VaWRKY65 contributes to cold tolerance through dual regulation of soluble sugar accumulation and reactive oxygen species scavenging in Vitis amurensis

Abstract

Although the significance of some plant WRKYs in response to cold stress have been identified, the molecular mechanisms of most WRKYs remain unclear in grapevine. In this study, we demonstrate that cold-induced expression of VaBAM3 in Vitis amurensis executes a beneficial role in enhancing resistance by the regulating starch decomposition. VaWRKY65 was identified as an upstream transcriptional activator of VaBAM3 through yeast one-hybrid library screening and validated to directly interact with the W-box region inside the VaBAM3 promoter. Transgenic Arabidopsis thaliana plants and grapevine roots overexpression VaWRKY65 exhibited improved cold tolerance along with higher BAM activity and soluble sugar levels, whereas opposite changes were observed in VaWRKY65 knockdown lines created by virus-induced gene silencing (VIGS) in grapevine plants and in the knockout wrky65 mutants generated by CRISPR/Cas9 technology in grapevine roots. The transcriptome data show that overexpression of VaWRKY65 led to significant alteration of a diverse set of stress-related genes at the transcriptional level. One of the genes, Peroxidase 36 (VaPOD36), was further verified as a direct target of VaWRKY65. Consistently, VaWRKY65-overexpressing plants had higher VaPOD36 transcript levels and POD activity but a reduced ROS level, while silencing VaWRKY65 results in contrary changes. Collectively, these results reveal that VaWRKY65 enhanced cold tolerance through modulating soluble sugars produced from starch breakdown and ROS scavenging.

Acknowledgements

This work was supported by the Project funded by the China Postdoctoral Science Foundation (No. 2023 M733717) and the Natural Science Foundation of China (Nos 32302517 and 32025032). We thank Dr Chunying Kang (Huazhong Agricultural University, Wuhan, China) for kindly providing CRISPR/Cas9 vector pKSE401.

Author contributions

L.M., H.M.Z., and L.S.T. conducted the experiments. L.M., H.M.Z., Y.J.H, S.K., W.J.L., and Q.Y.L. collected the data. H.P.X. and L.M. designed the experiments. L.M. wrote the paper, H.P.X., Z.C.L., J.L., and R.A. revised the manuscript.

Data availability

The leaves of V. amurensis under chilling stress RNA-seq data were deposited into NCBI GEO database with the accession number PRJNA934965.

Conflict of interest statement

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Wan Y, Schwaninger H, Li D. et al. The eco-geographic distri-bution of wild grape germplasm in China. Vitis J Grapevine Res. 2008; 47:77-80

[2] Fennell A. Freezing tolerance and injury in grapevines. JCrop Improv. 2004; 10:201-35

[3] Xu W, Li R, Zhang N. et al. Transcriptome profiling of Vitis amurensis, an extremely cold-tolerant Chinese wild Vitis species, reveals candidate genes and events that potentially connected to cold stress. Plant Mol Biol. 2014; 86:527-41

[4] Wang H, Chen W, Xu Z. et al. Functions of WRKYs in plant growth and development. Trends Plant Sci. 2023; 28:630-45

[5] Jiang J, Ma S, Ye N. et al. WRKY transcription factors in plant responses to stresses. J Integr Plant Biol. 2017; 59:86-101

[6] Rushton PJ, Somssich IE, Ringler P. et al. WRKY transcription factors. Trends Plant Sci. 2010; 15:247-58

[7] Yokotani N, Sato Y, Tanabe S. et al. WRKY76 is a rice transcrip-tional repressor playing opposite roles in blast disease resistance and cold stress tolerance. JExp Bot. 2013; 64:5085-97

[8] Kim CY, Vo KTX, Nguyen CD. et al. Functional analysis of a cold-responsive rice WRKY gene, OsWRKY71. Plant Biotechnol Rep. 2016; 10:13-23

