Pu-miR172d regulates stomatal density and water-use efficiency via targeting PuGTL1 in poplar

miRNAs play essential regulatory roles in many aspects of plant development and in responses to abiotic and biotic stresses. Here, we characterize Pu-miR172d, which acts as a negative regulator of stomatal density by directly repressing the expression of PuGTL1 in Populus ussuriensis. Quantitative real-time PCR and GUS reporter analyses showed that Pu-miR172d was strongly expressed in the guard cells of young leaves. Overexpression of Pu-miR172d significantly decreased stomatal density, resulting in increases in water use efficiency (WUE) and drought tolerance by reducing net photosynthetic rate, stomatal conductance, and transpiration. Molecular analysis showed that PuGTL1 was a major target of Pu-miR172d cleavage. Moreover, PuGTL1-SRDX plants, in which PuGTL1 is suppressed, phenocopied Pu-miR172d-overexpression lines with reduced stomatal density and enhanced WUE. The expression of PuSDD1, a negative regulator of stomatal development, was significantly increased in young leaves of both Pu-miR172d-overexpression and PuGTL1-SRDX plants. RNA-seq analysis of mature leaves indicated that overexpression of Pu-miR172d decreased the expression of many genes related to photosynthesis. Our findings show that the Pu-miR172d/PuGTL1/PuSDD1 module plays an important role in stomatal differentiation, and hence it is a potential target for engineering improved drought tolerance in poplar.