Pu-miR172d negatively regulates stomatal differentiation response to drought stress in poplar

We characterized Pu-miR172d, a miRNA that negatively regulates stomatal differentiation in Populus ussuriensis. Overexpression of Pu-miR172d significantly decreased stomatal density in P. ussuriensis. Molecular analysis indicated that PuGTL1 as a major target of Pu-miR172d by cleavage. Moreover, chimeric dominant repressor version of PuGTL1 (PuGTL1-SRDX) lines resembled the Pu-miR172d overexpression (Pu-miR172d-OE) lines, resulting in reduced stomatal density, net photosynthetic rate, stomatal conductance, transpiration and enhanced instantaneous water use efficiency, and thus improving tolerance to drought stress. QRT-PCR analysis showed that PuSDD1 transcript abundance in young leaves of PuGTL1-SRDX and Pu-miR172d-OE plants compared to WT plants, suggesting that Pu-miR172d positively regulates PuSDD1 expression through repression of PuGTL1. RNA-seq analysis showed Pu-miR172d overexpression significantly reduced the expression of many genes related to photosynthesis under drought stress. Overall, the present results demonstrate that Pu-miR172d/PuGTL1/PuSDD1 module plays an important role in stomatal differentiation and regulate WUE, illustrating its capacity for engineering drought tolerance improvement in poplar under future drier environments. Overall design: The 7th and 8th leaves from shoot tips were harvested from 70-day-old Pu-miR172d-OE and WT plants transplanted in soil growing under well-watered and drought treatment (4 days) conditions were used for transcriptome analysis. Total RNA isolation, library construction and sequencing were performed by Annoroad Gene Technology Corporation (Beijing, China). A total amount of 2 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. The library preparations were sequenced on an Illumina Hiseq 2500 platform and paired-end reads were generated. About 4 Gb of high-quality 150-bp paired-end reads were generated from each library. The DEGs were defined according to the following thresholds: fold change = 1, false discovery rate (FDR) < 0.05.