Application and Comparison of Two Label-free Quantitative Techniques for Screening Differentially Expressed Proteins in Strawberry (Fragaria ananassa) Gynoecium Development
QIU Jie-Ren, CHAI Wei-Guo, ZHOU Li-Ping, WANG Shu-Zhen*
Institute of Biotechnology, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China
Abstract：Studying the developmental process of gynoecium is very important for breeding. Proteomic technique is a powerful tool for system biology research, and proteomics in strawberry (Fragaria ananassa) gynoecium development has not been reported to date. In order to select a suitable method for large-scale proteomics analysis of strawberry gynoecium, DDA (data-dependent acquisition) and DIA (data-independent acquisition) techniques were applied for label-free quantitative proteomics analysis of strawberry gynoecium at 3 developmental stages of small bud stage, early blooming stage and full blooming stage. The two techniques were evaluated in terms of protein identification quantity, coefficient of variation (CV) of peptide ion peak intensity and the proportion of missing data. The result shows that the coverage, reproducibility, and accuracy of DIA data were higher than those of DDA. A total of 3 687 proteins were identified in DDA and 5 341 proteins were identified in DIA; The average CV value of peptide ion peak intensity for the 3 periods were 28.4%, 33.3% and 28.4% in the DDA data, respectively, while in DIA data the CV values were only 10.4%, 10.9% and 11.9%. Gene ontology (GO) function analysis was performed in the differentially expressed proteins (DEPs) screened by DIA. The DEPs between small bud stage and early blooming stage were mainly annotated to mitosis, methylation, and secondary metabolite synthesis, while the DEPs between early and full blooming stage were annotated to energy metabolism, photosynthesis, and primary metabolism. KEGG enrichment analysis showed that multiple peroxidases including peroxidase 4 (POD4) were involved in lignin synthesis, and their expression increased simultaneously with the flower developmental process. Further analysis of methylation-related proteins tapped into multiple proteins with important physiological functions, including flowering-regulated protein arginine methyltransferase 5 (PRMT5), transposon silencing related DNA (cytosine 5)-methyltransferase 3 (CMT3), and aromatic compounds synthesis related orcinol O-methyltransferase (OOMT). Moreover, some essential proteins such as PRMT5, CMT3 and POD4 were identified only in DIA data, which further demonstrated the superiority of DIA over DDA. This study established DIA as a superior method for large-scale proteomics analysis of strawberry gynoecium, and preliminarily explored the changes in gynoecium protein expression levels during 3 developmental periods of small bud stage, early blooming stage and full blooming stage, which could provide basic information for the research on the mechanism of strawberry gynoecium development process.
[1] 侯毅枫,邓娴,曹晓风,等.2015.蛋白质精氨酸甲基化参与基因转录后调控的研究进展[J].生命科学,27(3):351-362.
(Hou Y F,Deng X,Cao X F,et al.2015.Research progress of protein arginine methylation in post-transcriptional regulation[J].Chinese Bulletin of Life Sciences,27(3):351-362.)
[2] 裘劼人,王淑珍,柴伟国.2018.蛋白质组学技术在草莓上应用研究进展[J].杭州农业与科技,(6):16-19.
(Qiu J R,Wang S Z,Chai W G.2018.Advances in application of proteomics in Strawberry[J].Hangzhou Agricultural Science and Technology,(6):16-19.)
[3] 王瑞娴,徐建红.2014.基因组DNA甲基化及组蛋白甲基化[J].遗传,36(3):191-199.
(Wang R X,Xu J H.2014.Genomic DNA methylation and histone methylation[J].Hereditas (Beijing),36(3):191-199.)
[4] 张亚惠,周历萍,王淑珍,等.2018.60Co-γ射线辐射草莓红颊诱变选育新品系的研究[J].核农学报,32(8):1457-1465.
(Zhang Y H,Zhou L P,Wang S Z,et al.2018.Study on selection of a new strawberry strain of Benihoppe developed by 60Co-γ irradiation induced mutation breeding[J].Journal of Nuclear Agricultural Sciences,32(8):1457-1465.)
[5] 赵密珍,王静,袁华招,等.2019.草莓育种新动态及发展趋势[J].植物遗传资源学报,20(02):15-23.
(Zhao M Z,Wang J,Yuan H Z,et al.2019.Situation and perspectives of strawberry breeding[J].Journal of Plant Genetic Resources,20(02):15-23.)
