Transcriptomic analysis provides insights into the abscisic acid mediates brassinosteroid-induced cold resistance of grapevine (Vitis vinifera L.)

Brassinosteroids (BRs) and abscisic acid (ABA) both play positive roles in plant resistance to cold stress. Despite the recent report on the involvement of ABA in BR-induced enhanced tolerance to cold stress, the underlying molecular mechanisms of stress tolerance remain unclear. Moreover, whether there are ABA-independent pathways for BR-induced enhancement of cold stress tolerance in grapevines needs to be clarified. Herein, the potential involvement of ABA in BR-induced cold resistance in grapevines was investigated by contrasting the different responses among ABA, BR, and the combination of BR and NDGA (an inhibitor of endogenous ABA biosynthesis) treatments under cold stress. Results showed BR and ABA foliar application alone increased the chlorophyll fluorescence parameters, regulated the antioxidant system, and alleviated oxidative damage induced by cold stress. Interestingly, NDGA blocked the BR-induced cold resistance by increasing reactive oxygen species content and reducing antioxidant enzyme activity. Transcriptomic analysis suggested that exposure to cold stress resulted in very different patterns of gene expression and enriched pathway responses. Among them, ERF transcription factors were observed to be up-regulated in both BR and ABA treatment, calcium-binding protein genes were up-regulated only under BR treatment alone, and xyloglucosyl transferase genes were up-regulated only under ABA treatment. Overall, we concluded that ABA was involved in BR-induced cold resistance in grapevines, but there was also a different candidate pathway between ABA and BR treatments under cold stress.


Introduction
Low temperature, an abiotic stressor, adversely endangers grapevine growth and development as well as limits the species' geographical distribution and output (Wang et al. 2021a).Cold stress causes the accumulation of reactive oxygen species (ROS).ROS are overproduced under cold stress, which directly oxidizes DNA, RNA, protein, and lipid, impairing photosynthesis and plant cell membrane (Guo et al. 2018;Mittler 2017).To withstand cold stress, a set of comprehensive molecular, physiological, and biochemical events, including perception and transduction of plant cold-signaling, scavenging of ROS, and accumulation of osmoprotectants, occur in plants (Ding et al. 2019).ROS detoxification is a crucial part of the ascorbate-glutathione (ASA-GSH) cycle.In the process, antioxidases such as superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR) work with nonenzymatic antioxidants (ascorbate and glutathione) to remove excessive ROS and protect the cell membrane from lipid peroxidation (Miller et al. 2010).Cold stress regulates several transcription factors (TFs) and cold-regulated genes at the molecular level, preparing plants for cold tolerance.C-REPEAT BINDING FACTOR (CBF) genes, the central nodes in the cold stress pathway, are immediately and strongly stimulated by cold stress, which encoded proteins trigger transcription of cold-responsive (COR) genes (Shi and Yang 2014).Furthermore, these changes are usually mediated by the level of phytohormones, including BR, ABA, and jasmonic acid (JA) (Devireddy et al. 2021).
Plant responses to unfavorable conditions such as drought, excessive salt, and extreme temperature are thought to be aided by ABA (Devireddy et al. 2021;Knight et al. 2004).Increasing endogenous ABA levels in different plants are associated with cold resistance (An et al. 2023;Wang et al. 2016;Zhang et al. 2022).Exogenous ABA could greatly improve cold resistance in Chorispora bungeana, Bermudagrass, and Elymus nutans (Fu et al. 2017;Huang et al. 2017;Liu et al. 2011).The resistance induced by ABA is thought to be mediated through enhancing antioxidase activity, modulating phenolic metabolism, lowering ROS levels, and reducing oxidative stress (Tian and Li 2018).Furthermore, ABA promotes cold-regulated gene expression by inducing CBF gene transcription (Knight et al. 2004).However, besides the CBF signaling pathway, the cold-regulated genes in the CBF-independent pathway regulated by ABA largely remain unclear.
BR, a steroid phytohormone family, regulates plant growth and development and protects plants from a variety of abiotic stresses such as heavy metals, salinity, drought, and temperature extremes (Fang et al. 2019;Li et al. 2022;Wang et al. 2022;Zhou et al. 2018).BRASSINOSTER-OID INSENSITIVE1 (BRI1) is a receptor kinase that perceives BRs and subsequently recruits BRI1-ASSOCI-ATED RECEPTOR KINASE1 (BAK1) to form a functional receptor complex.The complex initiates a series of phosphorylation cascades, activating BRASSINAZOLER-ESISTANT 1 (BZR1) and BRI1EMSSUPPRESSOR1 (BES1).Subsequently, activation of BES1/BZR1 binds to the promoter and induces the expression of BR-responsive genes (Clouse 2011).BZR1 improves Arabidopsis plant resistance to low temperatures via directly binding to and regulating the expression of CBF genes (Li et al. 2017).Aside from the CBF/DREB1 signaling pathway, BZR1 alleviates cold stress-induced photoinhibition in tomatoes via apoplastic H 2 O 2 production mediated by RESPIRA-TORY BURST OXIDASE HOMOLOG1 (RBOH1) (Fang et al. 2019).Plant hormone interactions are also important in the stress response induced by BR.In the regulation of stomatal closure in Arabidopsis, BRs and ABA have synergistic and antagonistic effects (Ha et al. 2016).Exogenous BR induces ABA biosynthesis, elevates ABA levels, and increases heat and paraquat (PQ) oxidative stress tolerance (Zhou et al. 2014).However, it has been shown that BR and ABA have an antagonistic connection that regulates plant growth and stress tolerance.For instance, brassinosteroid reduces ABA-mediated inhibition of early seedling development via suppressing ABA signaling output (Ryu et al. 2014).Reduced endogenous brassinosteroid biosynthesis or signaling in Arabidopsis thaliana could increase plant responsiveness to abscisic acid and, in turn, promote drought tolerance (Northey et al. 2016).The findings indicate that ABA participates in BR-induced processes of plant growth and stress response.Nevertheless, the interaction between BR and ABA in the cold stress response remains unclear and contentious.
Previous studies have shown that single applications of ABA or BR increase cold tolerance in plants through their regulating antioxidant systems (Chen et al. 2019;Fu et al. 2017).Moreover, BR-induced cold stress tolerance via ABA biosynthesis in tomatoes (An et al. 2023).However, whether ABA is involved in BR-induced regulation of the antioxidant system in response to cold stress is poorly understood, and whether there is an ABA-independent pathway for BRinduced improvement of plant cold resistance is also uncertain.Herein, we investigated the potential function of ABA in cold tolerance induced by BR of grapevines, contrasting the different responses in the antioxidant system to cold stress under pretreatment with ABA, BR, and the combination of BR and NDGA.Furthermore, the genes associated with potentially different functions between BR and ABA under cold stress were explored by analyzing the transcriptomic landscape in grapevine.Accordingly, we will reveal the underlying mechanisms of BR-mediated cold tolerance in grapevines.

