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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2023, Vol. 49 Issue (1): 23-30    DOI: 10.3785/j.issn.1008-9209.2022.01.112
Biological sciences & biotechnologies     
Heat shock transcription factor HSFB2b negatively regulates plant thermomorphogenesis in Arabidopsis
Ziwei YAO1(),Jingliang SUN2,Jianxiang LIU1(),Haiping LU1()
1.College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China
2.College of Environment and Bioresources, Dalian Minzu University, Dalian 116600, Liaoning, China
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Abstract  

In order to explore whether heat shock transcription factor (HSF) known to be involved in plant adaptation to extreme heat stress is also involved in plant thermomorphogenesis at warm temperatures, the result of CRISPR/Cas9 gene editing, physiological and biochemical, genetic experiments, and effector-reporter assay demonstrated that the heat shock transcription factor HSFB2b was induced by the warm temperature and played an important role in the process of plant thermomorphogenesis. Under the warm temperature (29 ℃), the Arabidopsis mutant hsfb2b exhibited a longer hypocotyl than the wild type, suggesting that HSFB2b functioned as a negative regulator in thermomorphogenesis. Subcellular localization results showed that the HSFB2b protein was localized in the nucleus. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed that the heat shock proteins (HSPs) gene, the heat shock transcription factor HSFA2, and the jasmonic acid degradation gene ST2A were up-regulated in the wild type under the warm temperature relative to the normal temperature (22 ℃), but these genes were more up-regulated by the warm temperature in the hsfb2b mutant than that in the wild type. Furthermore, effector-reporter assay demonstrated that HSFB2b could inhibit ST2A expression by binding to the heat shock element (HSE). In conclusion, the heat shock transcription factor HSFB2b induced by the warm temperature played a negative regulatory role in the hypocotyl elongation and negatively regulated the expression of gene ST2A by recognizing the HSE in molecular mechanism, thusnegatively regulated the plant thermomorphogenesis.

Key words： Arabidopsis      thermomorphogenesis      heat shock transcription factor      jasmonic acid catabolism      hypocotyl elongation     
Received: 11 January 2022      Published: 07 March 2023
CLC:  Q943.2  
Corresponding Authors: Jianxiang LIU,Haiping LU     E-mail: 21907011@zju.edu.cn;jianxiangliu@zju.edu.cn;luhaiping@zju.edu.cn
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Ziwei YAO
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Cite this article:

Ziwei YAO,Jingliang SUN,Jianxiang LIU,Haiping LU. Heat shock transcription factor HSFB2b negatively regulates plant thermomorphogenesis in Arabidopsis. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(1): 23-30.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.01.112     OR     https://www.zjujournals.com/agr/Y2023/V49/I1/23


拟南芥热激转录因子HSFB2b负调控植物热形态建成

为探究参与极端高温环境应答的热激转录因子（heat shock transcription factor, HSF）是否也参与了温和高温下的植物热形态建成，本研究通过CRISPR/Cas9基因编辑技术，结合一系列生理生化和遗传学实验，以及效应因子-报告基因系统（effector-reporter）等实验方法，发现热激转录因子HSFB2b受温和高温诱导，在热形态建成过程中发挥着重要作用。在温和高温（29 ℃）下，拟南芥突变体hsfb2b表现出比野生型更长的下胚轴表型，揭示HSFB2b作为负调控因子在热形态建成中发挥作用。亚细胞定位实验表明，HSFB2b蛋白在细胞核中发挥功能。实时荧光定量聚合酶链反应分析表明，温和高温下野生型植株中热激蛋白（heat shock proteins, HSPs）基因、热激转录因子HSFA2和茉莉酸降解基因ST2A相对于常温（22 ℃）时表达上调，但这些基因在突变体hsfb2b中上调倍数更大。此外，效应因子-报告基因系统实验证实HSFB2b可以结合ST2A启动子热激元件（heat shock element, HSE），从而抑制ST2A表达。综上所述，受温和高温诱导的热激转录因子HSFB2b在下胚轴伸长调控中起负调控作用，并且在分子机制上HSFB2b通过识别HSE元件，负调控茉莉酸降解相关基因ST2A的表达，从而负调控植物热形态建成。


关键词： 拟南芥,  热形态建成,  热激转录因子,  茉莉酸代谢,  下胚轴伸长 

基因

Gene

编号

Code

引物序列（5′→3′）

Primer sequence (5′→3′)

HSFB2aAT5G62020F: CTATCCCAACGCCGTTTCTC
R: CGAAATCTGTCGGATTCCATACGA
HSFB2bAT4G11660F: TTGTTCGTCAGCTCAATACTTACG
R: TTTCCGCCGTTGAATATCCCGAA
HSFA2AT2G26150F: GCGGCTTCTTCATCTGTA
R: CTGTTACGACCATTACTCCAA
ST2AAT5G07010F: ATCCCAAGTTTCTCCATGTG
R: AACATCTCTTGGAACTCGCA
HSP17.6AAT1G59860F: ATGGGATCCATTCAAGGAACTT
R: TTCTTTCTTCATCCCCGGTAAA
HSP17.8AT1G07400F: CTCAAGATCAGTGGAGAGAGAC
R: CTTCTTCTTAGCCTCTTCCACT
HSP23.6AT4G25200F: ATGGCATCTGCTCTCGCTCT
R: AGGAACAGAGCGGCGATATA
PP2AAT1G13320F: TAACGTGGCCAAAATGATGC
R: GTTCTCCACAACCGCTTGGT
Table 1 Primers used in qRT-PCR experiment
Fig. 1 Expression patterns of homology genes of HSFB2a and HSFB2b at the warm temperatureA. Homology alignment of protein sequences of HSFB subfamily in Arabidopsis; B-C. Expression levels of HSFB2a and HSFB2b at different temperatures. n=3.
Fig. 2 Negative regulatory role of HSFB2b in thermomorphogenesis of ArabidopsisA-B. Mutant sequences of hsfb2a and hsfb2b;C-D. Phenotype and length of hypocotyls in Arabidopsis. WT: Wild type. Different lowercase letters above bars indicate significant differences at the 0.05 probability level, and the same as below.
Fig. 3 Subcellular localizations of HSFB2a and HSFB2b proteins
Fig. 4 Expression patterns of downstream genes of HSFB2b at the warm temperature
Fig. 5 Negative regulation on thermomorphogenesis by HSFB2bA. Schematic diagram of the effector-reporter vectors; B. Relative luciferase activity; C. Working model for the regulatory role of HSFB2b in plant thermomorphogenesis. Single asterisk (*) indicates significant differences at the 0.05 probability level.
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