Genome-Wide Identification and Comparison of Cysteine Proteases in the Pollen Coat and Other Tissues in Maize

Yanhua Li; Liangjie Niu; Xiaolin Wu; Claudia Faleri; Fuju Tai; Man Zhang; Hui Liu; Wei Wang; Giampiero Cai

doi:10.3389/fpls.2021.709534

Genome-Wide Identification and Comparison of Cysteine Proteases in the Pollen Coat and Other Tissues in Maize

Front Plant Sci. 2021 Sep 23:12:709534. doi: 10.3389/fpls.2021.709534. eCollection 2021.

Authors

Yanhua Li¹, Liangjie Niu¹, Xiaolin Wu¹, Claudia Faleri², Fuju Tai¹, Man Zhang¹, Hui Liu¹, Wei Wang¹, Giampiero Cai²

Affiliations

¹ National Key Laboratory of Wheat and Maize Crop Science, College of Life Sciences, Henan Agricultural University, Zhengzhou, China.
² Department of Life Sciences, University of Siena, Siena, Italy.

Abstract

Cysteine proteases, belonging to the C1-papain family, play a major role in plant growth and development, senescence, and immunity. There is evidence to suggest that pollen cysteine protease (CP) (ZmCP03) is involved in regulating the anther development and pollen formation in maize. However, there is no report on the genome-wide identification and comparison of CPs in the pollen coat and other tissues in maize. In this study, a total of 38 homologous genes of ZmCP03 in maize were identified. Subsequently, protein motifs, conserved domains, gene structures, and duplication patterns of 39 CPs are analyzed to explore their evolutionary relationship and potential functions. The cis-elements were identified in the upstream sequence of 39 CPs, especially those that are related to regulating growth and development and responding to environmental stresses and hormones. The expression patterns of these genes displayed remarked difference at a tissue or organ level in maize based on the available transcriptome data in the public database. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that ZmCP03 was preferably expressed at a high level in maize pollen. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, immunofluorescence and immunogold electron microscopy all validated the cellular localization of ZmCP03 in both the pollen coat and pollen cytoplasm. In addition, 142 CP genes from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and cotton (Gossypium hirsutum), together with 39 maize CPs, were retrieved to analyze their evolution by comparing with orthologous genes. The results suggested that ZmCP03 was relatively conservative and stable during evolution. This study may provide a referential evidence on the function of ZmCP03 in pollen development and germination in maize.

Keywords: cysteine protease; expression analysis; gene duplication; immunofluorescence microscopy; immunogold electron microscopy; maize pollen; phylogenetic analysis.