Mechanism of GhWRKY21 Transcription Factor in Regulating Cotton High Temperature Resistance

doi:10.13560/j.cnki.biotech.bull.1985.2025-1061

Abstract

Abstract:

Objective To investigate the molecular mechanism of the cotton WRKY transcription factor GhWRKY21 under high-temperature stress and elucidate its functional role in cotton stress resistance. Method GhWRKY21-silenced cotton plants were constructed using virus-induced gene silencing (VIGS) and preliminarily functionally characterized. Concurrently, GhWRKY21-overexpressing transgenic tobacco lines were generated via Agrobacterium-mediated leaf disc transformation. Functional assessment of GhWRKY21 in heat-stress response was then conducted through a series of experiments spanning seed germination assays, seedling phenotypic analysis, microscopic examination of leaf guard cells, and measurement of reactive oxygen species (ROS) accumulation under high-temperature stress, thereby evaluating its role at phenotypic, cellular, and physiological levels. Result GhWRKY21 is a member of the IId subfamily of WRKY transcription factors in cotton. Its expression can be induced by temperature, H₂O₂, and signaling molecules. Functional analysis revealed that silencing GhWRKY21 in cotton significantly enhanced thermotolerance. Conversely, its overexpression in tobacco increased sensitivity to heat stress, as evidenced by reduced seed germination rates, suppressed root elongation, and impaired stomatal closure in leaf guard cells. Further physiological and molecular evidence suggests that GhWRKY21 may participate in the plant response to high-temperature stress by modulating the accumulation of ROS. Conclusion GhWRKY21 functions as a negative regulator in plant high-temperature stress responses by modulating ROS homeostasis, thereby influencing plant stress resistance.

Key words: Gossypium hirsutum, WRKY transcription factor, GhWRKY21, tobacco, heat, ROS, stomata

WANG Jia-yu, WANG Chen. Mechanism of GhWRKY21 Transcription Factor in Regulating Cotton High Temperature Resistance[J]. Biotechnology Bulletin, 2026, 42(10): 134-141.

Figures/Tables 5

Fig. 1 Amino acid sequence alignment (A) and phylogenetic analysis (B) of WRKY21 from different speciesA: Amino acid sequence homology comparison of GhWRKY21 from Gossypium hirsutum, AtWRKY21 from Arabidopsis thaliana, PmWRKY21 from pine, PtWRKY21 from poplar, and TcWRKY21 from Taxus chinensis. The black regions indicate the same amino acids, where the 60-amino-acid WRKY domain is marked by a double-headed arrow. The boxed sequences are the highly conserved amino-acid sequence WRKYGQK and the conserved C-terminal motif. The putative nuclear localization signal, KKRK, is marked by asterisks (****). B: The phylogenetic tree shows the seven subgroups of the WRKY family. The position of GhWRKY21 is marked with a black rectangular box. The tree was constructed using MEGA 5.0

Fig. 2 Expression pattern analysis of GhWRKY21 in cottonData are presented as the mean±standard error (SE) of three independent experiments. Different letters above the error bars indicate statistically significant differences among groups (P<0.05) based on Tukey’s HSD test. The same below

Fig. 3 Silencing of GhWRKY21 enhances heat tolerance in cottonA: Representative phenotypes of GhWRKY21-silenced cotton plants after heat stress (42 ℃, 24 h). B: Relative expression of GhWRKY21 in GhWRKY21-silenced cotton. C: Representative leaf phenotypes of GhWRKY21-silenced cotton plants after heat treatment (42 ℃, 24 h)

Fig. 4 Overexpression of GhWRKY21 increases the sensitivity to heat stress in transgenic plantsA, B: Seed germination phenotype and germination rates of Vec and OE tobacco lines exposed to heat stress (42 ℃, 6 h). C, D: Root length phenotype and statistical analysis of empty vector control and transgenic seeds after germination, exposed to heat stress (42 ℃) for 6 days. E: Phenotypes of Vec and OE plants grown to 8 weeks after 12 h high-temperature treatment (42 ℃). F: Survival rates of 8-week-old Vec and OE after a 12 h high-temperature treatment (42 ℃) followed by recovery under normal growth conditions. G, H: Stomatal closure degree of Vec and OE lines under heat stress (42 ℃, 12 h)

Fig. 5 Overexpression of GhWRKY21 increases the sensitivity of transgenic tobacco plants to oxidative stressA: Representative phenotypes of leaves from Vec and OE plants showing DAB and NBT staining after heat treatment. B: Phenotypes of leaf discs obtained from Vec and OE plants after being incubated in different concentrations of MV. C: The chlorophyll content of tobacco leaf discs from empty vector control and transgenic plants under different concentrations was quantified in Fig. B. Tukey’s HSD test, * P<0.05, ** P<0.01, and *** P<0.001