Plant materials and treatments
One-year-old hardwood cuttings of "Cabernet Sauvignon" (Vitis vinifera L.) were obtained from the experimental vineyard of the College of Enology, Northwest A&F University, Yangling Shaanxi, China.The cuttings were grown in pots containing a mixture of soil, humus and perlite (1:1:1 by volume) and placed in growth chambers.The light incubator had a photoperiod of 16 h/8 h light/dark, a day/ night temperature of 25 °C/20 °C, and a light intensity of 100 µmol•m −2 •s −1 .Uniformly grown grape seedlings with four to five fully expanded leaves were selected for further experiments.
Control plants were given distilled water laced with 0.1% ethanol.After eight hours of spraying, the seedlings were kept at 4 °C for 72 h.Each treatment included 3 replicates, and each replicate consisted of 18 plants.The samples of leaves were frozen using liquid nitrogen and then stored at − 80 °C until further analysis.

Detection of electrolyte leakage
Electrolyte leakage (EL) was examined in accordance with Janda et al. (1999).

Superoxide anion content detection
The micro-superoxide anion (O 2 − ) production rate was determined using a micro-superoxide anion assay kit (Solarbio, Beijing, China).

Detection of reduced ascorbate and glutathione levels
Reduced ascorbate (AsA) content was evaluated using an AsA content test kit (Solarbio, Beijing, China).The GSH content of reduced glutathione was assayed using a GSH content test kit (Solarbio, Beijing, China).

Detection of antioxidant enzymes' activities
Leaf tissues (0.1 g) were ground with 1 mL of cold 0.1 M phosphate buffer (pH 7.8, containing 1 mM EDTA and 2% insoluble polyvinylpyrrolidone) for extraction of SOD, CAT, APX, DHAR, and GR or with 1 mL of cold 50 mM MES/ KOH buffer (pH 6.0, containing 1 mM ascorbate, 40 mM KCl, 2 mM CaCl 2 and 2% insoluble polyvinylpyrrolidone) for extraction of MDHAR.The homogenates were centrifuged at 15,000 rpm at 4 °C for 10 min, and the supernatants were collected for further use to determine antioxidant enzyme activities.SOD and CAT activity were tested separately with the SOD assay kit (Solarbio, Beijing, China) and the CAT assay kit.APX, GR, DHAR, and MDHAR activity were assessed in accordance with Noctor et al. (2016).

