The GAs-RhMYB70 feedback loop fine-tunes cell expansion and petal size by modulating cellulose content in rose

Abstract

Cell expansion in petals plays a crucial role in flower opening and final size in rose (Rosa hybrida), which largely determines its market value. While cell expansion is known to be closely associated with gibberellins (GAs), the underlying molecular mechanism remains elusive. Here, we measured the levels of GAs during flower opening and demonstrated that GA₃ treatment significantly increases petal size. Moreover, we identified RhMYB70, an R2R3 MYB transcription factor, whose expression was inhibited by GA₃ treatment. RhMYB70 silencing resulted in larger petals and petal cell size than those of TRV control. Through transcriptome analysis and biochemical identification, RhMYB70 could directly bind to the promoter of the cellulose synthase gene RhCESA8 and repress its transcription, thereby resulting in decreased cellulose content of petals and final size. In addition, we also identified the GA biosynthesis gene RhGA3ox3 as an RhMYB70 target and demonstrated that RhMYB70 directly binds to and inhibits the promoter activity of RhGA3ox3, leading to decreased cellulose content of petals and petal size. Besides, knocking down RhMYB70 expression not only resulted in increasing GA₁ and GA₃ levels in petals compared to TRV but also elevated cellulose content. Together, our findings reveal that the feedback regulation of GAs and RhMYB70 signaling fine-tunes cell expansion and petal size by modulating cellulose content of rose petals, providing genetic targets for improving rose flower quality.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant number 32202530), Talent Introduction and Training Project of Yunnan Academy of Agricultural Sciences (Grant number 2024RCYP-09), Yunnan Province Agricultural Joint Key Project (Grant number 202401BD070001-016), and Fundamental Research Project (Grant number 202401CF070046), and Xingdian Talent Support Program (Grant number XDYC-QNRC-2023-0457).

Author contributions

W.J. designed the research plans, F.G. X.W., and Q.Z. performed the experiments. J.W., Q.W., D.W., X.Q., H.J., Y.Z., and H.W. provided technical support. Q.W., D.W., and H.J. provided conceptual advice. W.J., F.G., and Q.Z. performed data analysis. W.J. and F.G. wrote the manuscript.

Data availability

The transcriptome data were deposited in the NCBI Sequence Read Archive database under accession numbers PRJNA1162946. All data supporting the findings of this study are available within the paper and within its supplementary data published online.

Conflict of interest statement

The authors declare no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Ma N, Xue J, Li Y. et al. Rh-PIP2;1, a rose aquaporin gene, is involved in ethylene-regulated petal expansion. Plant Physiol. 2008; 148:894-907

[2] Pei H, Ma N, Tian J. et al. An NAC transcription factor controls ethylene-regulated cell expansion in flower petals. Plant Physiol. 2013; 163:775-91

[3] Cosgrove DJ. Structure and growth of plant cell walls. Nat Rev Mol Cell Biol. 2024; 25:340-58

[4] McFarlane HE, Döring A, Persson S. The cell biology of cellulose synthesis. Annu Rev Plant Biol. 2014; 65:69-94

[5] Chen S, Ehrhardt DW, Somerville CR. Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase. Proc Natl Acad Sci USA. 2010; 107:17188-93

[6] Doi RH, Kosugi A. Cellulosomes: plant-cell-wall-degrading enzyme complexes. Nat Rev Microbiol. 2004; 2:541-551

[7] Zhang Y, Yu J, Wang X. et al. Molecular insights into the com-plex mechanics of plant epidermal cell walls. Science. 2021; 372: 706-11

[8] Taylor NG, Laurie S, Turner SR. Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Ara-bidopsis. Plant Cell. 2000; 12:2529-39

[9] Beeckman T, Przemeck GKH, Stamatiou G. et al. Genetic com-plexity of cellulose synthase A gene function in Arabidopsis embryogenesis. Plant Physiol. 2002; 130:1883-93

[10] Pedersen GB, Blaschek L, Frandsen KEH. et al. Cellulose synthesis in land plants. Mol Plant. 2023; 16:206-31

[11] Yang W, Cai Y, Hu L. et al. PhCESA3 silencing inhibits elongation and stimulates radial expansion in petunia. Sci Rep. 2017; 7:41471

[12] Fujioka S, Yamane H, Spray CR. et al. The dominant non-gibberellin-responding dwarf mutant (D8) of maize accumulates native gibberellins (GC-MS/internal standards/quantifica-tion/Zea mays). Proc Natl Acad Sci USA. 1988; 85:9031-5

[13] Yamaguchi S. Gibberellin metabolism and its regulation. Annu Rev Plant Biol. 2008; 59:225-51

