Physiological And Protein Expression Regulation Of The Formation Of Leaf Variegated Phenotype In Catalpa Fargesii

Variegated leaves are a type of leaf color variation that is ubiquitous in nature.It has outstanding scientific research value on the analyses of leaf color variation mechanism,new gene discovery,functional protein interaction and other biological events and processes.Currently,the research on the regulatory mechanism of variegation is mainly based on artificially created mutant materials,but few researches on natural leaf color variation was reported.Catalpa fargesii Bur.is a precious timber species and a famous ornamental tree species cultivated in China's warm temperate zone and subtropical zone.“Maiyuan” is a new natural variety selected from C.fargesii,and its leaves present typical variegated phenotype with yellow margin and green middle.In this study,the yellow and green sectors of Maiyuan were used as materials,and the corresponding sectors of C.fargesii as controls.Integrative analysis of anatomical,physiological,proteomics and modified omics were conducted,the aim is to reaveal the physiological and molecular mechanisms of leaf variegation.The results are as follows:Firstly,the contents of chlorophyll and carotenoid in Maiyuan were significantly lower than those in C.fargesii,however,the contents of lutein were higher than that in C.fargesii.The measurement of chlorophyll synthesis precursors showed that the chlorophyll synthesis had been blocked from the first step of?-aminolevulinic acid(ALA),which might be related to the chloroplasts and thylakoid structure defects.Compared with C.fargesii,the structure was degraded and the matrix was ambiguous in the yellow sectors.In contrast,the untrastructure of the green sectors was between the yellow sectors and the control leaves.Secondly,the measurement of photosynthetic fluorescence parameters showed that the photosynthetic capacity of the yellow sectors in Maiyuan was significantly lower than that in the green sectors,especially the PSII electron transport rate.In the yellow tissue,more energy was distributed to PSI under medium and high light,which was confirmed by the 77 K low temperature fluorescence experiment.The results of OJIP curves showed that the electron transfer process from QA to QB was blocked in Maiyuan leaves,and its redox balance was disturbed.Meanwhile,the oxygen-evolving complex(OEC)was seriously damaged in Maiyuan.and the the damage degree of the yellow tissue was higher than that of the green tissue.The increased SOD,APX enzyme activities and MDA content in the yellow sectors suggested that the yellow leaves had been damaged by oxidative stress or other stress.The hormone content determination showed that the distribution of hormones in different cells played an important role in the formation of variegated leaves.Thirdly,the PPIs regulatory network of proteomics implied that the imbalance of protein synthesis and degradation was the important reason for the variegated leaf phenotype.GO enrichment analyses showed that metabolic processes including isoprenoid biosynthesis,carotenoid synthesis,terpene biosynthesis and photosynthesis were disorder in Maiyuan.The lack of isoprenoids in the yellow sectors lead to a decrease in the function of photosynthesis and development-related proteins,while an increased expression in oxidative stress proteins.The green sectors can achieve a relative balance of protein synthesis and degradation through a compensation mechanism.Proteomics also found that the MEP pathway,which is located in the upstream of the chlorophyll and carotenoid synthesis,which was a definitive pathway for distinguishing the yellow and green sectors of the Maiyuan.DXS enzyme inhibitor treatment experiments further confirmed this finding.Fourthly,GO enrichment of differentially phosphorylated proteins in each comparison were analyzed,the results showed that the processes of photosynthesis,regulation of the generation of precursor metabolites,response to stress,homeostasis,amino acid metabolism,transport–related processes and most of the energy metabolisms might contribute to leaf color.KEGG enrichment analysis was performed on the phosphorylated proteins specifically between the yellow sectors and the green sectors in Maiyuan.The results showed that most enriched pathways through GO enrichment analysis were mostly located in the chloroplast and cytosol.Fifthly,functional enrichment of acetylated proteins in each comparison showed that the acetylation on photosynthesis,pigment metabolism,energy metabolism,carbon fixation,oxidative phosphorylation,protein synthesis and degradation and antioxidant related pathways regulated the yellow leaf color formation,and the acetylation of pathways such as pigment metabolism,glycometabolism,amino acid synthesis and metabolism,glutathione metabolism,and carbon fixation affected the formation of the green sectors of Maiyuan.Furthermore,KEGG enrichment analysis was carried out on the specific acetylated proteins in the yellow and green parts of Maiyuan,and it was found that the acetylated proteins located in chloroplast,cytosal,mitochondria and nucleus of different organs jointly regulated the formation of leaf variegation.Sixthly,function enrichment analysis of the overlap proteins between acetylation and phosphorylation in each comparison revealed that carbon metabolism,carbon fixation,secondary metabolism,glyoxylate metabolism,photosynthesis,ascorbate metabolism,and glycolysis/gluconeogenesis were significantly enriched.Protein modification on these pathways might have a more complex regulatory relationship with the formation of yellow leaf color.In addition to the above pathways,the acetylation and phosphorylation modification of glycometabolism and amino acid metabolism played important roles in the formation of the green sectors of Maiyuan.PPIs regulatory network analyses found that the acetylation and phosphorylated modification of pathways,such as porphyrin and chlorophyll metabolism,carbon metabolism,protein synthesis and degradation,and antioxidant were involved in the formation of yellow leveas in Maiyuan.In contrast,the processes of photosynthesis,protein synthesis and degradation,and antioxidant –related pathways regulated the formation of green leaves in Maiyuan.In additon,the acetylation and phosphorylation modifications of Psb O and Psb P located on chloroplast might be a response mechanism to OEC complex damage.A hypothesis model of leaf variegation was proposed through crosstalk analyses of proteome,phosphorylome and acetylome.