VvGRX28 interacting with VvZNF10 modulates cold tolerance via eliminating excessive ROS in grapevine

VvGRX28 interacting with VvZNF10 modulates cold tolerance via eliminating excessive ROS in grapevine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article VvGRX28 interacting with VvZNF10 modulates cold tolerance via eliminating excessive ROS in grapevine Guojie Nai, Congcong Zhang, Haokai Yan, Lei Ma, Zhihui Pu, Jingrong Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8260182/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Glutaredoxins (GRXs) are small oxidoreductases that play a crucial role in responses to abiotic stress. Although the GRX gene family has been characterized in several species, the knowledge of their evolution relationship, diversification and function in grape are still limited. Results In this study, 32 VvGRX genes were identified and clustered into CC-, CGFS-, GRL- and CPYC-type categories. The structure and motifs of VvGRXs were similar in genes clustered into close branches, indicating highly conserved during the evolutional process. Cis -acting elements mainly were involved in stress response and hormone regulation. Tissue-specific expression showed that VvGRXs was differentially expressed in different grape tissues. RT-PCR indicated that VvGRX28 expression can actively be induced by cold stress. Furthermore, VvGRX28 were functionally characterized and cloned to verify the cold tolerance function. Through Agrobacterium -mediated to overexpression and interfere VvGRX28 , the result demonstrated that the VvGRX28 overexpression can enhance the content of proline, soluble sugar, glutathione and peroxidase activities, and reduced the content of MDA and H 2 O 2 , and upregulated the expression of ICE , CBF and COR in Arabidopsis thaliana and grape callus, while exhibiting an opposite trend after RNAi. VvZNF10, as the interaction protein of VvGRX28, overexpression and co-transformation with VvGRX28 could improve the cold tolerance in grape callus. Conclusions The result demonstrated that VvGRX28 was a positive regulator to enhance cold tolerance interacting with VvZNF10 mainly in nucleus in grape callus. Collectively, this study provides a comprehensive analysis of the VvGRX gene family, offering novel insights into the regulation mechanism of VvGRX28 under cold stress in grape. Glutaredoxin Cold stress VvGRX28 Overexpression RNAi Grape Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Grape ( Vitis vinifera L.), as an important perennial fruit crop, has been widely cultivated all over the word[ 1 ]. During the growth process of grapes, grape often suffers from various abiotic and biotic stresses, such as drought, salinity, heat, cold, and diseases and insect pests[ 2 ]. These adverse environmental factors cause the decreasing in production and quality of grape. In particular, low temperature (LT) is a critical environmental factor that influences metabolism, growth, and development of grape in northwestern China, where winter is extremely cold and the grapevine is very vulnerable to freezing damage[ 3 ]. Although the application of soil covering and exogenous substances have been employed to address, the problem still cannot be addressed at source[ 4 , 5 ]. Therefore, it is urgent for salt-tolerance genes mining, the cold-resistant grapes breeding and cold-resistant cultivation model in future. Cold stress, including chilling (> 0°C) and freezing (< 0°C) stresses, has an adverse effect on plant growth and development, causing the changing of membrane lipids composition, the decreasing of intracellular enzymes activities, the accumulation of reactive oxygen species (ROS), the decreasing of photosynthetic capacity in grape[ 6 ]. Plant must cope with cold stress by coordinating cell molecular, metabolic, and physiological responses in different signal transduction pathways. When plants were subjected to cold stress, plants accumulate a large amount of ROS, leading to the imbalanced antioxidant defense system, triggering oxidative stress and causing damage to cells and tissues[ 7 ]. The antioxidant and redox systems which are composed of oxidoreductase proteins transferring electrons from input elements to downstream target proteins contribute to reduce ROS levels. Transmitters oxidoreductase proteins mainly include thioredoxins (TRXs) and glutaredoxins (GRXs)[ 8 , 9 ]. The ascorbate–glutathione (AsA–GSH) cycle plays a key role in efficiently scavenging ROS within the chloroplast through the action of enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR)[ 10 ]. Among them, GR uses glutathione (GSH) as the substrate to catalyze the reduction of H₂O₂ and organic peroxides, protecting cells from oxidative damage. Research has found that glutaredoxin (GRX) in plants can utilize glutathione (GSH) to reduce the disulfide bonds produced by oxidation, effectively protecting intracellular proteins from the damage of ROS and playing an important regulatory role in ROS signaling[ 8 ]. GRXs, an important oxidoreductase protein gene family, is widely present in prokaryotes and eukaryotes, and plays a significant role in plant development and abiotic stress[ 11 ]. Based on conserved residues in their active sites, plant GRX genes are classified into four types, namely CPYC, CGFS, CC and GRX-like (GRL) type[ 12 ]. Among them, the CC-type has only been occured in higher plants[ 13 ]. The characteristic of the CPYC type Grx has Cxx(C/S) active sites, and is a classic dithiol Grx. It has the function of oxidoreductase and participates in the assembly of iron-sulfur clusters, severing as an important redox regulatory factor for intracellular metabolism[ 14 ]. Research showed that the overexpression of CPYC-type OsGrxC2.2 could regulate embryo development during embryogenesis, and increased grain weight, which interfere with the normal embryogenesis of rice embryo[ 15 ]. Interestingly, CPYC type OsGrx20 has a positive regulatory effect on adverse stress in rice. Overexpression of OsGRX20 can significantly enhance the resistance of rice to white leaf blight, as well as its tolerance to methyl violet and salt stress[ 16 ]. The CGFS type is generally a monothiol Grx with a strictly conserved CGFS motif. The CGFS-type Grx mainly functions as a ferredoxin transferase, which is determined by the unique ring-like structure near the active site[ 14 ]. SlGrxS1 is widely expressed in tomato leaves, roots, stems and flowers, and its expression is induced by oxidation, drought and salt stress[ 17 ]. AtGrxS17 can interact with NF-YC11/NC2α and transmit the redox signals generated by the photoperiod, thereby maintaining the function of the meristem[ 18 ]. AtGRXS17 may serve as a fundamental protection against moderate heat stress through redox dependent companion activity, thereby preventing heat stress-induced reactions injury[ 19 ]. In tomatoes, the absence of CGFS-type SlGrxS15 can cause embryo death, Slgrxs14 and Slgrxs17 mutants showed hypersensitivity to heat, chilling, drought, heavy metal toxicity, nutrient deficiency, and short photoperiod stresses. Slgrxs16 mutants were affected by chilling stress [ 20 ]. The CC-type Grx has a special CCxx active site, usually in the form of CCxC or CCxS, which is a unique motif found in terrestrial plants. It is the first identified members that AtROXY1 and AtROXY2 belong to the CC-type Grx family in Arabidopsis , as well as their homologous genes OsROXY1 and OsROXY2 in rice, play a crucial role in the differentiation process of petals and anthers[ 21 , 22 ]. The knockout of GrxS13 impaired the tolerance to light, delaying plant growth in Arabidopsis thaliana [ 23 ]. The sensitivity of transgenic RNAi CC type OsGRX8 to various abiotic stresses is enhanced in rice plants[ 24 ]. In maize, the CC type GRX can regulate the redox state of other proteins and plays an important role in the growth and meristems development[ 25 ]. The GRL type GRX has three domains, namely an N-terminal GRX domain and two domains whose functions are unknown[ 11 , 26 ]. But so far, there have been no reports on the functions of the GRL type. A growing evidences showed GRX genes plays in regulating the apical meristem of plants[ 27 ], flower development[ 28 ], abiotic and biotic stress[ 9 , 29 ]. GRX gene family has been identified in many plant species, mainly including Arabidopsis thaliana [ 30 ], Oryza sativa L.[ 13 ], Triticum aestivum L.[ 31 ], Quercus glauca [ 32 ], Camellia sinensis[ 33 ], Musa acuminata [ 34 ], Puccinellia tenuiflora [ 35 ], populus[ 34 ]. And the biological function of single GRX genes were performed by transgenic techniques approaches in plant. ROXY18 (GRXS13) and ROXY19 (GRXC9) interacted with class II TGAs to regulate detoxification response and pathogen defense[ 36 , 37 ]. The MeGRXC3 negatively regulated drought tolerance in cassava[ 38 ]. Ectopic overexpressed SlGRX1 actively responded to abiotic tolerance against oxidative, drought, and salt stresses in Arabidopsis thaliana [ 17 ]. Silencing of GhGRL28 increased the sensitivity to salt stress in cotton[ 39 ]. Overexpressing PagGRXC9 enhanced salt tolerance in poplar[ 40 ]. At present, the biological function of GRX gene in cold stress mainly focuses on the basic expression levels by qRT-PCR, while the concrete knowledge of single GRX genes under cold stress remains a few in plants. However, it has yet been reported on systematic identification of VvGRX gene family member, structure analysis, responses to various abiotic stresses, and adaptive evolution of GRX genes in grape. Therefore, in this study, we identified VvGRX gene members, performed the in-depth characterization of its molecular, explored the expression pattern of VvGRXs in different organs and stress-inducible by Real-time fluorescent quantitative PCR (RT-PCR). Furthermore, the VvGRX28 gene related to cold stress was selected, we constructed transgenic plants ectopically expressing VvGRX28 in Arabidopsis thaliana , which were subjected to cold stress for functional analysis. the protein-protein interaction between VvGRX2 8 and its counterpart was analyzed by yeast two-hybrid (Y2H). The function of VvGRX28 and its counterpart were analyzed in grape callus. The findings will serve as a guide to the analysis and discovery of gene function in woody plant, and contribute to understand the genetic and molecular mechanisms of plant response to cold stress in grape and to provide candidate genes with improved stress tolerance via innovative molecular breeding strategies. Materials and method Plant materials and cultivation conditions The ‘Pinot Noir’ plantlets in vitro were used as test materials for RT-PCR under salt, drought and cold stresses, which were reserved in the plant stress physiology laboratory of the college of life sciences, Gansu Agricultural University, China. The cuttings (2–3 cm) with a single bud were inoculated in GS medium (pH, 5.8–6.0) supplemented with 20 g/L sucrose and 5.0 g/L agar to propagate a large number of aseptic seedlings. The plantlets in vitro were cultivated in light incubator with a light intensity of 120 µmol·m-2·s-1 at 26°C, 16 h/light, 8 h/dark growth condition. for various stress treatments. After grew for 35 days, the plantlets in vitro with uniform growth and no pollution were selected as experimental materials for RT-PCR assay. Arabidopsis thaliana (Columbia, Col-0) was used as heterologous transformation material. The Arabidopsis thaliana plants grew in the substrate containing a mixture of soil and vermiculite (3:1), and then placed in a growth chamber at 22°C (16 h light/8 h dark, 65% − 75% relative humidity, and 120 µmol·m − 2 ·s − 1 light intensity). ‘Pinot Noir’ callus was aseptically cultured in B5 medium (30 g/L sucrose + 6 g/L agar + 0.5 mg/L 1-Naphthaleneacetic acid (NAA) + 1 mg/L kinetin (KT) + 300 mg/L polyvinyl pyrrolidone (PVP) + 1 mg/L myo-inositol), and then sub-cultured every 30 days in a dark incubator at 25°C. The callus of ‘Pinot Noir’ with better growth was selected as transformation materials for Agrobacterium infestation and cold stress. Nicotiana benthamiana was planted in seedling substrate (nutritious soil and vermiculite = 3:1), and then placed in a growth chamber under a 16-h-light/8-h-dark photoperiod at 25°C. After cultivated for 25 days, tobacco leaves were used for following experiment on subcellular localization, GUS staining, firefly luciferase fragment complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC). Identification of VvGRX family members and phylogenetic tree construction Arabidopsis thaliana GRX protein sequences were downloaded from the TAIR database. Rice, tomato, maize GRX protein sequences were derived from the Phytozome version 13 database ( https://phytozomenext.jgi.doe.gov ) and used as the query to perform a BLASTP search against the grape protein database[ 41 ]. Reference sequence genome files of grape were downloaded from the Ensembl database ( http://plants.ensembl.org/index.html ). Hidden Markov Models (HMMs) for the GRX (Accession number: PF00462) were searched from the Pfam database ( http://pfam-legacy.xfam.org/ ) and used as queries to search the grape protein database by the HMMsearch tool, with the threshold set as E < 1e − 5 [ 42 ]. Proteins identified by both methods were combined and manually curated using the Conserved Domain Database (CDD)[ 43 ], removing redundancies and retaining only those proteins containing GRX domains as members of GRX family in grape. According to the location of genes on chromosomes, VvGRXs were named respectively. To investigate the phylogenetic relationships for the GRX family in grape, GRX family protein sequences of grape, tomato, barley and Arabidopsis thaliana were downloaded from the Phytozome and TAIR databases, respectively. Comparative analysis of amino acid sequences of GRX proteins using the software Clustal X. The phylogenetic tree was established by the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in the MEGA 7.0. The phylogenetic tree was visualized using the iTOL tool ( https://itol.embl.de/ ), and GRX protein were classified based on the active-site motif (Boubakri et al. 2022). Physicochemical properties and the secondary structure prediction of VvGRX protein The physical and chemical properties of grape GRX protein were analyzed by Expasy online tool ( https://web.expasy.org/protparam/ ), including amino acid number, molecular weight (MW), isoelectric point (PI), instability coefficient and grand average of hydrophilicity. The subcellular localization was predicted by WoLFPSORT ( https://wolfpsort.hgc.jp/ )[ 44 ]. The prediction of protein secondary structure was completed online by SOPMA ( http://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.plpage =/NPSA/npsa_hnn.html). Gene structure, conserved motifs, cis-elements and collinearity analysis of VvGRXs CDS sequences of grape GRX gene family were extracted from grape whole genome and gene annotation file, and the structure of this gene family was analyzed by GSDS ( http://gsds.gao-lab.org/index.php )[ 45 ]. MEME Suite 5.4.1 ( https://meme-suite.org/meme/ ) was used to predict the conservative motifs of grape GRX protein, and the motifs number was set to 10. The online website PlantCARE [ 46 ]. TBtools was used to visually analyze the conservative motifs of the gene family[ 47 ] ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) was used to analyze the distribution of cis -acting elements in the 2 kb base fragment upstream of the initiation codon of the grape GRX gene. The collinearity was analyzed among grape, Arabidopsis and tomato GRX gene family members. Stresses treatment For the preliminary function exploration of GRX genes under abiotic stresses, after grew for 35 days, the grape plantlets in vitro were treated NaCl, PEG and cold stress. Some uniform growth and no pollution plantlets were transformed into GS medium which contained 200 mmol·L − 1 NaCl, 10% PEG for 24 h, respectively. Sterile water was used as a control (CK) treatment. Plantlets were treated with 4°C cold stress for 24 h by gradient cooling in Ultra-low temperature alternating temperature incubator. Leaves tissues of grape were sampled for RT-PCR assay. For cold stress of Arabidopsis , 3 weeks-old seedlings were treated at -4°C for 5 h, and normal temperature conditions were used as a control. In the freezing tolerance of Arabidopsis , seedlings were grown at 22°C for 2 week and then treated at 4°C for 3 days to examine frost tolerance of Arabidopsis plants. And then the seedlings were treated at − 6°C for 5 h through gradient cooling, and then transferred to 22°C for 7 days, observing phenotype changing. Arabidopsis leaves after treated with − 4°C were collected at 0 h and 5 h, quickly frozen in liquid nitrogen and stored at − 80°C for RT-PCR and determination of physiological indexes related to cold tolerance. For cold stress of grape callus , grape callus after cultivated 30 days were selected to treat with 4°C for 15 d. Grape calluses grew in 25°C were used as control. Grape calluses were sampled, and quickly frozen in liquid nitrogen and stored at − 80°C for RT-PCR and determination of physiological indexes. Three biological replicates were applied in this experiment. Measurement of stress-related physiological parameters, histochemical staining After treated with cold stress, Arabidopsis and grape callus were collected to determine physiological indexes related to cold tolerance. The relative electrolyte leakage (REL) of plant materials was determined by a conductivity meter (DDSJ-318, Shanghai, China)[ 50 ]. The content of malondialdehyde (MDA), Proline (Pro) and hydrogen peroxide (H 2 O 2 ), soluble sugar (SS), glutathione (GSH) and peroxidase (POD) activities were determined by Comin Biotechnology Kit (Suzhou, Jiangsu, China) according to the manufacturers’ protocols, respectively. Three biological replicates were set in the experiment. 