Jasmonate enhances cold acclimation in jojoba by promoting flavonol synthesis

Abstract Jojoba is an industrial oil crop planted in tropical arid areas, and its low-temperature sensitivity prevents its introduction into temperate areas. Studying the molecular mechanisms associated with cold acclimation in jojoba is advantageous for developing breeds with enhanced cold tolerance. In this study, metabolomic analysis revealed that various flavonols accumulate in jojoba during cold acclimation. Time-course transcriptomic analysis and weighted correlation network analysis (WGCNA) demonstrated that flavonol biosynthesis and jasmonates (JAs) signaling pathways played crucial roles in cold acclimation. Combining the biochemical and genetic analyses showed that ScMYB12 directly activated flavonol synthase gene (ScFLS). The interaction between ScMYB12 and transparent testa 8 (ScTT8) promoted the expression of ScFLS, but the negative regulator ScJAZ13 in the JA signaling pathway interacted with ScTT8 to attenuate the transcriptional activity of the ScTT8 and ScMYB12 complex, leading to the downregulation of ScFLS. Cold acclimation stimulated the production of JA in jojoba leaves, promoted the degradation of ScJAZ13, and activated the transcriptional activity of ScTT8 and ScMYB12 complexes, leading to the accumulation of flavonols. Our findings reveal the molecular mechanism of JA-mediated flavonol biosynthesis during cold acclimation in jojoba and highlight the JA pathway as a promising means for enhancing cold tolerance in breeding efforts.


Introduction
Simmondsia chinensis, commonly known as jojoba, is a dioecious shrub belonging to the family Simmondsiaceae within the genus Simmondsia [1].It is one of the three most important non-edible oils (Jatropha curcas and Camelina sativa) [2].Jojoba originates in the Sonoran Desert in southwestern North America and exhibits strong resistance to high temperatures and drought stress.Jojoba oil is widely used in cosmetics, pharmaceuticals, and industrial lubricants owing to its exceptional resistance to high temperatures and pressures [3,4].Jojoba oil exhibits physical and chemical properties similar to those of sperm whale oil; thus, it is a suitable substitute, especially because of the prohibition of sperm whale oil.Studies have indicated that jojoba cultivation can help prevent land desertification [5], highlighting its substantial ecological value as a desert shrub.
Low temperatures significantly inf luence the growth, development, and geographical distribution of plants, resulting in adverse effects on crop quality and yield [6]. Plants underwent various physiological and biochemical changes in response to low-temperature stress-induced damage, including changes in enzyme activities, synthesis of plant hormones, and maintenance of ROS balance [7].Jojoba exhibits tolerance to high temperatures and drought and is susceptible to low temperatures.Temperatures below 0 • C inhibited the growth and development of jojoba [8].The sensitivity of jojoba to low temperatures significantly impedes its cultivation in temperate and arid regions.Therefore, investigating methods to enhance the cold tolerance of jojoba is crucial.Notably, cold acclimation (i.e., exposure to a prolonged period of low non-lethal temperatures) is an effective method for improving the cold tolerance of most plants [9].
Cold acclimation involves a complex regulatory network encompassing a vast array of genes.Several cold-responsive genes, including transcription factors (TFs) and functional genes, have been identified in various plants [10].CBF TFs played significant roles in the cold acclimation of plants.CBFs interacted with CRT/DREs in the promoter regions of various cold-responsive genes, activating their expression [11,12].One of the outcomes of complex gene expression regulation was that changes in metabolites were crucial for the cold acclimation of plants.Studies have shown that many metabolic pathways, such as the biosynthesis of f lavonoids and sugars, were triggered during cold acclimation, leading to changes in the contents of related metabolites, which partly contributed to increased plant cold tolerance [13][14][15].Studies have also shown that plant hormones played key roles in regulating the responses to low temperatures.Additionally, cold stress induced the accumulation of various plant hormones, such as abscisic acid (ABA) and JAs [16].Omics analysis has been used to reveal the regulatory networks of plant stress responses [17].Transcriptomic and metabolomic analyses have been employed to identify gene regulatory networks associated with cold stress responses in various plants, including wheat [18], tobacco [19], and peanut [20].Previous research demonstrated that cold acclimation improved the cold tolerance of jojoba [21].Nevertheless, the molecular mechanisms underlying cold acclimation remain unclear.
