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Maize, a monoecious crop, is a model plant for studying the regulation of floral organ development. In a previous study, we found that ZmMYB150 is significantly downregulated in sterile anthers by analyzing transcriptome data of the cytoplasmic male sterility (CMS) line C48-2 and its maintainer line 48-2. To elucidate the functional mechanism of ZmMYB150 in maize reproductive development, we cloned ZmMYB150, and sequence analysis showed it is a typical R2R3-MYB transcription factor (TF). Tissue-specific analysis revealed ZmMYB150 to be specifically highly in anthers. The subcellular localization in tobacco leaves proved that ZmMYB150 is located in the nucleus. Co-expression analysis identified 876 genes that may be coexpressed with ZmMYB150, and sequence alignment analysis showed that the coexpressed genes ZmMYB116 and ZmPIP5K6 are homologous to Arabidopsis male fertility-related genes. Furthermore, a yeast two-hybrid (Y2H) assay revealed that ZmMYB150 interacts with ZmPIP5K6. qRT-PCR analysis showed that ZmMYB150 is specifically highly expressed at the late stage of normal anther development, but no expression during the whole stages of sterile anther development. Tobacco transient expression system was used to confirm that ZmMYB150 can be regulated by miR159e-3p at the translational level. Our results lay a foundation for further understanding of the regulatory mechanism of MYB TFs in maize reproductive development.

Background The tricarboxylic acid (TCA) cycle is crucial for cellular energy metabolism and carbon skeleton supply. However, the detailed functions of the maize TCA cycle genes remain unclear. Results In this study, 91 TCA genes were identified in maize by a homology search, and they were distributed on 10 chromosomes and 1 contig. Phylogenetic results showed that almost all maize TCA genes could be classified into eight major clades according to their enzyme families. Sequence alignment revealed that several genes in the same subunit shared high protein sequence similarity. The results of cis-acting element analysis suggested that several TCA genes might be involved in signal transduction and plant growth. Expression profile analysis showed that many maize TCA cycle genes were expressed in specific tissues, and replicate genes always shared similar expression patterns. Moreover, qPCR analysis revealed that some TCA genes were highly expressed in the anthers at the microspore meiosis phase. In addition, we predicted the potential interaction networks among the maize TCA genes. Next, we cloned five TCA genes located on different TCA enzyme complexes, Zm00001d008244 (isocitrate dehydrogenase, IDH), Zm00001d017258 (succinyl-CoA synthetase, SCoAL), Zm00001d025258 (α-ketoglutarate dehydrogenase, αKGDH), Zm00001d027558 (aconitase, ACO) and Zm00001d044042 (malate dehydrogenase, MDH). Confocal observation showed that their protein products were mainly localized to the mitochondria; however, Zm00001d025258 and Zm00001d027558 were also distributed in the nucleus, and Zm00001d017258 and Zm00001d044042 were also located in other unknown positions in the cytoplasm. Through the bimolecular fluorescent complimentary (BiFC) method, it was determined that Zm00001d027558 and Zm00001d044042 could form homologous dimers, and both homologous dimers were mainly distributed in the mitochondria. However, no heterodimers were detected between these five genes. Finally, Arabidopsis lines overexpressing the above five genes were constructed, and those transgenic lines exhibited altered primary root length, salt tolerance, and fertility. Conclusion Sequence compositions, duplication patterns, phylogenetic relationships, cis-elements, expression patterns, and interaction networks were investigated for all maize TCA cycle genes. Five maize TCA genes were overexpressed in Arabidopsis, and they could alter primary root length, salt tolerance, and fertility. In conclusion, our findings may help to reveal the molecular function of the TCA genes in maize.

OsEAT1 (DTD), a stamen-specific bHLH transcription factor, is a master regulator in rice male reproduction. To elucidate its functional mechanism in maize male reproduction, the maize orthologue ZmbHLH122 of OsEAT1 was isolated in this study. Sequence analysis revealed that the ZmbHLH122 coding sequence consists of 1422 bp which encode a protein of 473 amino acids. Bioinformatic analysis confirmed that ZmbHLH122 belongs to the bHLH TF family. The subcellular localization of ZmbHLH122-eGFP in rice protoplasts showed that ZmbHLH122 is a nuclear-localized protein. Yeast one-hybrid assays demonstrated that ZmbHLH122 exhibits weak transactivation ability. Genome-wide coexpression analysis identified 751 potential collaborators of ZmbHLH122, most of which are highly expressed in maize anthers and tassels, and sequence alignment analysis revealed that 43 coexpressed genes are homologous to Arabidopsis male fertility-related genes. Expression pattern analysis confirmed that ZmbHLH122 and its potential collaborators Ms23 (ZmbHLH16), ZmbHLH51, ZmbHLH66 (Ms32) and ZmMYB74 are preferentially expressed in the maize male reproductive organs, especially those at the uninucleate and early binucleate stages. Yeast two-hybrid assays revealed that ZmMYB74 directly interacts with two maize stamen development regulators, Ms23 (ZmbHLH16) and ZmbHLH51, and indirectly collaborates with ZmbHLH122. Our study lays the foundation for further understanding of the ZmbHLH122 molecular function in maize male reproduction.