[9] Zou C, Jiang W, Yu D. Male gametophyte-specific WRKY34 tran-scription factor mediates cold sensitivity of mature pollen in arabidopsis. JExp Bot. 2010; 61:3901-14

[10] Zhang L, Zhao T, Sun X. et al. Overexpression of VaWRKY12, a transcription factor from Vitis amurensis with increased nuclear localization under low temperature, enhances cold tolerance of plants. Plant Mol Biol. 2019; 100:95-110

[11] Sun X, Zhang L, Wong DCJ. et al. The ethylene response factor VaERF092 from Amur grape regulates the transcription factor VaWRKY33, improving cold tolerance. Plant J. 2019; 99:988-1002

[12] Huang X, Cao L, Fan J. et al. CdWRKY2-mediated sucrose biosyn-thesis and CBF-signalling pathways coordinately contribute to cold tolerance in bermudagrass. Plant Biotechnol J. 2022; 20:660-75

[13] Ding ZJ, Yan JY, Xu XY. et al. Transcription factor WRKY46 regulates osmotic stress responses and stomatal movement independently in arabidopsis. Plant J. 2014; 79:13-27

[14] Chen THH, Murata N. Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol. 2002; 5:250-7

[15] Zhao M, Zhang N, Gao T. et al. Sesquiterpene glucosylation mediated by glucosyltransferase UGT91Q2 is involved in the modulation of cold stress tolerance in tea plants. New Phytol. 2020; 226:362-72

[16] Ming R, Zhang Y, Wang Y. et al. The JA-responsive MYC2-BADH-like transcriptional regulatory module in Poncirus trifoliata contributes to cold tolerance by modulation of glycine betaine biosynthesis. New Phytol. 2021; 229:2730-50

[17] Zhang Y, Zhu J, Khan M. et al. Transcription factors ABF4 and ABR1 synergistically regulate amylase-mediated starch catabolism in drought tolerance. Plant Physiol. 2023; 191:591-609

[18] Song J, Sun P, Kong W. et al. SnRK2.4-mediated phosphory-lation of ABF2 regulates arginine decarboxylase expression and putrescine accumulation under drought stress. New Phytol. 2023; 238:216-36

[19] Zhu J-K. Abiotic stress signaling and responses in plants. Cell. 2016; 167:313-24

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

[21] Thalmann M, Santelia D. Starch as a determinant of plant fitness under abiotic stress. New Phytol. 2017; 214:943-51

[22] Sami F, Yusuf M, Faizan M. et al. Role of sugars under abiotic stress. Plant Physiol Biochem. 2016; 109:54-61

[23] Yuanyuan M, Yali Z, Jiang L. et al. Roles of plant soluble sugars and their responses to plant cold stress. Afr J Biotechnol. 2009; 8: 2004-10

[24] Krasensky J, Jonak C. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. JExp Bot. 2012; 63:1593-608

[25] Fulton DC, Stettler M, Mettler T. et al. β-Amylase4, a noncatalytic protein required for starch breakdown, acts upstream of three active β-amylases in arabidopsis chloroplasts. Plant Cell. 2008; 20: 1040-58

[26] Stitt M, Zeeman SC. Starch turnover: pathways, regulation and role in growth. Curr Opin Plant Biol. 2012; 15:282-92

[27] Reinhold H, Soyk S, Simková K. et al. B-amylase-like proteins function As transcription factors in arabidopsis, controlling shoot growth and development. Plant Cell. 2011; 23:1391-403

[28] Yue C, Cao H, Lin H. et al. Expression patterns of alpha-amylase and beta-amylase genes provide insights into the molecular mechanisms underlying the responses of tea plants (Camellia sinensis) to stress and postharvest processing treatments. Planta. 2019; 250:281-98

[29] Monroe JD, Storm AR. Review: the arabidopsis β-amylase (BAM) gene family: diversity of form and function. Plant Sci. 2018; 276: 163-70

