Plant Growth Regulation

, Volume 85, Issue 1, pp 113–122 | Cite as

Characterization of the ZmbHLH122 transcription factor and its potential collaborators in maize male reproduction

  • Yongming Liu
  • Zhuofan Zhao
  • Gui Wei
  • Peng Zhang
  • Hai Lan
  • Suzhi Zhang
  • Chuan Li
  • Moju CaoEmail author
Original paper


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.


bHLH transcription factor Yeast hybrids Coexpression analysis Male reproduction Maize 


Author contributions

MC and YL designed the research and wrote the article. YL performed most of the experiments. GW, ZZ, and PZ performed some of the experiments. HL, SZ, and CL assisted in the data analysis. All authors have read and approved the final manuscript.


This work was supported by Grants from the National Key Research and Development Program of China (No. 2016YFD0101206) and the Platform for Mutation Breeding by Radiation in Sichuan (No. 2016NZ0106).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

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Analysis of transcriptional activity of ZmMYB74 in yeast. The recombinant vector pGBKT7-ZmMYB74 was transformed into an AH109 competent yeast cell and then was cultivated on the corresponding selective medium at 28°C for 48-72h (TIF 172 KB)
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Supplementary material 6 (XLSX 16 KB)


  1. Achard P, Herr A, Baulcombe DC, Harberd NP (2004) Modulation of floral development by a gibberellin-regulated microRNA. Development 131:3357–3365CrossRefPubMedGoogle Scholar
  2. Ba ANN, Pogoutse A, Provart N, Moses AM (2009) NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinform 10:202CrossRefGoogle Scholar
  3. Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bohra A, Jha UC, Adhimoolam P, Bisht D, Singh NP (2016) Cytoplasmic male sterility (CMS) in hybrid breeding in field crops. Plant Cell Rep 35:967–993CrossRefPubMedGoogle Scholar
  5. Chang Z, Chen Z, Wang N, Xie G, Lu J, Yan W, Zhou J, Tang X, Deng XW (2016) Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proc Natl Acad Sci USA 113:14145–14150CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chen L, Liu Y (2014) Male sterility and fertility restoration in crops. Annu Rev Plant Biol 65:579–606CrossRefPubMedGoogle Scholar
  7. Chen X, Huang H, Qi T, Liu B, Song S (2016) New perspective of the bHLH-MYB complex in jasmonate-regulated plant fertility in arabidopsis. Plant Signal Behav 11:e1135280CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cigan AM, Singh M, Benn G, Feigenbutz L, Kumar M, Cho MJ, Svitashev S, Young J (2017) Targeted mutagenesis of a conserved anther-expressed P450 gene confers male sterility in monocots. Plant Biotechnol J 15:379–389CrossRefPubMedGoogle Scholar
  9. Du X, Wang G, Ji J, Shi L, Guan C, Jin C (2017) Comparative transcriptome analysis of transcription factors in different maize varieties under salt stress conditions. Plant Growth Regul 81:183–195CrossRefGoogle Scholar
  10. Dukowic-Schulze S, Harris A, Li J, Sundararajan A, Mudge J, Retzel EF, Pawlowski WP, Chen C (2014) Comparative transcriptomics of early meiosis in Arabidopsis and maize. J Genet Genom 41:139–152CrossRefGoogle Scholar
  11. Feller A, Machemer K, Braun EL, Grotewold E (2011) Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J 66:94–116CrossRefPubMedGoogle Scholar
  12. Fu F, Xue H (2010) Coexpression analysis identifies Rice Starch Regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator. Plant Physiol 154:927–938CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Method Enzymol 350:87–96CrossRefGoogle Scholar
  14. Gómez JF, Talle B, Wilson ZA (2015) Anther and pollen development: a conserved developmental pathway. J Integr Plant Biol 57:876–891CrossRefPubMedPubMedCentralGoogle Scholar
  15. Huang J, Smith AR, Zhang T, Zhao D (2016) Creating completely both male and female sterile plants by specifically ablating microspore and megaspore mother cells. Front Plant Sci 7:30PubMedPubMedCentralGoogle Scholar
  16. Ji C, Li H, Chen L, Xie M, Wang F, Chen Y, Liu YG (2013) A novel rice bHLH transcription factor, DTD, acts coordinately with TDR in controlling tapetum function and pollen development. Mol Plant 6:1715–1718CrossRefPubMedGoogle Scholar
  17. Jiang Y, Zeng B, Zhao H, Zhang M, Xie S, Lai J (2012) Genome-wide transcription factor gene prediction and their expressional tissue-specificities in Maize. J Integr Plant Biol 54:616–630CrossRefPubMedGoogle Scholar
  18. Kim Y, Zhang D (2017) Molecular control of male fertility for crop hybrid breeding. Trends Plant Sci 23:53–65CrossRefPubMedGoogle Scholar
  19. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9:299–306CrossRefPubMedPubMedCentralGoogle Scholar
  20. Li D, Wang L, Liu X, Cui D, Chen T, Zhang H, Jiang C, Xu C, Li P, Li S (2013) Deep sequencing of maize small RNAs reveals a diverse set of microRNA in dry and imbibed seeds. PLoS ONE 8:e55107CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li H, Wang L, Yang ZM (2015) Co-expression analysis reveals a group of genes potentially involved in regulation of plant response to iron-deficiency. Gene 554:16–24CrossRefPubMedGoogle Scholar