Flavonol is a polyphenolic compound synthesized through the f lavonoid biosynthetic pathway and possesses various biological functions, including anti-ultraviolet [22], anti-insect [23], antioxidant [24,25], and anti-abiotic stress [26].Flavonol biosynthesis is a segment of the f lavonoid metabolic pathway, and the enzymes in this pathway contain chalcone synthase (CHS), chalcone isomerase (CHI), and f lavonol synthase (FLS), which catalyze chalcone to produce the intermediate naringenin and then synthesize f lavonol from dihydrof lavonol catalyzed by FLS [27].In most plants, the transcriptional regulation of functional genes associated with f lavonol biosynthesis is governed by MYB TFs or the MYB/bHLH/WD repeat (MBW) complex [28,29].AtMYB11, AtMYB12, and AtMYB111 have been found to independently activate the expression of f lavonol synthesis-related genes such as CHS, CHI, and FLS [30][31][32].
JAs, comprising jasmonic acid and its oxylipin derivatives, are crucial oxylipins in plants.They played significant roles in regulating plant growth, development, and stress response [33][34][35].The components of the JA signaling pathway have been identified, including the SCF COI1 complex, JAZs, and MYC2 TFs [36,37].The JA-ZIM-domain (JAZ) proteins serve as substrates for the SCF COI1 complex and act as negative regulators by inhibiting multiple transcriptional regulators [38].Upon sensing JA signals, COI1 recruits JAZs for ubiquitination and subsequent degradation to activate downstream signaling cascades that initiate the production of defense compounds against biotic and abiotic stresses [39].Studies have shown that JA positively regulated plant cold tolerance.Cold stress induced the expression of genes involved in JA biosynthesis, increasing JA content [40,41], and JA addition significantly improved the cold tolerance of orange [42], banana [43], and tomato [44].Molecular studies found that JA enhanced cold tolerance in Arabidopsis by activating the ICE-CBF module [45].These results demonstrated the importance of JA in the response of plants to cold stress.The regulatory mechanism of JA signaling pathway in jojoba response to cold acclimation has not been studied yet.
Jojoba genome sequencing has provided relevant data for further molecular research [46,47].Metabolomic and transcriptomic analyses showed that cold acclimation activated the biosynthesis of f lavonol and JA.By combining biochemical and genetic analyses, we found that ScMYB12 directly activated the expression of the ScFLS gene; the interaction between ScMYB12 and ScTT8 promoted the expression of ScFLS, but the negative regulator ScJAZ13 interacted with ScTT8 to attenuate the transcriptional activity of the ScTT8 and ScMYB12 complex, leading to downregulation of ScFLS gene; and cold acclimation-induced JA promoted the transcriptional activity of the ScTT8/ScMYB12 complex through the degradation of ScJAZ13, further promoting the accumulation of f lavonol in jojoba.

Accumulation of flavonoids in jojoba after cold acclimation
Studies have shown that cold acclimation enhanced the cold tolerance of jojoba [21].In this study, phenotypic analysis revealed no significant changes in jojoba after 0, 6, 24, 48, and 72 h and 1 week of cold acclimation at 15 • C/10 • C (day/night) (Fig. S1, see online supplementary material).We speculated that the increased cold tolerance in jojoba may be associated with the accumulation of certain metabolites.To reveal the alterations in metabolite pre-cold and post-cold acclimation in jojoba, we used widely targeted metabolomics to assess the variances in metabolites between the control group (28  C, day/night, 1 week, CA).PCA and the heat map of all metabolites revealed a significant separation in the metabolic profiles of CK and CA, indicating that substantial change in metabolites occurred after cold acclimation in jojoba leaves (Fig. S2a and b, see online supplementary material).A total of 918 metabolites were detected in jojoba leaves (Table S1, see online supplementary material) and were categorized into 12 groups.The most enriched category was f lavonoids (261 metabolites), followed by phenolic acids (134 metabolites), lipids (125 metabolites), and amino acids and their derivatives (75 metabolites) (Fig. S2c, see online supplementary material).A replacement test of the PLS-DA model revealed a significant difference in the abundances of some metabolites between CK and CA (Fig. S2d, see online supplementary material).VIP analysis revealed that the metabolites with the highest VIP values were f lavonoids, including anthocyanins, f lavonols, and f lavones (Table S2, see online supplementary material).