RNA extraction and real-time quantitative PCR analysis
Total RNA was isolated from grape leaves using an RNA extraction kit according to the manufacturer's instructions (TaKaRa, Dalian, China).Next, the RNA was quantified with an ultra low volume spectrometer-BioDrop Touch (Biochrom, England), and its integrity was confirmed using 1.0% agarose gel electrophoresis.The isolated RNA was reverse-transcribed to cDNA using a PrimeScript First-Strand cDNA Synthesis Kit (Takahashi, Japan).qRT-PCR was conducted utilizing an SYBR Green qPCR kit (TaKaRa, Dalian, China) and a Bio-Rad IQ5 light cycler system (Applied Biosystems, USA).Supplemental Table S6 lists the gene-specific primer pairs used in qRT-PCR.Relative expression levels were normalized using the actin gene as an internal reference gene and calculated as per Livak and Schmittgen (2001).

Transcriptome investigation
To explore the potential mechanisms that brassinosteroid enhanced cold stress tolerance in grapevine, transcript abundance was compared among BR treatment, ABA treatment, and the control treatment plants.The total RNA of leaves was extracted using an RNA extraction kit.RNA quantity and quality were detected as described previously.RNA-seq libraries were prepared using the above RNA samples according to the method described by Miao et al. (2016), and then sequenced using the Illumina Hiseq 2500 platform (Biomarker, Beijing, China).The clean reads were obtained by deleting residual adapters and low-quality sequences of raw reads using Perl scripts developed by Biomarker Technologies Co. Ltd. (Beijing, China) and were then aligned to the V. vinifera reference genome (12 ×) (Jaillon et al. 2007).Calculating gene expression levels using pieces per kilobase of exon per million fragments (FPKM).Differentially expressed genes (DEGs) were identified using a false discovery rate (FDR) < 0.01 and an |log2FoldChange|> 1. Gene ontology (GO) enrichment analysis was performed with AgiGO 2.0.The statistical enrichment of DEGs was examined using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
There were three repetitions of each experiment.Oneway ANOVA was used to assess the differences between treatments.SPSS version 20.0 was used to conduct Duncan's multiple range test for post-hoc multiple comparisons (P < 0.05).The values are presented as mean standard deviation (SD).Graphs were plotted using SigmaPlot 8.0 (Systat Software).

ABA contributed to BR-induced cold tolerance in grapevine plant
Following 72 h of exposure to 4 °C, the seedlings in control exhibited leaf drooping and withering, a significant increase in O 2 − accumulations and electrolyte leakage (EL), and a decrease in the effective quantum yield of photosystem II (ϕPSII) and the nonphotochemical quenching (NPQ).However, ABA treatment attenuated the cold-induced damage of leaves as indicated by higher chlorophyll fluorescence parameters (ϕPSII and NPQ) and lower oxidative damage (EL and O 2 − accumulations) relative to the control plants.BR pretreatment, like ABA, significantly reduced O 2 − accumulations and EL while increasing ϕPSII and NPQ levels in comparison to control seedlings (Fig. 1).Notably, NDGA application obviously inhibited BR-induced ϕPSII parameters at 24 h of cold stress.In addition, NDGA pretreatment blocked BR-induced decreases in EL.For instance, EL in NDGA-pretreated plants increased by 34.9% compared to the BR treatment alone at 24 h after cold stress.These data suggested that BR and ABA positively regulated cold tolerance in grapevines, and ABA contributed to BR-induced cold tolerance in grapevine plants.

ABA were involved in BR-induced ASA-GSH pathway regulation in response to cold stress
To assess the function of ABA in cold tolerance induced by BR in grapevine seedlings, the changes of antioxidant substance contents and antioxidant enzyme activities in the AsA-GSH cycle were analyzed.As shown in Fig. 2, Cold stress treatment led to a gradual decrease of AsA and GSH contents in grape seedlings.AsA contents in both ABA and BR treatment increased first and then decreased during cold stress in grape leaves, peaking 24 h and 48 h after treatment, respectively.Similar to AsA contents, GSH contents gradually increased in ABA treatment, and increased first and then decreased in BR treatment during cold stress in grape leaves.Compared to the cold treatment alone, BR and ABA pretreatment increased AsA and GSH levels during cold stress.However, NDGA pretreatment blocked BR-induced increase in ASA and GSH contents within 48 h and 72 h after cold stress treatment, respectively (Fig. 2g-h).
The activities of SOD, CAT, APX, MDHAR, and GR increased and then decreased after cold treatment in grape leaves, peaking 24 h after treatment.DHAR activities increased and then decreased after cold treatment in grape leaves, peaking 48 h after treatment.Compared to the cold treatment alone, ABA pretreatment increased SOD, MDHAR and GR activities during cold stress, APX and DHAR activities within 24 h of cold stress, and CAT activities within 48 h of cold stress.BR pretreatment increased SOD and MDHAR activities during cold stress, as CAT, APX, DHAR and GR activities within 48 h of cold stress.However, NDGA pretreatment attenuated the rise in SOD, CAT, and DHAR activities at 24 h after cold treatment, GR activities at 48 h after cold treatment, as well as APX activities within 48 h of cold treatment induced by BR (Fig. 2a-f).Results revealed that BR-induced improvement of antioxidative capacity under cold stress was mediated by ABA.Interestingly, in comparison to those measured in control plants, the application of NDGA combined with BR elevated AsA and GSH contents, SOD, APX, DHAR, and MDHAR activities to different degrees (Fig. 2).The result suggested that BR also activated the AsA-GSH cycle to scavenge excess ROS under cold stress in grapevines via ABA-independent regulatory systems.

