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Growth-regulating factor 5 (GRF5)-mediated gene regulatory network promotes leaf growth and expansion in poplar.

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.

SUBMITTER: Wu W

PROVIDER: S-EPMC8048564 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionHybrid poplars are industrial trees in China. An understanding of the molecular mechanism underlying wood formation in hybrid poplars is necessary for molecular breeding. Although the division and differentiation of vascular cambial cells is important for secondary growth and wood formation, the regulation of this process is largely unclear.MethodsIn this study, mPagGRF15 OE and PagGRF15-SRDX transgenic poplars were generated to investigate the function of PagGRF15. RNA-seq and qRT-PCR were conducted to analyze genome-wide gene expression, while ChIP‒seq and ChIP-PCR were used to identified the downstream genes regulated by PagGRF15.Results and discussionWe report that PagGRF15 from hybrid poplar (Populus alba × P. glandulosa), a growth-regulating factor, plays a critical role in the regulation of vascular cambium activity. PagGRF15 was expressed predominantly in the cambial zone of vascular tissue. Overexpression of mPagGRF15 (the mutated version of GRF15 in the miR396 target sequence) in Populus led to decreased plant height and internode number. Further stem cross sections showed that the mPagGRF15 OE plants exhibited significant changes in vascular pattern with an increase in xylem and a reduction in phloem. In addition, cambium cell files were decreased in the mPagGRF15 OE plants. However, dominant suppression of the downstream genes of PagGRF15 using PagGRF15-SRDX showed an opposite phenotype. Based on the RNA-seq and ChIP-seq results, combining qRT-PCR and ChIP-PCR analysis, candidate genes, such as WOX4b, PXY and GID1.3, were obtained and found to be mainly involved in cambial activity and xylem differentiation. Accordingly, we speculated that PagGRF15 functions as a positive regulator mediating xylem differentiation by repressing the expression of the WOX4a and PXY genes to set the pace of cambial activity. In contrast, PagGRF15 mediated the GA signaling pathway by upregulating GID1.3 expression to stimulate xylem differentiation. This study provides valuable information for further studies on vascular cambium differentiation mechanisms and genetic improvement of the specific gravity of wood in hybrid poplars.

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Growth-regulating factor 5 (GRF5)-mediated gene regulatory network promotes leaf growth and expansion in poplar.

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