[30] Livingston DP, Hincha DK, Heyer AG. Fructan and its relationship to abiotic stress tolerance in plants. Cell Mol Life Sci. 2009; 66: 2007-23

[31] Keunen E, Peshev D, Vangronsveld J. et al. Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept. Plant Cell Environ. 2013; 36: 1242-55

[32] Lao NT, Schoneveld O, Mould RM. et al. An arabidopsis gene encoding a chloroplast-targeted β-amylase. Plant J. 1999; 20: 519-27

[33] Monroe JD, Storm AR, Badley EM. et al. β-amylase1 and β-amylase3 are plastidic starch hydrolases in arabidopsis that seem to be adapted for different thermal, pH, and stress con-ditions. Plant Physiol. 2014; 166:1748-63

[34] Zeeman SC, Thorneycroft D, Schupp N. et al. Plastidial α-glucan phosphorylase is not required for starch degradation in ara-bidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiol. 2004; 135:849-58

[35] Peng T, Zhu X, Duan N. et al. PtrBAM1, a β-amylase-coding gene of Poncirus trifoliata, is a CBF regulon member with function in cold tolerance by modulating soluble sugar levels. Plant Cell Environ. 2014; 37:2754-67

[36] Zhao L, Yang T, Xing C. et al. The β-amylase PbrBAM3 from pear (Pyrus betulaefolia) regulates soluble sugar accumulation and ROS homeostasis in response to cold stress. Plant Sci. 2019; 287: 110184

[37] Sun S, Hu C, Qi X. et al. The AaCBF4-AaBAM3.1 module enhances freezing tolerance of kiwifruit (Actinidia arguta). Hortic Res. 2021; 8:1-15

[38] Hao X-Y, Yue C, Tang H. et al. Cloning of β-amylase gene ( CsBAM3 ) and its expression model response to cold stress in tea plant. Acta Agron Sin. 2017; 43:1417

[39] Nielsen TH, Deiting U, Stitt M. A β-amylase in potato tubers is induced by storage at low temperature. Plant Physiol. 1997; 113: 503-10

[40] Hou J, Zhang H, Liu J. et al. Amylases StAmy23, StBAM1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways. JExp Bot. 2017; 68:2317-31

[41] Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem. 2010; 48:909-30

[42] Baxter A, Mittler R, Suzuki N. ROS as key players in plant stress signalling. JExp Bot. 2014; 65:1229-40

[43] Lee DH, Lee CB. Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: In gel enzyme activity assays. Plant Sci. 2000; 159:75-85

[44] Zhang Y, Ming R, Khan M. et al. ERF9 of Poncirus trifoliata (L.) Raf. Undergoes feedback regulation by ethylene and modulates cold tolerance via regulating a glutathione S-transferase U17 gene. Plant Biotechnol J. 2022; 20:183-200

[45] Wang M, Dai W, Du J. et al. ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) contributes to cold tolerance by directly regu-lating expression of Prx1 involved in antioxidative process. Plant Biotechnol J. 2019; 17:1316-32

[46] Morsy MR, Jouve L, Hausman JF. et al. Alteration of oxidative and carbohydrate metabolism under abiotic stress in two rice (Oryza sativa L.) genotypes contrasting in chilling tolerance. J Plant Physiol. 2007; 164:157-67

[47] Kaur S, Gupta AK, Kaur N. et al. Antioxidative enzymes and sucrose synthase contribute to cold stress tolerance in chickpea. J Agron Crop Sci. 2009; 195:393-7

[48] Couée I, Sulmon C, Gouesbet G. et al. Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. JExp Bot. 2006; 57:449-59

[49] Xin H, Zhu W, Wang L. et al. Genome wide transcriptional profile analysis of Vitis amurensis and Vitis vinifera in response to cold stress. PLoS One. 2013; 8:1-14

[50] Chai F, Liu W, Xiang Y. et al. Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation. Hortic Res. 2019; 6:8

