Alternative Splicing Provides a Mechanism to Regulate LlHSFA3 Function in Response to Heat Stress in Lily

Heat stress transcription factors (HSFs) are central regulators of plant responses to heat stress. Their heat-induced transcriptional regulation has been extensively studied; however, their posttranscriptional and posttranslational regulation is poorly understood. In a previous study, we established that there were at least two HSFA3 homologs, LlHSFA3A and LlHSFA3B, in lily (Lilium spp.) and that these genes played distinct roles in thermotolerance. Here, we demonstrate that LlHSFA3B is alternatively spliced under heat stress to produce the heat-inducible splice variant LlHSFA3B-III. We further show that LlHSFA3B-III protein localizes in the cytoplasm and nucleus, has no transcriptional activity, and specifically disturbs the protein interactions of intact HSFA3 orthologs LlHSFA3A-I and LlHSFA3B-I. Heterologous expression of LlHSFA3B-III in Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana increased plant tolerance of salt and prolonged heat at 40 degrees C, yet reduced plant tolerance of acute heat shock at 45 degrees C. Conversely, heterologous expression of LlHSFA3A-I caused opposing phenotypes, which were substantially ameliorated by coexpression of LlHSFA3B-III. LlHSFA3B-III interacted with LlHSFA3A-I to limit its transactivation function and temper the function of LlHSFA3A-I, thus reducing the adverse effects of excessive LlHSFA3A-I accumulation. Based on these observations, we propose a regulatory mechanism of HSFs involving heat-inducible alternative splicing and protein interaction, which might be used in strategies to promote thermotolerance and attenuate the heat stress response in crop plants. A genetic buffering mechanism of lily through alternative splicing counteracts the side effect of LlHSFA3 in response to heat stress.