3, 3, 9-Diaminobenzidine (DAB) and Nitroblue tetrazolium (NBT) staining were used to detect the accumulation of H 2 O 2 and superoxide anion (O 2 ·− ) of Arabidopsis referring to the methods as described previously[ 51 , 52 ]. Trypan blue dye was used to detect the integrity of the cell membrane and the survival rate of the cells under cold stress by the previous method[ 53 ]. The detached leaves were put into DAB, NBT and After stained by solution, respectively, and vacuumed for 5–10 min making the leaves completely immersed in the solution. After stained by DAB and NBT, the leaves were immersed in 95% ethanol for decolorized in a boiling water about 20 min. After stained by stained, the leaves were immersed in 2.5 g/L chloral hydrate solution. After the color of stained leaves gradually faded, the samples were placed in 30% glycerol solution and the dyed leaves were photographed and recorded using a camera. Subcellular localization assay and GUS staining and activity in Nicotiana benthamiana leaves For performing subcellular localization, 4-week-old tobaccos were used to observe EGFP signaling in leaves cell. The pART-CAM-EGFP- VvGRX28 and pART-CAM-EGFP empty vector Agrobacterium liquids were infiltrated into the epidermal cells on the reverse side of the tobacco leaf, and tobaccos were kept in dark condition for 12 h. Empty vector Agrobacterium liquids was used the control. The infiltrated tobacco plants were cultivated in a growth chamber under a 16-h-light/8-h-dark photoperiod at 25°C. After infiltrated for 48–72 h, green fluorescent protein (GFP) signals were detected with a laser scanning confocal microscope (Olympus FV1000 viewer) in tobacco leaves. To construct the pro- VvGRX28 ::GUS vector, the VvGRX28 promoter fragment at 2000 bp upstream of the start codon was cloned into the pBI121-GUS vector to replace the 35S promoter. The recombinant vector liquid was injected into tobacco leaves using the Agrobacterium-mediated transformation method for transient expression. The injected plants were cultivated under normal conditions of 25°C, 16 h light/8 h dark for 2 days, and then subjected to a 4°C for 6 h. Tobaccos under normal conditions were used as the control. Subsequently, the injected leaves were detached from tobacco and made small round pieces using for GUS staining and the expression level of pro-VvGRX28 determination. Yeast two-hybrid (Y2H) assay In Y2H assay, VvGRX28 protein was used as the bait, and the CDS of VvGRX28 was cloned into the pGBKT7 vector to gain the recombinant plasmid of pGBKT7-VvGRX28. The recombinant bait and pGADT7 empty vector plasmids were co-transformed into Y2HGold strain in synthetic dropout minimal base (SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade) to detect self-activating toxicity detection. Through the mating hybridization method, potential interacting proteins were screened from AD library related to cold stress which was conserved in the plant stress physiology laboratory. Mixed yeast solution was coated on SD/-Leu/-Trp/-His/-Ade medium and the plates were cultured at 30°C for 4–6 days. Finally, these plaques were transferred to SD/-Leu/-Trp/-His/-Ade + X-α-gal medium for further screening. The blue plaques were screened, and identified by PCR. After sequenced, the protein sequences were blasted in NCBI. The screened potential proteins sequences were cloned into pGADT7 to generate the prey construct. The prey and bait vectors were co-transformed in SD/-Leu/-Trp, SD/-Leu/-Trp/-His/-Ade and SD/-Leu/-Trp/-His/-Ade + X-α-gal to examinate interaction relationship between VvGRX28 protein and potential interaction proteins. pGBKT7-p53 + pGADT7-T and pGBKT7-Lam + pGADT7-T were used as positive and negative controls, respectively. The amplification primers are listed in Table S1 . LCI and BiFC assay LCI assay was conducted as described in the previous method [ 54 ]. The CDS fragment of VvGRX28 and VvZNF10 (deleted the stop codon) were cloned and inserted into pCAMBIA1300-cLUC and pCAMBIA1300-nLUC to gain the recombinants plasmids, respectively. Subsequently, the VvGRX28 -cLUC and VvZNF10 -nLUC recombinants plasmids were transformed into A.tumefaciens GV3101. 3-weeks-old tobaccos were used to injected with the combination of VvGRX28 -cLUC and VvZNF10 -nLUC, cLUC and VvZNF10 -nLUC, VvGRX28 -cLUC and VvZNF10 , cLUC and nLUC Agrobacterium suspension (volume ratio = 1:1) in the same leaf, resuspension solution was distilled water (1 M MES (pH = 5.6), 2.5 M MgCl 2 , 100 mM Acetosyringone). The injected tobaccos were incubated in the dark for 12 h, and then transferred in the light for 48 h. D-luciferin potassium salt (beyotime biotechnology, Shanghai, China) was applied to the injection sites, and the samples were kept in the dark for 5 min. LUC activity was measured using the FusionFX Vilber Lourma Imaging System (Vilber, France). The cDNA sequences of VvGRX28 and VvZNF10 were cloned into pC1300-mVYCE and pC1300-mVYNE vectors respectively to construct fusions expressing YFPN and YFPC protein. The fusion expression vectors plasmids were transformed into A.tumefaciens GV3101 and the mixture of two different plasmids ( VvGRX28 -YCE and VvZNF10 -YNE, VvGRX28 and VvZNF10 -YNE, VvGRX28 -YCE and VvZNF10 , YCE and YNE) of equal volume was trans-formed instantaneously into N. benthamiana leaves. Resuspension solution was as the same to LCI experiment. After in the dark for 12 h, the injected plants were cultured for 2 days at 28°C for 8-hour light/16-hourdarkness conditions, and the fluorescence of fluorescent protein signaling was observed in the area of tobacco leaves injected with Agrobacterium using a Laser confocal microscopy (LSM80, Zeiss, Germany) RNA extraction and RT-qPCR analysis The total RNA was extracted from the leaves and callus of grape, and Arabidopsis thaliana using RNAprep Pure Plant Plus Kit (DP441, TIANGEN biotechnology, Beijing, China) under different treatments, referring to the manual for detailed steps. Genes primers for RT-qPCR and cloning were designed and synthesized in the online software (the Shanghai Sangon Biotech Company). Extracted RNA was used as a template to synthesize cDNA using the prime reverse transcriptase Kit FastKing gDNA Dispelling RT SuperMix (KR118, TIANGEN biotechnology, Beijing, China). RT-PCR was performed by Light Cycler® 96 real-time PCR system (Roche, Switzerland). The VvGAPDH and AtActin gene were used as the reference genes, a sample of cDNA (3 µL) was subjected to RT-PCR in an eventual of 20 µL, which contained 1.6 µL primers (upstream primer was 0.8 µL, downstream primer was 0.8 µL ), 10 µL SYBR Green Master Mix Reagent (Takara Bio, Shiga, Japan) and 5.4 µL ddH 2 O. PCR amplification procedure was 40 cycles of 95°C for 30 s, 95°C for 10 s, 58°C for 30 s, 72°C for 20 s. The relative expression of each gene was analyzed by the 2 −ΔΔCt method. All RT-PCR analyses were performed with three biological repeats. All primers were listed in Supplementary Table S1 in this study. Statistical analysis All statistical data were analyzed by SPSS software (IBM, Chicago, IL, USA), and significance was evaluated at p < 0.05. The GraphPad Prism 10.1.2 software was used for mapping. Error bars indicate mean SE of three biological replicates. The relative expression of each gene was analyzed by the 2 −ΔΔCt method. Result Members identification, physicochemical properties and phylogenetic relationships of GRXs family Arabidopsis thaliana , rice, tomato, maize GRX protein sequences were used as the query to perform a BLASTP search against the grape protein database. As shown in Supplementary Table S2 , a total of 33 GRX genes containing GRX domain (PF00462) were obtained in the whole grape genome, and named VvGRX1 - VvGRX33 based on the position of each gene on the chromosome distribution. The analysis of protein physical and chemical properties showed that grape GRX gene family had some differences in amino acid number, molecular weight, isoelectric point, instability coefficient, GRAVY. The number of amino acids in grape GRX gene was 102 (VvGRX8) to 546 aa (VvGRX1), and the molecular weight was 11.06 to 62.94 kDa, which was consistent with the change trend of amino acids. The theoretical isoelectric points were 5.05 (VvGRX19) to 9.48 (VvGRX13), with an average of 7.13. Among them, 18 proteins were basic proteins (PI > 7.5) and 15 proteins were acidic proteins (PI < 7.5). When the instability index is less than 40, it is a stable protein, of which 75.76% VvGRX were unstable proteins. The aliphatic index ranged from 69.07 to 110.22. Hydrophilic GRX proteins accounted for 63.64% (GRAY 0), indicated that VvGRX family members are mainly hydrophilic proteins. The subcellular localization prediction indicated that grape GRX gene family proteins were mainly located in chloroplast (42.4%), cytoplasm (30.3%), mitochondrion (12.1%) and nucleus (9.1%). In order to explore the evolutionary relationship between grape and Arabidopsis, tomato and barley GRX genes, 132 GRX proteins were analyzed for phylogenetic evolution (Fig. 1A). The results showed that 132 GRX family genes could be divided into four subgroups, including CC type, CGFS type, CPYC type and GRL type. There were differences in species categories and the number of GRX genes in different subgroups. In addition, among grape GRX genes, the most of VvGRX family members in GRL type (14 VvGRX genes), the second in CC type (11 VvGRX genes), the third in CPYC type (5 VvGRX genes), and the least in CGFS type (3 VvGRX genes). CC type members account for a large proportion in the phylogenetic trees of the four species, and one branch was divided into two subbranches of monocotyledons and dicotyledons, indicating that CC type GRX forms new family members with the differentiation of monocotyledons and dicotyledons. In addition, according to the phylogenetic tree, the homology of VvGRX in dicotyledons is greater than that of monocotyledons, and barley has unique evolutionary branches, which indicating the genetic relationship of VvGRX genes are in line with the evolutionary relationship of plants. Structures, motifs, cis-elements and collinearity analysis of VvGRXs Through the analysis of the gene structure of VvGRX family members, there were certain differences in the VvGRX gene structure in different subfamilies (Fig. 1B). Among them, the 3 members of the CGFS subfamily had 2, 3, and 6 exons, and the CC subfamily had 11 exons. All members had 1 exon, 5 members of CPYC subfamily had 4 exons, and 14 members of GRL subfamily had 1 to 7 exons. The secondary structure prediction analysis showed that grape GRX proteins were mainly composed of α-helix, β-turn and random coil are three structural elements, of which random coil accounts for the largest proportion, followed by α-helix, β-turn proportion is relatively small (Fig. 1C). Subsequently, for analyze the diversity of VvGRX protein in grapes, the conserved motifs were analyzed using MEME online tool (Fig. 1B). The results showed that proteins clustered in one subfamily had similar motifs, and different motifs were distributed in different subfamilies. Motifs 1, 3, 6, and 7 were mainly distributed in CC, CPYC, and CGFS subfamilies, while motifs 2, 4, 5, 8, 9, and 10 were only distributed in GRL subfamily, the result suggesting that the presence of different motifs were necessary for the execution of VvGRX protein function. In addition, motif 1 involved the LxxLL active site, most of grape GRX (94%) had this motif. Motif 2 existed in all GRL types except VvGRX1 and VvGRX32 . Motif 3 contained the active CxxC site in GRX, and motif 6 is distributed in CGFS, CC and CPYC types. Motif 8, 9 and 10 were unique to VvGRX30 and VvGRX33 of GRL type, which was different from other motifs. The cis -acting elements can interact with the corresponding trans-regulators to regulate gene expression. The analysis of cis -acting elements in the VvGRX revealed that 39 cis -acting elements were identified (Fig. 1D), which could be divided into three categories, including growth and development, plant hormone responsive, abiotic and biotic stress responsive elements. There were 7 types of growth and development elements, namely AACA_motif, ARE, CAT-box, GCN4_motif, HD-Zip 1, O2-site and RY-element, which were related to anaerobic induction (ARE) and meristem expression (CAT-box), Zein metabolism (O 2 -site) is the most relevant. There are 10 kinds of plant hormone responsive elements, including ABRE, AuxRR-core, CGTCA-motif, GARE-motif, P-box, TATC-box, TCA-element, TGA-box, TGACG-motif and TGA-element, which are combined with abscisic acid (ABRE), methyl jasmonate (CGTCA-motif, TAGG-motif), gibberellin (P-box), salicylic acid (TCA-element) and other metabolic pathway-related elements. There were 21 kinds of biotic/abiotic stress-related elements, namely LTR, MBS, TC-rich repeats, ACE, AE-box, AT1-motif, ATCT -motif, Box 4, chs-CMA1a, GA-motif, Gap-box, GATA-motif, G-box, GT1-motif, GTGGC-motif, I-box, LAMP-element, MRE, Sp1, TCCC-motif and TCT-motif, which had low temperature (LTR), drought (MBS), defense and stress (TC-rich repeats) and light response (G-box, GT1-motif, BOX 4) components occupy an important position. Based on the above analysis, we speculated that VvGRX genes largely participated in regulating plant growth, development and abiotic stress response. In order to better understand the collinearity mode of grape GRX gene, collinearity analysis was carried out on grape GRX gene. It was found that there were 5 pairs of genes in the family with collinearity, including VvGRX2 and VvGRX27 , VvGRX8 and VvGRX11 , VvGRX9 and VvGRX24 , VvGRX20 and VvGRX23 , VvGRX28 and VvGRX31 (Fig. 1E). At the same time, the result found that the genes with collinearity had close phylogenetic relationship. The analysis examined the collinear interactions among members of the VvGRX gene family and GRX genes in other species (Fig. 1F). Representative species were selected, including A. thaliana and Solanum lycopersicum . In total, the analysis of collinear maps revealed 29 collinear blocks in A. thaliana and grape, 26 collinear blocks in tomato and grape. Greater VvGRX collinearity was observed in A. thaliana. The result supported the hypothesis that the VvGRX gene family is more closely related to GRXs in A. thaliana. Tissue-specific expression profile of VvGRXs under drought, salt and cold stress The expression levels of GRX genes in grape were analyzed in different tissues during the different developmental stages. The results showed that the expression levels of 33 VvGRX genes had significantly differences in different grape plant tissues of different development stages (Fig. 2A). VvGRX1 , VvGRX17 , VvGRX18 , VvGRX19 , VvGRX20 , VvGRX22 , VvGRX23 , VvGRX26 , VvGRX29 , VvGRX32, and VvGRX33 gene showed relatively high expression level in all grape tissues, such as tendrils, stems, buds, roots, flowers, leaves, seeds, seedlings, and other organs, other genes are expressed specifically in some tissues. VvGRX1 was expressed specifically in leaf, VvGRX6 was expressed specifically in bud, VvGRX1 was expressed specifically in rachis. Figure 1 The evolutionary relationship and sequences analysis of GRX genes in grape. (A) Phylogenetic analysis of GRX genes in Vitis vinifera , Arabidopsis thaliana , Solanum lycopersicum , and Hordeum vulgare . (B) The structure and motif analysis of GRX genes, yellow column represents CDS, purple column represents upstream/downstream, black line represents intron, the motifs number was set to 10. (C) The secondary structure prediction of VvGRX protein, mainly including alpha helix, beta turn, random coil. (D) Cis -acting regulatory element predictions of VvGRXs at the 2000 bp upstream of VvGRX, divided into growth and development, hormones, and abiotic and biotic stress responsive elements. (E) The synteny analysis of VvGRXs in grape, the collinear blocks within the genomes of grape were marked with gray lines, while the syntenic VvGRX gene pairs are highlighted in red. (F) The comparative analysis of the VvGRX gene synteny between grape and two other representative plant species, the collinear blocks within the genomes of grape and other species are marked with gray lines, while the syntenic VvGRX gene pairs are highlighted in red. The expression pattern analysis of VvGRXs family members under drought, salt and cold stress Based above tissue-specific expression analysis, a total of 11 gene were selected, which were highly expressed in almost all grape tissues, including VvGRX1 , VvGRX17 , VvGRX18 , VvGRX19 , VvGRX20 , VvGRX22 , VvGRX23 , VvGRX25 , VvGRX28 , VvGRX32 , and VvGRX33 . To preliminary explore the expression pattern of VvGRX gene family under abiotic stress, the grape plantlets in vitro were subjected to NaCl, drought and cold stress. As shown in (Fig. 2B), RT-PCR analysis showed that relative expression levels of VvGRXs were significantly upregulated than control, of which VvGRX19, VvGRX20, VvGRX32 gene were highly expressed about 10–30 than control in grape, the expression of VvGRX32 was the highest under drought stress. The expression levels of VvGRX1 , VvGRX18 , VvGRX20 and VvGRX32 gene were relatively high, VvGRX20 gene was the highest under slat stress. The expression levels of VvGRX17 , VvGRX18 , VvGRX23 , VvGRX28 and VvGRX32 were relatively high, VvGRX28 gene was the highest under cold stress. Taking into account the overall expression level of the GRX gene under salt, drought and cold stress, we find that the expression levels of VvGRX gene were highly expressed under the cold treatment than salt and drought stress. Therefore, VvGRX28 gene with high expression level was selected to further explore cold function in grape. VvGRX28 could be a very important responsive gene in regulating cold tolerance in grape. Figure 2 Gene chip and RT-PCR analysis under different tissues and stresses. (A) The tissue-specific expression profile analysis heatmap of VvGRXs, the redder the square color is, the higher the expression level is. (B) The relative expression levels of VvGRXs in grape leaves under drought, salt and cold stress, VvGRX28 was selected for the function verification in cold tolerance. Overexpression of VvGRX28 improved cold tolerance in transgenic Arabidopsis thaliana To further understand cold response pattern of VvGRX28 in grape, different low temperature treatment times were set. RT-PCR analysis showed the expression level of VvGRX28 was upregulated with the elongation of the processing time, and up to a maximum in 72 h (Fig. 3A). For cold function verification of VvGRX28 , the CDS fragment of VvGRX28 was successfully clone (Fig. 3B) and inserted into the pART-CAM-EGFP plant overexpression vector driven by the CaMV35S (Fig. 3C). The subcellular localization analysis indicated VvGRX28 was located in nucleus and cell membrane with a significant green fluorescence signal in tobacco epidermal cells (Fig. 3D). Owing to the constraints of grape genetic transformation, VvGRX28 was heterologously transformed into Arabidopsis thaliana by floral dip method. Transgenic plants were screened in 1/2 MS plate medium (50mg/mL Kan), green plants were planted in the substrate containing a mixture of soil and vermiculite, harvested seeds after maturity (Fig. 3E). Transgenic Arabidopsis thaliana was further identified by PCR (Fig. 3F). Subsequently, T3 homozygous and stable lines (OE3, OE8, OE13) were used to carry out cold stress assay. WT and three overexpression stable T3 lines (OE3, OE8, OE13) were subjected to -6 ℃ cold stress for 5 h, and then placed in normal growth temperature 23 ℃. After the recovery of 12 d, phenotypic observation result showed most WT plants leaves showed severely wilting, yellow and even death, while the growth status of VvGRX28 overexpressed plants were significantly better than that of WT plants (Fig. 4A). Under − 4 ℃ cold stress for 5 h, the REL of transgenic lines was relatively lower than that of WT plants, indicating that the damage degree of transgenic lines cell membrane was weak than that of WT (Fig. 4B). MDA and H 2 O 2 content of OEs was decreased than WT, indicating a slight degree of membrane lipid peroxidation (Fig. 4C, Fig. 4E). Under cold stress conditions, plant tissues produce various ROS, H 2 O 2 and O 2 . − is two main types of ROS. DAB and NBT staining were used to evaluate the content of H 2 O 2 and O 2 . − in leaves. Hydrogen peroxide can rapidly react with DAB to form a brown compound under the catalysis of peroxidase, thereby locating hydrogen peroxide in leaves. Oxygen free radicals can reduce NBT to insoluble blue methazan compounds, thereby locating superoxide anion in leaves. Under cold stress, WT and OE plants can produce a lot of H 2 O 2 and O 2 . − compared with the normal temperature. DAB and NBT staining result showed the brown and blue color of the OE leaves were relatively light than WT leaves under cold stress, demonstrating that OE plants suffered relatively minor damage from the low temperature (Fig. 4D). Trypan blue staining indicated that the blue areas of leaves were small in OE than WT, indicating the number of dead cells was little (Fig. 4D). Furthermore, the content of H 2 O 2 and POD activity were determined. The result showed the content of H 2 O 2 and POD activity of OE lines were relatively high than that of WT, maintaining the oxidative balance in plants and decreasing the production of ROS (Fig. 4E and Fig. 4I). Under normal growth conditions, there was no significant difference in proline (Fig. 4F), soluble sugar (Fig. 4G) and glutathione (Fig. 4H) content between OEs and WT plants, while the content of proline, soluble sugar and glutathione were increased in OE plants under cold stress. Overall, the overexpression of VvGRX28 enhanced the cold tolerance by promoting the ability to removing ROS, maintaining cell membrane integrity and increasing the contents of osmotic adjustment substances in transgenic Arabidopsis thaliana . The overexpression of VvGRX28 has led to a series of physiological changes in Arabidopsis thaliana , while gene expression of some key cold responsive genes was unknown. In this study, the expression of some key cold responsive genes which includes inducer of CBF expression 1 ( AtICE1 ), inducer of CBF expression 2 ( AtICE2 ), cold-regulated 15A ( AtCOR15A ), C-repeat Binding Factor 1 ( AtCBF1 ) and C-repeat Binding Factor 2 ( AtCBF2 ) was detected in WT and OE plants of Arabidopsis thaliana under low temperature stress. AtICE , AtCOR and AtCBF genes are the core associated gene group of cold stress response in Arabidopsis thaliana , together forming the ICE-CBF-COR signaling axis, regulates the adaptive response of plants to cold stresses. The three undergo a cascade reaction of transcriptional activation - gene expression - functional execution. AtActin gene was used as intern reference gene to calculate relative expression levels. RT-PCR analysis indicated that the relative expression levels of VvGRX28 , AtICE1 , AtICE2 , AtCOR15A , AtCBF1 and AtCBF2 were significantly upregulated in transgenic plants by induced by cold stress (Fig. 4J). Altogether, the finding demonstrated that VvGRX28 gene could actively response to cold stress by regulating some key cold gene expression levels. Figure 3. The relative expression level under cold stress, subcellular localization and heterologous expression of VvGRX28 . (A) The expression pattern of VvGRX28 in grape leaves under cold stress at different time point. (B) The clone of VvGRX28 , fragment size was 345 bp, and marker was 2000bp. (C) Overexpression vector schematic diagram of VvGRX28 , the vector carried the EGFP (enhanced green fluorescent protein) tag. (D) The subcellular localization of VvGRX28 in Nicotiana tabacum . 