[51] Zhou H, Li Q, Gichuki DK. et al. Dynamics of starch degrada-tion and expression of related genes during chilling stress in grapevine. Hortic Adv. 2023; 1:1-16

[52] Rushton DL, Tripathi P, Rabara RC. et al. WRKY transcription fac-tors: key components in abscisic acid signalling. Plant Biotechnol J. 2012; 10:2-11

[53] Guo M, Yang F, Liu C. et al. A single-nucleotide polymorphism in WRKY33 promoter is associated with the cold sensitivity in cultivated tomato. New Phytol. 2022; 236:989-1005

[54] Zhang Y, Yu H, Yang X. et al. CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner. Plant Physiol Biochem. 2016; 108:478-87

[55] Daryanavard H, Postiglione AE, Mühlemann JK. et al. Flavonols modulate plant development, signaling, and stress responses. Curr Opin Plant Biol. 2023; 72:102350

[56] Habibollahi M, Kavousi HR, Lohrasbi-Nejad A. et al. Cloning, characterization and expression of a phenylalanine ammonia-lyase gene (CcPAL) from cumin (Cuminum cyminum L.). J Appl Res Med Aromat Plants. 2020; 18:100253

[57] Ishihama N, Yoshioka H. Post-translational regulation of WRKY transcription factors in plant immunity. Curr Opin Plant Biol. 2012; 15:431-7

[58] Fiil BK, Petersen K, Petersen M. et al. Gene regulation by MAP kinase cascades. Curr Opin Plant Biol. 2009; 12:615-21

[59] Adachi H, Nakano T, Miyagawa N. et al. Wrky transcription fac-tors phosphorylated by mapk regulate a plant immune nadph oxidase in Nicotiana benthamiana. Plant Cell. 2015; 27:2645-63

[60] Kaplan F, Guy CL. B-amylase induction and the protective role of maltose during temperature shock. Plant Physiol. 2004; 135: 1674-84

[61] Liu K, Zou W, Gao X. et al. Young seedlings adapt to stress by retaining starch and retarding growth through ABA-dependent and -independent pathways in Arabidopsis. Biochem Biophys Res Commun. 2019; 515:699-705

[62] Hu M, Shi Z, Zhang Z. et al. Effects of exogenous glucose on seed germination and antioxidant capacity in wheat seedlings under salt stress. Plant Growth Regul. 2012; 68:177-88

[63] Horrer D, Flütsch S, Pazmino D. et al. Blue light induces a distinct starch degradation pathway in guard cells for stomatal opening. Curr Biol. 2016; 26:362-70

[64] Leprince O, Hendry GAF, McKersie BD. The mechanisms of desiccation tolerance in developing seeds. Seed Sci Res. 1993; 3: 231-46

[65] Bolouri-moghaddam MR, Le RK, Xiang L. et al. Sugar signalling and antioxidant network connections in plant cells. FEBS J. 2022; 277:2022-37

[66] Yano R, Nakamura M, Yoneyama T. et al. Starch-related α-glucan/water dikinase is involved in the cold-induced develop-ment of freezing tolerance in Arabidopsis. Plant Physiol. 2005; 138: 837-46

[67] Kaplan F, Guy CL. RNA interference of Arabidopsis beta-amylase8 prevents maltose accumulation upon cold shock and increases sensitivity of PSII photochemical efficiency to freezing stress. Plant J. 2005; 44:730-43

[68] Liang G, He H, Nai G. et al. Genome-wide identification of BAM genes in grapevine (Vitis vinifera L.) and ectopic expres-sion of VvBAM1 modulating soluble sugar levels to improve low-temperature tolerance in tomato. BMC Plant Biol. 2021; 21:1-15

[69] Ma QJ, Sun MH, Lu J. et al. Transcription factor AREB2 is involved in soluble sugar accumulation by activating sugar transporter and amylase genes1. Plant Physiol. 2017; 174:2348-62

