Banana MabHLH28 positively regulates the expression of softening-related genes to mediate fruit ripening independently or via cooperating with MaWRKY49/111

Abstract

Texture softening is a physiological indicator of fruit ripening, which eventually contributes to fruit quality and the consumer’s acceptance. Despite great progress having been made in identification of the genes related to fruit softening, the upstream transcriptional regulatory pathways of these softening-related genes are not fully elucidated. Here, a novel bHLH gene, designated as MabHLH28, was identified because of its significant upregulation in banana fruit ripening. DAP-Seq analysis revealed that MabHLH28 bound to the core sequence of ‘CAYGTG’ presented in promoter regions of fruit softening-associated genes, such as the genes related to cell wall modification (MaPG3, MaPE1, MaPL5, MaPL8, MaEXP1, MaEXP2, MaEXPA2, and MaEXPA15) and starch degradation (MaGWD1 and MaLSF2), and these bindings were validated by EMSA and DLR assays. Transient overexpression and knockdown of MabHLH28 in banana fruit resulted in up- and down-regulation of softening-related genes, thereby hastening and postponing fruit ripening. Furthermore, overexpression of MabHLH28 in tomato accelerated the ripening process by elevating the accumulation of softening-associated genes. In addition, MabHLH28 showed interaction with MaWRKY49/111 and itself to form protein complexes, which could combinatorically strengthen the transcription of softening-associated genes. Taken together, our findings suggest that MabHLH28 mediates fruit softening by upregulating the expression of softening-related genes either alone or in combination with MaWRKY49/111.

Acknowledgements

This work was financially supported by the National Key R & D Program of China (2022YFD2100102), China Postdoctoral Science Foundation (2023 M731146), Innovative Team Project of Guangdong Universities (2022KCXTD051), and China Agriculture Research System of MOF and MARA (CARS-31).

Author contributions

J.K. and X.S. conceived the research and designed the experiments; C.W. performed most of the experiments and analysed the data; D.C., J.L., Z.L., and W.W. performed some of the experiments; J.K., C.W., and X.S. wrote the manuscript; W.S., J.C., and W.L. gave advice and revised the manuscript. All authors read and approved the final manuscript.

Data availability

All data from this study are included in the submitted article.

Conflict of interest statement

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Peng Z, Liu G, Li H. et al. Molecular and genetic events determining the softening of fleshy fruits: a comprehensive review. Int J Mol Sci. 2022; 23:12482

[2] Shi Y, Li BJ, Su G. et al. Transcriptional regulation of fleshy fruit texture. J Integr Plant Biol. 2022; 64:1649-72

[3] Tucker G, Yin XR, Zhang AD. et al. Ethylene and fruit softening. Food Qual Saf. 2017; 1:253-67

[4] Xiao YY, Kuang JF, Qi XN. et al. A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening. Plant Biotechnol J. 2018; 16:151-64

[5] Zhang AD, Wang WQ, Tong Y. et al. Transcriptome analysis identifies a zinc finger protein regulating starch degradation in kiwifruit. Plant Physiol. 2018; 178:850-63

[6] Lai X, Chahtane H, Martin-Arevalillo R. et al. Contrasted evolutionary trajectories of plant transcription factors. Curr Opin Plant Biol. 2020; 54:101-7

[7] Smaczniak C, Muiño JM, Chen D. et al. Differences in DNA binding specificity of floral homeotic protein complexes predict organ-specific target genes. Plant Cell. 2017; 29:1822-35

[8] Li C, Hou X, Qi N. et al. Insight into ripening-associated transcription factors in tomato: a review. Sci Hortic. 2021; 288:110363

[9] Li X, Wang X, Zhang Y. et al. Regulation of fleshy fruit ripening: from transcription factors to epigenetic modifications. Hortic Res. 2022;9:uhac013

[10] Wu C, Deng W, Shan W. et al. Banana MKK1 modulates fruit ripening via the MKK1-MPK6-3/11-4-bZIP21 module. Cell Rep. 2023; 42:112832

[11] Zhu L, Chen L, Wu C. et al. Methionine oxidation and reduction of the ethylene signaling component MaEIL9 are involved in banana fruit ripening. J Integr Plant Biol. 2023; 65:150-66

[12] Zhu L, Shan W, Cai D. et al. High temperature elevates carotenoid accumulation of banana fruit via upregulation of MaEIL9 module. Food Chem. 2023; 412:135602

[13] Buck MJ, Atchley WR. Phylogenetic analysis of plant basic helix-loop-helix proteins. J Mol Evol. 2003; 56:742-50

[14] Toledo-Ortiz G, Huq E, Quail PH. The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell. 2003; 15:1749-70

[15] Zuo ZF, Lee HY, Kang HG. Basic helix-loop-helix transcription factors: regulators for plant growth development and abiotic stress responses. Int J Mol Sci. 2023; 24:1419

