VaMIEL1-mediated ubiquitination of VaMYB4a orchestrates cold tolerance through integrated transcriptional and oxidative stress pathways in grapevine

Abstract

Cold stress poses a significant threat to viticulture, particularly under the increasing pressures of climate change. In this study, we identified VaMIEL1, a RING-type E3 ubiquitin ligase from Vitis amurensis, as a negative regulator of cold tolerance. Under normal temperature conditions, VaMIEL1 facilitates the ubiquitination and subsequent proteasomal degradation of the cold-responsive transcription factor VaMYB4a, thereby attenuating its regulatory role in the CBF-COR signaling cascade. However, under cold stress, VaMIEL1 expression is downregulated, leading to the stabilization of VaMYB4a and the activation of CBF-COR signaling. Through a combination of biochemical assays and functional analysis in Arabidopsis thaliana and grapevine calli, we demonstrate that VaMIEL1 overexpression reduces cold tolerance, as evidenced by increased oxidative stress, excessive reactive oxygen species (ROS) accumulation, and downregulated expression of cold-responsive genes. Conversely, silencing of VaMIEL1 enhances cold tolerance by stabilizing VaMYB4a and boosting antioxidant defenses. These findings uncover a previously unrecognized regulatory mechanism by which VaMIEL1 modulates cold tolerance through transcriptional and oxidative stress pathways, offering potential targets for the development of climate-resilient grapevine cultivars and other crops.

Acknowledgements

The gift of the Arabidopsis mutant miel1 (SALK_087883) from Zheng Yuan’s team at the State Key Laboratory of Crop Adaptation and Improvement, School of Life Sciences, Henan University, is greatly appreciated. This work is supported by Ningxia Hui Autonomous Region Key R&D Program (Grant no. 2023BCF01003), National Natural Science Foundation of China (Grant no. 32472711 and 32060672), and Agricultural Breeding Project of Ningxia Hui Autonomous Region (Grant no. NXNYYZ202101).

Author contributions

W.X. conceived and designed the research; Y.X., K.L., Q.Y., and J.W. performed the experiments; N.Z., J.Z., J.L., and H.Z. analyzed the data; and Y.X. wrote the manuscript. W.X. reviewed and edited the manuscript. All authors read and approved the manuscript.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Conflict of interests

The authors declare no conflict of interest.

Supplementary information

Supplementary data is available at Horticulture Research online.

References

[1] Kerbler SM, Wigge PA. Temperature sensing in plants. Annu Rev Plant Biol. 2023; 74:341-66

[2] Kidokoro S, Shinozaki K, Yamaguchi Shinozaki K. Transcriptional regulatory network of plant cold-stress responses. Trends Plant Sci. 2022; 27:922-35

[3] Habib S, Dang VC, Ickert Bond SM. et al. Robust phylogeny of Tetrastigma (Vitaceae) based on ten plastid DNA regions: implications for infrageneric classification and seed character evolution. Front Plant Sci. 2017; 8:255350

[4] Chai F, Liu W, Xiang Y. et al. Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation. Horticul-ture Research. 2019; 6:6

[5] Gong Z, Xiong L, Shi H. et al. Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci. 2020; 63:635-74

[6] Zhang H, Zhao Y, Zhu J-K. Thriving under stress: how plants balance growth and the stress response. Dev Cell. 2020; 55:529-43

[7] Li Z, Yang L, Wu Y. et al. TOR balances plant growth and cold tolerance by orchestrating amino acid-derived metabolism in tomato. Horticulture Research. 2024;11:uhae253

[8] Zhang J, Dong K-L, Ren M-Z. et al. Coping with alpine habi-tats: genomic insights into the adaptation strategies of Triploste-gia glandulifera (Caprifoliaceae). Horticulture Research. 2024;11: uhae077

[9] Tokheim C, Wang X, Timms RT. et al. Systematic characterization of mutations altering protein degradation in human cancers. Mol Cell. 2021; 81:1292-1308.e11

[10] Shu K, Yang W. E 3 ubiquitin ligases: ubiquitous actors in plant development and abiotic stress responses. Plant Cell Physiol. 2017; 58:1461-76

[11] Vierstra RD. The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol. 2009; 10:385-97

[12] Loix M, Zelcer N, Bogie JF. et al. The ubiquitous role of ubiquiti-nation in lipid metabolism. Trends Cell Biol. 2023; 34:416-29