35s::EGFP was used positive control, DAPI was nuclear location marker. (E) Screening of transgenic Arabidopsis thaliana in plates (1/2 MS with 50 mg/L Kana), green plants were cultivated until harvested seed. (F) Transgenic Arabidopsis thalianas were identified by PCR, marker was 2000bp, T3 plants were used to verify cold function. Figure 4 Function analysis of VvGRX28 overexpression under cold stress in Arabidopsis thalianas . (A) Phenotypic observation of VvGRX28 overexpression under cold stress in Arabidopsis thalianas , 23 ℃ as control. (B) Relative electrolytic leakage of OE and WT plants. (C) MDA content of OE and WT plants. (D) Histochemical stain of OE and WT plant leaves, from left to right DAB, NBT and trypan blue, respectively. DAB, NBT and trypan blue staining were employed to evaluate the content of H 2 O 2 , O 2 . − and death cells in leaves respectively, indicating the degree of cell damage under cold stress. (E) H 2 O 2 content of OE and WT plants. (F) Proline content of OE and WT plants. (G) Soluble sugar content of OE and WT plants. (H) Glutathione content of OE and WT plants. (I) Pod activity of OE and WT plants. (J) The relative expression levels of VvGRX28 and cold responsive gene, including AtICE1 , AtCOR15A , AtCBF1 , AtCBF2. Data are expressed as the mean ± standard error of three independent biological replicates. Different asterisks indicate significant differences, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001. VvGRX28 and VvZNF10 proteins exists an interaction relationship The initial function verification showed that VvGRX28 gene plays a crucial role in responding and adapting to low temperatures by heterologous transformation. For detecting the promoter activity of VvGRX28 , pro VvGRX28 -GUS, 35S::GUS and P0-GUS agrobacterium solution were injected into tobacco leaves, GUS staining showed the blue color became more prominent, indicating GUS activity was higher than the controls under cold stress, and the lower the temperature, the higher the GUS activity (Fig. 5A). Relative expression of pro VvGRX28 were upregulated with decreasing of cold stress temperature (Fig. 5B). To further gain a thorough understanding of the regulatory mechanism of VvGRX28 , Y2H technique was employed to find the potential interaction protein of VvGRX28 protein. VvGRX28, as a bait protein, was cloned and linked to pGBKT7 vector. Recombinant plasmid of VvGRX28 and empty pGADT7 plasmid were co-transformed into Y2Hgold yeast strain in SD/-Trp-Leu and SD/-Trp-Leu-Ade-His medium to detect self-activating toxicity. Result showed there is no self-activating toxicity of VvGRX28 protein compared the controls (Supplementary Fig. S1 ). Through screen of mating hybrid from AD library related to cold, AD library and Y2Hgold of BD-VvGRX28 yeast mixed liquid were coated in plates with SD/–Trp/–Leu/–His/–Ade medium. After grew for 3–4 d, single clonal plaques were spread in plates with SD/–Trp/–Leu/–His/–Ade + X-α‐gal. Blue monoclonal yeast plaques were selected for PCR identification and sequence (Supplementary Fig. S2 ). After sequenced successfully, sequences were blast in NCBI, potential interaction proteins which includes VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19 were used to verify their interaction relationship by point to point. VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19, as prey proteins, were cloned and inserted into pGADT7. pGADT7-VvSRC2, VvZNF10, VvRPSA, Vv2OGDD19-pGADT7 and VvGRX28-pGBDT7 plasmid were co-transformed into Y2Hgold and then transferred in SD/–Trp/–Leu/–His/–Ade and SD/–Trp/–Leu/–His/–Ade + X‐α‐gal. Result showed that VvSRC2, VvRPSA and Vv2OGDD19 couldn’t growth in DDQ, only VvZNF10 normally grew and became blue in DDQ + X‐α‐gal, indicating VvZNF10 and VvGRX28 existed interaction relationship with VvGRX28 in vitro (Fig. 5C). The interaction between VvGRX28 and VvZNF10 proteins was further verified by BiFC and LCI LCI and BiFC experiments were employed to further confirm the interaction between VvZNF10 and VvGRX28 proteins. The CDS of VvZNF10 and VvGRX28 (delete stop codon) were cloned and linked to PCAMBIA1300-cLUC and PCAMBIA1300-nLUC, respectively. The Agrobacterium liquid with VvGRX28-nLUC and VvZNF10-cLUC (V:V = 1:1) were transiently expressed in tobacco leaves. The strong luciferase complementation fluorescence signals were observed in tobacco cells co-transformed with VvGRX28-nLUC and VvZNF10-cLUC (Fig. 5D). Negative controls with nLUC and cLUC, nLUC and VvZNF10-cLUC, VvGRX28-nLUC and cLUC had no luciferase complementation, thereby confirming the two proteins interactions. Additionally, we constructed fusion proteins of VvZNF10 with the N-terminal half of yellow fluorescent protein (mYNE) and VvGRX28 (delete stop codon) with the C‐terminal half of YFP (mYCE). The Agrobacterium liquid with VvGRX28-mYCE and VvZNF10-mYNE (V:V = 1:1) were transiently expressed in tobacco leaves. A stronger YFP fluorescence signals were obseved in nucleus and cell membrane in tobacco cells co-transformed with VvGRX28-mYCE + VvZNF10-mYNE (Fig. 5E). Negative controls with VvGRX28-mYCE + mYNE and mYCE + VvZNF10-mYNE had no yellow fluorescence signals, thus confirming the two proteins interactions in vivo. Overall, these results indicated that VvGRX28 interacted with VvZNF10 in vivo . Figure 5 Analysis of pro VvGRX28 transcriptional activity and the interaction verification between VvGRX28 and VvZNF10 by Y2H, LCI and BiFC. (A) The GUS staining of pro VvGRX28 in Nicotiana tabacum under cold stress. 35s-GUS was the positive control. P0-GUS was the negative control. (B) The relative expression levels of pro VvGRX28 under cold stress, NtGUS were used as the internal gene. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences at p < 0.05. (C) Yeast two-hybrid (Y2H) assay demonstrates that VvGRX28 interacts with VvZNF10 in Y2Hgold yeast. The interaction verification of VvGRX28 and potential interaction protein (VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19) by point to point in SD/–Trp/–Leu/–His/–Ade and SD/–Trp/–Leu/–His/–Ade + X-α‐gal, pGBKT7-p53 + pGADT7-T and pGBKT7-Lam + pGADT7-T were used as positive and negative controls, respectively. Co-transformation of VvZNF10 and VvGRX28 can normally grew and become blue in DDQ + X‐α‐gal, indicating VvZNF10 and VvGRX28 protein exist interaction relationship. (D) Luciferase complementarity assay showing that fluorescence signal was only observed in sites co-injected with VvGRX28-nLUC and VvZNF10-cLUC in tobacco cell. (E) BiFC assay shows that VvGRX28 interacts with VvZNF10 mainly in the nucleus. The subcellular localization and expression pattern analysis of VvZNF10 under cold stress in grape ZNF gene was a C2H2-type zinc fingers protein functioning as a transcriptional regulator in plant growth and development. VvZNF10 with 912 bp size was located in 8th chromosome, which had ZnF_C2H2 domain (Fig. 6 A). VvZNF10 (with EGFP tag) was cloned and transformed into pART-CAM-EGFP. Compared with the control, a strong green fluorescence signal was observed in tobacco cells, indicating VvZNF10 was located in nucleus (Fig. 6 B). For clarifying expression pattern of VvZNF10 under cold stress in grape, RT-PCR were employed to detect its expression. RT-PCR analysis showed that under cold stress, the relative expression of VvZNF10 was significantly upregulated, and expression level showed a peak at 48 h (Fig. 6 C). We suggested that VvZNF10 might be a positive regulator in response to low temperature. VvGRX28 improved cold tolerance by interacted with VvZNF10 in grape callus To further investigate the cold regulation mechanism of VvGRX28 and VvZNF10 in grape callus, overexpression vector of VvGRX28 (with EGFP tag) and VvZNF10 (with Flag tag), and RNAi vector of VvGRX28 were constructed. Transgenic calluses were obtained by Agrobacterium -mediated genetic transformation and were identified by PCR amplify (Supplementary Fig. 3), namely OE- VvGRX28 , OE- VvGRX28/VvZNF10 , RNAi- VvGRX28 (RNAi transformation based on co-transformed of OE- VvGRX28/VvZNF10 ). Transgenic calluses were subjected to 4 ℃ for 15 d. As shown in Fig. 6 E, compared with WT and only overexpression VvGRX28 , co-transformed calluses of OE- VvGRX28/VvZNF10 had a better and quick growth status, after interfered expression of VvGRX28 , growth of transformed calluses were inhibited under normal and cold stress condition. Under cold stress, all type calluses occurred browning appearance, especially severed in WT and RNAi. The fresh weight of all type calluses was decreased, indicating water loss after cold stress. The fresh weight of transformed calluses of OE- VvGRX28 and OE- VvGRX28/VvZNF10 was higher than WT and RNAi callus, suggesting a low water loss rate under cold stress (Fig. 6 G). Under cold stress, the content of MDA (Fig. 6 H) and H 2 O 2 (Fig. 6 L) in co-transformed calluses of OE- VvGRX28/VvZNF10 was decreased, while after interfering VvGRX28 sequence, the two oxidation indexes content was increased. Additionally, the content of proline (Fig. 6 I), soluble sugar (Fig. 6 J) and glutathione (Fig. 6 K), POD activity (Fig. 6 M) in co-transformed calluses of OE- VvGRX28/VvZNF10 was significantly higher than other calluses type. RT-PCR analysis indicated that VvZNF10 expression levels of co-transformed callus was significantly upregulated under cold stress, and the relative expression levels of VvGRX28 , VvICE1 , VvICE2 , VvCOR413 , VvCBF1 and VvCBF2 were also significantly upregulated in overexpressed callus induced by cold stress, while RNAi callus was downregulated under cold stress (Fig. 6 N). Comprehensive above findings, these results demonstrated that VvGRX28 promoting cold tolerance by interacted with VvZNF10 in grape callus. Discussion Plants are subjected to a wide array of biotic and abiotic stress conditions, which trigger signal transduction pathways and elicit molecular, metabolic, and physiological responses to regulate the ability to adapt various environment[ 55 , 56 ]. This is particularly concerning in the context of a growing global population, especially as low temperature constrains plant productivity and yield. ROS, as part of the signal transduction pathways, refers to a class of highly reactive oxygen-containing molecules, including superoxide anions, hydrogen peroxide, hydroxyl radicals, which participates in various physiological processes in the body, but in excessive ROS can cause oxidative stress and damage cells[ 57 ]. In order to maintain the oxidative homeostasis of cells, the antioxidant system and oxidative-reductive system composed of proteins that can transfer electrons from the input component to the downstream target protein remove excessive ROS. These mediators belong to a large family of oxidative-reductive protein enzymes in plants, including so-called oxidoreductase, including thioredoxins (TRXs), TRX-like proteins, and glutaredoxins (GRXs)[ 8 , 11 ]. GRX is a small disulfide oxidoreductase that catalyzes the reversible reduction of disulfide bridges and glutathione (GSH)-containing disulfides using glutathione as the electron donor[ 58 ]. GRX proteins by coded by GRX genes that play a very important in removing excessive ROS to adapt to constantly changing survival environment. Previous researches have been reported GRX gene family members numbers in 31 Arabidopsis [ 59 ], 48 rice[ 13 ], 58 tomato[ 60 ], and 86 wheat[ 31 ]. In this study, 32 gene family members were identified in grape. VvGRXs were classified into four clades including CPYC, CGFS, and CC subfamilies by phylogenetic tree analysis, consistent with previous results in other species[ 61 – 63 ]. Most members of the same subfamily had similar motif compositions and gene structural features, indicating that these members may have similar evolution relationship and functions. VvGRX28 was clustered in CPYC type, and had the similar structure and motifs with VvGRX31 , demonstrating that an extremely close relationship in function and evolution. Structure differences between the four clades were observed, of which CC-type VvGRX genes were mainly characterized by intron-less genes. Consistently, the genomic feature type genes that could quickly respond to various stresses, as also revealed for other genes with fewer exons[ 64 , 65 ]. Additionally, complex structures in exon/intron and motifs composition were found within CPYC-, GRL- and CGFS-types, indicating that these genes might be subjected to fine-tuning regulatory events such as alternative splicing (AS)[ 66 ]. Although there existed significant differences in structure composition, the highly conserved motif distribution pattern between CC and CPYC-types demonstrated the fact that CC-type gene might have evolved from the CPYC clade, before undergoing gene expansion during land plant evolution[ 67 ]. Interestingly, among the 5 VvGRX gene pairs that were co-linear in grape, and the two genes with synteny had the same branch in phylogenetic tree and similar structures. Consistently, previous studies in reported species found that GRX members in same subfamily exhibited similar motifs and structure arrangements, but the motif patterns differed across subgroups[ 68 ]. These results suggested that GRX genes exhibited highly conserved and variable motif and structure patterns in different subfamilies, indicating functional conservation and diversification. Upstream genes could bound to the cis -regulatory element in the promoter of target genes to regulate the expression of the target genes to perform specific biological functions. cis -regulator elements of VvGRX gene promoters had been analyzed and divided into three categories, including growth and development, plant hormone responsive, abiotic and biotic stress responsive elements. Core stress-responsive elements had ABRE, MYB, and W-box in GRX promoters, indicating evolutionary conservation of stresses regulation. Similarly, the unique fiber development-associated motifs of GRX and stress responsive elements were found in cottons, playing its specialized role in cotton fiber formation[ 61 ]. Common bean of GRX exhibited nodulation-specific motifs, associating with the symbiotic nitrogen-fixing capacity[ 62 ]. Banana had fruit ripening-related and responsive elements regulatory elements promoting fruit mature and regulating stress tolerance[ 69 ]. ROXY1 and ROXY2 gene could regulate anther development[ 22 ]. The OsGRXC2.2 gene overexpression promoted embryonic development to increase grain weight in rice[ 15 ]. These results speculated that VvGRX genes maybe play important roles in hormone regulation, morphogenesis, and stress response in grape. Tissue-specific expression analysis revealed distinct developmental patterns for the VvGRXs, of which VvGRX1, VvGRX17, VvGRX18, VvGRX19, VvGRX20, VvGRX22, VvGRX23, VvGRX26, VvGRX29, VvGRX32 , and VvGRX33 gene showed relatively high expression level in all grape tissues, especially highly expressly in root, stem and leaves. Consistent with the finding, TaGRX73-7D exhibited high expression levels in leaves, while TaGRX29-3B was highly expressed in root tissues[ 31 ]. The expression levels of GRX were relatively higher in leaves in Quercus glauca[ 32 ]. Additionally, previous studies have reported that GRX genes play important roles in response to stresses. RT-PCR analysis indicated expression of VvGRX s were different induced by salt, drought and low temperature. The expression levels of VvGRX17 , VvGRX20 and VvGRX32 were all upregulated salt, drought and low temperature stress. The expression of VvGRX32 was the highest under PEG stress, the expression of VvGRX20 was the highest under salt stress, the expression of VvGRX28 was the highest under cold stress. In others species, there were numerous GRX genes participating in response to various stresses with similar function of VvGRXs . Consistently, there were similar findings in previous studies. OsGRX20 gene could positively regulate plant responses to bacterial and fungal attacks, and overexpression of OsGRX20 significantly enhanced resistance to bacterial blight and ability to oxidative and salt stresses[ 16 ]. The Silencing of OsGRXS17 improves drought stress tolerance by removing excessive ROS and stomatal closure in rice[ 70 ]. The silencing of GhGRL28 improved the sensitivity to salt stress in cotton[ 39 ]. Most researches on abiotic stress focused on drought and salt, the function analysis of GRX genes was limited under cold stress. Therefore, VvGRX28 was cloned to investigated its role under cold stress. Initial findings showed the result demonstrated that the VvGRX28 overexpression enhanced the