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Proteomic analysis of differential anther development from sterile/fertile lines in Capsicum annuum L.

ABSTRACT: Background: Pepper (Capsicum annuum L.) is a major cash crop throughout the world. Male sterility is an important characteristic in crop species that leads to a failure to produce functional pollen, and it has crucial roles in agricultural breeding and the utilization of heterosis. Objectives: In this study, we identified many crucial factors and important components in metabolic pathways in anther and pollen development, and elucidated the molecular mechanism related to pollen abortion in pepper. Methods: Pepper pollen was observed at different stages to detect the characteristics associated with male sterility and fertility. The phytohormone and oxidoreductase activities were detected in spectrophotometric and redox reaction assays, respectively. Proteins were extracted from male sterile and fertile pepper lines, and identified by TMT/iTRAQ (Tandem mass tags/isobaric tags for relative and absolute quantitation) and LC-MS/MS (liquid chromatograph-mass spectrometer) analysis. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes database according to |fold change)| > 1.3 and P value < 0.05. DAPs were quantified in the meiosis, tetrad, and binucleate stages by parallel reaction monitoring (PRM). Results: In this study, we screened and identified one male sterile pepper line with abnormal cytological characteristics in terms of pollen development. The peroxidase and catalase enzyme activities were significantly reduced and increased, respectively, in the male sterile line compared with the male fertile line. Phytohormone analysis demonstrated that the gibberellin, jasmonic acid, and auxin contents changed by different extents in the male sterile pepper line. Proteome analysis screened 1645 DAPs in six clusters, which were mainly associated with the chloroplast and cytoplasm based on their similar expression levels. According to proteome analysis, 45 DAPs were quantitatively identified in the meiosis, tetrad, and binucleate stages by PRM, which were related to monoterpenoid biosynthesis, and starch and sucrose metabolism pathways. Conclusions: We screened 1645 DAPs by proteomic analysis and 45 DAPs were related to anther and pollen development in a male sterile pepper line. In addition, the activities of peroxidase and catalase as well as the abundances of phytohormones such as gibberellin, jasmonic acid, and auxin were related to male sterility. The results obtained in this study provide insights into the molecular mechanism responsible for male sterility and fertility in pepper.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Capsicum Annuum

TISSUE(S): Anther

SUBMITTER: hongxia pei

LAB HEAD: Hongxia pei

PROVIDER: PXD031331 | Pride | 2022-05-19

REPOSITORIES: Pride

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Project description:Gametophytic self-incompatibility is the primary cause of low fruit set in almond. The mechanism of recognition that determines whether the gametophyte is successfully fertilized between pollen tube SCF (F-box-SSK1-Cul1-Rbx1) protein and pistil S-RNase protein during fertilization is unclear. In this study, the pistils of two almond cultivars 'Wanfeng' and 'Nonpareil' were used as the experimental materials after selfing- and nonselfing/cross-pollination, and pistils from the stamen-removed flowers were used as controls. We used fluorescence microscopy to observe the development of pollen tubes after pollination and 4-dimensional label-free quantitation (4D-LFQ) to detect the protein expression profiles of 'Wanfeng' and 'Nonpareil' pistils and in controls. The results showed that it took 24-36 h for the development of the pollen tube to 1/3 of the pistil, and a total of 7684 differentially accumulated proteins (DAPs) were identified in the pistil after pollinating 36 h, of which 7022 were quantifiable. The up- and down-regulation of the 12 differential protein expressions identified by parallel reaction monitoring (PRM) was the same as that identified by 4D-LFQ, with an average fold-change difference of 14.34%, a maximum of 31.37% and a minimum of 3.68%. Bioinformatics analysis based on the function of the differential proteins, including classification, enrichment, and clustering, identified RNA polymerases (4 DAPs), autophagy (3 DAPs), oxidative phosphorylation (3 DAPs), and homologous recombination (2 DAPs) pathways associated with the self-incompatibility process. The interaction between the serine/threonine kinase (MARK2) protein E3 ubiquitin ligase and the microtubule protein component microtubule-associated protein tau (MAPT/ACT) was found using the STRING database, which demonstrated the involvement of the MARK2 protein in the reaction of pollen tube recognition the nonself- and the self-S-RNase protein. It provides a new way to reveal the mechanism by which almond pollen tubes recognize the self and nonself S-RNase enzyme protein.

Project description:1α,25(OH)2VD3 is the most active form of VD3 in animals and it plays an important role in regulating mineral metabolism but also reproduction and immunity. Testes are the main reproductive organs of male mammals. In order to study the effect of 1α,25(OH)2VD3 on early testicular development, 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 μg.kg-1 of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides, 60 days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labeling technique was used to analyze the protein profile of the testes in each group. Western blotting was used to verify the target of differentially abundant proteins (DAPs). The results showed that of the identified 132715 peptides, 122755 were specific peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs was focused on proteins closely related to the regulation of the testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results also indicate that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets’ testes. Significance: The early development of the testes of breeding boars is closely related to their lifetime fertility. Our research identifies potential signaling pathways and differentially abundant proteins associated with testicular development. These findings indicate that plants containing 1α,25(OH)2VD3-glycosides used as a nutritional ingredient may help early testicular development in breeding boars and reveal the regulatory mechanism of 1α,25(OH)2VD3 on testicular development.

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Proteomic analysis of differential anther development from sterile/fertile lines in Capsicum annuum L.

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