ABA was involved in BR-induced CBF pathway regulation in response to cold stress
CBFs, as an essential transcription factor, play an important role in cold stress response are well known (Shi and Yang 2014).In the current research, cold treatment significantly increased the expression of VvCBF1, VvCBF2, VvCBF3, and VvCBF4 in grape seedlings.Among them, VvCBF1 expression was elevated following exposure to cold, and the largest peak occurred 72 h following cold stress in control plants.VvCBF2, VvCBF3, and VvCBF4 expression increased rapidly after cold treatment, peaked at 3 h after cold stress, and then progressively lowered to the level at 0 h (Fig. 3a-d).ABA pretreatment had no significant effect on VvCBF1, VvCBF2, and VvCBF3 expression during cold stress compared with cold treatment alone in grape seedlings (Fig. 3a-c).Whereas ABA treatment upregulated the expression of VvCBF4 gene by 1.57 and 0.92-fold at 1 h and 3 h after cold treatment, respectively, compared with cold treatment alone (Fig. 3d).There was no significant difference in VvCBF3 and VvCBF4 expression BR treatment and the control (Fig. 3c-d).However, VvCBF1 transcripts levels in BR-pretreated plants increased by 1.10 and 4.29-folds at 3 h and 96 h after cold treatment, respectively, compared to the control plants (Fig. 3a).VvCBF2 transcripts levels in BRpretreated plants increased by 0.34-folds compared to the − generation (c), ϕPSII (d), and NPQ (e) in the four types of plants were measured after being subjected to cold (4 °C) for the specified time.The data were represented as mean ± SD (n = 3).Variations between treatments that are statistically significant (P < 0.05) are denoted by distinct lowercase letters ◂ control plants at 24 h after cold treatment (Fig. 3b).Compared with BR treatment alone, co-application of NDGA and BR caused a significant decrease in the expression level of VvCBF1 compared with BR treatment alone at 3 h after cold treatment, decreased by 0.55-folds (Fig. 3a).

Transcriptome profile of BR and ABA under cold stress
Based on the relative expression level of CBF genes (Fig. 3), the seedlings of 3 h-cold stress were chosen for the transcriptomic analysis.Each sample was sequenced in triplicate.Using the Illumina Hiseq 2500 platform, a total of twelve samples were sequenced.After QC and alignment, the clean data were obtained with a Q30 percentage > 93% and a GC percentage between 46.3-47.0%(Table S1).Spearman's correlations among the replicates of each treatment were examined.The significantly high correlation suggested good reproducibility of samples (Fig. S1).
Differentially expressed genes (DEGs) were identified by comparing the global gene expression profiles of the various treatments.1359 and 2968 DEGs were identified in ABA (Control_3 versus ABA) and BR (Control_3 versus BR) based on the criteria of padj < 0.05 and |log2 (fold change) |> 1 (Fig. 4b, c).These results demonstrated that BR had a more marked effect on the transcription of a group of genes than ABA treatment under cold stress.Further analysis showed that 4156 DEGs were identified in COLD (Con-trol_0 versus Control_3), which contained 2672 upregulated genes and 1484 downregulated genes (Fig. 4a).For upregulated genes, only 3 DEGs were commonly identified in three pairwise comparisons, BR and ABA shared 199 upregulated (h) contents in the four types of plants were measured after being subjected to cold (4 °C) for the specified time.Data were represented as mean ± SD (n = 3).Variations between treatments that are statistically significant (P < 0.05) are denoted by distinct lowercase letters genes, BR and COLD shared 46 upregulated genes, ABA and COLD shared 15 upregulated genes.Moreover, 2608, 1033, and 329 DEGs were uniquely upregulated in cold treatment, BR treatment, and ABA treatment, respectively (Fig. 5a).For downregulated genes, a total of 3 DEGs were commonly identified in three pairwise comparisons, BR and ABA shared 297 downregulated genes, BR and COLD shared 27 downregulated genes, ABA and COLD shared 23 downregulated genes.Moreover, 1431, 1360, and 490 DEGs were uniquely down-regulated in COLD, BR, and ABA treatments, respectively (Fig. 5b).