content of proline, soluble sugar, glutathione and peroxidase activities, and reduced the content of MDA and H 2 O 2 , and upregulated the expression of AtICE , AtCBF and AtCOR related to cold stress in transgenic Arabidopsis thaliana . The findings was similar with the works that BrMDHAR heterologous overexpression improved cold tolerance in the mini Chinese cabbage[ 71 ]. VvZNF10 could interacted with VvGRX28 by Y2H, LCI and BiFC in vivio and vitro. VvZNF10 was a C2H2 type zinc finger protein composed of 2 Cys and 2 His residues surrounding Zn 2+ , widely distributed in eukaryotes, and occupied in response to various stresses. It has been reported that in response to low-temperature stress, for instance, the overexpression of OsZFP245 can enhance the tolerance of rice to low temperature, drought and oxidative stress[ 72 ]. Overexpression of OsCTZFP8 , a novel zinc finger protein was found to enhance cold tolerance in transgenic rice[ 73 ]. A line with previous study, further investigation demonstrated that the overexpression VvGRX28 could improve the ability to resist low temperature by interacting with VvZNF10 in transgenic grape callus, while interfered VvGRX28 exhibited a opposite result. Similarly, previous studies also indicated that the OsGRX15 protein improved disease resistance to bacterial and fungal pathogens interacting with the transcription factor OsWRKY65 in the nucleus by upregulating the expression of the defense-related gene OsPR1 [ 74 ]. QgROXY1 could physically interact with AtTGA2 , and transgenic Arabidopsis thaliana ectopically overexpressing QgROXY1 is hypersensitive to exogenously applied salicylic acid[ 32 ]. Taken together, the findings suggested that VvGRX28 was a regulator gene in contributing to cold stress tolerance regulation in grape. However, the results only preliminary verified the cold function in ectopically Arabidopsis thaliana and grape callus levels not in grape plant, the specific molecular mechanisms regulating these putative VvGRX-mediated stress responses require further investigation. Conclusion As crucial oxidoreductases, plant GRXs play vital roles in developmental regulation and stress responses. In this study, we successfully identified 32 VvGRX genes family members in grape. Comprehensive bioinformatics analyses revealed that the structure, motifs, cis -acting elements of VvGRX were similar in genes clustered into close branches. Most CC-type genes were less introns and highly conserved. Cis -acting elements mainly were involved in stress response and hormone regulation. VvGRXs with a high expression level almost in all tissues were induced by salt, drought and cold stress to differentially express. Furthermore, VvGRX28 were functionally characterized to detect the cold tolerance function. The result suggested that the VvGRX28 , as positive regulator, could improve the ability to resist low temperature by interacting with VvZNF10 in nucleus and cytomembrane in transgenic Arabidopsis thaliana and grape callus (Fig. 7 ). Collectively, the findings provide important insights into the redox regulatory mechanisms mediated by VvGRXs in grape, contributing to provide candidates genes for functional validation to future breeding for grape cold stress improvement. Declarations Acknowledgements Not applicable. Author contributions Conceptualization: GJN, SL, BHC and SM Methodology: GJN, ZHP, JRZ, ZLL, XXQ validation: CCZ, LM and HKY Data curation: formal analysis: GJN, SL, BHC Software: GJN, JRZ, ZLL, XXQ Investigation: ZHP, JRZ Writing—original draft: GJN. Writing—review & editing: GJN, SL, BHC and SM. Funding This research was supported by the Project funded by the Central Government - Guided Local Scientific and Technological Development Funds (25ZYJA033), Gansu Provincial Key Talent Project (2023RCXM23), and Gansu Province Graduate Innovation Star Program (2025CXZX-803). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. 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(A) Phylogenetic analysis of \u003cem\u003eGRX\u003c/em\u003e genes in \u003cem\u003eVitis vinifera\u003c/em\u003e,\u003cem\u003e Arabidopsis thaliana\u003c/em\u003e,\u003cem\u003eSolanum lycopersicum\u003c/em\u003e, and \u003cem\u003eHordeum vulgare\u003c/em\u003e. (B) The structure and motif analysis of \u003cem\u003eGRX \u003c/em\u003egenes, yellow column represents CDS, purple column represents upstream/downstream, black line represents intron, the motifs number was set to 10. (C) The secondary structure prediction of VvGRX protein, mainly including alpha helix, beta turn, random coil. (D) \u003cem\u003eCis\u003c/em\u003e-acting regulatory element predictions of VvGRXs at the 2000 bp upstream of VvGRX, divided into growth and development, hormones, and abiotic and biotic stress responsive elements. (E) The synteny analysis of VvGRXs in grape, the collinear blocks within the genomes of grape were marked with gray lines, while the syntenic VvGRX gene pairs are highlighted in red. (F) The comparative analysis of the VvGRX gene synteny between grape and two other representative plant species, the collinear blocks within the genomes of grape and other species are marked with gray lines, while the syntenic VvGRX gene pairs are highlighted in red.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/c122f05552663d73f04af9d0.png"},{"id":98187006,"identity":"a95c43ab-9f86-440a-a4e8-0b39309879bc","added_by":"auto","created_at":"2025-12-15 03:50:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11184373,"visible":true,"origin":"","legend":"\u003cp\u003eGene\u003cstrong\u003e \u003c/strong\u003echip\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eRT-PCR analysis under different tissues and stresses. (A) The tissue-specific expression profile analysis heatmap of VvGRXs, the redder the square color is, the higher the expression level is. (B) The relative expression levels of VvGRXs in grape leaves under drought, salt and cold stress, \u003cem\u003eVvGRX28\u003c/em\u003e was selected for the function verification in cold tolerance.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/6b1e5b502de655c47306990b.png"},{"id":98187013,"identity":"a36a7ac5-c609-4c27-866c-7c5bfb50baf5","added_by":"auto","created_at":"2025-12-15 03:50:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11475738,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression level under cold stress, subcellular localization and heterologous expression of \u003cem\u003eVvGRX28\u003c/em\u003e. (A) The expression pattern of \u003cem\u003eVvGRX28 \u003c/em\u003ein grape leaves under cold stress at different time point. (B) The clone of \u003cem\u003eVvGRX28\u003c/em\u003e, fragment size was 345 bp, and marker was 2000bp. (C) Overexpression vector schematic diagram of \u003cem\u003eVvGRX28\u003c/em\u003e, the vector carried the EGFP (enhanced green fluorescent protein) tag. (D) The subcellular localization of VvGRX28 in \u003cem\u003eNicotiana tabacum\u003c/em\u003e. 35s::EGFP was used positive control, DAPI was nuclear location marker. (E) Screening of transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e in plates (1/2 MS with 50 mg/L Kana), green \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cbr\u003e\n plants were cultivated until harvested seed. (F) Transgenic \u003cem\u003eArabidopsis thalianas\u003c/em\u003e were identified by PCR, marker was 2000bp, T3 plants were used to verify cold function.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/8cee5395ee114e6f9dc8666a.png"},{"id":98430623,"identity":"5a5179e1-b6fe-4e61-b686-466858f2397e","added_by":"auto","created_at":"2025-12-17 16:45:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10376856,"visible":true,"origin":"","legend":"\u003cp\u003eFunction analysis of \u003cem\u003eVvGRX28\u003c/em\u003e overexpression under cold stress in \u003cem\u003eArabidopsis thalianas\u003c/em\u003e. (A) Phenotypic observation of \u003cem\u003eVvGRX28\u003c/em\u003e overexpression under cold stress in \u003cem\u003eArabidopsis thalianas\u003c/em\u003e, 23 ℃ as control. (B) Relative electrolytic leakage of OE and WT plants. (C) MDA content of OE and WT plants. (D) Histochemical stain of OE and WT plant leaves, from left to right DAB, NBT and trypan blue, respectively. DAB, NBT and trypan blue staining were employed to evaluate the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e and death cells in leaves respectively, indicating the degree of cell damage under cold stress. (E) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content of OE and WT plants. (F) Proline content of OE and WT plants. (G) Soluble sugar content of OE and WT plants. (H) Glutathione content of OE and WT plants. (I) Pod activity of OE and WT plants. (J) The relative expression levels of \u003cem\u003eVvGRX28 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003ecold responsive gene, including \u003cem\u003eAtICE1\u003c/em\u003e, \u003cem\u003eAtCOR15A\u003c/em\u003e, \u003cem\u003eAtCBF1\u003c/em\u003e, \u003cem\u003eAtCBF2.\u003c/em\u003e Data are expressed as the mean ± standard error of three independent biological replicates. Different asterisks indicate significant differences, ** represents \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** represents \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, **** represents \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/93416f8006d0c60d6dab9497.png"},{"id":98187018,"identity":"be93767e-463b-42f4-b297-459692c9d36e","added_by":"auto","created_at":"2025-12-15 03:50:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28254037,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of pro\u003cem\u003eVvGRX28\u003c/em\u003e transcriptional activity and the interaction verification between VvGRX28 and VvZNF10 by Y2H, LCI and BiFC. (A) The GUS staining of pro\u003cem\u003eVvGRX28 \u003c/em\u003ein \u003cem\u003eNicotiana tabacum\u003c/em\u003e under cold stress. 35s-GUS was the positive control. P0-GUS was the negative control. (B) The relative expression levels of pro\u003cem\u003eVvGRX28 \u003c/em\u003eunder cold stress,\u003cem\u003e NtGUS\u003c/em\u003e were used as the internal gene. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. (C) Yeast two-hybrid (Y2H) assay demonstrates that VvGRX28 interacts with VvZNF10 in Y2Hgold yeast. The interaction verification of VvGRX28 and potential interaction protein (VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19) by point to point in SD/–Trp/–Leu/–His/–Ade and SD/–Trp/–Leu/–His/–Ade + X‐α‐gal, pGBKT7-p53 + pGADT7-T and pGBKT7-Lam + pGADT7-T were used as positive and negative controls, respectively. Co-transformation of VvZNF10 and VvGRX28 can normally grew and become blue in DDQ + X‐α‐gal, indicating VvZNF10 and VvGRX28 protein exist interaction relationship. (D) Luciferase complementarity assay showing that fluorescence signal was only observed in sites co-injected with VvGRX28-nLUC and VvZNF10-cLUC in tobacco cell. (E) BiFC assay shows that VvGRX28 interacts with VvZNF10 mainly in the nucleus.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/1013747da8a2708c93cc07a5.png"},{"id":98430922,"identity":"3c40bd8b-fcc4-40dd-8107-530fbd676831","added_by":"auto","created_at":"2025-12-17 16:46:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12271195,"visible":true,"origin":"","legend":"\u003cp\u003eVvGRX28\u003cem\u003e \u003c/em\u003eimproved cold tolerance by interacted with VvZNF10 in grape callus. (A) The distribution of chromosomal location and domain of VvZNF10. (B) The location vector schematic diagram of VvZNF10, the vector carried the EGFP (enhanced green fluorescent protein) tag. (C) The subcellular localization of VvZNF10 in tobacco. (D) The expression pattern of \u003cem\u003eVvZNF10 \u003c/em\u003ein grape leaves under cold stress at different time point. (E) Phenotypic observation of overexpression\u003cem\u003e VvGRX28\u003c/em\u003e, co-transformation with overexpression\u003cem\u003e VvZNF10\u003c/em\u003e and RNAi \u003cem\u003eVvGRX28 \u003c/em\u003eunder cold stress in grape callus, 25 ℃ as control. (F) The relative expression levels of \u003cem\u003eVvZNF10 \u003c/em\u003ein WT and OE callus. Measurement of physiological indicators: (G) The fresh weight; (H) MDA content; (I) Proline content; (J) Soluble sugar content; (K) Glutathione content; (L) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content; (M) Pod activity in WT overexpression\u003cem\u003e VvGRX28\u003c/em\u003e, co-transformation with overexpression\u003cem\u003e VvZNF10\u003c/em\u003e and RNAi \u003cem\u003eVvGRX28\u003c/em\u003e. (M) The relative expression levels of overexpression\u003cem\u003e VvGRX28\u003c/em\u003e, co-transformation with overexpression\u003cem\u003e VvZNF10\u003c/em\u003e and RNAi \u003cem\u003eVvGRX28 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003ecold responsive gene, including \u003cem\u003eVvICE1\u003c/em\u003e, \u003cem\u003eVvCOR413\u003c/em\u003e, \u003cem\u003eVvCBF1\u003c/em\u003e, \u003cem\u003eVvCBF2.\u003c/em\u003e Data are expressed as the mean ± standard error of three independent biological replicates. Different asterisks indicate significant differences, ** represents \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** represents \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, **** represents \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/8236ed790de1e54697a6bb8a.png"},{"id":98187023,"identity":"bb7ec0b3-80b7-4b41-8cc3-16254872569d","added_by":"auto","created_at":"2025-12-15 03:50:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16704790,"visible":true,"origin":"","legend":"\u003cp\u003eA working model of the redox regulatory mechanisms of \u003cem\u003eVvGRX28 \u003c/em\u003einteracted with \u003cem\u003eVvZNF10 \u003c/em\u003eto regulate cold tolerance in grape. \u003cem\u003eVvGRX28 \u003c/em\u003epositively regulates cold tolerance by forming a complex with \u003cem\u003eVvZNF10\u003c/em\u003e, which keeps ROS homeostasis, maintains cell membrane integrity, increase the contents of osmotic adjustment substances and upregulated the expression of cold responsive genes, protecting plants from cold stress damage.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/658ff4052dc2df8ecd0eaf8b.png"},{"id":98774601,"identity":"3039ef5d-53ca-44e1-bfee-71fd0c2d24e5","added_by":"auto","created_at":"2025-12-22 12:03:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":91086060,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/163d5c47-cfab-4d92-ab81-d0a4dc0afa0e.pdf"},{"id":98431350,"identity":"8a3b0a0e-b0a5-48ce-82bd-186a49f7839d","added_by":"auto","created_at":"2025-12-17 16:47:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":33411,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementlaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/9e1d6c07bbee2e0b77e3a7e0.docx"},{"id":98430840,"identity":"7aa1c481-f8c9-4609-9508-85b2e48948b2","added_by":"auto","created_at":"2025-12-17 16:46:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11875923,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementlaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8260182/v1/a2d825a1a2fcb2d414d7be1d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"VvGRX28 interacting with VvZNF10 modulates cold tolerance via eliminating excessive ROS in grapevine","fulltext":[{"header":"Background","content":"\u003cp\u003eGrape (\u003cem\u003eVitis vinifera\u003c/em\u003e L.), as an important perennial fruit crop, has been widely cultivated all over the word[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During the growth process of grapes, grape often suffers from various abiotic and biotic stresses, such as drought, salinity, heat, cold, and diseases and insect pests[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These adverse environmental factors cause the decreasing in production and quality of grape. In particular, low temperature (LT) is a critical environmental factor that influences metabolism, growth, and development of grape in northwestern China, where winter is extremely cold and the grapevine is very vulnerable to freezing damage[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although the application of soil covering and exogenous substances have been employed to address, the problem still cannot be addressed at source[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, it is urgent for salt-tolerance genes mining, the cold-resistant grapes breeding and cold-resistant cultivation model in future.\u003c/p\u003e\u003cp\u003eCold stress, including chilling (\u0026gt;\u0026thinsp;0\u0026deg;C) and freezing (\u0026lt;\u0026thinsp;0\u0026deg;C) stresses, has an adverse effect on plant growth and development, causing the changing of membrane lipids composition, the decreasing of intracellular enzymes activities, the accumulation of reactive oxygen species (ROS), the decreasing of photosynthetic capacity in grape[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Plant must cope with cold stress by coordinating cell molecular, metabolic, and physiological responses in different signal transduction pathways. When plants were subjected to cold stress, plants accumulate a large amount of ROS, leading to the imbalanced antioxidant defense system, triggering oxidative stress and causing damage to cells and tissues[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The antioxidant and redox systems which are composed of oxidoreductase proteins transferring electrons from input elements to downstream target proteins contribute to reduce ROS levels. Transmitters oxidoreductase proteins mainly include thioredoxins (TRXs) and glutaredoxins (GRXs)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The ascorbate\u0026ndash;glutathione (AsA\u0026ndash;GSH) cycle plays a key role in efficiently scavenging ROS within the chloroplast through the action of enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among them, GR uses glutathione (GSH) as the substrate to catalyze the reduction of H₂O₂ and organic peroxides, protecting cells from oxidative damage. Research has found that glutaredoxin (GRX) in plants can utilize glutathione (GSH) to reduce the disulfide bonds produced by oxidation, effectively protecting intracellular proteins from the damage of ROS and playing an important regulatory role in ROS signaling[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGRXs, an important oxidoreductase protein gene family, is widely present in prokaryotes and eukaryotes, and plays a significant role in plant development and abiotic stress[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Based on conserved residues in their active sites, plant GRX genes are classified into four types, namely CPYC, CGFS, CC and GRX-like (GRL) type[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among them, the CC-type has only been occured in higher plants[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The characteristic of the CPYC type Grx has Cxx(C/S) active sites, and is a classic dithiol Grx. It has the function of oxidoreductase and participates in the assembly of iron-sulfur clusters, severing as an important redox regulatory factor for intracellular metabolism[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Research showed that the overexpression of CPYC-type \u003cem\u003eOsGrxC2.2\u003c/em\u003e could regulate embryo development during embryogenesis, and increased grain