References 24

[1]	Yamasaki K, Kigawa T, Inoue M, et al. Structures and evolutionary origins of plant-specific transcription factor DNA-binding domains [J]. Plant Physiol Biochem, 2008, 46(3): 394-401.
[2]	Eulgem T, Rushton PJ, Robatzek S, et al. The WRKY superfamily of plant transcription factors [J]. Trends Plant Sci, 2000, 5(5): 199-206.
[3]	Jiang YJ, Liang G, Yu DQ. Activated expression of WRKY57 confers drought tolerance in Arabidopsis [J]. Mol Plant, 2012, 5(6): 1375-1388.
[4]	Yan HR, Jia HH, Chen XB, et al. The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production [J]. Plant Cell Physiol, 2014, 55(12): 2060-2076.
[5]	Yan Y, Jia HH, Wang F, et al. Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana [J]. Front Physiol, 2015, 6: 265.
[6]	胡启瑞, 宋桂成, 王雪姣, 等. 高温对陆地棉花粉萌发及棉铃发育的影响 [J]. 新疆农业大学学报, 2017, 40(1): 1-7.
	Hu QR, Song GC, Wang XJ, et al. Effects of high temperature on pollen germination and boll development in upland cotton [J]. J Xinjiang Agric Univ, 2017, 40(1): 1-7.
[7]	刘群, 陈振, 张巨松, 等. 高温胁迫对海岛棉光合生理及棉铃发育的影响 [J]. 西北植物学报, 2020(9): 1574-1581.
	Liu Q, Chen Z, Zhang JS, et al. Effect of high temperature stress on photosynthetic physiology and boll development of island cotton [J]. Acta Bot Boreali Occidentalia Sin, 2020(9): 1574-1581.
[8]	Cottee NS, Tan DKY, Bange MP, et al. Multi-level determination of heat tolerance in cotton (Gossypium hirsutum L.) under field conditions [J]. Crop Sci, 2010, 50(6): 2553-2564.
[9]	Song GC, Wang MM, Zeng B, et al. Anther response to high-temperature stress during development and pollen thermotolerance heterosis as revealed by pollen tube growth and in vitro pollen vigor analysis in upland cotton [J]. Planta, 2015, 241(5): 1271-1285.
[10]	Min L, Li YY, Hu Q, et al. Sugar and auxin signaling pathways respond to high-temperature stress during anther development as revealed by transcript profiling analysis in cotton [J]. Plant Physiol, 2014, 164(3): 1293-1308.
[11]	Pettigrew WT. The effect of higher temperatures on cotton lint yield production and fiber quality [J]. Crop Sci, 2008, 48(1): 278-285.
[12]	王佳玉. 棉花转录因子GhWRKY21调控植株抗旱和耐高温的分子机理研究 [D]. 泰安: 山东农业大学, 2020.
	Wang JY. Moleculor mechanism of cotton GhWRKY21 transcription factor regulating plant drought and high temperature resistance [D]. Tai’an: Shandong Agricultural University, 2020.
[13]	Journot-Catalino N, Somssich IE, Roby D, et al. The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana [J]. Plant Cell, 2006, 18(11): 3289-3302.
[14]	Cai RH, Zhao Y, Wang YF, et al. Overexpression of a maize WRKY58 gene enhances drought and salt tolerance in transgenic rice [J]. Plant Cell Tiss Organ Cult, 2014, 119(3): 565-577.
[15]	Wang CT, Ru JN, Liu YW, et al. Maize WRKY transcription factor ZmWRKY106 confers drought and heat tolerance in transgenic plants [J]. Int J Mol Sci, 2018, 19(10): 3046.
[16]	Ma JL, Wang YY, Hong YH, et al. SlWRKY55 coordinately acts with SlVQ11 to enhance tomato thermotolerance by activating SlHsfA2 [J]. Plant J, 2024, 119(6): 2904-2918.
[17]	Zhang ZJ, Yang C, Xi J, et al. The MdHSC70-MdWRKY75 module mediates basal apple thermotolerance by regulating the expression of heat shock factor genes [J]. Plant Cell, 2024, 36(9): 3631-3653.
[18]	Shi WN, Hao LL, Li J, et al. The Gossypium hirsutum WRKY gene GhWRKY39-1 promotes pathogen infection defense responses and mediates salt stress tolerance in transgenic Nicotiana benthamiana [J]. Plant Cell Rep, 2014, 33(3): 483-498.
[19]	Wang JY, Wang LJ, Yan Y, et al. GhWRKY21 regulates ABA-mediated drought tolerance by fine-tuning the expression of GhHAB in cotton [J]. Plant Cell Rep, 2021, 40(11): 2135-2150.
[20]	Mittler R, Zandalinas SI, Fichman Y, et al. Reactive oxygen species signalling in plant stress responses [J]. Nat Rev Mol Cell Biol, 2022, 23(10): 663-679.
[21]	Averill-Bates D. Reactive oxygen species and cell signaling. Review [J]. Biochim Biophys Acta BBA Mol Cell Res, 2024, 1871(2): 119573.
[22]	Li SH, Liu S, Zhang Q, et al. The interaction of ABA and ROS in plant growth and stress resistances [J]. Front Plant Sci, 2022, 13: 1050132.
[23]	Waszczak C, Carmody M, Kangasjärvi J. Reactive oxygen species in plant signaling [J]. Annu Rev Plant Biol, 2018, 69: 209-236.
[24]	Miller G, Suzuki N, Ciftci-Yilmaz S, et al. Reactive oxygen species homeostasis and signalling during drought and salinity stresses [J]. Plant Cell Environ, 2010, 33(4): 453-467.