The GATA8-GRF5-XTH9 feed-forward loop regulates cell size in poplar

Abstract Although triploid poplars have larger cells and leaves than their diploid counterparts, the molecular mechanisms underlying this disparity remain elusive. Here, we found that PpnGATA8 and PpnGRF5 were significantly up-regulated in triploid poplars through differential gene expression analysis between diploid and triploid poplars. Furthermore, through genetic transformation in poplar, it was found that both PpnGATA8 and PpnGRF5 positively regulated poplar cell size, resulting in increased leaf size and improved photosynthetic efficiency. RNA-sequencing of PpnGATA8-overexpressing poplars showed that PpnGATA8 promotes expression of PagGRF5 and PagXTH9. Yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay were employed to substantiate that PpnGATA8 directly regulated PagGRF5 and PagXTH9 expression. Meanwhile, PpnGRF5 positively regulates the expression of PagXTH9. Poplar protoplast co-transformation assays further proved that co-expression of PpnGATA8 and PpnGRF5 had the strongest effect on promoting PagXTH9 expression. Moreover, overexpression of PpnXTH9 also significantly increased poplar cell and leaf size. Therefore, GATA8, GRF5 and XTH9 formed a feed-forward regulatory loop to regulate plant cell size. Our results are of major significance for revealing the molecular regulatory mechanisms of plant cell size and leaf development, especially the genetic basis of giant variation in cells and leaves in polyploid plants.