Transcriptome Profiles of hormone biosynthesis and signaling transduction under cold stress
In this study, two NCED genes (NCED1) and one ZEP gene, key enzymes that catalyze ABA biosynthetic pathway, were upregulated in control treatment at 3 h, which suggested cold stress-induced ABA synthesis.Signaling of ABA is perceived by PYR/PYL/RCAR receptors (PYLs), which inhibit group-A protein phosphatases 2C (PP2Cs) by binding to them.Inhibition of PP2Cs activates the serine/threonine-protein kinase SRK2 (SnRK2) family, activating the ABA response pathway downstream effectors (Finkelstein 2013).In the control treatment, ten PP2C genes largely increased during cold stress.Among them, six PP2C genes were down-regulated in BR treatment.Besides, two PYL genes and one ABI gene upregulated in BR treatment, and these genes showed no significant change in the control treatment (Fig. 6).
In addition, cold stress significantly enhanced the BR pathway's transcriptional landscape, including BR synthesis (VvROT3 and VvBAS1) and signaling activation (VvBAK1 and VvBZR1-2).Meanwhile, ABA treatments led to an upregulation of genes in the BR pathway (VvBR6OX1 and VvBKI1) (Fig. 7), further confirming the synergistic and BR.Control_0, the grape seedlings were transported to 4 °C for 0 h after distilled water treatment.Control_3, the grape seedlings were transported to 4 °C for 3 h after distilled water treatment.BR, the grape seedlings were transported to 4 °C for 3 h after BR treatment.ABA, the grape seedlings were transported to 4 °C for 3 h after ABA treatment effect of ABA and BR on the response of grapes under cold stress.

KEGG analyses of the DEGs in response to cold treatment
In total, 4156 DEGs were identified under cold stress.Among these, 2672 genes were upregulated, and 1484 were downregulated (Fig. 4a).KEGG analysis showed that these upregulated genes were involved in 104 distinct pathways, in which there were 6 pathways with P-value less than 0.05, including plant hormone signal transduction (KO04075), phenylpropanoid biosynthesis (KO00940), plant-pathogen interaction (KO04626), starch and sucrose metabolism (KO00500), glycerophospholipid metabolism (KO00564) and pentose and glucuronate interconversions (KO00040).KEGG analysis of the downregulated genes revealed that they were involved in 86 different pathways, of which four 4 °C for 0 h after distilled water treatment.Control_3, the grape seedlings were transported to 4 °C for 3 h after distilled water treatment.BR, the grape seedlings were transported to 4 °C for 3 h after BR treatment.ABA, the grape seedlings were transported to 4 °C for 3 h after ABA treatment had P values less than 0.05, including glycine, serine, and threonine metabolism (KO00260), flavonoid biosynthesis (KO00941), plant hormone signal transduction (KO04075) and tropane, piperidine, and pyridine alkaloid biosynthesis (KO00960) (Table S5).

GO and KEGG analyses of the DEGs exclusively in response to BR under cold treatment
The potential biological functions and signal transduction pathways of the DEGs were identified using GO and KEGG analysis.Results of the GO analysis demonstrated that 46 upregulated DEGs in BR and COLD treatments were primarily focused on biological processes, which were annotated into 31 GO classifications.According to the corresponding -log 10 Pvalue of each term, the top 20 significant GO terms were sorted from large to small.Of these, respiratory burst involved in defense response was the most significantly enriched term with 16 genes.In this term, six DEGs were annotated as 'calcium-binding protein'.The KEGG analysis of elevated DEGs in BR and COLD treatments revealed enrichment in six distinct pathways.Three pathways (KO00940, KO00941, KO00945) were involved in the biosynthesis of other secondary metabolites, two (KO00053 and KO00562) in carbohydrate metabolism and one (KO04626) in environmental Fig. 6 Heat maps of the differentially expressed genes associated with ABA synthesis and signaling pathway in response to Control_0, Control_3, BR, and ABA treatments.Control_0, the grape seedlings were transported to 4 °C for 0 h after distilled water treatment.Con-trol_3, the grape seedlings were transported to 4 °C for 3 h after distilled water treatment.BR, the grape seedlings were transported to 4 °C for 3 h after BR treatment.ABA, the grape seedlings were transported to 4 °C for 3 h after ABA treatment adaptation.KO04626 'plant-pathogen interaction' pathway was specifically enriched in BR and COLD treatments (Fig. 5a).In this pathway, the six calcium-binding proteins were upregulated (Fig. 8).

