Cloning, Molecular Evolution And Functional Characterization Of ZmbHLH16, The Maize Ortholog Of OsTIP2 (OsbHLH142)

Abstract Basic helix-loop-helix (bHLH) transcription factors play key roles in plant male reproduction. More than 14 bHLH proteins related to pollen development have been cloned from rice and Arabidopsis. However, little is known about the role of the bHLH family in maize microspore development. In this study, the bHLH transcription factor ZmbHLH16 was cloned. ZmbHLH16 shares high similarity with the OsTIP2 (OsbHLH142) protein, a master regulator of the developmental coordination of male reproduction in rice. Expression characterization analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs and is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 SNPs and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. A yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. To identify potential ZmbHLH16 interactors, 395 genes that shared similar expression patterns in a genome-wide search were obtained through coexpression analysis. Among these genes, the transcription factor ZmbHLH51 had an expression pattern and subcellular localization similar to those of ZmbHLH16. The interaction between ZmbHLH51 and ZmbHLH16 was verified in yeast cells. In addition to the typical bHLH domain, other regions of ZmbHLH16 were necessary and adequate for its heterodimerization with ZmbHLH51. Our results contribute to a solid foundation for further understanding the functions and mechanisms of ZmbHLH16.