Mechanism of GhWRKY21 Transcription Factor in Regulating Plant 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 Plant High Temperature Resistance[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1061.

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

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