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

Abstract

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°C, yet reduced plant tolerance of acute heat shock at 45°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.

TeaAS: a comprehensive database for alternative splicing in tea plants (Camellia sinensis)

“…In addition, the gene involved in the AS of an MYB transcription factor affects anthocyanin biosynthesis in tomato fruits, and mutations in the splice sites cause a complete loss of function in the wild-type protein [ 6 ]. Moreover, LlHSFA3B-III , an AS isoform of LlHSFA3B (heat stress transcription factors), was also found to regulate abiotic heat stress in lily plants [ 7 ]. In tea plants, an AS-associated analysis of different tissues showed that it could regulate the flavonoid pathway through certain transcription factors and functional genes [ 8 ].…”

RNA-seq Analysis Reveals Alternative Splicing Under Heat Stress in Rainbow Trout (Oncorhynchus mykiss)

“…For example, mouse hsf1 was alternatively spliced to form four HSF1 variants, which differentially regulated the expression of chaperones (Neueder et al 2014). The LlHSFA3B gene of lily was alternatively spliced under heat stress to produce the heat-inducible splice variant LlHSFA3B-III (Wu et al 2019). Another extremely important example of AS is the Dscam gene of Drosophila melanogaster, with more than 38,000 splice variants (Schmucker et al 2000).…”

A Novel R2R3-MYB Gene LoMYB33 From Lily Is Specifically Expressed in Anthers and Plays a Role in Pollen Development

“…In transgenic lines, the expression of AtMYB33 and AtMYB65 was also up-regulated, which might stem from their self-activating activity. The expression of AtMYB33, AtMYB65 , and AtCYP704B1 is independent of LoMYB33 expression, and the expression pattern of AtCYP703A2 is opposite that of LoMYB33 (Wu et al, 2019 ; Ding et al, 2021 ). We speculate that this might be explained by a negative feedback regulation mechanism involving LoMYB33 , wherein the high expression of LoMYB33 activates the feedback pathway to control the expression of these genes at a level appropriate for anther development.…”

“…The cells were then cultured with LB medium at 28 • C and 200 rpm for 3-5 h. After centrifugation and resuspending the bacteria, they were evenly spread on solid LB medium (containing 50 µg•µL −1 Kan and 100 µg•µL −1 Rif) and cultured at 28 • C for 2 days. The appropriate clones were then selected and cultured with liquid LB medium (containing 50 µg•µL −1 Kan and 100 µg•µL −1 Rif) for 12 h. The bacterial solution was resuspended with the injection buffer (10 mM MgCl 2 , 100 mM 2-morpholinoethanesulfonic acid, and 200 µM acetosyringone, pH = 5.8) and injected into tobacco leaves (Wu et al, 2019). After 48 h, the GFP signal was observed with a laser scanning confocal microscope (LSM800, Zeiss, Germany).…”