[16] Waseem M, Li Z. Overexpression of tomato SlbHLH22 transcription factor gene enhances fruit sensitivity to exogenous phytohormones and shortens fruit shelf-life. J Biotechnol. 2019; 299:50-6

[17] Zhang L, Kang J, Xie Q. et al. The basic helix-loop-helix transcription factor bHLH95 affects fruit ripening and multiple metabolisms in tomato. J Exp Bot. 2020; 71:6311-27

[18] Wang W, Yu J, Du M. et al. Basic helix-loop-helix (bHLH) transcription factor MdbHLH3 negatively affects the storage performance of postharvest apple fruit. Hortic Plant J. 2022; 8:700-12

[19] Zhou D, Shen Y, Zhou P. et al. Papaya CpbHLH1/2 regulate carotenoid biosynthesis-related genes during papaya fruit ripening. Hortic Res. 2019; 6:80

[20] Wang L, Guo D, Zhao G. et al. Group IIc WRKY transcription factors regulate cotton resistance to Fusarium oxysporum by promoting GhMKK2-mediated flavonoid biosynthesis. New Phytol. 2022; 236:249-65

[21] Xie Z, Zhang ZL, Zou X. et al. Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol. 2005; 137:176-89

[22] Gan Z, Yuan X, Shan N. et al. AcWRKY40 mediates ethylene biosynthesis during postharvest ripening in kiwifruit. Plant Sci. 2021; 309:110948

[23] Zhang WW, Zhao SQ, Gu S. et al. FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca. Plant Physiol. 2022; 189:1037-49

[24] Zhang L, Ma B, Wang C. et al. MdWRKY126 modulates malate accumulation in apple fruit by regulating cytosolic malate dehydrogenase (MdMDH5). Plant Physiol. 2022; 188:2059-72

[25] Wu C, Su X, Shan W. et al. Banana MaWRKY49 and MaWRKY 111 cooperate with MabZIP21 to activate the transcription of MaACS1 and MaACO1 during fruit ripening. Postharvest Biol Technol. 2022; 194:112087

[26] Braun P, Aubourg S, Leene JV. et al. Plant protein interactomes. Annu Rev Plant Biol. 2013; 64:161-87

[27] Li SJ, Yin XR, Wang WL. et al. Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3. J Exp Bot. 2017; 68:3419-26

[28] Jiang G, Li Z, Song Y. et al. LcNAC13 physically interacts with LcR1MYB1 to coregulate anthocyanin biosynthesis-related genes during litchi fruit ripening. Biomol Ther. 2019; 9:135

[29] Zhang T, Li W, Xie R. et al. CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and regulate fruit ripening in papaya. Plant J. 2020; 103:1318-37

[30] Wang X, Pan L, Wang Y. et al. PpIAA1 and PpERF4 form a positive feedback loop to regulate peach fruit ripening by integrating auxin and ethylene signals. Plant Sci. 2021; 313:111084

[31] Wei W, Yang YY, Wu CJ. et al. MaMADS1-MaNAC083 transcriptional regulatory cascade regulates ethylene biosynthesis during banana fruit ripening. Hortic Res. 2023;10:uhad177

[32] Hao Y, Zong X, Ren P. et al. Basic helix-loop-helix (bHLH) transcription factors regulate a wide range of functions in Arabidopsis. Int J Mol Sci. 2021; 22:7152

[33] Gao F, Dubos C. The Arabidopsis bHLH transcription factor family. Trends Plant Sci. 2023. https://doi.org/10.1016/j.tplants.2023.11.022

[34] Wang L, Tang W, Hu Y. et al. A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang. Plant J. 2019; 99:359-78

[35] Liu S, Liu X, Gou B et al. The interaction between CitMYB52 and CitbHLH2 negatively regulates citrate accumulation by activating CitALMT in citrus fruit. Front Plant Sci 2022; 13:848869

[36] Li Y, Xu P, Chen G. et al. FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis by forming a HY5-bHLH9 transcription complex in strawberry fruits. Plant Cell Physiol. 2020; 61:826-37

[37] Sidhu JS, Zafar TA. Bioactive compounds in banana fruits and their health benefits. Food Qual Saf. 2018; 2:183-8

[38] Song CB, Shan W, Kuang JF. et al. The basic helix-loop-helix transcription factor MabHLH7 positively regulates cell wall-modifying-related genes during banana fruit ripening. Postharvest Biol Technol. 2020; 161:111068

[39] Wang Z, Jia C, Wang JY. et al. Genome-wide analysis of basic helix-loop-helix transcription factors to elucidate candidate genes related to fruit ripening and stress in banana Musa acuminata L. AAA Group, cv. Cavendish. Front Plant Sci. 2020; 11:650

[40] Kuang JF, Wu CJ, Guo YF. et al. Deciphering transcriptional regulators of banana fruit ripening by regulatory network analysis. Plant Biotechnol J. 2021; 19:477-89