weight, which interfere with the normal embryogenesis of rice embryo[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Interestingly, CPYC type \u003cem\u003eOsGrx20\u003c/em\u003e has a positive regulatory effect on adverse stress in rice. Overexpression of \u003cem\u003eOsGRX20\u003c/em\u003e can significantly enhance the resistance of rice to white leaf blight, as well as its tolerance to methyl violet and salt stress[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The CGFS type is generally a monothiol Grx with a strictly conserved CGFS motif. The CGFS-type Grx mainly functions as a ferredoxin transferase, which is determined by the unique ring-like structure near the active site[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eSlGrxS1\u003c/em\u003e is widely expressed in tomato leaves, roots, stems and flowers, and its expression is induced by oxidation, drought and salt stress[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eAtGrxS17\u003c/em\u003e can interact with NF-YC11/NC2α and transmit the redox signals generated by the photoperiod, thereby maintaining the function of the meristem[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eAtGRXS17\u003c/em\u003e may serve as a fundamental protection against moderate heat stress through redox dependent companion activity, thereby preventing heat stress-induced reactions injury[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In tomatoes, the absence of CGFS-type \u003cem\u003eSlGrxS15\u003c/em\u003e can cause embryo death, \u003cem\u003eSlgrxs14\u003c/em\u003e and \u003cem\u003eSlgrxs17\u003c/em\u003e mutants showed hypersensitivity to heat, chilling, drought, heavy metal toxicity, nutrient deficiency, and short photoperiod stresses. \u003cem\u003eSlgrxs16\u003c/em\u003e mutants were affected by chilling stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The CC-type Grx has a special CCxx active site, usually in the form of CCxC or CCxS, which is a unique motif found in terrestrial plants. It is the first identified members that \u003cem\u003eAtROXY1\u003c/em\u003e and \u003cem\u003eAtROXY2\u003c/em\u003e belong to the CC-type Grx family in \u003cem\u003eArabidopsis\u003c/em\u003e, as well as their homologous genes \u003cem\u003eOsROXY1\u003c/em\u003e and \u003cem\u003eOsROXY2\u003c/em\u003e in rice, play a crucial role in the differentiation process of petals and anthers[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The knockout of \u003cem\u003eGrxS13\u003c/em\u003e impaired the tolerance to light, delaying plant growth in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The sensitivity of transgenic RNAi CC type \u003cem\u003eOsGRX8\u003c/em\u003e to various abiotic stresses is enhanced in rice plants[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In maize, the CC type GRX can regulate the redox state of other proteins and plays an important role in the growth and meristems development[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The GRL type GRX has three domains, namely an N-terminal GRX domain and two domains whose functions are unknown[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. But so far, there have been no reports on the functions of the GRL type.\u003c/p\u003e\u003cp\u003eA growing evidences showed GRX genes plays in regulating the apical meristem of plants[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], flower development[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], abiotic and biotic stress[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. GRX gene family has been identified in many plant species, mainly including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], \u003cem\u003eOryza sativa\u003c/em\u003e L.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], \u003cem\u003eTriticum aestivum\u003c/em\u003e L.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eQuercus glauca\u003c/em\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], Camellia sinensis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], \u003cem\u003eMusa acuminata\u003c/em\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], \u003cem\u003ePuccinellia tenuiflora\u003c/em\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], populus[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. And the biological function of single GRX genes were performed by transgenic techniques approaches in plant. \u003cem\u003eROXY18 (GRXS13)\u003c/em\u003e and \u003cem\u003eROXY19 (GRXC9)\u003c/em\u003e interacted with class II TGAs to regulate detoxification response and pathogen defense[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The \u003cem\u003eMeGRXC3\u003c/em\u003e negatively regulated drought tolerance in cassava[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Ectopic overexpressed \u003cem\u003eSlGRX1\u003c/em\u003e actively responded to abiotic tolerance against oxidative, drought, and salt stresses in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Silencing of \u003cem\u003eGhGRL28\u003c/em\u003e increased the sensitivity to salt stress in cotton[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Overexpressing \u003cem\u003ePagGRXC9\u003c/em\u003e enhanced salt tolerance in poplar[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt present, the biological function of GRX gene in cold stress mainly focuses on the basic expression levels by qRT-PCR, while the concrete knowledge of single GRX genes under cold stress remains a few in plants. However, it has yet been reported on systematic identification of VvGRX gene family member, structure analysis, responses to various abiotic stresses, and adaptive evolution of GRX genes in grape. Therefore, in this study, we identified VvGRX gene members, performed the in-depth characterization of its molecular, explored the expression pattern of \u003cem\u003eVvGRXs\u003c/em\u003e in different organs and stress-inducible by Real-time fluorescent quantitative PCR (RT-PCR). Furthermore, the \u003cem\u003eVvGRX28\u003c/em\u003e gene related to cold stress was selected, we constructed transgenic plants ectopically expressing \u003cem\u003eVvGRX28\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, which were subjected to cold stress for functional analysis. the protein-protein interaction between VvGRX2\u003cem\u003e8\u003c/em\u003e and its counterpart was analyzed by yeast two-hybrid (Y2H). The function of \u003cem\u003eVvGRX28\u003c/em\u003e and its counterpart were analyzed in grape callus. The findings will serve as a guide to the analysis and discovery of gene function in woody plant, and contribute to understand the genetic and molecular mechanisms of plant response to cold stress in grape and to provide candidate genes with improved stress tolerance via innovative molecular breeding strategies.\u003c/p\u003e"},{"header":"Materials and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and cultivation conditions\u003c/h2\u003e\u003cp\u003eThe \u0026lsquo;Pinot Noir\u0026rsquo; plantlets in vitro were used as test materials for RT-PCR under salt, drought and cold stresses, which were reserved in the plant stress physiology laboratory of the college of life sciences, Gansu Agricultural University, China. The cuttings (2\u0026ndash;3 cm) with a single bud were inoculated in GS medium (pH, 5.8\u0026ndash;6.0) supplemented with 20 g/L sucrose and 5.0 g/L agar to propagate a large number of aseptic seedlings. The plantlets in vitro were cultivated in light incubator with a light intensity of 120 \u0026micro;mol\u0026middot;m-2\u0026middot;s-1 at 26\u0026deg;C, 16 h/light, 8 h/dark growth condition. for various stress treatments. After grew for 35 days, the plantlets in vitro with uniform growth and no pollution were selected as experimental materials for RT-PCR assay. Arabidopsis thaliana (Columbia, Col-0) was used as heterologous transformation material. The Arabidopsis thaliana plants grew in the substrate containing a mixture of soil and vermiculite (3:1), and then placed in a growth chamber at 22\u0026deg;C (16 h light/8 h dark, 65% \u0026minus;\u0026thinsp;75% relative humidity, and 120 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity). \u0026lsquo;Pinot Noir\u0026rsquo; callus was aseptically cultured in B5 medium (30 g/L sucrose\u0026thinsp;+\u0026thinsp;6 g/L agar\u0026thinsp;+\u0026thinsp;0.5 mg/L 1-Naphthaleneacetic acid (NAA)\u0026thinsp;+\u0026thinsp;1 mg/L kinetin (KT)\u0026thinsp;+\u0026thinsp;300 mg/L polyvinyl pyrrolidone (PVP)\u0026thinsp;+\u0026thinsp;1 mg/L myo-inositol), and then sub-cultured every 30 days in a dark incubator at 25\u0026deg;C. The callus of \u0026lsquo;Pinot Noir\u0026rsquo; with better growth was selected as transformation materials for Agrobacterium infestation and cold stress. \u003cem\u003eNicotiana benthamiana\u003c/em\u003e was planted in seedling substrate (nutritious soil and vermiculite\u0026thinsp;=\u0026thinsp;3:1), and then placed in a growth chamber under a 16-h-light/8-h-dark photoperiod at 25\u0026deg;C. After cultivated for 25 days, tobacco leaves were used for following experiment on subcellular localization, GUS staining, firefly luciferase fragment complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIdentification of VvGRX family members and phylogenetic tree construction\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e GRX protein sequences were downloaded from the TAIR database. Rice, tomato, maize GRX protein sequences were derived from the Phytozome version 13 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozomenext.jgi.doe.gov\u003c/span\u003e\u003cspan address=\"https://phytozomenext.jgi.doe.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and used as the query to perform a BLASTP search against the grape protein database[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Reference sequence genome files of grape were downloaded from the Ensembl database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"http://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Hidden Markov Models (HMMs) for the GRX (Accession number: PF00462) were searched from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam-legacy.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam-legacy.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and used as queries to search the grape protein database by the HMMsearch tool, with the threshold set as E\u0026thinsp;\u0026lt;\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Proteins identified by both methods were combined and manually curated using the Conserved Domain Database (CDD)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], removing redundancies and retaining only those proteins containing GRX domains as members of GRX family in grape. According to the location of genes on chromosomes, VvGRXs were named respectively.\u003c/p\u003e\u003cp\u003eTo investigate the phylogenetic relationships for the GRX family in grape, GRX family protein sequences of grape, tomato, barley and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were downloaded from the Phytozome and TAIR databases, respectively. Comparative analysis of amino acid sequences of GRX proteins using the software Clustal X. The phylogenetic tree was established by the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in the MEGA 7.0. The phylogenetic tree was visualized using the iTOL tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and GRX protein were classified based on the active-site motif (Boubakri et al. 2022).\u003c/p\u003e\n\u003ch3\u003ePhysicochemical properties and the secondary structure prediction of VvGRX protein\u003c/h3\u003e\n\u003cp\u003eThe physical and chemical properties of grape GRX protein were analyzed by Expasy online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including amino acid number, molecular weight (MW), isoelectric point (PI), instability coefficient and grand average of hydrophilicity. The subcellular localization was predicted by WoLFPSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The prediction of protein secondary structure was completed online by SOPMA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.plpage\u003c/span\u003e\u003cspan address=\"http://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.plpage\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e =/NPSA/npsa_hnn.html).\u003c/p\u003e\n\u003ch3\u003eGene structure, conserved motifs, cis-elements and collinearity analysis of VvGRXs\u003c/h3\u003e\n\u003cp\u003eCDS sequences of grape GRX gene family were extracted from grape whole genome and gene annotation file, and the structure of this gene family was analyzed by GSDS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/index.php\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. MEME Suite 5.4.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the conservative motifs of grape GRX protein, and the motifs number was set to 10. The online website PlantCARE [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. TBtools was used to visually analyze the conservative motifs of the gene family[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the distribution of \u003cem\u003ecis\u003c/em\u003e-acting elements in the 2 kb base fragment upstream of the initiation codon of the grape GRX gene. The collinearity was analyzed among grape, \u003cem\u003eArabidopsis\u003c/em\u003e and tomato GRX gene family members.\u003c/p\u003e\n\u003ch3\u003eStresses treatment\u003c/h3\u003e\n\u003cp\u003eFor the preliminary function exploration of GRX genes under abiotic stresses, after grew for 35 days, the grape plantlets in vitro were treated NaCl, PEG and cold stress. Some uniform growth and no pollution plantlets were transformed into GS medium which contained 200 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, 10% PEG for 24 h, respectively. Sterile water was used as a control (CK) treatment. Plantlets were treated with 4\u0026deg;C cold stress for 24 h by gradient cooling in Ultra-low temperature alternating temperature incubator. Leaves tissues of grape were sampled for RT-PCR assay.\u003c/p\u003e\u003cp\u003eFor cold stress of \u003cem\u003eArabidopsis\u003c/em\u003e, 3 weeks-old seedlings were treated at -4\u0026deg;C for 5 h, and normal temperature conditions were used as a control. In the freezing tolerance of \u003cem\u003eArabidopsis\u003c/em\u003e, seedlings were grown at 22\u0026deg;C for 2 week and then treated at 4\u0026deg;C for 3 days to examine frost tolerance of Arabidopsis plants. And then the seedlings were treated at \u0026minus;\u0026thinsp;6\u0026deg;C for 5 h through gradient cooling, and then transferred to 22\u0026deg;C for 7 days, observing phenotype changing. Arabidopsis leaves after treated with \u0026minus;\u0026thinsp;4\u0026deg;C were collected at 0 h and 5 h, quickly frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for RT-PCR and determination of physiological indexes related to cold tolerance. For cold stress of grape \u003cem\u003ecallus\u003c/em\u003e, grape \u003cem\u003ecallus\u003c/em\u003e after cultivated 30 days were selected to treat with 4\u0026deg;C for 15 d. Grape \u003cem\u003ecalluses\u003c/em\u003e grew in 25\u0026deg;C were used as control. Grape \u003cem\u003ecalluses\u003c/em\u003e were sampled, and quickly frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for RT-PCR and determination of physiological indexes. Three biological replicates were applied in this experiment.\u003c/p\u003e\n\u003ch3\u003eMeasurement of stress-related physiological parameters, histochemical staining\u003c/h3\u003e\n\u003cp\u003eAfter treated with cold stress, \u003cem\u003eArabidopsis\u003c/em\u003e and grape callus were collected to determine physiological indexes related to cold tolerance. The relative electrolyte leakage (REL) of plant materials was determined by a conductivity meter (DDSJ-318, Shanghai, China)[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The content of malondialdehyde (MDA), Proline (Pro) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), soluble sugar (SS), glutathione (GSH) and peroxidase (POD) activities were determined by Comin Biotechnology Kit (Suzhou, Jiangsu, China) according to the manufacturers\u0026rsquo; protocols, respectively. Three biological replicates were set in the experiment. 3, 3, 9-Diaminobenzidine (DAB) and Nitroblue tetrazolium (NBT) staining were used to detect the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e) of Arabidopsis referring to the methods as described previously[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Trypan blue dye was used to detect the integrity of the cell membrane and the survival rate of the cells under cold stress by the previous method[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The detached leaves were put into DAB, NBT and After stained by solution, respectively, and vacuumed for 5\u0026ndash;10 min making the leaves completely immersed in the solution. After stained by DAB and NBT, the leaves were immersed in 95% ethanol for decolorized in a boiling water about 20 min. After stained by stained, the leaves were immersed in 2.5 g/L chloral hydrate solution. After the color of stained leaves gradually faded, the samples were placed in 30% glycerol solution and the dyed leaves were photographed and recorded using a camera.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSubcellular localization assay and GUS staining and activity in Nicotiana benthamiana leaves\u003c/h2\u003e\u003cp\u003eFor performing subcellular localization, 4-week-old tobaccos were used to observe EGFP signaling in leaves cell. The pART-CAM-EGFP-\u003cem\u003eVvGRX28\u003c/em\u003e and pART-CAM-EGFP empty vector Agrobacterium liquids were infiltrated into the epidermal cells on the reverse side of the tobacco leaf, and tobaccos were kept in dark condition for 12 h. Empty vector Agrobacterium liquids was used the control. The infiltrated tobacco plants were cultivated in a growth chamber under a 16-h-light/8-h-dark photoperiod at 25\u0026deg;C. After infiltrated for 48\u0026ndash;72 h, green fluorescent protein (GFP) signals were detected with a laser scanning confocal microscope (Olympus FV1000 viewer) in tobacco leaves.