  22. Li J, Zhang H, Si X, Tian Y, Chen K, Liu J, Chen H, Gao C (2017) Generation of thermosensitive male-sterile maize by targeted knockout of the ZmTMS5 gene. J Genet Genom 44:465–468CrossRefGoogle Scholar
  23. Liu Y, Zhang L, Zhou J, Cao M (2015) Research progress of the bHLH transcription factors involved in genic male sterility in plants. Hereditas 37:1194–1203PubMedGoogle Scholar
  24. Liu Y, Li J, Wei G, Sun Y, Lu Y, Lan H, Li C, Zhang S, Cao M (2017) Cloning, molecular evolution and functional characterization of ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142). Biol Open 6:1654–1663CrossRefPubMedPubMedCentralGoogle Scholar
  25. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  26. Ma J, Skibbe DS, Fernandes J, Walbot V (2008) Male reproductive development: gene expression profiling of maize anther and pollen ontogeny. Genome Biol 9:R181CrossRefPubMedPubMedCentralGoogle Scholar
  27. Moon J, Skibbe D, Timofejeva L, Wang CR, Kelliher T, Kremling K, Walbot V, Cande WZ (2013) Regulation of cell divisions and differentiation by MALE STERILITY32 is required for anther development in maize. Plant J 76:592–602CrossRefPubMedPubMedCentralGoogle Scholar
  28. Nan G, Zhai J, Arikit S, Morrow D, Fernandes J, Mai L, Nguyen N, Meyers BC, Walbot V (2017) MS23, a master basic helix-loop-helix factor, regulates the specification and development of tapetum in maize. Development 144:163–172CrossRefPubMedGoogle Scholar
  29. Niu N, Liang W, Yang X, Jin W, Wilson ZA, Hu J, Zhang D (2013) EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat Commun 4:1445CrossRefPubMedGoogle Scholar
  30. Pires N, Dolan L (2010) Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol 27:862–874CrossRefPubMedGoogle Scholar
  31. Qi T, Huang H, Song S, Xie D (2015) Regulation of jasmonate-mediated stamen development and seed production by a bHLH-MYB complex in Arabidopsis. Plant Cell 27:1620–1633CrossRefPubMedPubMedCentralGoogle Scholar
  32. Qi S, Liu K, Gao C, Li D, Jin C, Duan S, Ma H, Hai J, Chen M (2017) The effect of BnTT8 on accumulation of seed storage reserves and tolerance to abiotic stresses during Arabidopsis seedling establishment. Plant Growth Regul 82:271–280CrossRefGoogle Scholar
  33. Ranum P, Peña Rosas JP, Garcia Casal MN (2014) Global maize production, utilization, and consumption. Ann N Y Acad Sci 1312:105–112CrossRefPubMedGoogle Scholar
  34. Rao GS, Deveshwar P, Sharma M, Kapoor S, Rao KV (2017) Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties. Plant Mol Biol. Google Scholar
  35. Sekhon RS, Lin H, Childs KL, Hansey CN, Buell CR, de Leon N, Kaeppler SM (2011) Genome-wide atlas of transcription during maize development. Plant J 66:553–563CrossRefPubMedGoogle Scholar
  36. Srivastava S, Singh G, Singh N, Srivastava G, Sharma A (2016) Analysis of bHLH coding genes using gene co-expression network approach. Mol Biol Rep 43:677–685CrossRefPubMedGoogle Scholar
  37. Svitashev S, Young J, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169:931–945CrossRefPubMedPubMedCentralGoogle Scholar
  38. Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7:13274CrossRefPubMedPubMedCentralGoogle Scholar
  39. Tsuji H, Aya K, Ueguchi Tanaka M, Shimada Y, Nakazono M, Watanabe R, Nishizawa NK, Gomi K, Shimada A, Kitano H (2006) GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers. Plant J 47:427–444CrossRefPubMedGoogle Scholar
  40. Wan S, Wang W, Zhou T, Zhang Y, Chen J, Xiao B, Yang Y, Yu Y (2018) Transcriptomic analysis reveals the molecular mechanisms of Camellia sinensis in response to salt stress. Plant Growth Regul. Google Scholar
  41. Wu Y, Fox TW, Trimnell MR, Wang L, Xu RJ, Cigan AM, Huffman GA, Garnaat CW, Hershey H, Albertsen MC (2016) Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnol J 14:1046–1054CrossRefPubMedGoogle Scholar
  42. Zhai J, Zhang H, Arikit S, Huang K, Nan G, Walbot V, Meyers BC (2015) Spatiotemporally dynamic, cell-type–dependent premeiotic and meiotic phasiRNAs in maize anthers. Proc Natl Acad Sci USA 112:3146–3151CrossRefPubMedPubMedCentralGoogle Scholar
  43. Zhang D, Wu S, An X, Xie K, Zhou Y, Xu L, Fang W, Liu S, Liu S, Zhu T (2017) Construction of a multi-control sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor. Plant Biotechnol J. Google Scholar
  44. Zhou H, He M, Li J, Chen L, Huang Z, Zheng S, Zhu L, Ni E, Jiang D, Zhao B, Zhuang C (2016) Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci Rep 6:37395CrossRefPubMedPubMedCentralGoogle Scholar
  45. Zou T, He Z, Qu L, Liu M, Zeng J, Liang Y, Wang T, Chen D, Xiao Q, Zhu J, Liang Y, Deng Q, Wang S, Zheng A, Wang L, Li P, Li S (2017) Knockout of OsACOS12 caused male sterility in rice. Mol Breed 37:126CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Yongming Liu
    • 1
  • Zhuofan Zhao
    • 1
  • Gui Wei
    • 1
  • Peng Zhang
    • 1
  • Hai Lan
    • 1
  • Suzhi Zhang
    • 1
  • Chuan Li
    • 1
  • Moju Cao
    • 1
    Email author
  1. 1.Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region of Ministry of Agriculture, Maize Research InstituteSichuan Agricultural UniversityChengduChina

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