[13] Chen X, Raiff A, Li S. et al. Mechanism of Ψ-pro/C-degron recog-nition by the CRL2FEM1B ubiquitin ligase. Nat Commun. 2024; 15: 3558

[14] Sun C, Yao GF, Li LX. et al. E 3 ligase BRG3 persulfidation delays tomato ripening by reducing ubiquitination of the repressor WRKY71. Plant Physiol. 2023; 192:616-32

[15] Dong C, Agarwal M, Zhang Y. et al. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci. 2006; 103:8281-6

[16] Chinnusamy V, Zhu J, Zhu J-K. Cold stress regulation of gene expression in plants. Trends Plant Sci. 2007; 12:444-51

[17] Wang D, Zhang X, Xu R. et al. Apple U-box-type E3 ubiqui-tin ligase MdPUB23 reduces cold-stress tolerance by degrading the cold-stress regulatory protein MdICE1. Horticulture. Research. 2022;9:uhac171

[18] Wang DR, Zhang X, Xu RR. et al. Apple U-box-type E3 ubiqui-tin ligase MdPUB23 reduces cold-stress tolerance by degrading the cold-stress regulatory protein MdICE1. Horticulture Research. 2022;9:uhac171

[19] Su Y, Ngea GLN, Wang K. et al. Deciphering the mechanism of E3 ubiquitin ligases in plant responses to abiotic and biotic stresses and perspectives on PROTACs for crop resistance. Plant Biotechnol J. 2024; 22:2811-43

[20] Yi H, Shi H, Mao W. et al. E 3 ubiquitin ligase IPI1 controls rice immunity and flowering via both E3 ligase-dependent and-independent pathways. Dev Cell. 2024; 59:2719-2730.e4

[21] Zhang C-Y, Liu H-C, Zhang X-S. et al. VcMYB4a, an R2R3-MYB transcription factor from Vaccinium corymbosum, neg-atively regulates salt, drought, and temperature stress. Gene. 2020; 757:144935

[22] Yu Q. Response Mechanism of Amur Grape VaMYB4a in Response to Cold Stress. Ningxia University; Ningxia. 2023

[23] An JP, Zhang CL, Li HL. et al. Apple SINA E3 ligase MdSINA3 neg-atively mediates JA-triggered leaf senescence by ubiquitinating and degrading the MdBBX37 protein. Plant J. 2022; 111:457-72

[24] Wang X, Zhang X, Song C-P. et al. PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis. Plant Cell. 2023; 35:3585-603

[25] An J, Wang X, Zhang X. et al. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J. 2020; 18:337-53

[26] Mittler R, Zandalinas SI, Fichman Y. et al. Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol. 2022; 23:663-79

[27] Liu XJ, Liu X, Zhao Q. et al. Calmodulin-like protein MdCML15 interacts with MdBT2 to modulate iron homeostasis in apple. Horticulture Research. 2024;11:uhae081

[28] Liu X, Bulley SM, Varkonyi-Gasic E. et al. Kiwifruit bZIP transcrip-tion factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress. Plant Physiol. 2023; 192:982-99

[29] Wang Z, Zhang Y, Hu H. et al. CabHLH 79 acts upstream of CaNAC035 to regulate cold stress in pepper. Int J Mol Sci. 2022; 23:2537

[30] Lee HG, Seo PJ. The Arabidopsis MIEL1 E3 ligase negatively reg-ulates ABA signalling by promoting protein turnover of MYB96. Nat Commun. 2016; 7:12525

[31] Doroodian P, Hua Z. The ubiquitin switch in plant stress response. Plan Theory. 2021; 10:246

[32] Yu P, Hua Z. The ubiquitin-26S proteasome system and autophagy relay proteome homeostasis regulation during silique development. Plant J. 2022; 111:1324-39

[33] Stone SL. Ubiquitin ligases at the nexus of plant responses to biotic and abiotic stresses. Essays Biochem. 2022; 66:123-33

[34] Dubiella U, Serrano I. The ubiquitin proteasome system as a double agent in plant-virus interactions. Plan Theory. 2021; 10:928

[35] He Y, Zhang K, Georgiev MI. et al. Ubiquitin proteins and the orchestration of transcription factors activity. Crit Rev Plant Sci. 2021; 40:366-77