GO and KEGG analyses of the DEGs exclusively in response to ABA under cold stress
In ABA and COLD treatments, a total of 15 upregulated DEGs were significantly enriched in five GO terms.Within the biological process category, the term remarkably enriched was xyloglucosyl transferase activity (GO:0016762).Within the cellular component category, the terms significantly enriched included extracellular space (GO: 0005615) and cell wall (GO: 0005618).Within the molecular function category, the term that was significantly enriched included aging (GO: 0007568) and cellular glucan metabolic process (GO: 0006073).The KEGG analysis of upregulated DEGs in ABA and COLD treatments was uniquely enriched in three KEGG pathways, including carotenoid biosynthesis, amino sugar and nucleotide sugar metabolism, and plant hormone signal transduction.'Cell wall' term with 5 genes was the most enriched GO term in ABA and COLD treatments (Fig. 5b).

Transcriptome profiles of transcription factors
TFs are crucial regulators of cold signal transduction (Chen and Zhu 2004).In our experiment, cold stress greatly enriched numerous transcription factor families, including the AP2/ERF, HLH, WRKY, MYB, GRAS, and C2H2-ZFP.TFs that were elevated in cold stress notably enriched in the AP2/ERF families.Cold stress significantly upregulated the expression of 32 ERF genes, of which VvERF81, VvERF86, and VvERF88 were upregulated in Cold, BR, and ABA treatments.VvERF60 was upregulated in BR and cold treatments.Moreover, cold treatment significantly upregulated the expression of 12 bHLH genes, 14 WRKY genes, 12 MYB genes, 6 GRAS genes, and 4 C 2 H 2 -ZFP genes.Notably, among these DEGs, VvSCL14.2,VvZAT10.1, and VvZAT10.2 were upregulated in BR treatment (Fig. 9).

Effects of cold acclimation on gene expression patterns of grape seedlings
Low temperature is one of the most common environmental stresses that seriously affect the growth and Fig. 7 Heat maps of the differentially expressed genes associated with BR synthesis and signaling pathway in response to Control_0, Control_3, BR, and ABA treatments.Control_0, the grape seedlings were transported to 4 °C for 0 h after distilled water treatment.
Control_3, the grape seedlings were transported to 4 °C for 3 h after water treatment.BR, the grape seedlings were transported to 4 °C for 3 h after BR treatment.ABA, the grape seedlings were transported to 4 °C for 3 h after ABA treatment development of plants.However, when plants are exposed to nonfreezing low temperatures in advance, their freezing resistance increases, known as cold acclimation (Thomashow 1999).CBF/DREB1 genes play central roles in cold acclimation.However, only 10-20% of COR genes are regulated by CBFs, and cbfs triple mutants are still capable of cold acclimation (Jia et al. 2016).Current results show that cold acclimation enhanced the expression of AP2/ERF, HLH, WRKY, MYB, GRAS, and C2H2-ZFP families in grape seedlings, suggesting these TF families play vital roles in cold acclimation-induced improvement of freezing resistance in grape seedlings.More appreciably, cold acclimation-induced genes were mainly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, plant-pathogen interaction, starch and sucrose metabolism, glycerophospholipid metabolism, and pentose and glucuronate interconversions pathways.Many researchers reported that plant hormone signal transduction, phenylpropanoid biosynthesis and starch, and sucrose metabolism positively regulate plant freezing resistance (Londo et al. 2018;Ming et al. 2021;Wang et al. 2021b), supported our results.Taken together, cold acclimation mainly improves the frost resistance of grapes by modulating the pathway of plant hormone signal transduction, phenylpropanoid biosynthesis, plant-pathogen interaction, starch and sucrose metabolism, glycerophospholipid metabolism, and pentose and glucuronate interconversions, the expression of AP2/ERF, HLH, WRKY, MYB, GRAS, and C2H2-ZFP TF families.

