Abstract
Although polyploid plants have larger leaves than their diploid counterparts, the molecular mechanisms underlying this difference (or trait) remain elusive. Differentially expressed genes (DEGs) between triploid and full-sib diploid poplar trees were identified from two transcriptomic data sets followed by a gene association study among DEGs to identify key leaf growth regulators. Yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay were employed to substantiate that PpnGRF5-1 directly regulated PpnCKX1. The interactions between PpnGRF5-1 and growth-regulating factor (GRF)-interacting factors (GIFs) were experimentally validated and a multilayered hierarchical regulatory network (ML-hGRN)-mediated by PpnGRF5-1 was constructed with top-down graphic Gaussian model (GGM) algorithm by combining RNA-sequencing data from its overexpression lines and DAP-sequencing data. PpnGRF5-1 is a negative regulator of PpnCKX1. Overexpression of PpnGRF5-1 in diploid transgenic lines resulted in larger leaves resembling those of triploids, and significantly increased zeatin and isopentenyladenine in the apical buds and third leaves. PpnGRF5-1 also interacted with GIFs to increase its regulatory diversity and capacity. An ML-hGRN-mediated by PpnGRF5-1 was obtained and could largely elucidate larger leaves. PpnGRF5-1 and the ML-hGRN-mediated by PpnGRF5-1 were underlying the leaf growth and development.