Analysis Of Thermotolerance And Salt Tolerance Mechanism Regulated By Heat Shock Transcription Factor LlHsfA3 From Lily(Lilium Longiflorum)

Lily prefers cool climate,does not tolerate high temperatures,and high temperature in summer severely limits the year-round production of lily.Lily has more themrotolerance germplasm,so it is feasible to use genetic engineering methods to improve commercial lily germplasm.Heat stress transcription factors(Hsfs)are central regulators of plant heat stress responses.Their heat-induced transcription regulation has been extensively studied,but their post-transcriptional and post-translational regulation is largely unknown.In this study,two homologous HsfA3 genes was isolated,the function and regulation of them were stuied.1.With a homologous-cloning method,two HsfA3 genes,LlHsfA3A and LlHsfA3B,were isolated from lily(Lilium longiflorum).Both genes were induced by heat stress,but not by salt stress.Under mannitol stress,LlHsfA3A was not induced,but LlHsfA3B was slightly upregulated.2.Overexpressing LlHsfA3A in Arabidopsis enhanced its basal and acquired thermotolerance,while overexpressing LlHsfA3B just enhanced its acquired thermotolerance.In both cases,overexpressing plants showed hypersensitivity to salt stress,and a lack of sucrose exacerbated this salt sensitivity.Using a transient assay,the opposite effects were observed in lily.3.Further analysis revealed that either LlHsfA3A or LlHsfA3B overexpression altered normal proline accumulation.During heat treatments,proline increased in wild-type Arabidopsis plants,but no such increase was detected in transgenic plants that showed better basal or acquired thermotolerance.Under salt stress,proline accumulation was decreased in Arabidopsis and lily with the overexpression of LlHsfA3A or LlHsfA3B.Proline catabolism was activated by overexpression,and both L1HsfA3A and L1HsfA3B affected proline oxidation via regulation of AtbZIP11,AtbZIP44,and AtbZIP53 to activate AtproDHl and AtproDH2 in transgenic Arabidopsis.4.Splice variants of lily LIHsfA3A and LlHsfA3B were isolated and their transcriptional activity determined.The interactions of L1HsfA3A and L1HsfA3B and their effects on heat and salt stress were investigated.Alternative splicing of LlHsfA3B produced a heat-inducible splice variant,LlHsfA3B2.Its protein localized in the cytoplasm and nucleus,had no transcriptional activity and specifically disturbed the protein interactions of intact HsfA3 orthologs L1HsfA3A1 and L1HsfA3B1.5.Overexpression of LlHsfA3B2 in Arabidopsis thaliana and Nicotiana benthamiana increased salt tolerance,reduced tolerance to sudden heat shock at 45?,but increased tolerance to prolonged heat at 40?.However,overexpression of LlHsfA3A1 caused opposing phenotypes,which were significantly reversed by co-expression of LlHsfA3B2.L1HsfA3B2 interacted with L1HsfA3Al to limit its transactivation function and temper the function of L1HsfA3A1 to reduce adverse effects of excessive L1HsfA3A1 accumulation.Taken together,our results suggested that overexpression of LlHsfA3A or LlHsfA3B caused opposite effects on heat and salt tolerance,which may implicate proline catabolism.In addition,a new regulation mechanism of Hsfs involving heat-inducible alternative splicing and protein interaction is proposed,which might be conducive to the establishment of thermotolerance and attenuation of the heat stress response.