\u003c/p\u003e\u003cp\u003eTo construct the pro-\u003cem\u003eVvGRX28\u003c/em\u003e::GUS vector, the \u003cem\u003eVvGRX28\u003c/em\u003e promoter fragment at 2000 bp upstream of the start codon was cloned into the pBI121-GUS vector to replace the 35S promoter. The recombinant vector liquid was injected into tobacco leaves using the Agrobacterium-mediated transformation method for transient expression. The injected plants were cultivated under normal conditions of 25\u0026deg;C, 16 h light/8 h dark for 2 days, and then subjected to a 4\u0026deg;C for 6 h. Tobaccos under normal conditions were used as the control. Subsequently, the injected leaves were detached from tobacco and made small round pieces using for GUS staining and the expression level of pro-VvGRX28 determination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eYeast two-hybrid (Y2H) assay\u003c/h2\u003e\u003cp\u003eIn Y2H assay, VvGRX28 protein was used as the bait, and the CDS of VvGRX28 was cloned into the pGBKT7 vector to gain the recombinant plasmid of pGBKT7-VvGRX28. The recombinant bait and pGADT7 empty vector plasmids were co-transformed into Y2HGold strain in synthetic dropout minimal base (SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade) to detect self-activating toxicity detection. Through the mating hybridization method, potential interacting proteins were screened from AD library related to cold stress which was conserved in the plant stress physiology laboratory. Mixed yeast solution was coated on SD/-Leu/-Trp/-His/-Ade medium and the plates were cultured at 30\u0026deg;C for 4\u0026ndash;6 days. Finally, these plaques were transferred to SD/-Leu/-Trp/-His/-Ade\u0026thinsp;+\u0026thinsp;X-α-gal medium for further screening. The blue plaques were screened, and identified by PCR. After sequenced, the protein sequences were blasted in NCBI. The screened potential proteins sequences were cloned into pGADT7 to generate the prey construct. The prey and bait vectors were co-transformed in SD/-Leu/-Trp, SD/-Leu/-Trp/-His/-Ade and SD/-Leu/-Trp/-His/-Ade\u0026thinsp;+\u0026thinsp;X-α-gal to examinate interaction relationship between VvGRX28 protein and potential interaction proteins. pGBKT7-p53\u0026thinsp;+\u0026thinsp;pGADT7-T and pGBKT7-Lam\u0026thinsp;+\u0026thinsp;pGADT7-T were used as positive and negative controls, respectively. The amplification primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eLCI and BiFC assay\u003c/h2\u003e\u003cp\u003eLCI assay was conducted as described in the previous method [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The CDS fragment of \u003cem\u003eVvGRX28\u003c/em\u003e and \u003cem\u003eVvZNF10\u003c/em\u003e (deleted the stop codon) were cloned and inserted into pCAMBIA1300-cLUC and pCAMBIA1300-nLUC to gain the recombinants plasmids, respectively. Subsequently, the \u003cem\u003eVvGRX28\u003c/em\u003e-cLUC and \u003cem\u003eVvZNF10\u003c/em\u003e-nLUC recombinants plasmids were transformed into \u003cem\u003eA.tumefaciens\u003c/em\u003e GV3101. 3-weeks-old tobaccos were used to injected with the combination of \u003cem\u003eVvGRX28\u003c/em\u003e-cLUC and \u003cem\u003eVvZNF10\u003c/em\u003e-nLUC, cLUC and \u003cem\u003eVvZNF10\u003c/em\u003e-nLUC, \u003cem\u003eVvGRX28\u003c/em\u003e-cLUC and \u003cem\u003eVvZNF10\u003c/em\u003e, cLUC and nLUC Agrobacterium suspension (volume ratio\u0026thinsp;=\u0026thinsp;1:1) in the same leaf, resuspension solution was distilled water (1 M MES (pH\u0026thinsp;=\u0026thinsp;5.6), 2.5 M MgCl\u003csub\u003e2\u003c/sub\u003e, 100 mM Acetosyringone). The injected tobaccos were incubated in the dark for 12 h, and then transferred in the light for 48 h. D-luciferin potassium salt (beyotime biotechnology, Shanghai, China) was applied to the injection sites, and the samples were kept in the dark for 5 min. LUC activity was measured using the FusionFX Vilber Lourma Imaging System (Vilber, France).\u003c/p\u003e\u003cp\u003eThe cDNA sequences of \u003cem\u003eVvGRX28\u003c/em\u003e and \u003cem\u003eVvZNF10\u003c/em\u003e were cloned into pC1300-mVYCE and pC1300-mVYNE vectors respectively to construct fusions expressing YFPN and YFPC protein. The fusion expression vectors plasmids were transformed into A.tumefaciens GV3101 and the mixture of two different plasmids (\u003cem\u003eVvGRX28\u003c/em\u003e-YCE and \u003cem\u003eVvZNF10\u003c/em\u003e-YNE, \u003cem\u003eVvGRX28\u003c/em\u003e and \u003cem\u003eVvZNF10\u003c/em\u003e-YNE, \u003cem\u003eVvGRX28\u003c/em\u003e-YCE and \u003cem\u003eVvZNF10\u003c/em\u003e, YCE and YNE) of equal volume was trans-formed instantaneously into N. benthamiana leaves. Resuspension solution was as the same to LCI experiment. After in the dark for 12 h, the injected plants were cultured for 2 days at 28\u0026deg;C for 8-hour light/16-hourdarkness conditions, and the fluorescence of fluorescent protein signaling was observed in the area of tobacco leaves injected with Agrobacterium using a Laser confocal microscopy (LSM80, Zeiss, Germany)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and RT-qPCR analysis\u003c/h2\u003e\u003cp\u003eThe total RNA was extracted from the leaves and callus of grape, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e using RNAprep Pure Plant Plus Kit (DP441, TIANGEN biotechnology, Beijing, China) under different treatments, referring to the manual for detailed steps. Genes primers for RT-qPCR and cloning were designed and synthesized in the online software (the Shanghai Sangon Biotech Company). Extracted RNA was used as a template to synthesize cDNA using the prime reverse transcriptase Kit FastKing gDNA Dispelling RT SuperMix (KR118, TIANGEN biotechnology, Beijing, China). RT-PCR was performed by Light Cycler\u0026reg; 96 real-time PCR system (Roche, Switzerland). The \u003cem\u003eVvGAPDH\u003c/em\u003e and \u003cem\u003eAtActin\u003c/em\u003e gene were used as the reference genes, a sample of cDNA (3 \u0026micro;L) was subjected to RT-PCR in an eventual of 20 \u0026micro;L, which contained 1.6 \u0026micro;L primers (upstream primer was 0.8 \u0026micro;L, downstream primer was 0.8 \u0026micro;L ), 10 \u0026micro;L SYBR Green Master Mix Reagent (Takara Bio, Shiga, Japan) and 5.4 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. PCR amplification procedure was 40 cycles of 95\u0026deg;C for 30 s, 95\u0026deg;C for 10 s, 58\u0026deg;C for 30 s, 72\u0026deg;C for 20 s. The relative expression of each gene was analyzed by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. All RT-PCR analyses were performed with three biological repeats. All primers were listed in Supplementary Table\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in this study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical data were analyzed by SPSS software (IBM, Chicago, IL, USA), and significance was evaluated at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The GraphPad Prism 10.1.2 software was used for mapping. Error bars indicate mean SE of three biological replicates. The relative expression of each gene was analyzed by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003eMembers identification, physicochemical properties and phylogenetic relationships of GRXs family\u003c/h2\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e, rice, tomato, maize GRX protein sequences were used as the query to perform a BLASTP search against the grape protein database. As shown in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, a total of 33 GRX genes containing GRX domain (PF00462) were obtained in the whole grape genome, and named \u003cem\u003eVvGRX1\u003c/em\u003e-\u003cem\u003eVvGRX33\u003c/em\u003e based on the position of each gene on the chromosome distribution. The analysis of protein physical and chemical properties showed that grape GRX gene family had some differences in amino acid number, molecular weight, isoelectric point, instability coefficient, GRAVY. The number of amino acids in grape GRX gene was 102 (VvGRX8) to 546 aa (VvGRX1), and the molecular weight was 11.06 to 62.94 kDa, which was consistent with the change trend of amino acids. The theoretical isoelectric points were 5.05 (VvGRX19) to 9.48 (VvGRX13), with an average of 7.13. Among them, 18 proteins were basic proteins (PI\u0026thinsp;\u0026gt;\u0026thinsp;7.5) and 15 proteins were acidic proteins (PI\u0026thinsp;\u0026lt;\u0026thinsp;7.5). When the instability index is less than 40, it is a stable protein, of which 75.76% VvGRX were unstable proteins. The aliphatic index ranged from 69.07 to 110.22. Hydrophilic GRX proteins accounted for 63.64% (GRAY\u0026thinsp;\u0026lt;\u0026thinsp;0), hydrophobic GRX proteins accounted for 36.36% (GRAY\u0026thinsp;\u0026gt;\u0026thinsp;0), indicated that VvGRX family members are mainly hydrophilic proteins. The subcellular localization prediction indicated that grape GRX gene family proteins were mainly located in chloroplast (42.4%), cytoplasm (30.3%), mitochondrion (12.1%) and nucleus (9.1%).\u003c/p\u003e\u003cp\u003eIn order to explore the evolutionary relationship between grape and Arabidopsis, tomato and barley GRX genes, 132 GRX proteins were analyzed for phylogenetic evolution (Fig.\u0026nbsp;1A). The results showed that 132 GRX family genes could be divided into four subgroups, including CC type, CGFS type, CPYC type and GRL type. There were differences in species categories and the number of GRX genes in different subgroups. In addition, among grape GRX genes, the most of VvGRX family members in GRL type (14 VvGRX genes), the second in CC type (11 VvGRX genes), the third in CPYC type (5 VvGRX genes), and the least in CGFS type (3 VvGRX genes). CC type members account for a large proportion in the phylogenetic trees of the four species, and one branch was divided into two subbranches of monocotyledons and dicotyledons, indicating that CC type GRX forms new family members with the differentiation of monocotyledons and dicotyledons. In addition, according to the phylogenetic tree, the homology of VvGRX in dicotyledons is greater than that of monocotyledons, and barley has unique evolutionary branches, which indicating the genetic relationship of VvGRX genes are in line with the evolutionary relationship of plants.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStructures, motifs, \u003cem\u003ecis-elements and collinearity analysis of VvGRXs\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThrough the analysis of the gene structure of VvGRX family members, there were certain differences in the VvGRX gene structure in different subfamilies (Fig.\u0026nbsp;1B). Among them, the 3 members of the CGFS subfamily had 2, 3, and 6 exons, and the CC subfamily had 11 exons. All members had 1 exon, 5 members of CPYC subfamily had 4 exons, and 14 members of GRL subfamily had 1 to 7 exons. The secondary structure prediction analysis showed that grape GRX proteins were mainly composed of α-helix, β-turn and random coil are three structural elements, of which random coil accounts for the largest proportion, followed by α-helix, β-turn proportion is relatively small (Fig.\u0026nbsp;1C). Subsequently, for analyze the diversity of VvGRX protein in grapes, the conserved motifs were analyzed using MEME online tool (Fig.\u0026nbsp;1B). The results showed that proteins clustered in one subfamily had similar motifs, and different motifs were distributed in different subfamilies. Motifs 1, 3, 6, and 7 were mainly distributed in CC, CPYC, and CGFS subfamilies, while motifs 2, 4, 5, 8, 9, and 10 were only distributed in GRL subfamily, the result suggesting that the presence of different motifs were necessary for the execution of VvGRX protein function. In addition, motif 1 involved the LxxLL active site, most of grape GRX (94%) had this motif. Motif 2 existed in all GRL types except \u003cem\u003eVvGRX1\u003c/em\u003e and \u003cem\u003eVvGRX32\u003c/em\u003e. Motif 3 contained the active CxxC site in GRX, and motif 6 is distributed in CGFS, CC and CPYC types. Motif 8, 9 and 10 were unique to \u003cem\u003eVvGRX30\u003c/em\u003e and \u003cem\u003eVvGRX33\u003c/em\u003e of GRL type, which was different from other motifs.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ecis\u003c/em\u003e-acting elements can interact with the corresponding trans-regulators to regulate gene expression. The analysis of \u003cem\u003ecis\u003c/em\u003e-acting elements in the VvGRX revealed that 39 \u003cem\u003ecis\u003c/em\u003e-acting elements were identified (Fig.\u0026nbsp;1D), which could be divided into three categories, including growth and development, plant hormone responsive, abiotic and biotic stress responsive elements. There were 7 types of growth and development elements, namely AACA_motif, ARE, CAT-box, GCN4_motif, HD-Zip 1, O2-site and RY-element, which were related to anaerobic induction (ARE) and meristem expression (CAT-box), Zein metabolism (O\u003csub\u003e2\u003c/sub\u003e-site) is the most relevant. There are 10 kinds of plant hormone responsive elements, including ABRE, AuxRR-core, CGTCA-motif, GARE-motif, P-box, TATC-box, TCA-element, TGA-box, TGACG-motif and TGA-element, which are combined with abscisic acid (ABRE), methyl jasmonate (CGTCA-motif, TAGG-motif), gibberellin (P-box), salicylic acid (TCA-element) and other metabolic pathway-related elements. There were 21 kinds of biotic/abiotic stress-related elements, namely LTR, MBS, TC-rich repeats, ACE, AE-box, AT1-motif, ATCT -motif, Box 4, chs-CMA1a, GA-motif, Gap-box, GATA-motif, G-box, GT1-motif, GTGGC-motif, I-box, LAMP-element, MRE, Sp1, TCCC-motif and TCT-motif, which had low temperature (LTR), drought (MBS), defense and stress (TC-rich repeats) and light response (G-box, GT1-motif, BOX 4) components occupy an important position. Based on the above analysis, we speculated that VvGRX genes largely participated in regulating plant growth, development and abiotic stress response.\u003c/p\u003e\u003cp\u003eIn order to better understand the collinearity mode of grape GRX gene, collinearity analysis was carried out on grape GRX gene. It was found that there were 5 pairs of genes in the family with collinearity, including\u003cem\u003eVvGRX2\u003c/em\u003e and \u003cem\u003eVvGRX27\u003c/em\u003e, \u003cem\u003eVvGRX8\u003c/em\u003e and \u003cem\u003eVvGRX11\u003c/em\u003e, \u003cem\u003eVvGRX9\u003c/em\u003e and \u003cem\u003eVvGRX24\u003c/em\u003e, \u003cem\u003eVvGRX20\u003c/em\u003e and \u003cem\u003eVvGRX23\u003c/em\u003e, \u003cem\u003eVvGRX28\u003c/em\u003e and VvGRX31 (Fig.\u0026nbsp;1E). At the same time, the result found that the genes with collinearity had close phylogenetic relationship. The analysis examined the collinear interactions among members of the VvGRX gene family and GRX genes in other species (Fig.\u0026nbsp;1F). Representative species were selected, including A. thaliana and \u003cem\u003eSolanum lycopersicum\u003c/em\u003e. In total, the analysis of collinear maps revealed 29 collinear blocks in A. thaliana and grape, 26 collinear blocks in tomato and grape. Greater VvGRX collinearity was observed in A. thaliana. The result supported the hypothesis that the VvGRX gene family is more closely related to GRXs in A. thaliana.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eTissue-specific expression profile of VvGRXs under drought, salt and cold stress\u003c/h2\u003e\u003cp\u003eThe expression levels of GRX genes in grape were analyzed in different tissues during the different developmental stages. The results showed that the expression levels of 33 VvGRX genes had significantly differences in different grape plant tissues of different development stages (Fig.\u0026nbsp;2A). \u003cem\u003eVvGRX1\u003c/em\u003e, \u003cem\u003eVvGRX17\u003c/em\u003e, \u003cem\u003eVvGRX18\u003c/em\u003e, \u003cem\u003eVvGRX19\u003c/em\u003e, \u003cem\u003eVvGRX20\u003c/em\u003e, \u003cem\u003eVvGRX22\u003c/em\u003e, \u003cem\u003eVvGRX23\u003c/em\u003e, \u003cem\u003eVvGRX26\u003c/em\u003e, \u003cem\u003eVvGRX29\u003c/em\u003e, VvGRX32, and \u003cem\u003eVvGRX33\u003c/em\u003e gene showed relatively high expression level in all grape tissues, such as tendrils, stems, buds, roots, flowers, leaves, seeds, seedlings, and other organs, other genes are expressed specifically in some tissues. \u003cem\u003eVvGRX1\u003c/em\u003e was expressed specifically in leaf, \u003cem\u003eVvGRX6\u003c/em\u003e was expressed specifically in bud, \u003cem\u003eVvGRX1\u003c/em\u003e was expressed specifically in rachis. \u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;1\u003c/b\u003e The evolutionary relationship and sequences analysis of \u003cem\u003eGRX\u003c/em\u003e genes in grape. (A) Phylogenetic analysis of \u003cem\u003eGRX\u003c/em\u003e genes in \u003cem\u003eVitis vinifera\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, and \u003cem\u003eHordeum vulgare\u003c/em\u003e. (B) The structure and motif analysis of \u003cem\u003eGRX\u003c/em\u003e genes, yellow column represents CDS, purple column represents upstream/downstream, black line represents intron, the motifs number was set to 10. (C) The secondary structure prediction of VvGRX protein, mainly including alpha helix, beta turn, random coil. (D) \u003cem\u003eCis\u003c/em\u003e-acting regulatory element predictions of VvGRXs at the 2000 bp upstream of VvGRX, divided into growth and development, hormones, and abiotic and biotic stress responsive elements. (E) The synteny analysis of VvGRXs in grape, the collinear blocks within the genomes of grape were marked with gray lines, while the syntenic VvGRX gene pairs are highlighted in red. (F) The comparative analysis of the VvGRX gene synteny between grape and two other representative plant species, the collinear blocks within the genomes of grape and other species are marked with gray lines, while the syntenic VvGRX gene pairs are highlighted in red.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eThe expression pattern analysis of VvGRXs family members under drought, salt and cold stress\u003c/h2\u003e\u003cp\u003eBased above tissue-specific expression analysis, a total of 11 gene were selected, which were highly expressed in almost all grape tissues, including \u003cem\u003eVvGRX1\u003c/em\u003e, \u003cem\u003eVvGRX17\u003c/em\u003e, \u003cem\u003eVvGRX18\u003c/em\u003e, \u003cem\u003eVvGRX19\u003c/em\u003e, \u003cem\u003eVvGRX20\u003c/em\u003e, \u003cem\u003eVvGRX22\u003c/em\u003e, \u003cem\u003eVvGRX23\u003c/em\u003e, \u003cem\u003eVvGRX25\u003c/em\u003e, \u003cem\u003eVvGRX28\u003c/em\u003e, \u003cem\u003eVvGRX32\u003c/em\u003e, and \u003cem\u003eVvGRX33\u003c/em\u003e. To preliminary explore the expression pattern of VvGRX gene family under abiotic stress, the grape plantlets in vitro were subjected to NaCl, drought and cold stress. As shown in (Fig.\u0026nbsp;2B), RT-PCR analysis showed that relative expression levels of VvGRXs were significantly upregulated than control, of which \u003cem\u003eVvGRX19, VvGRX20, VvGRX32\u003c/em\u003e gene were highly expressed about 10\u0026ndash;30 than control in grape, the expression of \u003cem\u003eVvGRX32\u003c/em\u003e was the highest under drought stress. The expression levels of \u003cem\u003eVvGRX1\u003c/em\u003e, \u003cem\u003eVvGRX18\u003c/em\u003e, \u003cem\u003eVvGRX20\u003c/em\u003e and \u003cem\u003eVvGRX32\u003c/em\u003e gene were relatively high, \u003cem\u003eVvGRX20\u003c/em\u003e gene was the highest under slat stress. The expression levels of \u003cem\u003eVvGRX17\u003c/em\u003e, \u003cem\u003eVvGRX18\u003c/em\u003e, \u003cem\u003eVvGRX23\u003c/em\u003e, \u003cem\u003eVvGRX28\u003c/em\u003e and \u003cem\u003eVvGRX32\u003c/em\u003e were relatively high, \u003cem\u003eVvGRX28\u003c/em\u003e gene was the highest under cold stress. Taking into account the overall expression level of the GRX gene under salt, drought and cold stress, we find that the expression levels of VvGRX gene were highly expressed under the cold treatment than salt and drought stress. Therefore, \u003cem\u003eVvGRX28\u003c/em\u003e gene with high expression level was selected to further explore cold function in grape. \u003cem\u003eVvGRX28\u003c/em\u003e could be a very important responsive gene in regulating cold tolerance in grape.