Limited information is available regarding the exact function of specific WRKY transcription factors in plant responses to heat stress. We analyzed the roles of WRKY25, WRKY26, and WRKY33, three types of group I WRKY proteins, in the regulation of resistance to heat stress. Expression of WRKY25 and WRKY26 was induced upon treatment with high temperature, whereas WRKY33 expression was repressed. Heat-treated WRKY single mutants exhibited small responses, while wrky25wrky26 and wrky25wrky33 double mutants and the wrky25wrky26wrky33 triple mutants showed substantially increased susceptibility to heat stress, showing reduced germination, decreased survival, and elevated electrolyte leakage, compared with wild-type plants. In contrast, constitutive expression of WRKY25, WRKY26, or WRKY33 enhanced resistance to heat stress. Expression studies of selected heat-defense genes in single, double, and triple mutants, as well as in over-expressing lines, were correlated with their thermotolerance phenotypes and demonstrated that the three WRKY transcription factors modulate transcriptional changes of heat-inducible genes in response to heat treatment. In addition, our findings provided evidence that WRKY25, WRKY26, and WRKY33 were involved in regulation of the heat-induced ethylene-dependent response and demonstrated positive cross-regulation within these three genes. Together, these results indicate that WRKY25, WRKY26, and WRKY33 positively regulate the cooperation between the ethylene-activated and heat shock proteins-related signaling pathways that mediate responses to heat stress; and that these three proteins interact functionally and play overlapping and synergetic roles in plant thermotolerance.
The WRKY family is one of the major groups of plant-specific transcriptional regulators. Arabidopsis thaliana WRKY25, which is induced by heat stress, is one of the group I WRKY proteins and responds to both abiotic and biotic stress. This study has examined the regulatory role of WRKY25 using wrky25 mutant and over-expressing WRKY25 transgenic A. thaliana. After 45 degrees C for different time periods, wrky25 null mutants showed a moderate increase in thermosensitivity with decreased germination, reduced hypocotyl and root growth, and enhanced conductivity compared to those of wide-type, while WRKY25 over-expressed transgenic seeds exhibited enhanced thermotolerance. Northern blot analysis of wrky25 mutants and WRKY25 over-expressing plants identified putative genes regulated by WRKY25. In consistence with the implication of WRKY25 in heat tolerance, the expression level of six heat-inducible genes and two oxidative stress-responsive genes was more or less down-regulated in wrky25 mutants during heat stress. Among them, heat shock protein Hsp101, heat shock transcription factor HsfB2a, and cytosolic ascrobate peroxidase APX1 were reduced more obviously than other detected genes. Meanwhile, over-expression of WRKY25 increased the expression of HsfA2, HsfB1, HsfB2a, and Hsp101 slightly or moderately. Together, these findings reveal that WRKY25 plays a partial role in thermotolerance.
Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.
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