By comparing the metabolite chromatograms between CK and CA, 207 differentially accumulated metabolites (DAMs) were identified, with 155 showing upregulation and 52 downregulation (Table S3, Fig. S2e, see online supplementary material).The DAMs were divided into 11 categories, including 68 f lavonoids, 25 lipids and phenolic acids, and 19 nucleotides and their derivatives.Notably, most metabolites in the f lavonoid category showed increased abundance after cold acclimation.The f lavonoids were further divided into nine subcategories: f lavonol, f lavone, dihydrof lavonol, chalcone, f lavanol, dihydrof lavone, dihydroisof lavone, anthocyanin, and f lavone glycoside, with f lavonol accounting for the largest proportion (Fig. 1a).In the f lavonol category, certain metabolites such as quercetin and spiraeoside showed noticeable accumulation after cold acclimation.KEGG enrichment analysis of the DAMs showed that the top three most enriched pathways were galactose metabolism; f lavonoid and f lavonol biosynthesis; and alanine, aspartate, and glutamate metabolism (Fig. 1b).These results indicated that cold acclimation induced f lavonoid accumulation in jojoba leaves.

Time-course transcriptome analysis revealed biological processes and metabolic pathways associated with cold acclimation in jojoba
Time-course transcriptomic analysis was conducted to explore the dynamic changes in gene expression during cold acclimation in jojoba.The PCA and correlation analyses showed good reproducibility within the same group (Fig. S3a and b, see online supplementary material).We used 0 h as a control, and 406 (239 upregulated, 167 downregulated), 2723 (1864 upregulated, 859 downregulated), 5112 (4226 upregulated, 886 downregulated), 1767 (698 upregulated, 1069 downregulated), and 5936 (2957 upregulated, 2979 downregulated) differentially expressed genes (DEGs) were identified at 6, 24, 48, and 72 h and 1 week, respectively (Fig. S3c, see online supplementary material).The Venn diagram showed a low number of overlapping genes among the DEGs in different groups, indicating that distinct biological events may occur at different stages of cold acclimation in jojoba (Fig. S3d, see online supplementary material).The e Metabolites transformation and the corresponding enzyme gene changes in f lavonoid metabolism pathways under cold acclimation.The dots and rectangle, respectively, indicated metabolites and the genes encoding the corresponding enzymes.Red and blue represented the increase and decrease of metabolites/genes after cold acclimation, respectively.f The morphology changes of the treatment group (spiraeoside, hyperin, and quercetin) and the control group (H 2 O) after cold stress.g The delayed f luorescence imaging of the treatment groups and the control group.h MDA content of control group and treatment group.i REL measurements.Statistical analysis of the data was conducted using Duncan's multiple range test.j Exogenous addition of spiraeoside, hyperin, and quercetin enhanced the cold tolerance of yeast strain W303-1A.results of the qRT-PCR analysis were in substantial agreement with the transcriptome sequencing results, indicating the credibility of the transcriptome sequencing outcomes (Fig. S3e, see online supplementary material).
GO enrichment analysis of all DEGs (identified as DEG at least at one time point) revealed the enrichment of terms associated with stress responses, such as response to stimuli, response to hormones, and antioxidant activity (Fig. 1c).KEGG analysis indicated that photosynthesis, proteasomes, porphyrin and chlorophyll metabolism, N-glycan biosynthesis, and f lavonoid biosynthesis were the top five most enriched metabolic pathways (Fig. 1d).

