Study On Comparative Proteomics Of Floral Thermogenesis In Nelumbo Nucifera

Floral thermogenesis is a phenomenon in which some flowering plants produce heat in their floral organs,which is biologically important for the reproductive development,pollination and fertilization of floral organs.Nelumbo nucifera is an aquatic crop with a great deal of important economic and ornamental value.Researches have reported that N.nucifera is a thermogenic species that can regulate heat production.So far,the molecular mechanism of the regulation of floral thermogenesis and the proteome expression profile during thermogenesis are still unclear.In the present study,a cultivar‘E Zilian 1'(also named‘Mantianxing')of N.nucifera was used for comparative proteomics analysis of the receptacle tissue to reveal the proteins and their regulatory networks involved in floral thermogenesis.The main experimental results of this research are as follows:1.The total 6913 proteins with quantitative information were identified using data-independent acquisition(DIA)-based quantitative proteomics technology.The development of the flower of N.nucifera was divided into 5 sequential stages,among which stage 2,stage 3 and stage 4 were the thermogenic stages of the receptacles.The number of proteins identified from stage 1 to stage 5 were 6554,6591,6429,6365,and6508,respectively,of which 5864 proteins were identified in all five stages.Among the6913 proteins,5867 have Gene ontology(GO)annotation entries,most of which are concentrated in the catalytic activity and binding of the molecular function category and the cell process and metabolic process of the biological process category.2.The 10 different comparison groups with a total of 3513 significantly differentially expressed proteins(DEPs)were obtained in the differentially proteins analysis.When using stage 1 as a control,the number of DEPs in stage 2,3,4 and 5was 203,1373,1826 and 2107,respectively.When using stage 3 as a control,the number of DEPs in stage 1,2,4 and 5 was 1373,752,192 and 1262,respectively.The number of DEPs between adjacent stages was 203,752,192,and 1088,respectively,according to the order of receptacle development.3.The total 6 clusters of proteins with different expression patterns were obtained by time series analysis,of which the cluster 3,containing 640 proteins,was consistent with the thermogenesis pattern and highly expressed during thermogenesis,and cluster5,containing 429 proteins,was the cluster with low expression during thermogenesis.The results of KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis showed that cluster 3 was mainly enriched in tricarboxylic acid cycle,carbon metabolism,starch and sucrose metabolism,and cluster 5 was mainly enriched in photosynthesis,plant hormone signal transduction,MAPK signaling pathway.And,the results of GO enrichment analysis showed that tricarboxylic acid cycle,oxidoreductase activity and tricarboxylic acid cycle enzyme complex were the major enriched GO entries in cluster 3;photosynthesis,signal transduction activity and photosynthetic system were the major enriched GO entries in cluster 5.4.Using quantitative real-time PCR,we have verified the expression patterns of transcripts corresponding to 10 thermogenesis-related proteins,including alternative oxidase(AOX)and plant uncoupling protein(p UCP).The results showed that the transcript expression levels of these proteins were consistent with the quantitative results of the transcriptome and proteome.And then,our Western blot experiments further validated the expression patterns of AOX and p UCP,and the results were consistent with the quantitative results of proteome.By comparing the differences in the expression of AOX and p UCP in different tissues,it was found that AOX was specifically expressed in the thermogenic receptacles,while p UCP was highly expressed in non-thermogenic tissue.This further confirmed that AOX played an important role in floral thermogenesis of N.nucifera.5.Several hub proteins(proteins at the center of the regulatory network with high connectivity)involved in tricarboxylic acid cycle were identified by protein-protein interaction(PPI)network analysis of the proteins in cluster 3.Citrate synthase was identified as the most highly connected protein in the PPI network.Subsequently,a protein module highly correlated with the thermogenic phenotype was identified by weighted gene co-expression network analysis(WGCNA),which consisted of 78proteins mainly related to metabolic processes,fatty acid degradation,and biosynthesis of ubiquinone and other terpenoid quinones in the KEGG pathway.6.Our H₂O₂detection experiments revealed that the changes in H₂O₂ content during the five developmental stages of the receptacles were highly correlated with the expression pattern of AOX,which indicated a link between reactive oxygen species(ROS)and AOX expression.In the present study,we attempted to reveal the protein regulatory networks involved in floral thermogenesis of N.nucifera from the perspective of proteomics,and obtained the protein expression profiles of the receptacles at different developmental stages.These results can not only help us understand the molecular mechanism of floral thermogenesis in N.nucifera,but also provide candidate proteins for future research,which are beneficial to further reveal the mystery of plant thermogenesis.