Cloning, molecular evolution and functional characterization of ZmbHLHl6, 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.

approach. The transformants were cultivated on SD/-Trp medium for 2-3 days at 28°C. Bacterial For coexpression analysis, expression data of genome-wide maize genes in 20 tissues and 66 periods 1 5 3 were obtained from q-teller 5 , and the Pearson correlation coefficients (PCCs) between ZmbHLH16 1 5 4 and other genes were calculated. Cluster3.0 (de Hoon et al., 2004) was used for target gene The primers for the semi-quantitative expression analysis of ZmbHLH16 were gene in this experiment was used as the internal control, and its amplifying primers were  The localization patterns of the ZmbHLH16 and ZmbHLH51 proteins were investigated using recombinants, pCAMBIA2300-P 35S :ZmbHLH16-eGFP and pCAMBIA2300-P 35S :ZmbHLH51-eGFP, 1 7 7 were constructed to assess the localization of these proteins. The empty pCAMBIA2300-P 35S -eGFP  To confirm the interaction between ZmbHLH16 and ZmbHLH51, a yeast two-hybrid assay was 1 8 5 conducted. The CDSs of ZmbHLH16 and ZmbHLH51 were inserted into the pGBKT7 and pGADT7 vectors, respectively. The pGBKT7-ZmbHLH16 vector without autoactivation activity was 1 8 7 constructed as above. The pGADT7-ZmbHLH51 vector was constructed using the In-Fusion cloning pGBKT7-ZmbHLH16 and pGADT7-ZmbHLH51 were co-transformed into AH109 yeast competent at 28°C for 2-3 days, and positive clones were confirmed using PCR. Positive clones were further 1 9 2 cultured on SD/-Ade-His-Leu-Trp medium containing 50 mg/L χ-α-gal at 28°C for 2-3 days. The vectors pGBKT7-T and pGBKT7-Lam were used as positive and negative controls, respectively. After confirming the interaction between ZmbHLH16 and ZmbHLH51, to investigate the interaction bait vector pGBKT7 and then co-transformed with the prey vector pGADT7-ZmbHLH51 into the 1 9 7 AH109 yeast competent cells. that these bHLH TFs were highly conserved, with most bootstrap values >90%, and that ZmbHLH16 2 1 1 was the most closely related to OsTIP2 ( Fig 1B). Together, the above results indicated that 2 1 2 ZmbHLH16 protein included the typically conserved domain of bHLH TFs and might play a crucial 2 1 3 role in male reproduction in maize. To analyze its molecular evolution, the DNA sequences of ZmbHLH16 were amplified from 78 23 SNPs, 13 (57%) and 10 (43%) involved transitions and transversions, respectively. Further showed that nucleotide variations were not evenly distributed in ZmbHLH16, and introns had higher highest nucleotide diversity ratio (π=6.15×10 -3 ) in the first intron but no significant nucleotide 2 2 6 variation in the 3rd intron or 3'-UTR (Table 2).

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To investigate whether ZmbHLH16 has experienced selection pressure, various regions of 2 2 8 ZmbHLH16 were assessed ( ZmbHLH16 underwent directional selection, its LD patterns and LD decay were also calculated. In the LD matrix, no obvious LD block was found in the ZmbHLH16 genome sequence (Fig 2A). A 2 3 4 schematic diagram of LD decay represented by plots of r 2 showed that the LD level dropped to 0.1 at 2 3 5 7 approximately 1,300 bp (Fig 2B), indicating a more rapid decay rate than the average 1.5 kb of 2 3 6 several genes under selection in maize (Remington et al., 2001). Therefore, our above results also 2 3 7 reflected the conserved evolution of ZmbHLH16 in the maize germplasm. To identify the activating function of ZmbHLH16, eight fragments of ZmbHLH16 were analyzed in 2 4 0 yeast ( Fig 3A). Yeast cells with pGBKT7-ZmbHLH16(A) 1-80 a.a. or pGBKT7-ZmbHLH16(E) 1-160a.a.
2 4 1 grew normally on both SD/-Trp and SD/-His-Trp selective media and turned the indicator blue. yeast. In conclusion, the above results indicated that the first 80 amino acids in the N-terminus of 2 5 0 bHLH16 could activate transcription in yeast, whereas the full-length version did not. Functionally associated genes are more likely to share similar expression patterns (Fu & Xue, 2010).