BR positively regulates cold tolerance in grapevine plant
BR, a type of growth-promoting phytohormone, implicates plant responses to diverse environmental stressors (Li et al. 2022;Zhou et al. 2018;Wang et al. 2022).Cold tolerance in plants is associated with endogenous BR concentrations and BR signaling (Fang et al. 2019;Li et al. 2017).Here, cold stress was observed to stimulate BR biosynthetic gene transcripts and BR signaling gene transcripts (Fig. 7).Meanwhile, exogenous BR significantly attenuated the leaf wilting, the photoinhibition, and oxidative damages against cold stress in grapevine (Fig. 1).To scavenge excessive ROS and mitigate oxidative damage, plants develop a sophisticated antioxidant defense system composed of both non-enzymatic antioxidants and antioxidases (Miller et al. 2010).Indeed, exogenously applied BR dramatically boosted ASA and GSH levels, as well as SOD, CAT, APX, DHAR, and MDHAR activities under cold stress in grapevine plants (Fig. 2).This further supports the function of BR in responding to changes in the growing environment by regulating photosynthesis and antioxidant systems (Fang et al. 2019;Xia et al. 2018).
Ca 2+ participates in cold signal transduction in plants (Ding et al. 2019).Cold stress rapidly elevates the Fig. 8 Heat maps of the differentially expressed genes associated with calcium-binding protein in response to Con-trol_0, Control_3, BR, and ABA treatments.Control_0, the grape seedlings were transported to 4 °C for 0 h after distilled water treatment.Control_3, the grape seedlings were transported to 4 °C for 3 h after distilled water treatment.BR, the grape seedlings were transported to 4 °C for 3 h after BR treatment.ABA, the grape seedlings were transported to 4 °C for 3 h after ABA treatment intracellular calcium level, which modulates the activity of calcium-binding proteins and subsequently triggers the cold-signaling cascade, such as CBF/COR gene transcription (Guo et al. 2018;Straltsova et al. 2015).Ca 2+ channel activity can be affected by BR, and calcium/calmodulindependent protein kinases are implicated in the antioxidant defense and stress tolerance induced by BR (Straltsova et al. 2015;Liu et al. 2020).In this research, cold stress significantly increased transcription of 32 CML genes that encoded calcium-binding protein, of which 6 were upregulated by BR treatment, and the genes were not induced by ABA treatment (Fig. 8).The data suggested that calcium signaling might be triggered in BR-induced cold tolerance due to the upregulation of CaBPs.Transcriptional factors (TFs) are proteins that typically bind cis-acting elements in gene promoter elements and affect transcription in either a positive or negative fashion in cold stress, consisting of WRKYs, MYBs, NACs, bHLHs, and ZFPs (Chen and Zhu 2004).BZR1, a central positive regulator of BR signaling, governs BR-regulated gene expression and stress responses.BZR1 functions upstream of CBF1 and CBF2 to positively modulate cold responses in CBF-dependent manners.Moreover, BZR1 regulated cold stress response in plants by modulating other COR genes such as WKRY6, PYL6, SOC1, JMT, and SAG21 in CBF-independent ways (Li et al. 2017).Herein, BR and cold treatments all upregulated the expression of Fig. 9 Heat maps of the differentially expressed genes associated with NAC, WRKY, AP2/ERF, HLH, WRKY, MYB, GRAS, and C2H2-ZFP transcription factor families in response to Control_0, Control_3, BR and ABA treatments.Control_0, the grape seedlings were transported to 4 °C for 0 h after distilled water treatment.Con-trol_3, the grape seedlings were transported to 4 °C for 3 h after distilled water treatment.BR, the grape seedlings were transported to 4 °C for 3 h after BR treatment.ABA, the grape seedlings were transported to 4 °C for 3 h after ABA treatment VvSCL14.2,VvZAT10.1, and VvZAT10.2 in grapevine seedlings (Fig. 9).These findings indicated that TFs and second messengers might coordinate the network regulation of transcriptional activities, in multiple pathways, finally increase cold tolerance in BR-treated grapevine.

ABA positively regulates cold tolerance in grapevine plant
ABA is a primary stress-responsive hormone (Zhang et al. 2022).Here, cold elevated ABA biosynthetic gene transcript levels, indicating that cold stimulated ABA synthesis.Furthermore, exogenous ABA treatment reduced cold-induced oxidative damage via regulating the antioxidant system in grapevine seedlings.Similar conclusions were observed in Elymus nutans, ABA markedly reduced the oxidative damage caused by cold (Fu et al. 2017).Former research indicated that ABA signaling positively regulated cold tolerance by acting upstream of CBFs (Knight et al. 2004).This research demonstrated that ABA up-regulated genes were associated with cell wall-remodeling (Fig. 5a).Freezing damage cause cell dehydration due to the lower chemical potential and vapor pressure of ice, leading to cellular desiccation and rigidification of the cell membrane ultimately (Ramirez and Poppenberger 2020).Non-freezing low-temperature conditions induce a significant increase in total cell wall content and improve freezing tolerance (Takahashi et al. 2019).In this research, cold triggered the xyloglucan metabolism pathway in grapevine seedlings, which strengthened the cell wall by remodeling (Han et al. 2017).Interestingly, ABA up-regulated the genes that encoded xyloglucosyl transferase, indicating ABA-induced cold tolerance may be closely related to the modification of cell walls.It had been documented that BR and ABA played key roles in the tolerance of grapevine to cold stress.However, the relationship between BR and ABA in grapevine cold stress responses remains unknown.