[14] Pimenta Lange MJ, Lange T. Gibberellin biosynthesis and the regulation of plant development. Plant Biol. 2006; 8:281-90

[15] Davière JM, Achard P. Gibberellin signaling in plants. Develop-ment. 2013; 140:1147-51

[16] He J, Xin P, Ma X. et al. Gibberellin metabolism in flowering plants: an update and perspectives. Front Plant Sci. 2020; 11: 532

[17] Olszewski N, Sun TP, Gubler F. Gibberellin signaling: biosyn-thesis, catabolism, and response pathways. Plant Cell. 2002; 14: S61-80

[18] Otani M, Meguro S, Gondaira H. et al. Overexpression of the gibberellin 2-oxidase gene from Torenia fournieri induces dwarf phenotypes in the liliaceous monocotyledon Tricyrtis sp. JPlant Physiol. 2013; 170:1416-23

[19] Yang Y, Zhang L, Chen P. et al. UV-B photoreceptor UVR8 inter-acts with MYB73/MYB77 to regulate auxin responses and lateral root development. EMBO J. 2020; 39:e101928

[20] LuoJ, MaN, Pei H. et al. A della gene, RhGAI1, is a direct target of EIN3 and mediates ethylene-regulated rose petal cell expansion via repressing the expression of RhCesA2. JExp Bot. 2013; 64: 5075-84

[21] Chen C, Hussain N, Wang Y. et al. An ethylene-inhibited NF-YC transcription factor RhNF-YC9 regulates petal expansion in rose. Hortic Plant J. 2020; 6:419-27

[22] Ren G, Li L, Huang Y. et al. GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxin. New Phytol. 2018; 219:728-42

[23] Dubos C, Stracke R, Grotewold E. et al. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010; 15:573-81

[24] Zhang Y, Liang W, Shi J. et al. MYB56 encoding a R2R3 MYB transcription factor regulates seed size in Arabidopsis thaliana. J Integr Plant Biol. 2013; 55:1166-78

[25] Wu P, Peng M, Li Z. et al. DRMY1, a Myb-like protein, regulates cell expansion and seed production in Arabidopsis thaliana. Plant Cell Physiol. 2019; 60:285-302

[26] Sun X, Qin M, Yu Q. et al. Molecular understanding of postharvest flower opening and senescence. Mol Hortic. 2021; 1:7

[27] Jung C, Seo JS, Han SW. et al. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance intransgenic Arabidopsis. Plant Physiol. 2008; 146:623-35

[28] Cao H, Chen J, Yue M. et al. Tomato transcriptional repressor MYB70 directly regulates ethylene-dependent fruit ripening. Plant J. 2020; 104:1568-81

[29] Wan J, Wang R, Zhang P. et al. MYB70 modulates seed ger-mination and root system development in Arabidopsis. iScience. 2021; 24:103228

[30] Jing W, Gong F, Liu G. et al. Petal size is controlled by the MYB73/TPL/HDA19-miR159-CKX6 module regulating cytokinin catabolism in Rosa hybrida. Nat Commun. 2023; 14:7106

[31] Chen C, Hussain N, Ma Y. et al. The ARF2-MYB6 module mediates auxin-regulated petal expansion in rose (Rosa hybrida). JExp Bot. 2023; 74:4489-502

[32] Liu X, Wu J, Ji F. et al. Transcriptomic profiling of rose flower under treatment of various phytohormones and plant growth regulators. Sci Data. 2022; 9:669

[33] Han Y, Yu J, Zhao T. et al. Dissecting the genome-wide evolution and function of R2R3-MYB transcription factor family in Rosa chinensis. Genes (Basel). 2019; 10:823

[34] Salazar-Cerezo S, Martínez-Montiel N, García-Sánchez J. et al. Gibberellin biosynthesis and metabolism: a convergent route for plants, fungi and bacteria. Microbiol Res. 2018; 208: 85-98

[35] Xie Y, Chen L. Epigenetic regulation of gibberellin metabolism and signaling. Plant Cell Physiol. 2020; 61:1912-8

[36] Ueguchi-Tanaka M. Gibberellin metabolism and signaling. Biosci Biotechnol Biochem. 2023; 87:1093-101

[37] CaoY, LiK, LiY. et al. MYB transcription factors as reg-ulators of secondary metabolism in plants. Biology (Basel). 2020;9:

[38] Naing AH, Kim CK. Roles of R2R3-MYB transcription factors in transcriptional regulation of anthocyanin biosynthesis in horti-cultural plants. Plant Mol Biol. 2018; 98:1-18

