A transposable element-derived regulatory variation in RsOFP2.3 underlies morphological diversification of radish taproots through the conserved OVATE family protein-TONNEAU1-recruiting motif module

Abstract

Radish (Raphanus sativus L.) represents a major root vegetable crop exhibiting remarkable variation in taproot morphology, yet the underlying genetic and molecular mechanisms remain poorly understood. Through a structural variant (SV)-based genome-wide association study (GWAS), we identified RsOFP2.3 , encoding an OVATE Family Protein (OFP), as a major determinant of fleshy taproot shape. Comprehensive expression profiling and RNA in situ hybridization revealed that RsOFP2.3 is broadly expressed, with the highest transcript accumulation in cambial tissues. Notably, RsOFP2.3 expression was markedly higher in the round-rooted accession than in the long-rooted one. Population-wide analysis showed that RsOFP2.3 expression was negatively correlated with taproot length and shape index, but positively correlated with taproot width. A 312-bp transposable element (TE) insertion in the RsOFP2.3 promoter repressed its expression and was strongly associated with taproot shape variation, revealing a TE-mediated cis-regulatory mechanism underlying morphological divergence. Functional analyses showed that RsOFP2.3 overexpression (OE) promotes radial expansion by enhancing cambial cell division and xylem differentiation, resulting in thicker and shorter taproots, whereas silencing RsOFP2.3 produced opposite phenotypic effects. Mechanistically, RsOFP2.3 physically interacts with the TONNEAU1-recruiting motif (TRM) protein RsTRM4, recruiting it from microtubules to the cytoplasm. Downregulation of RsTRM4 reduced taproot length, while RsTRM4 OE partially alleviated the shortened organ phenotypes caused by RsOFP2.3 OE, indicating an antagonistic relationship that fine-tunes organ morphology. These findings uncover two coordinated regulatory mechanisms involving TE-mediated cis-regulatory variation and a conserved OFP-TRM interaction module that jointly shape the balance between radial and longitudinal growth during radish taproot development, providing valuable molecular targets for precision breeding of storage root crops.

References

[1] Goldman IL, Wang Y, Alfaro AV, et al. Form and contour: breeding and genetics of organ shape from wild relatives to modern vegetable crops. Front Plant Sci. 2023; 14: 1257707

[2] Che G, Pan Y, Liu X, et al. Natural variation in CRABS CLAW contributes to fruit length divergence in cucumber. Plant Cell. 2023; 35: 738-55

[3] Guan J, Xu Y, Yu Y, et al. Genome structure variation analyses of peach reveal population dynamics and a 1.67 Mb causal inversion for fruit shape. Genome Biol. 2021; 22: 13

[4] Mao L, Shen Y, Cui Q, et al. The IQ67-domain protein IQD1 regulates fruit shape through complex multiprotein interactions in pepper (Capsicum annuum L.) . Plant Biotechnol J. 2025; 23: 2651-66

[5] Song W, Xie Y, Liu B, et al. Single nucleotide polymorphisms in SEPALLATA2 underlie fruit length variation in cucurbits. Plant Cell. 2024; 36: 4607-21

[6] Tsaballa A, Pasentsis K, Darzentas N, et al. Multiple evidence for the role of an ovate-like gene in determining fruit shape in pepper. BMC Plant Biol. 2011; 11: 46

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

[8] Zhang B, Li Q, Keyhaninejad N, et al. A combinatorial TRM-OFP module bilaterally fine-tunes tomato fruit shape. New Phytol. 2023; 238: 2393-409

[9] Zhou H, Ma R, Gao L, et al. A 1.7-Mb chromosomal inversion downstream of a PpOFP1 gene is responsible for flat fruit shape in peach. Plant Biotechnol J. 2021; 19: 192-205

[10] Shi H, Li P, Yun P, et al. A DOF transcription factor GLW9/OsDOF25 regulates grain shape and tiller angle in rice. Plant Biotechnol J. 2025; 23: 2367-82

[11] 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

[12] Wang W, Pan Q, Tian B, et al. Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat. Plant J. 2019; 100: 251-64

[13] Xiao Y, Liu D, Zhang G, et al. Brassinosteroids regulate OFP1, a DLT-interacting protein, to modulate plant architecture and grain morphology in rice. Front Plant Sci. 2017; 8: 1698

[14] Zhang L, Fu M, Li W, et al. Genetic variation in ZmKW1 contributes to kernel weight and size in dent corn and popcorn. Plant Biotechnol J. 2024; 22: 1453-67

[15] Brainard SH, Ellison SL, Simon PW, et al. Genetic characterization of carrot root shape and size using genome-wide association analysis and genomic-estimated breeding values. Theor Appl Genet. 2022; 135: 605-22

[16] Dong J, Wang Y, Xu L, et al. RsLBD3 regulates the secondary growth of taproot by integrating auxin and cytokinin signaling in radish (Raphanus sativus L.) . J Integr Plant Biol. 2025; 67: 1823-42

[17] Ju A, Wang Y, Yan Y, et al. StOFP20 regulates tuber shape and interacts with TONNEAU1 recruiting motif proteins in potato. J Integr Agric. 2023; 22: 752-61

[18] Vega A, Oravec M, Goldman IL . Phenotypic, genetic and population structure analyses offer insights into the genetic architecture of root shape in Beta vulgaris. Hortic Res. 2025; 12: uhaf201

[19] Wang Y, Wang Q, Hao W, et al. Characterization of the OFP gene family and its putative involvement of tuberous root shape in radish. Int J Mol Sci. 2020; 21: 1293

[20] Wei Q, Wang J, Wang W, et al. Identification of QTLs controlling radish root shape using multiple populations. Horticulturae. 2022; 8: 931