Combined analysis of transcriptome and metabolome revealed the involvement of the flavonoid synthesis pathway in cold acclimation
Combined analysis of the transcriptome and metabolome identified 47 co-enriched metabolic pathways (Fig. S4a, see online supplementary material).These pathways were divided into three categories: amino acid metabolism (e.g., amino acid biosynthesis and alanine, aspartate, and glutamate metabolism), sugar metabolism (e.g., galactose metabolism and starch and sucrose metabolism), and f lavonoid metabolism (e.g., f lavonoid and f lavonol biosynthesis and phenylalanine biosynthesis) (Fig. S4b, see online supplementary material).These co-enriched metabolic pathways might play crucial roles in cold acclimation in jojoba.
Flavonoid compounds constituted the largest proportion of DAMs, with most f lavonoid contents increasing after cold acclimation.To further analyse the potential gene regulation mechanism in the f lavonoid metabolic pathway after cold acclimation, the differentially accumulated f lavonoids and the corresponding key enzyme-coding genes were analysed jointly.Within the phenylpropanoid biosynthesis pathway, the levels of three metabolites, including p-coumaric acid and ferulic acid, were elevated after cold acclimation.This trend was in line with the upregulation of genes encoding crucial enzymes such as PAL and 4CL.Within the f lavonoid biosynthesis pathway, the contents of 10 f lavonols, including spiraeoside, hyperin, and quercetin, increased.Most of the related enzyme-coding genes were also upregulated, including CHS and glucoside transferase (GT) (Fig. 1e; Table S4, see online supplementary material).These results suggested that an increase in f lavonoid biosynthesis was crucial for the cold acclimation of jojoba.
The combined metabolome and transcriptome analyses showed that f lavonols levels in jojoba increased significantly after cold acclimation.To evaluate the inf luence of f lavonol accumulation on the cold resistance of jojoba, we investigated the effects of spraying tobacco seedlings with 1 mM spiraeoside, hyperin, and quercetin to understand their impact on tobacco cold tolerance.The control group displayed noticeable wilting, and the tobacco leaves treated with f lavonols showed slight wilting after cold treatment (Fig. 1f), indicating that the exogenous application of f lavonols improved the cold tolerance of tobacco.The delayed f luorescence of tobacco leaves treated with f lavonols was substantially greater than that of the control group, indicating that the exogenous application of f lavonols reduced the damage to the photosynthetic apparatus caused by cold stress (Fig. 1g).Malondialdehyde (MDA) content and relative electrolytic leakage (REL) measurements also indicated that the exogenous application of f lavonols reduced cell membrane damage caused by cold stress (Fig. 1h and i).After the freezing stress treatment, yeast strain W303-1A exhibited a higher growth rate in the medium supplemented with spiraeoside, hyperin, and quercetin (Fig. 1j).These results indicated that the addition of spiraeoside, hyperin, and quercetin enhanced the cold tolerance of tobacco seedlings and yeast cells.

Identification of key modules and genes regulating cold acclimation of jojoba by WGCNA analysis
To identify the regulatory modules that played key roles in the cold acclimation process of jojoba, we conducted WGCNA for all DEGs.All DEGs were divided into 18 modules with a minimum height of 0.1992 (Fig. S5a, see online supplementary material).The number of genes in each module varied substantially, with 5098 and 30 genes in the largest and smallest modules, respectively.Correlation analysis between these modules and the different time points indicated that the genes in the three modules of MEblack, MEsalmon, and MEdarkorange showed similar expression patterns during cold acclimation, which were positively correlated at 0, 6, 24, and 48 h, and the five modules of MEdarkgreen, MEsteelblue, MEdarkred, MEgreen, and MEroyalblue were positively correlated at 24 and 48 h, and negatively correlated at 0, 6, and 72 h.MEdarkgreen, MEgreen, MEbrown, MEmagenta, MEpaleturquoise, and MEblack were positively correlated at 1 week.Notably, the MEbrown module exhibited the highest correlation at 1 week (r = 0.98) and was negatively correlated with the other time points (Fig. S5b, see online supplementary material), indicating that this module may be involved in the response to the late stages of cold acclimation in jojobas.
