Interaction among homeodomain transcription factors mediates ethylene biosynthesis during pear fruit ripening

Abstract

Fruit ripening is manipulated by the plant phytohormone ethylene in climacteric fruits. While the transcription factors (TFs) involved in ethylene biosynthesis and fruit ripening have been extensively studied in tomato, their identification in pear remains limited. In this study, we identified and characterized a HOMEODOMAIN TF, PbHB.G7.2, through transcriptome analysis. PbHB.G7.2 could directly bind to the promoter of the ethylene biosynthetic gene, 1-aminocyclopropane-1-carboxylic acid synthase (PbACS1b), thereby enhancing its activity and resulting in increased ethylene production during pear fruit ripening. Yeast-two-hybrid screening revealed that PbHB.G7.2 interacted with PbHB.G1 and PbHB.G2.1. Notably, these interactions disrupted the transcriptional activation of PbHB.G7.2. Interestingly, PbHB.G1 and PbHB.G2.1 also bind to the PbACS1b promoter, albeit different regions from those bound by PbHB.G7.2. Moreover, the regions of PbHB.G1 and PbHB.G2.1 involved in their interaction with PbHB.G7.2 differ from the regions responsible for binding to the PbACS1b promoter. Nonetheless, these interactions also disrupt the transcriptional activation of PbHB.G1 and PbHB.G2.1. These findings offer a new mechanism of ethylene biosynthesis during climacteric fruit ripening.

Acknowledgements

This work was supported by the Bioinformatics Center of Nanjing Agricultural University, the Jiangsu seed industry revitalization project (JBGS[2021]022), the Key Research and Development Program (Modern Agriculture) of Jiangsu Province (BE2022381), the Jiangsu Agricultural Science and Technology Innovation Fund (CX(20)2020), and the Earmarked Fund for China Agriculture Research System (CARS-28).

Author contributions

C.G., S.-L.Z., and S.-F.S. conceived this study; S.-H.C. and Z.-H.G. completed most experiments with help from H.L., G.-M.W., and R.-P.T.; K.-J.Q., Z.-W.W., and S.-F.S. conducted sample collection and data analysis; C.G. and S.-H.C. drafted this manuscript.

Data availability statement

The data for this article are available in the article or in its supplementary material.

Conflict of interests

The authors declare that they have no competing interests.

Supplementary information

Supplementary data is available at Horticulture Research online.

References

[1] Giovannoni JJ. Genetic regulation of fruit development and ripening. Plant Cell. 2004; 16:S170-80

[2] Goff SA, Klee HJ. Plant volatile compounds: sensory cues for health and nutritional value? Science. 2006; 311:815-9

[3] Klee HJ, Giovannoni JJ. Genetics and control of tomato fruit ripening and quality attributes. Annu Rev Genet. 2011; 45:41-59

[4] Liu Y, Tang M, Liu M. et al. The molecular regulation of ethylene in fruit ripening. Small methods. 2020; 4:1900485

[5] Pech JC, Purgatto E, Bouzayen M. et al. Ethylene and fruit ripening. Annual Plant Reviews. 2012; 44:275-304

[6] Fujisawa M, Nakano T, Shima Y. et al. A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening. Plant Cell. 2013; 25:371-86

[7] Gao Y, Wei W, Zhao X. et al. A NAC transcription factor, NOR-like1, is a new positive regulator of tomato fruit ripening. Hortic Res. 2018; 5:75

[8] Karlova R, Chapman N, David K. et al. Transcriptional control of fleshy fruit development and ripening. J Exp Bot. 2014; 65:4527-41

[9] Li T, Xu Y, Zhang L. et al. The jasmonate-activated transcription factor MdMYC2 regulates ETHYLENE RESPONSE FACTOR and ethylene biosynthetic genes to promote ethylene biosynthesis during apple fruit ripening. Plant Cell. 2017; 29:1316-34

[10] Yue P, Lu Q, Liu Z. et al. Auxin-activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening. New Phytol. 2020; 226:1781-95

[11] Li T, Jiang Z, Zhang L. et al. Apple (Malus domestica) MdERF2 negatively affects ethylene biosynthesis during fruit ripening by suppressing MdACS1 transcription. Plant J. 2016; 88:735-48

[12] Han YC, Kuang JF, Chen JY. et al. Banana transcription factor MaERF11 recruits histone deacetylase MaHDA1 and represses the expression of MaACO1 and expansins during fruit ripening. Plant Physiol. 2016; 171:00301.2016-1084

[13] Ariel FD, Manavella PA, Dezar CA. et al. The true story of the HD-Zip family. Trends Plant Sci. 2007; 12:419-26

[14] Mukherjee K, Brocchieri L, Burglin TR. A comprehensive classification and evolutionary analysis of plant homeobox genes. Mol Biol Evol. 2009; 26:2775-94

[15] Horst NA, Katz A, Pereman I. et al. A single homeobox gene triggers phase transition, embryogenesis and asexual reproduction. Nature Plants. 2016; 2:15209

[16] Khan M, Ragni L, Tabb P. et al. Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis. Plant Physiol. 2015; 169:2166-86

[17] Nakata M, Matsumoto N, Tsugeki R. et al. Roles of the middle domain-specific WUSCHEL-RELATED HOMEOBOX genes in early development of leaves in Arabidopsis. Plant Cell. 2012; 24:519-35

[18] Romera-Branchat M, Ripoll JJ, Yanofsky MF. et al. The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit. Plant J. 2013; 73:37-49

[19] Rosin FM, Hart JK, Horner HT. et al. Overexpression of a knotted-like homeobox gene of potato alters vegetative development by decreasing gibberellin accumulation. Plant Physiol. 2003; 132:106-17

