Molecular Mechanism Of MiR165/166-PHB Module In Response To Heat Stress In Arabidopsis Thaliana

With the increasing global warming,extreme high-temperature climate has become one of the important factors that inhibit crop growth and affect the food security.Therefore,uncovering of the molecular mechanism of plants response to heat,identification of heat stress resistance genes,and improvement of plants thermotolerance have become the major scientific issue.As sessile organisms,plants have evolved strictly controlled regulatory mechanisms to adapt to heat stress.Increasing evidence show that microRNA(miRNA)is a vital regulator of heat stress response.Therefore,identification of miRNA involved in plants response to heat stress and elucidating the underlying molecular mechanisms will provide important theoretical basis and applied targets for improving plants thermotolerance in future.Short Tandem Target Mimic(STTM)is a miRNA knockdown technology that directly targets mature miRNAs with high specificity and efficiency.Utilizing STTM,we obtained a series of miRNA knockdown mutants in Arabidopsis thaliana.Among these mutants,miR165/166 knockdown lines,STTM165/166,were sensitive to heat stress.Consistent with STTM165/166,plants expressing a miR165/166-resistant form of PHABULOSA(PHB)were also sensitive to heat stress,whereas miR165/166 overexpression lines and miR165/166 target gene mutants exhibited enhanced thermotolerance.Further study revealed that the miR165/166-PHB module,formed by miR165/166 and its target gene PHB,regulated heat stress master regulator genes,Heat Shock transcription Factor A1s(HSFA1s),at both transcriptional and post-translational levels.At the transcriptional level,PHB directly binds to the HSFA1s promoters and represses its transcription.At the post-translational level,PHB physically interacts with HSFA1s and has an antagonistic effect on the transcriptional activity of HSFA1s.In addition,we also found that PHB and HSFA1s share a common target gene,Heat Shock transcription Factor A2(HSFA2),which is essential for activation of the heat stress response in plants.Transcriptome analysis identified that nearly 50%of the heat responsive genes regulated by HSFA1s were also controlled by PHB.Together,these results suggest that heat-triggered regulation of the miR165/166-PHB module controls the HSFA1s-mediated transcriptional reprogramming and plays a critical role during heat stress in Arabidopsis.In summary,we identified miR165/166 as an important factor in the regulation of thermotolerance,and uncovered the underlying molecular mechanism of miR165/166PHB regulator module in plants response to heat stress.This study provides further insight into heat stress regulatory network and will eventually facilitate the improvement of crops stress tolerance and yield stability in the future.