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;2\u003c/b\u003e Gene chip and RT-PCR analysis under different tissues and stresses. (A) The tissue-specific expression profile analysis heatmap of VvGRXs, the redder the square color is, the higher the expression level is. (B) The relative expression levels of VvGRXs in grape leaves under drought, salt and cold stress, \u003cem\u003eVvGRX28\u003c/em\u003e was selected for the function verification in cold tolerance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eOverexpression of VvGRX28 improved cold tolerance in transgenic Arabidopsis thaliana\u003c/h2\u003e\u003cp\u003eTo further understand cold response pattern of \u003cem\u003eVvGRX28\u003c/em\u003e in grape, different low temperature treatment times were set. RT-PCR analysis showed the expression level of \u003cem\u003eVvGRX28\u003c/em\u003e was upregulated with the elongation of the processing time, and up to a maximum in 72 h (Fig.\u0026nbsp;3A). For cold function verification of \u003cem\u003eVvGRX28\u003c/em\u003e, the CDS fragment of \u003cem\u003eVvGRX28\u003c/em\u003e was successfully clone (Fig.\u0026nbsp;3B) and inserted into the pART-CAM-EGFP plant overexpression vector driven by the CaMV35S (Fig.\u0026nbsp;3C). The subcellular localization analysis indicated VvGRX28 was located in nucleus and cell membrane with a significant green fluorescence signal in tobacco epidermal cells (Fig.\u0026nbsp;3D). Owing to the constraints of grape genetic transformation, \u003cem\u003eVvGRX28\u003c/em\u003e was heterologously transformed into \u003cem\u003eArabidopsis thaliana\u003c/em\u003e by floral dip method. Transgenic plants were screened in 1/2 MS plate medium (50mg/mL Kan), green plants were planted in the substrate containing a mixture of soil and vermiculite, harvested seeds after maturity (Fig.\u0026nbsp;3E). Transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was further identified by PCR (Fig.\u0026nbsp;3F). Subsequently, T3 homozygous and stable lines (OE3, OE8, OE13) were used to carry out cold stress assay.\u003c/p\u003e\u003cp\u003eWT and three overexpression stable T3 lines (OE3, OE8, OE13) were subjected to -6 ℃ cold stress for 5 h, and then placed in normal growth temperature 23 ℃. After the recovery of 12 d, phenotypic observation result showed most WT plants leaves showed severely wilting, yellow and even death, while the growth status of \u003cem\u003eVvGRX28\u003c/em\u003e overexpressed plants were significantly better than that of WT plants (Fig.\u0026nbsp;4A). Under \u0026minus;\u0026thinsp;4 ℃ cold stress for 5 h, the REL of transgenic lines was relatively lower than that of WT plants, indicating that the damage degree of transgenic lines cell membrane was weak than that of WT (Fig.\u0026nbsp;4B). MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content of OEs was decreased than WT, indicating a slight degree of membrane lipid peroxidation (Fig.\u0026nbsp;4C, Fig.\u0026nbsp;4E). Under cold stress conditions, plant tissues produce various ROS, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e is two main types of ROS. DAB and NBT staining were used to evaluate the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e in leaves. Hydrogen peroxide can rapidly react with DAB to form a brown compound under the catalysis of peroxidase, thereby locating hydrogen peroxide in leaves. Oxygen free radicals can reduce NBT to insoluble blue methazan compounds, thereby locating superoxide anion in leaves. Under cold stress, WT and OE plants can produce a lot of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e compared with the normal temperature. DAB and NBT staining result showed the brown and blue color of the OE leaves were relatively light than WT leaves under cold stress, demonstrating that OE plants suffered relatively minor damage from the low temperature (Fig.\u0026nbsp;4D). Trypan blue staining indicated that the blue areas of leaves were small in OE than WT, indicating the number of dead cells was little (Fig.\u0026nbsp;4D). Furthermore, the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and POD activity were determined. The result showed the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and POD activity of OE lines were relatively high than that of WT, maintaining the oxidative balance in plants and decreasing the production of ROS (Fig.\u0026nbsp;4E and Fig.\u0026nbsp;4I). Under normal growth conditions, there was no significant difference in proline (Fig.\u0026nbsp;4F), soluble sugar (Fig.\u0026nbsp;4G) and glutathione (Fig.\u0026nbsp;4H) content between OEs and WT plants, while the content of proline, soluble sugar and glutathione were increased in OE plants under cold stress. Overall, the overexpression of \u003cem\u003eVvGRX28\u003c/em\u003e enhanced the cold tolerance by promoting the ability to removing ROS, maintaining cell membrane integrity and increasing the contents of osmotic adjustment substances in transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe overexpression of \u003cem\u003eVvGRX28\u003c/em\u003e has led to a series of physiological changes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, while gene expression of some key cold responsive genes was unknown. In this study, the expression of some key cold responsive genes which includes inducer of CBF expression 1 (\u003cem\u003eAtICE1\u003c/em\u003e), inducer of CBF expression 2 (\u003cem\u003eAtICE2\u003c/em\u003e), cold-regulated 15A (\u003cem\u003eAtCOR15A\u003c/em\u003e), C-repeat Binding Factor 1 (\u003cem\u003eAtCBF1\u003c/em\u003e) and C-repeat Binding Factor 2 (\u003cem\u003eAtCBF2\u003c/em\u003e) was detected in WT and OE plants of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e under low temperature stress. \u003cem\u003eAtICE\u003c/em\u003e, \u003cem\u003eAtCOR\u003c/em\u003e and \u003cem\u003eAtCBF\u003c/em\u003e genes are the core associated gene group of cold stress response in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, together forming the ICE-CBF-COR signaling axis, regulates the adaptive response of plants to cold stresses. The three undergo a cascade reaction of transcriptional activation - gene expression - functional execution. \u003cem\u003eAtActin\u003c/em\u003e gene was used as intern reference gene to calculate relative expression levels. RT-PCR analysis indicated that the relative expression levels of \u003cem\u003eVvGRX28\u003c/em\u003e, \u003cem\u003eAtICE1\u003c/em\u003e, \u003cem\u003eAtICE2\u003c/em\u003e, \u003cem\u003eAtCOR15A\u003c/em\u003e, \u003cem\u003eAtCBF1\u003c/em\u003e and \u003cem\u003eAtCBF2\u003c/em\u003e were significantly upregulated in transgenic plants by induced by cold stress (Fig.\u0026nbsp;4J). Altogether, the finding demonstrated that \u003cem\u003eVvGRX28\u003c/em\u003e gene could actively response to cold stress by regulating some key cold gene expression levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;3.\u003c/b\u003e The relative expression level under cold stress, subcellular localization and heterologous expression of \u003cem\u003eVvGRX28\u003c/em\u003e. (A) The expression pattern of \u003cem\u003eVvGRX28\u003c/em\u003e in grape leaves under cold stress at different time point. (B) The clone of \u003cem\u003eVvGRX28\u003c/em\u003e, fragment size was 345 bp, and marker was 2000bp. (C) Overexpression vector schematic diagram of \u003cem\u003eVvGRX28\u003c/em\u003e, the vector carried the EGFP (enhanced green fluorescent protein) tag. (D) The subcellular localization of VvGRX28 in \u003cem\u003eNicotiana tabacum\u003c/em\u003e. 35s::EGFP was used positive control, DAPI was nuclear location marker. (E) Screening of transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e in plates (1/2 MS with 50 mg/L Kana), green\u003c/p\u003e\u003cp\u003eplants were cultivated until harvested seed. (F) Transgenic \u003cem\u003eArabidopsis thalianas\u003c/em\u003e were identified by PCR, marker was 2000bp, T3 plants were used to verify cold function.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;4\u003c/b\u003e Function analysis of \u003cem\u003eVvGRX28\u003c/em\u003e overexpression under cold stress in \u003cem\u003eArabidopsis thalianas\u003c/em\u003e. (A) Phenotypic observation of \u003cem\u003eVvGRX28\u003c/em\u003e overexpression under cold stress in \u003cem\u003eArabidopsis thalianas\u003c/em\u003e, 23 ℃ as control. (B) Relative electrolytic leakage of OE and WT plants. (C) MDA content of OE and WT plants. (D) Histochemical stain of OE and WT plant leaves, from left to right DAB, NBT and trypan blue, respectively. \u003cem\u003eDAB, NBT and\u003c/em\u003e trypan blue \u003cem\u003estaining were employed to evaluate the content of H\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e.\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eand death cells in leaves\u003c/em\u003e respectively, indicating the degree of cell damage under cold stress. (E) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content of OE and WT plants. (F) Proline content of OE and WT plants. (G) Soluble sugar content of OE and WT plants. (H) Glutathione content of OE and WT plants. (I) Pod activity of OE and WT plants. (J) The relative expression levels of \u003cem\u003eVvGRX28\u003c/em\u003e and cold responsive gene, including \u003cem\u003eAtICE1\u003c/em\u003e, \u003cem\u003eAtCOR15A\u003c/em\u003e, \u003cem\u003eAtCBF1\u003c/em\u003e, \u003cem\u003eAtCBF2.\u003c/em\u003e Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of three independent biological replicates. Different asterisks indicate significant differences, ** represents \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** represents \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** represents \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eVvGRX28 and VvZNF10 proteins exists an interaction relationship\u003c/h2\u003e\u003cp\u003eThe initial function verification showed that \u003cem\u003eVvGRX28\u003c/em\u003e gene plays a crucial role in responding and adapting to low temperatures by heterologous transformation. For detecting the promoter activity of \u003cem\u003eVvGRX28\u003c/em\u003e, pro\u003cem\u003eVvGRX28\u003c/em\u003e-GUS, 35S::GUS and P0-GUS agrobacterium solution were injected into tobacco leaves, GUS staining showed the blue color became more prominent, indicating GUS activity was higher than the controls under cold stress, and the lower the temperature, the higher the GUS activity (Fig.\u0026nbsp;5A). Relative expression of pro\u003cem\u003eVvGRX28\u003c/em\u003e were upregulated with decreasing of cold stress temperature (Fig.\u0026nbsp;5B).\u003c/p\u003e\u003cp\u003eTo further gain a thorough understanding of the regulatory mechanism of \u003cem\u003eVvGRX28\u003c/em\u003e, Y2H technique was employed to find the potential interaction protein of VvGRX28 protein. VvGRX28, as a bait protein, was cloned and linked to pGBKT7 vector. Recombinant plasmid of VvGRX28 and empty pGADT7 plasmid were co-transformed into Y2Hgold yeast strain in SD/-Trp-Leu and SD/-Trp-Leu-Ade-His medium to detect self-activating toxicity. Result showed there is no self-activating toxicity of VvGRX28 protein compared the controls (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Through screen of mating hybrid from AD library related to cold, AD library and Y2Hgold of BD-VvGRX28 yeast mixed liquid were coated in plates with SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade medium. After grew for 3\u0026ndash;4 d, single clonal plaques were spread in plates with SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade\u0026thinsp;+\u0026thinsp;X-α‐gal. Blue monoclonal yeast plaques were selected for PCR identification and sequence (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). After sequenced successfully, sequences were blast in NCBI, potential interaction proteins which includes VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19 were used to verify their interaction relationship by point to point. VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19, as prey proteins, were cloned and inserted into pGADT7. pGADT7-VvSRC2, VvZNF10, VvRPSA, Vv2OGDD19-pGADT7 and VvGRX28-pGBDT7 plasmid were co-transformed into Y2Hgold and then transferred in SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade and SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade\u0026thinsp;+\u0026thinsp;X‐α‐gal. Result showed that VvSRC2, VvRPSA and Vv2OGDD19 couldn\u0026rsquo;t growth in DDQ, only VvZNF10 normally grew and became blue in DDQ\u0026thinsp;+\u0026thinsp;X‐α‐gal, indicating VvZNF10 and VvGRX28 existed interaction relationship with VvGRX28 in \u003cem\u003evitro\u003c/em\u003e(Fig.\u0026nbsp;5C).\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eThe interaction between VvGRX28 and VvZNF10 proteins was further verified by BiFC and LCI\u003c/h2\u003e\u003cp\u003eLCI and BiFC experiments were employed to further confirm the interaction between VvZNF10 and VvGRX28 proteins. The CDS of VvZNF10 and VvGRX28 (delete stop codon) were cloned and linked to PCAMBIA1300-cLUC and PCAMBIA1300-nLUC, respectively. The Agrobacterium liquid with VvGRX28-nLUC and VvZNF10-cLUC (V:V\u0026thinsp;=\u0026thinsp;1:1) were transiently expressed in tobacco leaves. The strong luciferase complementation fluorescence signals were observed in tobacco cells co-transformed with VvGRX28-nLUC and VvZNF10-cLUC (Fig.\u0026nbsp;5D). Negative controls with nLUC and cLUC, nLUC and VvZNF10-cLUC, VvGRX28-nLUC and cLUC had no luciferase complementation, thereby confirming the two proteins interactions. Additionally, we constructed fusion proteins of VvZNF10 with the N-terminal half of yellow fluorescent protein (mYNE) and VvGRX28 (delete stop codon) with the C‐terminal half of YFP (mYCE). The Agrobacterium liquid with VvGRX28-mYCE and VvZNF10-mYNE (V:V\u0026thinsp;=\u0026thinsp;1:1) were transiently expressed in tobacco leaves. A stronger YFP fluorescence signals were obseved in nucleus and cell membrane in tobacco cells co-transformed with VvGRX28-mYCE\u0026thinsp;+\u0026thinsp;VvZNF10-mYNE (Fig.\u0026nbsp;5E). Negative controls with VvGRX28-mYCE\u0026thinsp;+\u0026thinsp;mYNE and mYCE\u0026thinsp;+\u0026thinsp;VvZNF10-mYNE had no yellow fluorescence signals, thus confirming the two proteins interactions in vivo. Overall, these results indicated that VvGRX28 interacted with VvZNF10 in \u003cem\u003evivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e Analysis of pro\u003cem\u003eVvGRX28\u003c/em\u003e transcriptional activity and the interaction verification between VvGRX28 and VvZNF10 by Y2H, LCI and BiFC. (A) The GUS staining of pro\u003cem\u003eVvGRX28\u003c/em\u003e in \u003cem\u003eNicotiana tabacum\u003c/em\u003e under cold stress. 35s-GUS was the positive control. P0-GUS was the negative control. (B) The relative expression levels of pro\u003cem\u003eVvGRX28\u003c/em\u003e under cold stress, \u003cem\u003eNtGUS\u003c/em\u003e were used as the internal gene. Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of three independent biological replicates. Different letters indicate significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. (C) Yeast two-hybrid (Y2H) assay demonstrates that VvGRX28 interacts with VvZNF10 in Y2Hgold yeast. The interaction verification of VvGRX28 and potential interaction protein (VvSRC2, VvZNF10, VvRPSA and Vv2OGDD19) by point to point in SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade and SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade\u0026thinsp;+\u0026thinsp;X-α‐gal, pGBKT7-p53\u0026thinsp;+\u0026thinsp;pGADT7-T and pGBKT7-Lam\u0026thinsp;+\u0026thinsp;pGADT7-T were used as positive and negative controls, respectively. Co-transformation of VvZNF10 and VvGRX28 can normally grew and become blue in DDQ\u0026thinsp;+\u0026thinsp;X‐α‐gal, indicating VvZNF10 and VvGRX28 protein exist interaction relationship. (D) Luciferase complementarity assay showing that fluorescence signal was only observed in sites co-injected with VvGRX28-nLUC and VvZNF10-cLUC in tobacco cell. (E) BiFC assay shows that VvGRX28 interacts with VvZNF10 mainly in the nucleus.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eThe subcellular localization and expression pattern analysis of VvZNF10 under cold stress in grape\u003c/h2\u003e\u003cp\u003eZNF gene was a C2H2-type zinc fingers protein functioning as a transcriptional regulator in plant growth and development. \u003cem\u003eVvZNF10\u003c/em\u003e with 912 bp size was located in 8th chromosome, which had ZnF_C2H2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). \u003cem\u003eVvZNF10\u003c/em\u003e (with EGFP tag) was cloned and transformed into pART-CAM-EGFP. Compared with the control, a strong green fluorescence signal was observed in tobacco cells, indicating VvZNF10 was located in nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). For clarifying expression pattern of \u003cem\u003eVvZNF10\u003c/em\u003e under cold stress in grape, RT-PCR were employed to detect its expression. RT-PCR analysis showed that under cold stress, the relative expression of \u003cem\u003eVvZNF10\u003c/em\u003e was significantly upregulated, and expression level showed a peak at 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). We suggested that \u003cem\u003eVvZNF10\u003c/em\u003e might be a positive regulator in response to low temperature.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eVvGRX28 improved cold tolerance by interacted with VvZNF10 in grape callus\u003c/h2\u003e\u003cp\u003eTo further investigate the cold regulation mechanism of \u003cem\u003eVvGRX28\u003c/em\u003e and \u003cem\u003eVvZNF10\u003c/em\u003e in grape callus, overexpression vector of \u003cem\u003eVvGRX28\u003c/em\u003e (with EGFP tag) and \u003cem\u003eVvZNF10\u003c/em\u003e (with Flag tag), and RNAi vector of \u003cem\u003eVvGRX28\u003c/em\u003e were constructed. Transgenic calluses were obtained by \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation and were identified by PCR amplify (Supplementary Fig.\u0026nbsp;3), namely OE-\u003cem\u003eVvGRX28\u003c/em\u003e, OE-\u003cem\u003eVvGRX28/VvZNF10\u003c/em\u003e, RNAi-\u003cem\u003eVvGRX28\u003c/em\u003e (RNAi transformation based on co-transformed of OE-\u003cem\u003eVvGRX28/VvZNF10\u003c/em\u003e). Transgenic calluses were subjected to 4 ℃ for 15 d. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, compared with WT and only overexpression \u003cem\u003eVvGRX28\u003c/em\u003e, co-transformed calluses of OE-\u003cem\u003eVvGRX28/VvZNF10\u003c/em\u003e had a better and quick growth status, after interfered expression of \u003cem\u003eVvGRX28\u003c/em\u003e, growth of transformed calluses were inhibited under normal and cold stress condition. Under cold stress, all type calluses occurred browning appearance, especially severed in WT and RNAi. The fresh weight of all type calluses was decreased, indicating water loss after cold stress. The fresh weight of transformed calluses of OE-\u003cem\u003eVvGRX28\u003c/em\u003e and OE-\u003cem\u003eVvGRX28/VvZNF10\u003c/em\u003e was higher than WT and RNAi callus, suggesting a low water loss rate under cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Under cold stress, the content of MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eH) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eL) in co-transformed calluses of OE-\u003cem\u003eVvGRX28/VvZNF10\u003c/em\u003e was decreased, while after interfering \u003cem\u003eVvGRX28\u003c/em\u003e sequence, the two oxidation indexes content was increased. Additionally, the content of proline (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), soluble sugar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ) and glutathione (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eK), POD activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eM) in co-transformed calluses of OE-\u003cem\u003eVvGRX28/VvZNF10\u003c/em\u003e was significantly higher than other calluses type. RT-PCR analysis indicated that \u003cem\u003eVvZNF10\u003c/em\u003e expression levels of co-transformed callus was significantly upregulated under cold stress, and the relative expression levels of \u003cem\u003eVvGRX28\u003c/em\u003e, \u003cem\u003eVvICE1\u003c/em\u003e, \u003cem\u003eVvICE2\u003c/em\u003e, \u003cem\u003eVvCOR413\u003c/em\u003e, \u003cem\u003eVvCBF1\u003c/em\u003e and \u003cem\u003eVvCBF2\u003c/em\u003e were also significantly upregulated in overexpressed callus induced by cold stress, while RNAi callus was downregulated under cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003eN). Comprehensive above findings, these results demonstrated that \u003cem\u003eVvGRX28\u003c/em\u003e promoting cold tolerance by interacted with VvZNF10 in grape callus.