[20] Sakamoto T, Sakakibara H, Kojima M. et al. Ectopic expression of KNOTTED1-like homeobox protein induces expression of cytokinin biosynthesis genes in rice. Plant Physiol. 2006; 142:54-62

[21] Johannesson H, Wang Y, Hanson J. et al. The Arabidopsis thaliana homeobox gene ATHB5 is a potential regulator of abscisic acid responsiveness in developing seedlings. Plant Mol Biol. 2003; 51:719-29

[22] Miao ZQ, Zhao PX, Mao JL. et al. HOMEOBOX PROTEIN52 mediates the crosstalk between ethylene and auxin signaling during primary root elongation by modulating auxin transport-related gene expression. Plant Cell. 2018; 30:2761-78

[23] Gu C, Guo ZH, Cheng HY. et al. A HD-ZIP II HOMEOBOX transcription factor, PpHB.G7, mediates ethylene biosynthesis during fruit ripening in peach. Plant Sci. 2019; 278:12-9

[24] Lin ZF, Hong YG, Yin MG. et al. A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening. Plant J. 2008; 55:301-10

[25] Wang G, Gao X, Wang X. et al. Transcriptome analysis reveals gene associated with fruit size during fruit development in pear. Scientia Horticuturae. 2022; 305:111367

[26] Ireland HS, Yao JL, Tomes S. et al. Apple SEPALLATA1/2-like genes control fruit flesh development and ripening. Plant J. 2013; 73:1044-56

[27] Xiao YY, Chen JY, Kuang JF. et al. Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes. J Exp Bot. 2013; 64:2499-510

[28] Wu C, Shan W, Liang S. et al. MaMPK2 enhances MabZIP93-mediated transcriptional activation of cell wall modifying genes during banana fruit ripening. Plant Mol Biol. 2019; 101:113-27

[29] Zhou H, Zhao L, Yang Q. et al. Identification of EIL and ERF genes related to fruit ripening in peach. Int J Mol Sci. 2020; 21:2846

[30] Wang WQ, Wang J, Wu YY. et al. Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164-NAC regulatory pathway for fruit ripening. New Phytol. 2020; 225:1618-34

[31] Yin XR, Allan AC, Chen KS. et al. Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiol. 2010; 153:1280-92

[32] Hao PP, Wang GM, Cheng HY. et al. Transcriptome analysis unravels an ethylene response factor involved in regulating fruit ripening in pear. Physiol Plant. 2018; 163:124-35

[33] Ji Y, Qu Y, Jiang Z. et al. The mechanism for brassinosteroids suppressing climacteric fruit ripening. Plant Physiol. 2021; 185:1875-93

[34] Li X, Guo W, Xu M. et al. PuWRKY31 affects ethylene production in response to sucrose signal in pear fruit. Hortic Res. 2022; 9:uhac156

[35] Guo ZH, Zhang YJ, Yao JL. et al. The NAM/ATAF1/2/CUC2 transcription factor PpNAC.A59 enhances PpERF.A16 expression to promote ethylene biosynthesis during peach fruit ripening. Hortic Res. 2021; 8:209

[36] Fu B, Wang W, Liu X. et al. An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening. New Phytol. 2021; 232:237-51

[37] Bellaoui M, Pidkowich MS, Samach A. et al. The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. Plant Cell. 2001; 13:2455-70

[38] Smith HM, Hake S. The interaction of two homeobox genes, BREVIPEDICELLUS and PENNYWISE, regulates internode patterning in the Arabidopsis inflorescence. Plant Cell. 2003; 15:1717-27

[39] Kim J, Harter K, Theologis A. Protein-protein interactions among the aux/IAA proteins. Proc Natl Acad Sci. 1997; 94:11786-91

[40] Xu C, Shen Y, He F. et al. Auxin-mediated aux/IAA-ARF-HB signaling cascade regulates secondary xylem development in Populus. New Phytol. 2019; 222:752-67

[41] Jeong JS, Park YT, Jung H. et al. Rice NAC proteins act as homodimers and heterodimers. Plant Biotechnol Rep. 2009; 3:127-34

[42] Yamaguchi M, Ohtani M, Mitsuda N. et al. VND-INTERACTING2, a NAC domain transcription factor, negatively regulates xylem vessel formation in Arabidopsis. Plant Cell. 2010; 22:1249-63

[43] de Folter S, Immink RG, Kieffer M. et al. Comprehensive interaction map of the Arabidopsis MADS Box transcription factors. Plant Cell. 2005; 17:1424-33

[44] Immink RG, Tonaco IA, de Folter S. et al. SEPALLATA3: the ’glue’ for MADS box transcription factor complex formation. Genome Biol. 2009; 10:R24

[45] Bu Q, Castillo A, Chen F. et al. Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis. Plant Mol Biol. 2011; 77:501-11

[46] Chi Y, Yang Y, Zhou Y. et al. Protein-protein interactions in the regulation of WRKY transcription factors. Mol Plant. 2013; 6:287-300

[47] Fan X, Argenta L, Mattheis JP. Inhibition of ethylene action by 1-methylcyclopropene prolongs storage life of apricots. Postharvest Biol Technol. 2000; 20:135-42

[48] Lin J, Sheng B, Li X. et al. A new Pyrus pyrifolia cultivar ‘Sucui 1’. Acta Horticulturae Sinica. 2013; 9:1849-50

[49] Bai S, Tao R, Yin L. et al. Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J. 2019; 100:1208-23

[50] Wang G, Guo L, Guo Z. et al. The involvement of Ein3-binding F-box protein PbrEBF 3 in regulating ethylene signaling during Cuiguan pear fruit ripening. Plant Sci. 2023; 329:111600