[36] Etherington RD, Bailey M, Boyer J-B. et al. Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases. Plant Physiol. 2023; 193:2086-104

[37] Parada F, Oklestkova J, Arce-Johnson P. Characterization of endogenous levels of brassinosteroids and related genes in grapevines. Int J Mol Sci. 2022; 23:1827

[38] Wu Y, Henderson SW, Walker RR. et al. Root-specific expression of Vitis vinifera VviNPF2. 2 modulates shoot anion concentration in transgenic Arabidopsis. Front Plant Sci. 2022; 13:863971

[39] Nie K, Zhao H, Wang X. et al. The MIEL1-ABI5/MYB30 regulatory module fine tunes abscisic acid signaling during seed germina-tion. J Integr Plant Biol. 2022; 64:930-41

[40] Zhang H, Wang Z, Li X. et al. The IbBBX24-IbTOE3-IbPRX17 module enhances abiotic stress tolerance by scavenging reac-tive oxygen species in sweet potato. New Phytol. 2022; 233: 1133-52

[41] Hilleary R. The Missing Link: The Role of the ROS Receptor HPCA1 in Systemic Signaling and Stress Acclimation in Plants. Plant Cell. Oxford University Press; American. 2022; 34:4128-9

[42] Wang P, Liu WC, Han C. et al. Reactive oxygen species: multidi-mensional regulators of plant adaptation to abiotic stress and development. J Integr Plant Biol. 2024; 66:330-67

[43] Xu W, Zhang N, Jiao Y. et al. The grapevine basic helix-loop-helix (bHLH) transcription factor positively modulates CBF-pathway and confers tolerance to cold-stress in Arabidopsis. Mol Biol Rep. 2014; 41:5329-42

[44] Xie X, Li S, Zhang R. et al. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell Environ. 2012; 35:1884-97

[45] Singhal R, Fernandez DE. Sorting of SEC translocase SCY compo-nents to different membranes in chloroplasts. JExp Bot. 2017; 68: 5029-43

[46] Anderson SA, Singhal R, Fernandez DE. Membrane-specific tar-geting of tail-anchored proteins SECE1 and SECE2 within chloro-plasts. Front Plant Sci. 2019; 10:1401

[47] Chung Y, Zhu Y, Wu M. et al. Auxin response factors pro-mote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS. Nat Commun. 2019; 10: 886

[48] An J, Liu X, Li H. et al. Apple RING E3 ligase MdMIEL1 inhibits anthocyanin accumulation by ubiquitinating and degrading MdMYB1 protein. Plant Cell Physiol. 2017; 58:1953-62

[49] Zhang L, Xue X, Zhai R. et al. Timing of calorie restriction in mice impacts host metabolic phenotype with correlative changes in gut microbiota. Msystems. 2019; 4:00348-19

[50] Lung S, Liao P, Yeung EC. et al. Acyl-CoA-binding protein ACBP1 modulates sterol synthesis during embryogenesis. Plant Physiol. 2017; 174:1420-35

[51] Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987; 6:3901-7

[52] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobac-terium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16:735-43

[53] Li J, Lv K, Wu J. et al. Exogenous melatonin promotes cold tolerance in grape seedlings: physiological, transcriptomic, and functional evidence. J Agric Food Chem. 2023; 71:19970-85

[54] Wu J, Zhang J, Hao X. et al. Establishment of an effi-cient callus transient transformation system for Vitis vinifera cv. ‘Chardonnay’. Protoplasma. 2023; 261:351-66

[55] Xie Q, Guo H, Dallman G. et al. SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals. Nature. 2002; 419:167-70

[56] Chen Z, Liu L, Cheng Q. et al. Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine-tune hypoxic mitophagy. EMBO Rep. 2017; 18:495-509

[57] Zhang LL, Shao YJ, Ding L. et al. XBAT31 regulates thermore-sponsive hypocotyl growth through mediating degradation of the thermosensor ELF3 in Arabidopsis. Sci Adv. 2021;7:eabf4427

[58] Yu Q, Zheng Q, Shen W. et al. Grape CIPK18 acts as a positive regulator of CBF cold signaling pathway by modulating ROS homeostasis. Environ Exp Bot. 2022; 203:105063

[59] Zheng Q, Yu Q, Yao W. et al. Decoding VaCOLD1 function in grapevines: a membrane protein enhancing cold stress toler-ance. J Agric Food Chem. 2023; 71:19357-71