKY45对SlPOD1和SlPOD启动子的激活作用 (A) SlPOD1和SlPOD启动子序列分析(加粗下划线表示W-box-like和W-box元件); (B) 报告基因和效应基因的载体示意图; (C) LUC/REN分析(以62SK+SlPOD1 pro和62SK+SlPOD pro为对照组。**表示在P<0.01水平上差异显著)

Figure 6 Dual-luciferase reporter assays analysis of SlWRKY45 on SlPOD1 and SlPOD promoters activation (A) Sequence analysis of the SlPOD1 and SlPOD promoters (the bold underlines represent W-box-like and W-box elements); (B) Schematic representation of reporter and effector vectors; (C) LUC/REN analysis (62SK+SlPOD1 pro and 62SK+SlPOD pro are the control groups. ** represents significant differences at P<0.01)

图7 酵母双杂交(Y2H) (A)和双分子荧光互补(BiFC) (B)检测SlWRKY45与SlWRKY46的相互作用 (A) pGBKT7-53+pGADT7-T为阳性对照, pGBKT7+pGADT7-SlWRKY45和pGBKT7-SlWRKY46+pGADT7为阴性对照; (B) 图片从左到右依次为黄色荧光信号、明场和叠加, nYFP-SlWRKY45+cYFP和nYFP+SlWRKY46-cYFP为阴性对照。Bars=20 µm

Figure 7 Interaction of SlWRKY45 and SlWRKY46 via yeast two-hybrid (Y2H) (A) and bimolecular fluorescence complementation (BiFC) (B) (A) pGBKT7-53+pGADT7-T represents a positive control, pGBKT7+pGADT7-SlWRKY45 and pGBKT7-SlWRKY46+pGADT7 represent negative controls; (B) From left to right, the yellow fluorescent signal, bright field image and merge image are shown, nYFP-SlWRKY45+cYFP and nYFP+SlWRKY46-cYFP represent negative controls. Bars=20 µm

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番茄SlWRKY45转录因子在响应低温和干旱胁迫中的功能(长英文摘要)

Functions of SlWRKY45 in Response to Low-temperature and Drought Stress in Tomato

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