In the heat of the moment: The miR165/166-PHB module mediates thermotolerance in Arabidopsis

Abstract

The increasing occurrence of heat waves due to climate change forms a major threat to global food security because high temperatures negatively impact plant growth and crop yield (Bailey-Serres et al. 2019). Hence, it is crucial to increase our knowledge of the molecular mechanisms underlying plant responses to heat stress to hasten the development of strategies to enhance plant thermotolerance. In Arabidopsis thaliana, class A1 heat stress transcription factors (HSFA1s) function as master regulators of heat stress responses. HSFA1s transcrip-tionally induce HSFA2, which in turn controls the expression of many heat-responsive genes, including those encoding heat shock proteins (HSPs) (Yoshida et al. 2011). In this issue, Jie Li and colleagues (Li et al. 2023) identify the role of a miR165/166-PHABULOSA (PHB) module in tran-scriptional and posttranslational regulation of HSFA1s (see Fig.). The highly conserved miRNAs miR165/166 regulate a variety of cellular processes by controlling transcript levels of Class III HD-ZIP transcription factors, such as PHB and PHAVOLUTA (PHV), which are important regulators of radial patterning in shoots (McConnell et al. 2001). In the first set of experiments, the authors showed that heat stress strongly induced the expression of miR165/166, leading to decreased transcript levels of PHB and PHV. To further explore the role of these 4 genes in heat stress responses, several transgenic lines were grown on agar plates or soil at ambient temperature and then subjected to a temperature of 37 °C. Whereas transgenic lines overexpressing miR165/ 166 and a phb phv double mutant displayed a higher thermo-tolerance compared with wild-type plants, the opposite was observed for miR165/166 knockdown lines and plants expressing a miR165/166 cleavage-resistant form of PHB. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that both HSFA1a and HSFA1b are direct PHB targets, and a dual-luciferase assay showed that their expression is negatively regulated by PHB. Together, these data suggest an important role of the miR165/ 166-PHB module in transcriptional control of plant heat stress responses. RNA-sequencing experiments then confirmed that many heat stress-responsive genes, including HSFA2, were downregulated in plants expressing a miR165/ 166 cleavage-resistant form of PHB and in miR165/166 knockdown lines and were upregulated in miR165/ 166-overexpressing lines. Interestingly, PHB was shown to directly bind to the HSFA2 promoter and negatively affect HSFA2 expression, indicating that HSFA2 is a common target of both PHB and HSFA1s. In the next part of the study, the authors demonstrated that PHB also controls HSFA1s at the posttranslational level. Using a combination of in vitro and in vivo approaches, they showed that PHB physically interacts with both HSFA1a and HSFA1b. PHB had an antagonistic effect on HSFA1b tran-scriptional activity by interfering with its DNA binding ability. Transcriptional responses to heat stress were similar between seedlings expressing a miR165/166 cleavage-resistant form of PHB and an hsfa1 quadruple mutant, indicating that PHB and HSFA1s coregulate transcriptional reprogramming in response to heat stress. A final set of experiments revealed that the miR165/166-PHB module functions upstream of HSFA1s because the enhanced thermotolerance of transgen-ic lines overexpressing miR165/166 was suppressed in a hsfa1a hsfa1b hsfa1d triple mutant. In conclusion, this study established the importance of the miR165/166-PHB module in plant heat stress responses and identified miRNAs as potential targets to enhance plant ther-motolerance. However, the molecular mechanisms functioning upstream of the miR165/166-PHB module remain to be In Brief