Proteomics And Physiology Analysis Of A Brassica Napus Chlorophyll-deficiency Mutant

Leaf is the primary photosynthetic organ of the plant,which may directly decide the biomass accumulation of plants.The chlorophyll participates in photosynthesis,and plays a key role in light capture and energy transfer.Synthesis,accumulation and degradation of chlorophyll are also associated with chloroplast development,photomorphogenesis and nucleo cytoplasmic signal.We recently isolated a chlorophyll-deficient mutant cde1 from ethyl methanesulfonate(EMS)mutagenized Brassica napus.In this study,iTRAQ-based proteomics and physiology analysis of the cdel and the corresponding wild type of B.napus were conducted.This work provides new insights into the regulation of chlorophyll biosynthesis and photosynthesis in higher plants and these findings may be applied to genetic engineering for high photosynthetic efficiency in crops.The main results of the study were as follows:Proteomics results:① A total of 5069 non-redundant proteins were detected from B.napus leaves,443 differentially accumulated proteins were detected,of which 228 presented up-accumulation,whereas 215 showed down-accumulation in cdel nutant plants.② Fluorescence quantitative PCR(qRT-PCR)analysis of the important differentially accumulated proteins showed consistency between the transcriptional level and the protein level of theses proteins.The differentially accumulated proteins were found to be enriched in several pathways,including photosynthesis,chlorophyll metabolism,porphyrin,biosynthesis of secondary metabolites,carbon fixation,spliceosome,mRNA surveillance and RNA degradation.The study showed that the decreased level of chlorophyll,the reduced photosynthesis,the impaired carbon fixation efficiency,and the disturbed redox homeostasis may explain for the decreased chlorophyll content,photosynthetic capacity and increased lipid peroxidation levels in this mutant.Moreover,Epigenetics may be involved in the regulation of gene expression in cd.el mutant,as proteins involved in DNA/RNA/histone methylation and methylation-dependent chromatin silencing were up-accumulated in the mutant.Physiological results:To study the physiological differences of chlorophyll defeciency mutant cdel and its corresponding wild-type,the photosynthetic rate,chlorophyll content,ascorbic acid peroxidase(APX)activity,thioredoxin reductase(TrxR)activity and malondialdehyde(MDA)content were measured.The results showed that:①the net photosynthetic rate of the cdel mutant decreased by 13.4%,while the stomatal conductance and intercellular CO2 concentration increased significantly,which suggested that the photosynthesis of the chlorophyll deficient mutant cdel was decreased.② The content of chlorophyll a,chlorophyll b and carotenoid of the cde1 mutant was significantly lower than that of the wild type.③ APX activity and TrxR content in mutant cdel was significantly lower than that of the wild typewhich may resulted in the damage of redox balance and the increase of reactive oxygen species,which may be one of the reasons for the abnormal metabolism of photosynthesis in tihe cdelmutamt.The content of MDA in the cdel mutant was significantly higher than that of the wild type.The degree of membrane lipid peroxidation in the mutant cdel was significantly higher than that of the wild type,suggesting that cdel mutant cellssuffer from oxidative damage.