[21] Fernie AR, Willmitzer L . Molecular and biochemical triggers of potato tuber development. Plant Physiol. 2001; 127: 1459-65

[22] Mitsui Y, Shimomura M, Komatsu K, et al. The radish genome and comprehensive gene expression profile of tuberous root formation and development. Sci Rep. 2015; 5: 10835

[23] Vanderschuren H, Agusti J . Storage roots. Curr Biol. 2022; 32: R607-9

[24] Wang Q, Fan J, Cong J, et al. Natural variation of ZmLNG1 alters organ shapes in maize. New Phytol. 2023; 237: 471-82

[25] Drevensek S, Goussot M, Duroc Y, et al. The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes. Plant Cell. 2012; 24: 178-91

[26] Spinner L, Gadeyne A, Belcram K, et al. A protein phosphatase 2A complex spatially controls plant cell division. Nat Commun. 2013; 4: 1863

[27] Lee YK, Kim GT, Kim IJ, et al. LONGIFOLIA1 and LONGIFOLIA2, two homologous genes, regulate longitudinal cell elongation in Arabidopsis. Development. 2006; 133: 4305-14

[28] Xie Y, Liu X, Sun C, et al. CsTRM5 regulates fruit shape via mediating cell division direction and cell expansion in cucumber. Hortic Res. 2023; 10: uhad007

[29] Liu D, Sun W, Yuan Y, et al. Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants. Ann Bot. 2014; 113: 1219-33

[30] Wang Z, Zhou Z, Wang L, et al. The CsHEC1-CsOVATE module contributes to fruit neck length variation via modulating auxin biosynthesis in cucumber. Proc Natl Acad Sci USA. 2022; 119: e2209717119

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

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

[33] Zhou Y, Zhang Z, Bao Z, et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature. 2022; 606: 527-34

[34] Jin S, Han Z, Hu Y, et al. Structural variation (SV)-based pangenome and GWAS reveal the impacts of SVs on the speciation and diversification of allotetraploid cottons. Mol Plant. 2023; 16: 678-93

[35] Gebrie A . Transposable elements as essential elements in the control of gene expression. Mob DNA. 2023; 14: 9

[36] Wang Y, Wang Q, Wu X, et al. A NAC transcription factor RsSND1 regulating secondary cell wall deposition involves in fleshy taproot formation in radish (Raphanus sativus L.) . J Plant Growth Regul. 2024; 43: 1844-57

[37] Kojima KK . Non-LTR retrotransposons from the radish genome. Repbase Rep. 2024; 24: 3460

[38] 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 USA. 2002; 99: 13302-6

[39] Wu Q, Sun J, Fu J, et al. Genome-wide identification of ovate family proteins in citrus and functional characterization of CitOFP19. Plant Sci. 2022; 321: 111328

[40] Wang S, Chang Y, Guo J, et al. Arabidopsis ovate family protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J. 2007; 50: 858-72

[41] Wang ZQ, Shan QY, Xu Q, et al. OVATE family protein 20 regulates fruit shoulder width by modulating auxin distribution in pepper. Plant Physiol. 2025; 199: kiaf404

[42] Schmitz AJ, Begcy K, Sarath G, et al. Rice ovate family protein 2 alters hormonal homeostasis and vasculature development. Plant Sci. 2015; 241: 177-88

[43] Alonge M, Wang X, Benoit M, et al. Major impacts of widespread structural variation on gene expression and crop improvement in tomato. Cell. 2020; 182: 145-161.e23

[44] Catlin NS, Josephs EB . The important contribution of transposable elements to phenotypic variation and evolution. Curr Opin Plant Biol. 2022; 65: 102140

[45] Borovsky Y, Raz A, Doron-Faigenboim A, et al. Pepper fruit elongation is controlled by Capsicum annuum ovate family protein 20 . Front Plant Sci. 2022; 12: 815589

[46] Xiao H, Jiang N, Schaffner E, et al. A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science. 2008; 319: 1527-30

[47] Studer A, Zhao Q, Ross-Ibarra J, et al. Identification of a functional transposon insertion in the maize domestication gene tb1. Nat Genet. 2011; 43: 1160-3

[48] Li Q, Luo S, Zhang L, et al. Molecular and genetic regulations of fleshy fruit shape and lessons from Arabidopsis and rice. Hortic Res. 2023; 10: uhad108

[49] Garrison E, Sirén J, Novak A, et al. Variation graph toolkit improves read mapping by representing genetic variation in the reference. Nat Biotechnol. 2018; 36: 875-9

[50] Wang J, Zhang Z . GAPIT version 3: boosting power and accuracy for genomic association and prediction. Genomics Proteomics Bioinformatics. 2021; 19: 629-40

[51] Xin X, Li P, Zhao X, et al. Temperature-dependent jumonji demethylase modulates flowering time by targeting H3K36me2/3 in Brassica rapa . Nat Commun. 2024; 15: 5470

[52] Zhang X, Henriques R, Lin SS, et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method . Nat Protoc. 2006; 1: 641-6

[53] Li P, Xin H, Li J, et al. The complex REDUCED COMPLEXITY locus contributes to leaf shape diversification in Brassica rapa . J Exp Bot. 2025; 76: 6853-67

[54] Tamura K, Stecher G, Kumar S . MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021; 38: 3022-7

[55] Bailey TL, Elkan C . Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol. 1994; 2: 28-36

[56] Li M, Li X, He Y, et al. The microtubule-associated protein CsTON2 interacts with CsTRM5 and CsSUN to regulate fruit shape development in cucumber. Plant Biotechnol J. 2025; 24: 2725-41

[57] Waadt R, Schmidt LK, Lohse M, et al. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J. 2008; 56: 505-16