[39] Zhang S, Zhao Q, Zeng D. et al. RhMYB108, an R2R3-MYB tran-scription factor, is involved in ethylene-and JA-induced petal senescence in rose plants. Hortic Res. 2019;6:

[40] He G, Zhang R, Jiang S. et al. The MYB transcription factor RcMYB1 plays a central role in rose anthocyanin biosynthesis. Hortic Res. 2023; 10:10

[41] Li K, Li Y, Wang Y. et al. Disruption of transcription fac-tor RhMYB123 causes the transformation of stamen to mal-formed petal in rose (Rosa hybrida). Plant Cell Rep. 2022; 41: 2293-303

[42] Yang T, Wang Y, Li Y. et al. The transcription factor RhMYB17 regulates the homeotic transformation of floral organs in rose (Rosa hybrida) under cold stress. JExp Bo.t 2024; 75: 2965-81

[43] Kelemen Z, Sebastian A, Xu W. et al. Analysis of the DNA-binding activities of the Arabidopsis R2R3-MYB transcription factor family by one-hybrid experiments in yeast. PLoS One. 2015; 10:e0141044

[44] Millard PS, Kragelund BB, Burow M. R2R3 MYB transcription factors-functions outside the DNA-binding domain. Trends Plant Sci. 2019; 24:934-46

[45] Ohtani M, Demura T. The quest for transcriptional hubs of lignin biosynthesis: beyond the NAC-MYB-gene regulatory network model. Curr Opin Biotechnol. 2019; 56:82-7

[46] Kim WC, Kim JY, Ko JH. et al. Transcription factor MYB46 is an obligate component of the transcriptional regulatory complex for functional expression of secondary wall-associated cellulose synthases in Arabidopsis thaliana. Plant Physiol. 2013; 170:1374-8

[47] Kim WC, Ko JH, Kim JY. et al. MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis. Plant J. 2013; 73:26-36

[48] Mitsuda N, Seki M, Shinozaki K. et al. The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell. 2005; 17:2993-3006

[49] Zhong R, Demura T, Ye ZH. SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell. 2006; 18:3158-70

[50] Zhong R, Lee C, Ye ZH. Functional characterization of poplar wood-associated NAC domain transcription factors. Plant Physiol. 2010; 152:1044-55

[51] Li A, Xia T, Xu W. et al. An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense. Planta. 2013; 237: 1585-97

[52] Cosgrove DJ. Growth of the plant cell wall. Nat Rev Mol Cell Biol. 2005; 6:850-61

[53] Huang D, Wang S, Zhang B. et al. A gibberellin-mediated DELLA-NAC signaling cascade regulates cellulose synthesis in rice. Plant Cell. 2015; 27:1681-96

[54] Teshome S, Kebede M. Analysis of regulatory elements in GA2ox, GA3ox and GA20ox gene families in Arabidopsis thaliana: an important trait. Biotechnol Biotechnol Equip. 2021; 35: 1603-12

[55] Woodger FJ, Millar A, Murray F. et al. The role of GAMYB tran-scription factors in GA-regulated gene expression. J Plant Growth Regul. 2003; 22:176-84

[56] Zhong R, Richardson EA, Ye ZH. The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosyn-thesis in Arabidopsis. Plant Cell. 2007; 19:2776-92

[57] Noda S, Koshiba T, Hattori T. et al. The expression of a rice secondary wall-specific cellulose synthase gene, OsCesA7, is directly regulated by a rice transcription factor, OsMYB58/63. Planta. 2015; 242:589-600

[58] Yamada K, Norikoshi R, Suzuki K. et al. Cell division and expan-sion growth during rose petal development. J Jpn Soc Hortic Sci. 2009; 78:356-62

[59] Jin W, Gong F, Zhang Y. et al. Cytokinin-responsive RhRR1-RhSCL28 transcription factor module positively regulates petal size by promoting cell division in rose. JExp Bot. 2025; 76:381-92

[60] Gong F, Jing W, Jin W. et al. RhMYC2 controls petal size through synergistic regulation of jasmonic acid and cytokinin signaling in rose. Plant J. 2024; 120:459-72

[61] Tian J, Pei H, Zhang S. et al. TRV-GFP: a modified tobacco rattle virus vector for efficient and visualizable analysis of gene func-tion. JExp Bot. 2014; 65:311-22

[62] Jiang C, Liang Y, Deng S. et al. The RhLOL1-RhILR3 module mediates cytokinin-induced petal abscission in rose. New Phytol. 2023; 237:483-96

[63] Lu P, Zhang C, Liu J. et al. RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida)petal senescence. Plant J. 2014; 78:578-90