Function Analysis Of Populus Tomentosa PtoCYCD3;3 Gene In Leaf And Stem Development

D-type cyclin(cyclin D,CYCD)combined with cyclin-dependent kinase(cyclin-dependent kinase,CDK)participates in the regulation of cell cycle G1/S transition,and it plays an important role in cell division and proliferation.In recent years,it has been reported that CYCD can affect the growth and development of herbaceous plants such as Arabidopsis thaliana by regulating the cell cycle process,but it has been less studied in woody plants(such as poplar).In this paper,using Populus tomentosa as the research material,the function of PtoCYCD3;3 gene on the development of leaves and stems of Populus tomentosa was studied.The main findings of this study are as follows:(1)The phylogenetic analysis of the poplar CYCD and CDK gene families with Arabidopsis showed that compared with Arabidopsis,the CYCD genes expanded to 24 members,and the CDK genes expanded to 18 members.Phylogenetic analysis showed that poplar CYCD genes were divided into 6 different subtypes,namely CYCD1,CYCD2/4,CYCD3,CYCD5,CYCD6 and CYCD7.Genomic localization analysis showed that large segment duplication is the main way of CYCD genes expansion in poplar.Cloning primers were designed according to the CDS regions of P.trichocarpa CYCD3 and CDK genes,and 5 PtoCYCD3 genes and 17 PtoCDK genes were amplified in P.tomentosa.The sequence alignment results showed that PtoCYCD3 genes has typical structural characteristics of CYCD sequence.Each subtype of PtoCDK genes also contains typical characteristic sequences.The tissue expression patterns of the five PtoCYCD3 genes were analyzed by RT-q PCR,and the results showed that the expression level of PtoCYCD3;3 gene was higher in the shoot tip and young leaf than the other PtoCYCD3 genes.The subcellular localization analysis of PtoCYCD3;3 showed that it was localized in the nucleus.(2)PtoCYCD3;3 transgenic plants had obvious morphological changes during the 170-day observation period.Compared with the wild type,PtoCYCD3;3 transgenic plants have increased plant height,increased number of internodes,increased length of internodes,increased stem thickness,enlarged and wrinkled leaves,and multiple branches.Through scanning electron microscopy,it was found that the number of cells per unit area of the epidermal cells and palisade tissue of mature leaves of PtoCYCD3;3transgenic plants was significantly more than that of the wild-type plants,and the cell area was significantly reduced.The photosynthesis was measured,and the results showed that the electron transfer rate of photosystemⅠand photosystemⅡper unit area of PtoCYCD3;3 transgenic plant leaves decreased,and the net assimilation rate decreased.Anatomical analysis of the stems showed that the thickness of secondary xylem,secondary phloem and cambium-derived tissues of the PtoCYCD3;3 transgenic plants were significantly increased compared with the wild-type plants;the area of the vessels in the secondary xylem was significantly increased.The endogenous hormone content determination results showed that the contents of brassinolide and abscisic acid in the leaves and stems of PtoCYCD3;3 transgenic plants increased,while the contents of auxin and cytokinin decreased.(3)Twelve PtoCDK proteins that interact with PtoCYCD3;3 in yeast have been identified through the yeast two-hybrid experiment.They are PtoCDKA;1,PtoCDKB1;1,PtoCDKB1;2,PtoCDKB2;1,PtoCDKB2;2,PtoCDKC;2,PtoCDKD;1,PtoCDKD;2,PtoCDKE;2,PtoCDKF;1,PtoCDKG;3,and PtoCDKG;4.Using bimolecular fluorescence complementation technology,the results showed that all 12 PtoCDKs could interact with PtoCYCD3;3 in tobacco leaf cells.(4)The phenotype of the perennial PtoCYCD3;3 transgenic plants was analyzed,and the results showed that the tree height,DBH(diameter at breast height)and base stem circumference of the transgenic plants were not significantly different from those of the wild-type plants.Compared with the wild-type plants,the top leaves of the transgenic plants were obviously wrinkled.Proteomics and phosphoproteomics analysis were performed on the leaves.In quantitative proteomics,a total of 648 proteins expressions were up-regulated and 381 proteins expression down-regulated.In phosphoproteomics,the expression of 31 phosphoproteins was up-regulated,and the expression of 37 phosphoproteins was down-regulated.In proteomics,the enriched differential proteins are mainly concentrated in the pathways of photosynthesis,spliceosome,oxidative phosphorylation and ribosomal biosynthesis.In phosphoproteomics,the enriched differential proteins are mainly concentrated in the synthesis of secondary metabolites,metabolic pathways,glycolysis and spliceosome pathways.These pathways participate in the regulation of PtoCYCD3;3 transgenic plant leaf development.In summary,this study studied the effect of PtoCYCD3;3 on leaf and stem development,and identified multiple interactions between PtoCDKs and PtoCYCD3;3.Through proteomics and phosphoproteomics analysis,it was found that the overexpression of PtoCYCD3;3 can cause multiple regulatory pathways to participate in leaf development.This study clarified the function of CYCD3;3 gene in the development of P.tomentosa leaves and stems,and illustrated its mechanism.