Summary 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.

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Adventitious root (AR) formation is critically important in vegetative propagation through cuttings in some plants, especially woody species. However, the underlying molecular mechanisms remain elusive. Here, we report the identification of a poplar homeobox gene, PuHox52, which was induced rapidly (within 15 min) at the basal ends of stems upon cutting and played a key regulatory role in adventitious rooting. We demonstrated that overexpression of PuHox52 significantly increased the number of ARs while suppression of PuHox52 had the opposite effect. A multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PuHox52 was reverse-engineered and demonstrated to govern AR formation. PuHox52 regulated AR formation through upregulation of nine hub regulators, including a jasmonate signaling pathway gene, PuMYC2, and an auxin signaling pathway gene, PuAGL12. We also identified coherent type 4 feed-forward loops within this ML-hGRN; PuHox52 repressed PuHDA9, which encodes a histone deacetylase, and led to an increase in acetylation and presumably expression of three hub regulators, PuWRKY51, PuLBD21 and PuIAA7. Our results indicate that the ML-hGRN mediated by PuHox52 governs AR formation at the basal ends of stem cuttings from poplar trees.

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Drought stress causes various negative impacts on plant growth and crop production. R2R3-MYB transcription factors (TFs) play crucial roles in the response to abiotic stress. However, their functions in Betula platyphylla haven’t been fully investigated. In this study, a R2R3 MYB transcription factor gene, BpMYB123, was identified from Betula platyphylla and reveals its significant role in drought stress. Overexpression of BpMYB123 enhances tolerance to drought stress in contrast to repression of BpMYB123 by RNA interference (RNAi) in transgenic experiment. The overexpression lines increased peroxidase (POD) and superoxide dismatase (SOD) activities, while decreased hydrogen peroxide (H2O2), superoxide radicals (O2–), electrolyte leakage (EL) and malondialdehyde (MDA) contents. Our study showed that overexpression of BpMYB123 increased BpLEA14 gene expression up to 20-fold due to BpMYB123 directly binding to the MYB1AT element of BpLEA14 promoter. These results indicate that BpMYB123 acts as a regulator via regulating BpLEA14 to improve drought tolerance in birch.