[6] Almagro L,Gómez ROS L V,Belchi-navarro S,et al.2009.Class Ⅲ peroxidases in plant defense reactions[J].Journal of Experimental Botany,60(2):377-390.
[7] Chan S W L,Henderson I R,Zhang X,et al.2006.RNAi,DRD1,and histone methylation actively target developmentally important non-CG DNA methylation in Arabidopsis[J].PLOS Genetics,2(6):791-797.
[8] Chen X,Mao X Z,Huang J J,et al.2011.KOBAS 2.0:A web server for annotation and identification of enriched pathways and diseases[J].Nucleic Acids Research,39(suppl 2):W316-W322.
[9] Collins B C,Hunter C L,Liu Y,et al.2017.Multi-laboratory assessment of reproducibility,qualitative and quantitative performance of SWATH-mass spectrometry[J].Nature Communications,8(1):291-302.
[10] Conesa A,Gotz S,Garcia-gomez J M,et al.2005.Blast2GO:A universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics,21(18):3674-3676.
[11] Dafni A,Maués M M.1998.A rapid and simple procedure to determine stigma receptivity[J].Sexual Plant Reproduction,11(3):177-180.
[12] Delannoy E,Jalloul A,Assigbetsé K,et al.2003.Activity of class Ⅲ peroxidases in the defense of cotton to bacterial blight[J].Molecular Plant-Microbe Interactions,16(11):1030-1038.
[13] Gabriel S,Claire L,Mickal L B,et al.2006.Role of petal-specific orcinol o-methyltransferases in the evolution of rose scent[J].Plant Physiology,140(1):18-29.
[14] Grabber J H,Ralph J,Hatfield R D,et al.1997.p-Hydroxyphenyl,guaiacyl,and syringyl lignins have similar inhibitory effects on wall degradability[J].Journal of Agricultural and Food Chemistry,45(7):2530-2532.
[15] Hong S,Song H R,Lutz K,et al.2010.Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana[J].Proceedings of the National Academy of Sciences of the USA,107(49):21211-21216.
[16] Isaacson T,Damasceno C M B,Saravanan R S,et al.2006.Sample extraction techniques for enhanced proteomic analysis of plant tissues[J].Nature Protocols,1(2):769-774.
[17] Klopfenstein D V,Liangsheng Z,Pedersen B S,et al.2018.GOATOOLS:A python library for gene ontology analyses[J].Scientific Reports,8(1):10872-10888.
[18] Lavid N,Wang J,Shalit M,et al.2002.O-Methyltransferases Involved in the biosynthesis of volatile phenolic derivatives in rose petals1[J].Plant Physiology,129(4):1899-1907.
[19] Lin J Y,Le B H,Chen M,et al.2017.Similarity between soybean and Arabidopsis seed methylomes and loss of non-CG methylation does not affect seed development[J].Proceedings of the National Academy of Sciences of the USA,114(45):E9730-E9739.
[20] Ludwig C,Gillet L,Rosenberger G,et al.2018.Data independent acquisition-based SWATH-MS for quantitative proteomics:A tutorial[J].Molecular Systems Biology,14(8):e8126-e8148.
[21] Pei Y,Niu L,Lu F,et al.2007.Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis[J].Plant Physiology,144(4):1913-1923.
[22] Sapir Y,Shmida A,Ne'eman G.2005.Pollination of Oncocyclus irises (Iris:Iridaceae) by night-sheltering male bees[J].Plant Biology,7(04):417-424.
[23] Szklarczyk D,Gable AL,Lyon D,et al.2019.STRING v11:Protein-protein association networks with increased coverage,supporting functional discovery in genome-wide experimental datasets[J].Nucleic Acids Research,47(D1):D607-613.
[24] Vogt T.2010.Phenylpropanoid biosynthesis[J].Molecular Plant,3(1):2-20.
[25] Walter W,Sánchez-cabo F,Ricote M.2015.GOplot:An R package for visually combining expression data with functional analysis[J].Bioinformatics,31(17):2912-2914.
[26] Wang Z,Baulcombe D C.2020.Transposon age and non-CG methylation[J].Nature Communications,11(1):1-9.
[27] Zhang H,Lang Z,Zhu J K.2018.Dynamics and function of DNA methylation in plants[J].Nature Reviews Molecular Cell Biology,19(8):489-506.
[28] ZhangY,FonslowB R,Shan B,et al.2013.Protein analysis by shotgun/bottom-up proteomics[J].Chemical Reviews,113(4):2343-2394.
