The miR319 / bHLH094 Module Regulates Creeping Bentgrass Thermotolerance by Modulating Auxin Biosynthesis and Signalling Pathway

MicroRNA319 (miR319) has been demonstrated to regulate plant development and responses to stress such as drought and salt. However, its role in thermotolerance, particularly in cool season grasses, remains unclear. Here we report that miR319 plays a negative role in heat tolerance of creeping bentgrass (Agrostis stolonifera). A basic helix-loop-helix (bHLH) transcription factor, AsbHLH094 was identified as the target gene of miR319, and its expression was significantly downregulated in the miR319-overexpressing (OE319) transgenic creeping bentgrass lines. Functional characterisation revealed that overexpression of AsbHLH094 enhanced heat tolerance of the transgenic tobacco plants. Furthermore, protein-protein interaction assays confirmed that AsbHLH094 physically interacts with AsIAA1, an Aux/IAA protein involved in auxin signalling. Transcriptomic analysis showed that auxin biosynthesis genes such as TARs, YUCCAs, along with auxin-response genes including Auxin/IAAs and ARFs were downregulated in the OE319 transgenic creeping bentgrass plants, leading to reduced auxin accumulation, while elevated auxin levels and induced changes in auxin biosynthesis- and response-related genes were observed in the AsbHLH094 overexpression tobacco. Endogenous indole-3-acetic acid (IAA) levels in creeping bentgrass were significantly increased under high-temperature conditions, and exogenous application of IAA at appropriate concentrations improved heat tolerance in creeping bentgrass. Together, our findings reveal a previously uncharacterized miR319-AsbHLH094 regulatory module that modulates auxin biosynthesis and signalling, thereby contributing to heat stress responses in creeping bentgrass.