[70] Han C, Hua W, Li J. et al. TOR promotes guard cell starch degra-dation by regulating the activity of β-AMYLASE1 in Arabidopsis. Plant Cell. 2022; 34:1038-53

[71] Rienth M, Torregrosa L, Sarah G. et al. Temperature desynchro-nizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome. BMC Plant Biol. 2016; 16: 1-23

[72] Sandalio LM, Rodríguez-Serrano M, Romero-Puertas MC. et al. Role of peroxisomes as a source of reactive oxygen species (ROS) signaling molecules. Subcell Biochem. 2013; 69: 231-55

[73] Sharma P, Jha AB, Dubey RS. et al. Reactive oxygen species, oxida-tive damage, and antioxidative defense mechanism in plants under stressful conditions. JBot. 2012; 2012:1-26

[74] Ma L, Li X, Zhang J. et al. MsWRKY33 increases alfalfa (Med-icago sativa L.) salt stress tolerance through altering the ROS scavenger via activating MsERF5 transcription. Plant Cell Environ. 2023; 46:3887-901

[75] Wang R, Shi CL, Wang X. et al. Tomato SlIDA has a critical role in tomato fertilization by modifying reactive oxygen species homeostasis. Plant J. 2020; 103:2100-18

[76] Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol. 2006; 141: 391-6

[77] Weise SE, Weber APM, Sharkey TD. Maltose is the major form of carbon exported from the chloroplast at night. Planta. 2004; 218: 474-82

[78] An D, Yang J, Zhang P. Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress. BMC Genomics. 2012; 13: 1-25

[79] Wang Z, Wong DCJ, Wang Y. et al. GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response. Plant Physiol. 2021; 186:1660-78

[80] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001; 25:402-8

[81] Geng J, Liu JH. The transcription factor CsbHLH18 of sweet orange functions in modulation of cold tolerance and homeosta-sis of reactive oxygen species by regulating the antioxidant gene. JExp Bot. 2018; 69:2677-92

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

[83] Tang T, Yu X, Yang H. et al. Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Front Plant Sci. 2018; 871:1-14

[84] Wang K. Agrobacterium Protocols. Vol. 2. third ed.ed. Humana Press, 2014:1-329

[85] Jiao B, Hao X, Liu Z. et al. Engineering CRISPR immune sys-tems conferring GLRaV-3 resistance in grapevine. Hortic Res. 2022; 9:uhab023

[86] Kurth EG, Peremyslov VV, Prokhnevsky AI. et al. Virus-derived gene expression and RNA interference vector for grapevine. JVirol. 2012; 86:6002-9

[87] Meng L, Zhang Q, Yang J. et al. PtrCDPK10 of Poncirus trifoli-ata functions in dehydration and drought tolerance by reduc-ing ROS accumulation via phosphorylating PtrAPX. Plant Sci. 2020; 291:110320

[88] Hu X, Sun T, Liang Z. et al. Transcription factors TgbHLH42-1 and TgbHLH42-2 positively regulate anthocyanin biosynthesis in tulip (Tulipa gesneriana L.). Physiol Plant. 2023; 175:e13939

[89] Shen Y, Sun T, Pan Q. et al. RrMYB5- and RrMYB10-regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in Rosa rugosa. Plant Biotechnol J. 2019; 17:2078-95

[90] Zou Y, Chang SKC, Gu Y. et al. Antioxidant activity and phenolic compositions of lentil (Lens culinaris var. Morton) extract and its fractions. J Agric Food Chem. 2011; 59:2268-76

[91] Meng L, Yang H, Xiang L. et al. NAC transcription factor TgNAP promotes tulip petal senescence. Plant Physiol. 2022; 190:1960-77

[92] Liu Q, Wang C, Jiao X. et al. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas sys-tems. Sci China Life Sci. 2019; 62:1-7