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. 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.