ABA contributes to the BR-induced cold tolerance of grapevines
Many reports have shown that ABA and BR interacted antagonistically in multiple physiological responses at different levels.BR signaling inhibits ABA responses by forming the BES1-TOPLESS-Histone Deacetylase 19 transcriptional repressor complex, mitigating early seedling development halted by ABA (Ryu et al. 2014).Furthermore, (Northey et al. 2016) reported that a decrease in endogenous brassinosteroid accumulations causes plants' hypersensitization to endogenous ABA and overall enhances drought resistance in Arabidopsis thaliana.However, the literature reports indicate that BR and ABA synergistically affect modulating plant growth and adaptation.BR-mediated resistance to cold, water stress, high temperature, and oxidative stresses is correlated with enhanced ABA synthesis in plants (Zhou et al. 2014;Zhang et al. 2011;Liu et al. 2011).Similarly, a low concentration of ABA triggers the BR signal in a rapid, restricted, and transient way, and the early-stage synergy between ABA and BR signaling is critical for the salt stress tolerance of rice seedlings (Li et al. 2021).These researches suggested that the relationship between ABA and BR in regulating plant growth and adaptation is more complex.In this research, we observed that BR up-regulated the expression of ABA biosynthetic genes.Furthermore, NDGA pretreatment blocked the BR-induced cold tolerance of grapevine plants.Mainly, NDGA pretreatment abolished BR-induced activities of antioxidant enzymes such as SOD, CAT, APX, DHAR, MDHAR, and GR, as well as the antioxidant levels of AsA and GSH under cold stress in different degrees (Fig. 2).Thus, it was apparent that ABA is intervened in BR-induced cold tolerance.
The AP2/ERF superfamily is among the biggest transcription factor families in plants and is distinguished by its conserved AP2 DNA-binding domain (Mizoi et al. 2012).In the grape genome, 149 AP2/ERF genes were identified, of which VaERF057 and VaERF092 play positive roles in cold tolerance (Sun et al. 2016(Sun et al. , 2019)).TINY, an AP2/ERF transcription factor, antagonistic interacts with BES1 and positively regulates drought responses in Arabidopsis via inhibiting BR-mediated growth (Xie et al. 2019).OsERF71 promotes drought tolerance in transgenic rice via modulating ABA signaling and proline biosynthesis (Jin et al. 2018).Herein, cold stress increased the expression of 32 ERF genes, of which 3 genes were upregulated by ABA treatment, and 4 genes were upregulated by BR treatment.These studies showed that BR and ABA promote stress tolerance were associated with the transcript levels of ERF.Transcriptome analyses indicated that cold, BR, and ABA treatments all upregulated the expression of ERF81, ERF86, and ERF88 in grapevine seedlings (Fig. 5).The results implied that ERF serves pivotal roles in cold tolerance mediated by BR and ABA.
In summary, we revealed that BR and ABA alleviated cold-induced leaf wilting, photoinhibition, and oxidative damage by increasing the chlorophyll fluorescence parameters and regulating the antioxidant system.Combining transcriptome profiling indicated that calcium-binding protein genes participated in the BR-induced cold tolerance in the grapevine.ABA treatment mainly up-regulated the genes associated with xyloglucosyl transferase.Meanwhile, ABA was involved in BR-induced cold tolerance.Transcriptomic data showed that ERF TFs serve essential functions in the cold tolerance co-mediated by BR and ABA.Taken together, these findings suggested that BR positively modulates the cold tolerance of grapevines by ABA-dependent and ABAindependent regulatory systems.

Fig. 1
Fig.1ABA contributed to BR-induced cold tolerance in grapevine plants.BR, ABA, or BR plus NDGA were applied separately to grapevine seedlings for 8 h at 25 °C before being transported to 4 °C for 72 h. a Phenotypes were recorded of the four types of plants at 72 h after cold stress.Electrolyte leakage (b), O 2 − generation (c), ϕPSII (d), and NPQ (e) in the four types of plants were measured after being subjected to cold (4 °C) for the specified time.The data were represented as mean ± SD (n = 3).Variations between treatments that are statistically significant (P < 0.05) are denoted by distinct lowercase letters

Fig. 2
Fig. 2 ABA was involved in BR-induced ASA-GSH pathway regulation in response to cold stress.BR, ABA, or BR plus NDGA were applied to grapevine seedlings for 8 h at 25 °C separately before being transported to 4 °C for 72 h.SOD (a), CAT (b), APX (c), DHAR (d), MDHAR (e) and GR (f) activities and AsA (g) and GSH

Fig. 3 Fig. 4
Fig. 3 The role of ABA in BR-induced CBF pathway regulation under cold stress.BR, ABA, or BR plus NDGA were applied to grapevine seedlings for 8 h at 25 °C separately before being transported to 4 °C for 72 h.VvCBF1 (a), VvCBF2 (b), VvCBF3 (c), and VvCBF4 (d) transcript levels in the four types of plants were meas-

Fig. 5
Fig. 5 GO and KEGG analyses of the DEGs exclusively in response to BR treatment and ABA treatment under cold stress.Venn diagram analysis of GO terms and KEGG pathways (P < 0.05) based on DEGs in Control_0 versus Control_3 (COLD), Control_3 versus BR (BR), and Control_3 versus ABA (ABA).a Up-regulated DEG.b Downregulated DEG.Control_0, the grape seedlings were transported to