[41] O’Malley RC, Huang SC, Song L. et al. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell. 2016; 165:1280-92

[42] Machanick P, Bailey TL. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics. 2011; 27:1696-7

[43] Kojima K. Softening of banana fruit: relationship between firmness and chemical composition. Jpn Agr Res Q. 1996; 30:269-74

[44] Shi YN, Vrebalov J, Zheng H. et al. A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening. Proc Natl Acad Sci USA. 2021; 118:e2102486118

[45] Fu CC, Han YC, Qi XY. et al. Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes CpPME1/2 and CpPG5 involved in fruit ripening. Plant Cell Rep. 2016; 35:2341-52

[46] Liu H, Liu L, Liang D. et al. SlBES1 promotes tomato fruit softening through transcriptional inhibition of PMEU1. iScience. 2021; 24:102926

[47] He Y, Liu H, Li H. et al. Transcription factors DkBZR1/2 regulate cell wall degradation genes and ethylene biosynthesis genes during persimmon fruit ripening. J Exp Bot. 2021; 72:6437-46

[48] Cheng C, Liu J, Wang X. et al. PpERF/ABR1 functions as an activator to regulate PpPG expression resulting in fruit softening during storage in peach (Prunus persica). Postharvest Biol Technol. 2022; 189:111919

[49] Cai J, Mo X, Wen C. et al. FvMYB79 positively regulates strawberry fruit softening via transcriptional activation of FvPME38. Int J Mol Sci. 2022; 23:101

[50] Zhai Z, Xiao Y, Wang Y. et al. Abscisic acid-responsive transcription factors PavDof2/6/15 mediate fruit softening in sweet cherry. Plant Physiol. 2022; 190:2501-18

[51] Bailey PC, Martin C, Toledo-Ortiz G. et al. Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell. 2003; 15:2497-502

[52] Peng HH, Shan W, Kuang JF. et al. Molecular characterization of cold-responsive basic helix-loop-helix transcription factors MabHLHs that interact with MaICE1 in banana fruit. Planta. 2013; 238:937-53

[53] Zhou Z, Yang Y, Shan W. et al. Ethylene attenuates chilling injury of banana fruit via the MabHLH060/183 module in controlling phosphatidic acid formation genes. Postharvest Biol Technol. 2021; 183:111724

[54] Zhao ML, Wang JN, Shan W. et al. Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit. Plant Cell Environ. 2013; 36:30-51

[55] Radoeva T, Lokerse AS, Llavata-Peris CI. et al. A robust auxin response network controls embryo and suspensor development through a basic helix loop helix transcriptional module. Plant Cell. 2019; 31:52-67

[56] Menkens AE, Schindler U, Cashmore AR. The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins. Trends Biochem Sci. 1995; 20:506-10

[57] Yang C, Huang S, Zeng Y. et al. Two bHLH transcription factors, bHLH48 and bHLH60, associate with phytochrome interacting factor 7 to regulate hypocotyl elongation in Arabidopsis. Cell Rep. 2021; 35:109054

[58] de Martin X, Sodaei R, Santpere G. Mechanisms of binding specificity among bHLH transcription factors. Int J Mol Sci. 2021; 22:9150

[59] Torres-Machorro AL. Homodimeric and heterodimeric interactions among vertebrate basic helix-loop-helix transcription factors. Int J Mol Sci. 2021; 22:12855

[60] An JP, Li HH, Song LQ. et al. The molecular cloning and functional characterization of MdMYC2, a bHLH transcription factor in apple. Plant Physiol Biochem. 2016; 108:24-31

[61] Hichri I, Heppel SC, Pillet J. et al. The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Mol Plant. 2010; 3:509-23

[62] Yang YY, Shan W, Yang TW. et al. MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening. Plant J. 2022; 110:1651-69

[63] Yang Y, Wu C, Shan W. et al. Mitogen-activated protein kinase 14-mediated phosphorylation of MaMYB4 negatively regulates banana fruit ripening. Hortic Res. 2023;10:uhac243

[64] Chen L, Zhong HY, Kuang JF. et al. Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta. 2011; 234:377-90

[65] Wu CJ, Shan W, Liu XC. et al. Phosphorylation of transcription factor bZIP21 by MAP kinase MPK6-3 enhances banana fruit ripening. Plant Physiol. 2022; 188:1665-85

[66] Hellens R, Allan A, Friel E. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Meth. 2005; 1:13

[67] Shan W, Kuang JF, Wei W. et al. MaXB3 modulates MaNAC2, MaACS1, and MaACO1 stability to repress ethylene biosynthesis during banana fruit ripening. Plant Physiol. 2020; 184:1153-71

[68] Fan ZQ, Ba LJ, Shan W. et al. A banana R2R3-MYB transcription factor MaMYB3 is involved in fruit ripening through modulation of starch degradation by repressing starch degradation-related genes and MabHLH6. Plant J. 2018; 96:1191-205