Yellowhorn Xso-miR5149-XsGTL1 enhances water-use efficiency and drought tolerance by regulating leaf morphology and stomatal density.

Yellowhorn (Xanthoceras sorbifolium) is a unique edible woody oil tree species in China. Drought stress is the major yield-limiting factor of yellowhorn. MicroRNAs play an important role in regulating the response of woody plants to drought stress. However, the regulatory function of miRNAs in yellowhorn remains unclear. Here, we first constructed coregulatory networks integrated with miRNAs and their target genes. According to GO function and expression pattern analysis, we selected the Xso-miR5149-XsGTL1 module for further study. Xso-miR5149 is a key regulator of leaf morphology and stomatal density by directly mediating the expression of the transcription factor XsGTL1. Downregulation of XsGTL1 in yellowhorn led to increased leaf area and reduced stomatal density. RNA-seq analysis indicated that downregulation of XsGTL1 increased the expression of genes involved in the negative control of stomatal density, leaf morphology, and drought tolerance. After drought stress treatments, the XsGTL1-RNAi yellowhorn plants were less damaged and had higher water-use efficiency than the WT plants, while destruction of Xso-miR5149 or overexpression of XsGTL1 had the opposite effect. Our findings indicated that the Xso-miR5149-XsGTL1 regulatory module plays a critical role in controlling leaf morphology and stomatal density; hence, it's a potential candidate module for engineering enhanced drought tolerance in yellowhorn.