Knockdown of Fa-miR159/160 reprograms plant development and improves heat-stress resistance in tall fescue.

MicroRNA159/160 (miR159/160) has been implicated in plant development and stress resistance in annual crops and Arabidopsis, yet their functional roles in perennial cool-season forage and turf grass remain elusive. Tall fescue (Festuca arundinacea Schreb.) is one main perennial cool-season forage and turfgrass species worldwide. Here, we found that Fa-miR159/160 has 7/4 copies in tall fescue, respectively. Knockdown of both Fa-miR159 and Fa-miR160 (STTM159/160) using the short tandem target mimics (STTM) system resulted in a dwarf phenotype with higher tiller density, lower growth rate levels, reduced leaf width and length compared with wild type (WT). The higher tiller density levels and lower growth rate in STTM159/160 lines will shorten the turfgrass establishment period and reduce the frequency of lawn mowing, which is one important cost-effective strategy that could bring remarkable economic benefits in lawn management. Compared with WT, STTM159/160 plants displayed altered vascular bundle organization, increased leaf trichomes, thicker cuticles, and higher wax accumulation. These reinforced epidermal structures reduce heat absorption and transpirational water loss, thereby enhancing water homeostasis and heat tolerance. STTM159/160 grasses displayed enhanced thermotolerance than WT. Transcriptomic profiling revealed 13 489 differentially expressed genes (5382 up- and 8107 down-regulated) in STTM159/160 versus WT in response to heat treatment. Natural variation in the Fa-miR159-MYB interaction across C3 and C4 monocots implies that the regulatory strength of this module has been repeatedly tuned during monocot evolution. Collectively, our findings unveil the novel function of Fa-miR159/160 in simultaneously regulating development and heat tolerance in tall fescue, and provide a new engineering strategy for genetic improvement in grass.