OVATE family gene CmOFP6-19b negatively regulates fruit size in melon (Cucumis melo L.)

Abstract

OVATE family proteins (OFPs) constitute a class of transcription factors regulating various developmental processes in plants. Nevertheless, their precise regulatory functions in melon (Cucumis melo L.) fruit development remain elusive. In this study, we identified expression profiling of melon OFP genes and revealed the molecular function of CmOFP6-19b gene mediating fruit size variation. Quantitative analysis revealed predominant CmOFP expression in reproductive organs (female/male flowers and ovaries), with distinct differential expression patterns observed among paralogs. Through melon genetic transformation, we revealed that CmOFP6-19b gene functions as a negative regulator in fruit enlargement. Overexpression of the CmOFP6-19b gene resulted in reduced fruit size, while its downregulation led to increased fruit size. Bimolecular fluorescence complementation and yeast two-hybrid assays confirmed nuclear-localized physical interaction between CmOFP6-19b and CmKNOX16. Overexpression of CmKNOX16 in melon produced smaller fruits, phenocopying the CmOFP6-19b-Oe lines. Quantitative real-time PCR (RT-qPCR) analysis showed negative correlation between CmOFP6-19b/CmKNOX16 expression level and fruit size, with peak expression levels observed in a cultivar displaying minimal longitudinal diameter. The results of histological section and expression analysis suggest that CmOFP6-19b and CmKNOX16 may affect melon fruit size by regulating genes related to cell division and cell expansion. In conclusion, our findings systematically characterized the phylogenetic architecture and expression divergence of CmOFP genes, and elucidated the function and molecular mechanism of CmOFP6-19b-CmKNOX16 regulatory module in mediating melon fruit development, providing a theoretical foundation for melon breeding.

Acknowledgements

This work was supported by the Applied Technology Research and Development Foundation of Inner Mongolia Autonomous Region, China (2021PT0001), the National Natural Science Foundation of China (32460769, 32202513), Inner Mongolia Autonomous Region universities ‘Young Science and Technology Talent Support Project’ (NJYT24067), and the Inner Mongolia Autonomous Region Department of Education First-class Scientific Research Project, China (YLXKZX-ND-030).

Author contributions

Agula Hasi and Gen Che designed the project. Junling Chi wrote this paper, performed the experiments, and prepared the figures. Haimei Yan and Wenjing Zhang analyzed the data. Dingfang Tian contributed plant materials and mapping tools. All authors have read the drafts of this paper.

Data availability

All experimental data are available in the main text and supplementary data. RNA-Seq data in this study can be found at the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra); the BioProject accession numbers are PRJNA543288 and PRJNA803327.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Adiguzel P, Nyirahabimana F, Shimira F. et al. Applied biotechno-logical approaches for reducing yield gap in melon grown under saline and drought stresses: an overview. J Soil Sci Plant Nutr. 2023; 23:139-51

[2] Yu X, Zhang J, Zhang X. et al. Identification and pathogenicity of fungi associated with leaf spot of muskmelon in eastern Shandong Province, China. Plant Dis. 2022; 106:872-90

[3] Tomason Y, Nimmakayala P, Reddy UK. Map based phylogenies, linkage disequilibrium and association mapping for fruit traits in melon. Mol Breeding. 2013; 31:829-41

[4] Pereira L, Ruggieri V, Pérez S. et al. QTL mapping of melon fruit quality traits using a high-density GBS-based genetic map. BMC Plant Biol. 2018; 18:324

[5] Pan Y, Wang Y, McGregor C. et al. Genetic architecture of fruit size and shape variation in cucurbits: a comparative perspective. Theor Appl Genet. 2020; 133:1-21

[6] Ma J, Li C, Zong M. et al. CmFSI8/CmOFP13 encoding an OVATE family protein controls fruit shape in melon. JExp Bot. 2022; 73: 1370-84

[7] Zhang S, Tan FQ, Chung CH. et al. The control of carpel determi-nacy pathway leads to sex determination in cucurbits. Science. 2022; 378:543-9

[8] Boualem A, Berthet S, Devani RS. et al. Ethylene plays a dual role in sex determination and fruit shape in cucurbits. Curr Biol. 2022; 32:2390-2401.e4

