Identification of crucial genes sustaining the curled leaf phenotype in perennial transgenic poplar via advanced proteomic and phosphoproteomic analyses

Populus tomentosa hybrid poplar 741 is a superior tree species in northern China. Due to its rapid growth, high productivity, and range of available genetic tools, it has always been a focus of forestry research. The perennial genetically modified Populus 741, exhibiting sustained overexpression of PtoCYCD3;3, consistently shows adaxial curvature and pronounced surface wrinkling. The curvature of leaves holds great significance for forestry production systems. Moderate leaf curling can optimize the angle of light reception, thereby enhancing the efficiency of light absorption and photosynthetic performance, shortening the wood maturation cycle, and improving economic feasibility. Protein phosphorylation modification is a major regulatory mechanism in the cell cycle process. To investigate these morphological changes, TMT quantitative proteomics and phosphoproteomics were performed on leaves of transgenic and wild-type plants. Among 6005 identified proteins, 648 showed increased abundance, whereas 386 were reduced. In phosphoproteomics, 68 proteins exhibited differential phosphorylation, with 31 increasing and 37 decreasing. Quantitative proteomics identified significant changes in protein abundance associated with photosynthesis, phytohormones, and cell proliferation. Notably, histone deacetylase 6 (HDA6), ANGUSTIFOLIA (AN), and cellulose synthase-like (CSL) proteins associated with leaf curling were significantly upregulated in transgenic poplar. Phosphoproteomics revealed enrichment of proteins such as HERK1, DGK, OST1, and BIG, which are involved in brassinosteroid (BR), abscisic acid (ABA), and other phytohormone signaling pathways. These analyses demonstrated the impact of exogenous gene PtoCYCD3;3 integration on photosynthetic pathways, hormone signaling, and cell proliferation, highlighting its role in modulating leaf morphogenesis in perennial Populus 741.

Significance

Current assessments of the effects of exogenously introduced genes in Populus species are largely limited to short-term studies in annual or semiannual genetically modified specimens. In this study, we collected mature leaves from both perennial wild-type and transgenic 741 poplar trees and conducted comprehensive proteomic and phosphoproteomic analyses. The results not only revealed alterations in the abundance of multiple proteins associated with leaf curling but also elucidated key plant hormones and signaling pathways involved in leaf morphogenesis. These findings complement the signaling network involved in leaf morphogenesis and provide a novel perspective for studying perennial transgenic plants.