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlants are subjected to a wide array of biotic and abiotic stress conditions, which trigger signal transduction pathways and elicit molecular, metabolic, and physiological responses to regulate the ability to adapt various environment[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This is particularly concerning in the context of a growing global population, especially as low temperature constrains plant productivity and yield. ROS, as part of the signal transduction pathways, refers to a class of highly reactive oxygen-containing molecules, including superoxide anions, hydrogen peroxide, hydroxyl radicals, which participates in various physiological processes in the body, but in excessive ROS can cause oxidative stress and damage cells[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In order to maintain the oxidative homeostasis of cells, the antioxidant system and oxidative-reductive system composed of proteins that can transfer electrons from the input component to the downstream target protein remove excessive ROS. These mediators belong to a large family of oxidative-reductive protein enzymes in plants, including so-called oxidoreductase, including thioredoxins (TRXs), TRX-like proteins, and glutaredoxins (GRXs)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGRX is a small disulfide oxidoreductase that catalyzes the reversible reduction of disulfide bridges and glutathione (GSH)-containing disulfides using glutathione as the electron donor[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. GRX proteins by coded by GRX genes that play a very important in removing excessive ROS to adapt to constantly changing survival environment. Previous researches have been reported GRX gene family members numbers in 31 \u003cem\u003eArabidopsis\u003c/em\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], 48 rice[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], 58 tomato[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and 86 wheat[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, 32 gene family members were identified in grape. VvGRXs were classified into four clades including CPYC, CGFS, and CC subfamilies by phylogenetic tree analysis, consistent with previous results in other species[\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Most members of the same subfamily had similar motif compositions and gene structural features, indicating that these members may have similar evolution relationship and functions. \u003cem\u003eVvGRX28\u003c/em\u003e was clustered in CPYC type, and had the similar structure and motifs with \u003cem\u003eVvGRX31\u003c/em\u003e, demonstrating that an extremely close relationship in function and evolution. Structure differences between the four clades were observed, of which CC-type \u003cem\u003eVvGRX\u003c/em\u003e genes were mainly characterized by intron-less genes. Consistently, the genomic feature type genes that could quickly respond to various stresses, as also revealed for other genes with fewer exons[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Additionally, complex structures in exon/intron and motifs composition were found within CPYC-, GRL- and CGFS-types, indicating that these genes might be subjected to fine-tuning regulatory events such as alternative splicing (AS)[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Although there existed significant differences in structure composition, the highly conserved motif distribution pattern between CC and CPYC-types demonstrated the fact that CC-type gene might have evolved from the CPYC clade, before undergoing gene expansion during land plant evolution[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Interestingly, among the 5 VvGRX gene pairs that were co-linear in grape, and the two genes with synteny had the same branch in phylogenetic tree and similar structures. Consistently, previous studies in reported species found that GRX members in same subfamily exhibited similar motifs and structure arrangements, but the motif patterns differed across subgroups[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. These results suggested that GRX genes exhibited highly conserved and variable motif and structure patterns in different subfamilies, indicating functional conservation and diversification.\u003c/p\u003e\u003cp\u003eUpstream genes could bound to the \u003cem\u003ecis\u003c/em\u003e-regulatory element in the promoter of target genes to regulate the expression of the target genes to perform specific biological functions. \u003cem\u003ecis\u003c/em\u003e-regulator elements of VvGRX gene promoters had been analyzed and divided into three categories, including growth and development, plant hormone responsive, abiotic and biotic stress responsive elements. Core stress-responsive elements had ABRE, MYB, and W-box in GRX promoters, indicating evolutionary conservation of stresses regulation. Similarly, the unique fiber development-associated motifs of GRX and stress responsive elements were found in cottons, playing its specialized role in cotton fiber formation[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Common bean of GRX exhibited nodulation-specific motifs, associating with the symbiotic nitrogen-fixing capacity[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Banana had fruit ripening-related and responsive elements regulatory elements promoting fruit mature and regulating stress tolerance[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. \u003cem\u003eROXY1\u003c/em\u003e and \u003cem\u003eROXY2\u003c/em\u003e gene could regulate anther development[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The \u003cem\u003eOsGRXC2.2\u003c/em\u003e gene overexpression promoted embryonic development to increase grain weight in rice[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These results speculated that VvGRX genes maybe play important roles in hormone regulation, morphogenesis, and stress response in grape.\u003c/p\u003e\u003cp\u003eTissue-specific expression analysis revealed distinct developmental patterns for the VvGRXs, of which \u003cem\u003eVvGRX1, VvGRX17, VvGRX18, VvGRX19, VvGRX20, VvGRX22, VvGRX23, VvGRX26, VvGRX29, VvGRX32\u003c/em\u003e, and \u003cem\u003eVvGRX33\u003c/em\u003e gene showed relatively high expression level in all grape tissues, especially highly expressly in root, stem and leaves. Consistent with the finding, TaGRX73-7D exhibited high expression levels in leaves, while TaGRX29-3B was highly expressed in root tissues[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The expression levels of GRX were relatively higher in leaves in Quercus glauca[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, previous studies have reported that GRX genes play important roles in response to stresses. RT-PCR analysis indicated expression of \u003cem\u003eVvGRX\u003c/em\u003es were different induced by salt, drought and low temperature. The expression levels of \u003cem\u003eVvGRX17\u003c/em\u003e, \u003cem\u003eVvGRX20\u003c/em\u003e and \u003cem\u003eVvGRX32\u003c/em\u003e were all upregulated salt, drought and low temperature stress. The expression of \u003cem\u003eVvGRX32\u003c/em\u003e was the highest under PEG stress, the expression of \u003cem\u003eVvGRX20\u003c/em\u003e was the highest under salt stress, the expression of \u003cem\u003eVvGRX28\u003c/em\u003e was the highest under cold stress. In others species, there were numerous GRX genes participating in response to various stresses with similar function of \u003cem\u003eVvGRXs\u003c/em\u003e. Consistently, there were similar findings in previous studies. \u003cem\u003eOsGRX20\u003c/em\u003e gene could positively regulate plant responses to bacterial and fungal attacks, and overexpression of \u003cem\u003eOsGRX20\u003c/em\u003e significantly enhanced resistance to bacterial blight and ability to oxidative and salt stresses[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The Silencing of \u003cem\u003eOsGRXS17\u003c/em\u003e improves drought stress tolerance by removing excessive ROS and stomatal closure in rice[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The silencing of \u003cem\u003eGhGRL28\u003c/em\u003e improved the sensitivity to salt stress in cotton[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Most researches on abiotic stress focused on drought and salt, the function analysis of GRX genes was limited under cold stress. Therefore, \u003cem\u003eVvGRX28\u003c/em\u003e was cloned to investigated its role under cold stress. Initial findings showed the result demonstrated that the \u003cem\u003eVvGRX28\u003c/em\u003e overexpression enhanced the content of proline, soluble sugar, glutathione and peroxidase activities, and reduced the content of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and upregulated the expression of \u003cem\u003eAtICE\u003c/em\u003e, \u003cem\u003eAtCBF\u003c/em\u003e and \u003cem\u003eAtCOR\u003c/em\u003e related to cold stress in transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The findings was similar with the works that \u003cem\u003eBrMDHAR\u003c/em\u003e heterologous overexpression improved cold tolerance in the mini Chinese cabbage[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. VvZNF10 could interacted with VvGRX28 by Y2H, LCI and BiFC in vivio and vitro. \u003cem\u003eVvZNF10\u003c/em\u003e was a C2H2 type zinc finger protein composed of 2 Cys and 2 His residues surrounding Zn\u003csup\u003e2+\u003c/sup\u003e, widely distributed in eukaryotes, and occupied in response to various stresses. It has been reported that in response to low-temperature stress, for instance, the overexpression of \u003cem\u003eOsZFP245\u003c/em\u003e can enhance the tolerance of rice to low temperature, drought and oxidative stress[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Overexpression of \u003cem\u003eOsCTZFP8\u003c/em\u003e, a novel zinc finger protein was found to enhance cold tolerance in transgenic rice[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. A line with previous study, further investigation demonstrated that the overexpression \u003cem\u003eVvGRX28\u003c/em\u003e could improve the ability to resist low temperature by interacting with VvZNF10 in transgenic grape callus, while interfered \u003cem\u003eVvGRX28\u003c/em\u003e exhibited a opposite result. Similarly, previous studies also indicated that the \u003cem\u003eOsGRX15\u003c/em\u003e protein improved disease resistance to bacterial and fungal pathogens interacting with the transcription factor \u003cem\u003eOsWRKY65\u003c/em\u003e in the nucleus by upregulating the expression of the defense-related gene \u003cem\u003eOsPR1\u003c/em\u003e[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. \u003cem\u003eQgROXY1\u003c/em\u003e could physically interact with \u003cem\u003eAtTGA2\u003c/em\u003e, and transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ectopically overexpressing \u003cem\u003eQgROXY1\u003c/em\u003e is hypersensitive to exogenously applied salicylic acid[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTaken together, the findings suggested that \u003cem\u003eVvGRX28\u003c/em\u003e was a regulator gene in contributing to cold stress tolerance regulation in grape. However, the results only preliminary verified the cold function in ectopically \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and grape callus levels not in grape plant, the specific molecular mechanisms regulating these putative VvGRX-mediated stress responses require further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAs crucial oxidoreductases, plant GRXs play vital roles in developmental regulation and stress responses. In this study, we successfully identified 32 VvGRX genes family members in grape. Comprehensive bioinformatics analyses revealed that the structure, motifs, \u003cem\u003ecis\u003c/em\u003e-acting elements of VvGRX were similar in genes clustered into close branches. Most CC-type genes were less introns and highly conserved. \u003cem\u003eCis\u003c/em\u003e-acting elements mainly were involved in stress response and hormone regulation. VvGRXs with a high expression level almost in all tissues were induced by salt, drought and cold stress to differentially express. Furthermore, \u003cem\u003eVvGRX28\u003c/em\u003e were functionally characterized to detect the cold tolerance function. The result suggested that the \u003cem\u003eVvGRX28\u003c/em\u003e, as positive regulator, could improve the ability to resist low temperature by interacting with \u003cem\u003eVvZNF10\u003c/em\u003e in nucleus and cytomembrane in transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and grape callus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Collectively, the findings provide important insights into the redox regulatory mechanisms mediated by VvGRXs in grape, contributing to provide candidates genes for functional validation to future breeding for grape cold stress improvement.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: GJN, SL, BHC and SM Methodology: GJN, ZHP, JRZ, ZLL, XXQ validation: CCZ, LM and HKY Data curation: formal analysis: GJN, SL, BHC Software: GJN, JRZ, ZLL, XXQ Investigation: ZHP, JRZ Writing\u0026mdash;original draft: GJN. Writing\u0026mdash;review \u0026amp; editing: GJN, SL, BHC and SM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Project funded by the Central Government - Guided Local Scientific and Technological Development Funds (25ZYJA033), Gansu Provincial Key Talent Project (2023RCXM23), and Gansu Province Graduate Innovation Star Program (2025CXZX-803).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eA College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eCollege of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eState Key Laboratory of Aridland Crop Science, Lanzhou 730070, China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eExperimental and Base Management Center, Gansu Agricultural University, Lanzhou 730070, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLongo R, Carew A, Sawyer S, Kemp B, Kerslake F. 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Biochem Biophys Res Commun. 2020;533:1385\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2020.10.027\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2020.10.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Glutaredoxin, Cold stress, VvGRX28, Overexpression, RNAi, Grape","lastPublishedDoi":"10.21203/rs.3.rs-8260182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8260182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e Glutaredoxins (GRXs) are small oxidoreductases that play a crucial role in responses to abiotic stress. Although the GRX gene family has been characterized in several species, the knowledge of their evolution relationship, diversification and function in grape are still limited.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e In this study, 32 VvGRX genes were identified and clustered into CC-, CGFS-, GRL- and CPYC-type categories. The structure and motifs of VvGRXs were similar in genes clustered into close branches, indicating highly conserved during the evolutional process. \u003cem\u003eCis\u003c/em\u003e-acting elements mainly were involved in stress response and hormone regulation. Tissue-specific expression showed that VvGRXs was differentially expressed in different grape tissues. RT-PCR indicated that \u003cem\u003eVvGRX28\u003c/em\u003e expression can actively be induced by cold stress. Furthermore, \u003cem\u003eVvGRX28\u003c/em\u003e were functionally characterized and cloned to verify the cold tolerance function. Through \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated to overexpression and interfere \u003cem\u003eVvGRX28\u003c/em\u003e, the result demonstrated that the \u003cem\u003eVvGRX28\u003c/em\u003e overexpression can enhance the content of proline, soluble sugar, glutathione and peroxidase activities, and reduced the content of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and upregulated the expression of \u003cem\u003eICE\u003c/em\u003e, \u003cem\u003eCBF\u003c/em\u003e and \u003cem\u003eCOR\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and grape callus, while exhibiting an opposite trend after RNAi. VvZNF10, as the interaction protein of VvGRX28, overexpression and co-transformation with \u003cem\u003eVvGRX28\u003c/em\u003e could improve the cold tolerance in grape callus.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e The result demonstrated that \u003cem\u003eVvGRX28\u003c/em\u003e was a positive regulator to enhance cold tolerance interacting with \u003cem\u003eVvZNF10\u003c/em\u003e mainly in nucleus in grape callus. Collectively, this study provides a comprehensive analysis of the VvGRX gene family, offering novel insights into the regulation mechanism of \u003cem\u003eVvGRX28\u003c/em\u003e under cold stress in grape.\u003c/p\u003e","manuscriptTitle":"VvGRX28 interacting with VvZNF10 modulates cold tolerance via eliminating excessive ROS in grapevine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 03:49:59","doi":"10.21203/rs.3.rs-8260182/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-15T08:52:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T11:58:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149202444498863599195686062647824003262","date":"2026-01-10T19:36:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225882597595115679511076397008494638273","date":"2026-01-01T06:31:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T07:49:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134719172440470542539858184566741421229","date":"2025-12-11T09:00:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T22:02:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-04T02:47:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-04T02:46:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical and Biological Technologies in Agriculture","date":"2025-12-02T11:47:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"403156c1-f87c-4db1-a04d-790c15f341bc","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-05T16:24:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 03:49:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8260182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8260182","identity":"rs-8260182","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}