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Coexpression analysis was therefore conducted to identify potential ZmbHLH16 cooperators. A total  ( Table 3). The similar expression pattern to a number of plant reproduction-related genes indicated 2 6 2 that ZmbHLH16 might be closely associated with maize male fertility.  gene OsTDR. Accordingly, ZmbHLH51 might be an important factor in maize pollen development.

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Some studies have indicated that the interactions among bHLH TFs are important for pollen 2 7 9 development (Niu et al., 2013;Zhu et al., 2015). Therefore, we next aimed to analyze the interaction 2 8 0 between ZmbHLH16 and ZmbHLH51. The expression patterns of ZmbHLH16 and ZmbHLH51 were simultaneously analyzed using showed a higher expression level in spikelets than organs (Fig 5A). This finding indicated that 2 8 5 ZmbHLH16 and ZmbHLH51 might be closely associated with maize male fertility. ZmbHLH16-eGFP and ZmbHLH51-eGFP were only located in the nucleus, whereas the control 2 9 0 eGFP was localized to both the cytoplasm and the nucleus. The similar expression profiles and 2 9 1 protein localization patterns between ZmbHLH16 and ZmbHLH51 supported their interaction. Because the aforementioned results indicated that the two bHLH TFs ZmbHLH16 and ZmbHLH51 2 9 4 had similar expression characteristics and subcellular localization patterns, a yeast two-hybrid assay 2 9 5 was used to verify the interaction between the two proteins. As shown in Fig 6A,  containing pGBKT7-ZmbHLH16 and pGADT7-ZmbHLH51 were able to grow on both confirmed the interaction between ZmbHLH16 and ZmbHLH51.

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To map the domains involved in the ZmbHLH16-ZmbHLH51 interaction, fragments without could grow normally on SD/-Ade-His-Leu-Trp media and turned the media blue ( Fig 6B).  based on homology analysis. Thus, here, we isolated ZmbHLH16 based on homology cloning from 3 1 5 OsTIP2, which has been reported to be a master regulator of pollen formation.

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In this study, the molecular evolution of ZmbHLH16 was investigated. In the analysis of selective 3 1 7 pressure, no significant signal was found in ZmbHLH16 according to Tajima's D and Fu and Li's 3 1 8 tests. Moreover, a lower nucleotide diversity ratio (π=2.58×10 -3 ) was observed in all regions of 3 1 9 ZmbHLH16 than in the average (π=6.3×10 -3 ) of 18 maize genes in previous reports (Ching et al.,  1,300 bp) was also less than the average intragenic level (r 2 <0.1 within 1,500 bp) (Remington et al., ZmbHLH16. The conserved molecular evolution of ZmbHLH16 further hinted at its crucial function 3 2 9 in maize male reproduction.  ability of transcriptional activation. Thus, we speculate that ZmbHLH16 might regulate target gene in addition to the typical bHLH and BIF domains.

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Coincidently, it was recently reported that ZmbHLH16 was a candidate gene for the ms23 mutant instead of tapetal differentiation (Chaubal et al., 2000). These results strongly support the hypothesis 3 5 0 that ZmbHLH16 is involved in tapetal specification and maturation. In Nan's paper (Nan et al., 2017), with proteomics data. These authors also confirmed the interaction between ZmbHLH16 and 3 5 3 ZmbHLH51, similar to our result. In contrast, we paid more attention to the ZmbHLH16 nucleotide      (MS)/reproduction (MR) genes.

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Query gene ID PCC-value Subject ID E-value Score Symbol Status Description