Genome-wide analyses on transcription factors and their potential microRNA regulators involved in maize male fertility

Anther development is a programmed biological process crucial to plant male reproduction. Genome-wide analyses on the functions of transcriptional factor (TF) genes and their microRNA (miRNA) regulators contributing to anther development have not been comprehensively performed in maize. Here, using published RNA-Seq and small RNA-Seq (sRNA-Seq) data from maize anthers at ten developmental stages in three genic male-sterility (GMS) mutants (ocl4, mac1, and ms23) and wild type W23, as well as newly sequenced maize anther transcriptomes of ms7-6007 and lob30 GMS mutants and their WT lines, we analyzed and found 1079 stage-differentially expressed (stage-DE) TF genes that can be grouped into six (premeiotic, meiotic, postmeiotic, premeiotic-meiotic, premeiotic-postmeiotic, and meiotic-postmeiotic clusters) expression clusters. Functional enrichment combined with cytological and physiological analyses revealed specific functions of genes in each expression cluster. In addition, 118 stage-DE miRNAs and 99 miRNA-TF gene pairs were identified in maize anthers. Further analyses revealed the regulatory roles of zma-miR319 and zma-miR159 as well as ZmMs7 and ZmLOB30 on ZmGAMYB expression. Moreover, ZmGAMYB and its paralog ZmGAMYB-2 were demonstrated as novel maize GMS genes by CRISPR/Cas9 knockout analysis. These results extend our understanding on the functions of miRNA-TF gene regulatory pairs and GMS TF genes contributing to male fertility in plants.