PagHB7/PagABF4-PagEPFL9 Module Regulates Stomatal Density and Drought Tolerance in Poplar

Epidermal patterning factor-like 9 (EPFL9) influences stomatal density and growth in poplar. There have been no reports on homeobox 7 (HB7) and ABRE binding factor 4 (ABF4) regulating stomatal density or drought tolerance by targeting EPFL9 in poplar. This study revealed that EPFL9 was specifically localised in guard cells in leaves and responded to drought stress. By constructing CRISPR/Cas9-mediated PagEPFL9 gene-edited lines, we found that epfl9 mutant plants showed significantly reduced stomatal density, inhibited growth and enhanced drought resistance. However, PagEPFL9 overexpression increased its drought stress sensitivity by increasing the stomatal density. PagHB7 was demonstrated to be an upstream regulator of PagEPFL9 by yeast one-hybrid screening library experiments, yeast one-hybrid experiments, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments. Yeast two-hybrid, bimolecular fluorescence complementation, split luciferase complementation assays, GST pull-down, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments further demonstrated that PagHB7 interacted with PagABF4 and that PagABF4 enhanced the inhibitory effect of PagHB7 on PagEPFL9. Knockout plants of PagHB7, a negative regulator of PagEPFL9, had a significantly increased stomatal density and reduced drought tolerance. Poplars overexpressing PagABF4 showed similar phenotypes to poplars knocking out PagEPFL9, with stomatal density significantly lower than that of WT, which may result in greater drought tolerance. Our study demonstrates that PagHB7 and PagABF4 interact with each other and regulate stomatal density by targeting PagEPFL9, thereby affecting drought resistance in poplar. This study provides new genetic resources for molecular design breeding of plant growth and drought tolerance.