[9] Wu H, Jia Y, Chen X. et al. Novel allelic gene variations in CmCLAVATA 3 (CmCLV3) were identified in a genetic population of melon (Cucumis melo L.). Int J Mol Sci. 2024; 25:6011

[10] Wang L, Wang Y, Luan F. et al. Biparental genetic mapping reveals that CmCLAVATA 3 (CmCLV3) is responsible for the vari-ation in carpel number in melon (Cucumis melo L.). Theor Appl Genet. 2022; 135:1909-21

[11] Switzenberg JA, Beaudry RM, Grumet R. Effect of CRC::etr1-1 transgene expression on ethylene production, sex expression, fruit set and fruit ripening in transgenic melon (Cucumis melo L.). Transgenic Res. 2015; 24:497-507

[12] Ma M, Liu S, Wang Z. et al. Genome-wide identification of the SUN gene family in melon (Cucumis melo) and functional characteri-zation of two CmSUN genes in regulating fruit shape variation. Int J Mol Sci. 2022; 23:16047

[13] Wu S, Zhang B, Keyhaninejad N. et al. A common genetic mecha-nism underlies morphological diversity in fruits and other plant organs. Nat Commun. 2018; 9:4734

[14] Snouffer A, van der Kraus C, Knaap E. The shape of things to come: ovate family proteins regulate plant organ shape. Curr Opin Plant Biol. 2020; 53:98-105

[15] Monforte AJ, Diaz A, Caño-Delgado A. et al. The genetic basis of fruit morphology in horticultural crops: lessons from tomato and melon. JExp Bot. 2014; 65:4625-37

[16] Liu J, Van Eck J, Cong B. et al. A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc Natl Acad Sci U S A. 2002; 99:13302-6

[17] Wang S, Chang Y, Ellis B. Overview of OVATE FAMILY PROTEINS, a novel class of plant-specific growth regulators. Front Plant Sci. 2016; 7:417

[18] Wang S, Chang Y, Guo J. et al. Arabidopsis ovate family proteins, a novel transcriptional repressor family, control multiple aspects of plant growth and development. PLoS One. 2011; 6:e23896

[19] Rodriguez GR, Muños S, Anderson C. et al. Distribution of SUN, OVATE, LC, and FAS in the tomato germplasm and the relation-ship to fruit shape diversity. Plant Physiol. 2011; 156:275-85

[20] Han L-j, Song X-f, Wang Z-y. et al. Genome-wide analysis of OVATE family proteins in cucumber (Cucumis sativus L.). J Integr Agric. 2022; 21:1321-31

[21] Dong Y, Huang L, Liu J. et al. Genome-wide identified VvOFP genes family and VvOFP4 functional characterization provide insight into fruit shape in grape. Int J Biol Macromol. 2024; 276: 133880

[22] Wu Q, Xia R, Yang J. et al. Identification and comprehensive analysis of OFP genes for fruit shape influence in mango. Genes (Basel). 2024; 15:823

[23] Xu X, Wang X, Zhou S. et al. Genome-wide identification and characterization of the OFP gene family in the wild strawberry Fragaria vesca. Agronomy. 2024; 14:569

[24] He C, Luo C, Yan J. et al. Genome-wide identification of the OVATE family proteins and functional analysis of BhiOFP1, BhiOFP5, and BhiOFP18 during fruit development in wax gourd (Benincasa hispida). Plant Physiol Biochem. 2024; 216:109135

[25] ZhouS, HuZ, LiF. et al. Overexpression of SlOFP20 affects floral organ and pollen development. Hortic Res. 2019; 6:125

[26] Wang Z, Zhou Z, Wang L. et al. The CsHEC1-CsOVATE mod-ule contributes to fruit neck length variation via modulat-ing auxin biosynthesis in cucumber. Proc Natl Acad Sci U S A. 2022; 119:e2209717119

[27] Yang C, Ma Y, He Y. et al. OsOFP19 modulates plant architecture by integrating the cell division pattern and brassinosteroid sig-naling. Plant J. 2018; 93:489-501

[28] Carolina PG, Hee-Ju Y, Venkatesan S. Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1. Plant Cell. 2007; 11:3578-92

[29] Zhao H, Zhang T, Meng X. et al. Genetic mapping and QTL analysis of fruit traits in melon (Cucumis melo L.). Curr Issues Mol Biol. 2023; 45:3419-33

[30] WuQ, SunJ, FuJ. et al. Genome-wide identification of ovate family in citrus and functional characterization of CitOFP19. Plant Sci. 2022; 321:111328

[31] Liu H, Lyu HM, Zhu K. et al. The emergence and evolution of intron-poor and intronless genes in intron-rich plant gene families. Plant J. 2021; 105:1072-82

[32] Parra G, Bradnam K, Rose AB. et al. Comparative and functional analysis of intron-mediated enhancement signals reveals con-served features among plants. Nucleic Acids Res. 2011; 39:5328-37

[33] Dongyuan L, Chouxian M, Weiguo H. et al. Construction and analysis of high-density linkage map using high-throughput sequencing data. PLoS One. 2014; 9:e98855

[34] Sun L, Rodriguez GR, Clevenger JP. et al. Candidate gene selection and detailed morphological evaluations of fs8.1, a quantitative trait locus controlling tomato fruit shape. JExp Bot. 2015; 66: 6471-82

[35] Wang Y, Clevenger JP, Illa-Berenguer E. et al. A compari-son of sun, ovate, fs8.1 and auxin application on tomato fruit shape and gene expression. Plant Cell Physiol. 2019; 60: 1067-81

[36] Colle M, Weng Y, Kang Y. et al. Variation in cucumber (Cucumis sativus L.) fruit size and shape results from multiple compo-nents acting pre-anthesis and post-pollination. Planta. 2017; 246: 641-58

[37] Dou J, Zhao S, Lu X. et al. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.). Theor Appl Genet. 2018; 131:947-58

[38] Harada T, Kurahashi W, Yanai M. et al. Involvement of cell proliferation and cell enlargement in increasing the fruit size of Malus species. Sci Hortic. 2005; 105:447-56

[39] Azzi L, Deluche C, Gévaudant F. et al. Fruit growth-related genes in tomato. JExp Bot. 2015; 66:1075-86

[40] Inzé D, Veylder LD. Cell cycle regulation in plant development. Annu Rev Genet. 2006; 40:77-105

[41] Marowa P, Ding A, Kong Y. Expansins: roles in plant growth and potential applications in crop improvement. Plant Cell Rep. 2016; 35:949-65

[42] Bernal-Gallardo JJ, González-Aguilera KL, Folter SD. EXPANSIN15 is involved in flower and fruit development in Arabidopsis. Plant Reprod. 2024; 37:259-70

[43] Van Sandt VS, Suslov D, Verbelen JP. et al. Xyloglucan endotrans-glucosylase activity loosens a plant cell wall. Ann Bot. 2007; 100: 1467-73

[44] Shin YK, Yum H, Kim ES. et al. BcXTH1, a Brassica campestris homologue of Arabidopsis XTH9, is associated with cell expan-sion. Planta. 2006; 224:32-41

[45] Schmitz AJ, Begcy K, Sarath G. et al. Rice Ovate Family Protein 2 (OFP2) alters hormonal homeostasis and vasculature develop-ment. Plant Sci. 2015; 241:177-88

[46] Li E, Wang S, Liu Y. et al. OVATE FAMILY PROTEIN4 (OFP4) interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana. Plant J. 2011; 67:328-41

[47] GaoJ, YangX, ZhaoW. et al. Evolution, diversification, and expression of KNOX proteins in plants. Front Plant Sci. 2015; 6:882

[48] Shtern A, Keren-Keiserman A, Mauxion JP. et al. Solanum lycopersicum CLASS-II KNOX genes regulate fruit anatomy via gibberellin-dependent and independent pathways. JExp Bot. 2023; 74:848-63

[49] Sheng M, Ma X, Wang J. et al. KNOX II transcription factor HOS59 functions in regulating rice grain size. Plant J. 2022; 110:863-80

[50] Hao J, Niu Y, Yang B. et al. Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway. Biotechnol Lett. 2011; 33:55-61

[51] Zeng J, Wu Y, Wang D. et al. Genetic expression in progeny of transgenic plants obtained by using pollen-tube pathway (or delivery) method and approach to the transformation mecha-nism. Chin Sci Bull. 1998; 43:798-803

[52] Zhou G, Weng J, Zeng Y. et al. Introduction of exogenous DNA into cotton embryos. Methods Enzymol. 1983; 101:433-81