To clarify the potential biological functions of the genes within the MEbrown module, we conducted comprehensive GO and KEGG analyses for all genes in the module.The results indicated significant enrichment of some biological processes and metabolic pathways associated with hormone signaling and f lavonoid biosynthesis, including responses to hormones; responses to jasmonic acid; and the biosynthesis of f lavonoids, f lavones, and f lavonols (Fig. 2a and b; Table S5, see online supplementary material).To further investigate the genes regulating f lavonoid synthesis in the MEbrown module, the core genes in the module were screened using module membership (MM) > 0.8 and gene significance (GS) > 0.3 as the criteria.The results showed that the key enzyme chalcone f lavanone isomerase encoding gene CFI and two bHLH TFs (ScTT2 and ScTT8) regulating f lavonoid metabolism were the hub genes in the network (Fig. 2c).

JA signaling pathway was crucial for cold acclimation of jojoba
Transcriptomic and WGCNA analyses indicated that hormone signaling pathways, particularly the JA signaling pathway, probably have significant regulatory functions in the cold acclimation of jojoba.LC-MS analysis showed a 90% increase in JA content in the leaves after cold acclimation (Fig. 2d), suggesting that cold acclimation induced JA production in jojoba.The changing trends of metabolites and corresponding genes in JA biosynthesis and signal transduction pathways were analysed based on transcriptomic and metabolomic data.In general, these metabolites and their corresponding genes in the JA signaling pathway were upregulated after cold acclimation.Some of these genes showed a gradual upward trend, such as LOX, CTS/PXA1, and OPCL1.Some genes exhibited an initial increase followed by a subsequent decrease, including AOS and AOC.The JA receptor gene COI1 in the JA signal transduction pathway was significantly upregulated, and multiple inhibitor JAZ proteins were downregulated (e.g., JAZ1 and JAZ2).The expression levels of crucial MYC2 TFs, which are responsible for regulating the downstream genes of the JA signaling pathway, exhibited a trend of first increasing and then decreasing (Fig. 2e).The qRT-PCR analysis also indicated that the changing trends of some key genes were in agreement with the transcriptome analysis (Fig. 2f).These results indicated that cold acclimation activated JA synthesis and signal transduction pathways in jojoba.
The impact of JA on the cold tolerance of jojoba was evaluated by measuring the physiological indexes of jojoba leaves after exogenous application of different concentrations (0, 10, and 100 μM) of methyl jasmonate (MeJA).The values of REL and MDA content in jojoba seedlings irrigated with 10 and 100 μM of MeJA solutions were significantly lower than that of jojoba seedlings irrigated with water under cold stress (Fig. 2g and h), and the delayed f luorescence imaging showed that MeJA treatment improved the tolerance of the leaf photosynthetic apparatus to damage induced via cold stress (Fig. 2i).These results suggested that the external application of MeJA alleviated the damage caused by cold stress to the cell membrane and photosynthetic apparatus.

ScJAZ13 physically interacted with ScTT8 in jojoba
The aforementioned results showed the crucial roles of f lavonol synthesis and the JA signaling pathway in the cold acclimation of jojoba.Studies have found that the key inhibitor JAZ proteins in the JA signaling pathway interacted with bHLH TFs such as TT8, GL3, and EGL3 in the MBW complex [33].WGCNA showed that ScTT8 was a key gene in a co-expression module that may promote f lavonol biosynthesis.Thus, we hypothesized that some ScJAZs may interact with ScTT8 to regulate f lavonol biosynthesis in jojoba.Thirteen ScJAZs were identified in jojoba (Fig. S6a, see online supplementary material).Phylogenetic analysis of JAZ proteins from jojoba and Arabidopsis showed that these JAZ proteins could be divided into five groups, with each group containing at least one AtJAZ protein, indicating that ScJAZ and AtJAZ proteins shared a certain degree of similarity (Fig. S6b, see online supplementary material).Studies have reported that AtTT8 interacted with AtJAZ1/2/5/6/8/9/10/11 in Arabidopsis [33].Based on the phylogenetic analysis, we selected the five JAZs genes ScJAZ1/6/7/8/13 to analyse whether there was an interaction between these proteins and ScTT8.The luciferase complementation assay (LCA) showed that ScJAZ13 interacted with ScTT8 and that ScJAZ1, ScJAZ6, ScJAZ7, and ScJAZ8 did not interact with ScTT8 (Fig. 3a).Next, we confirmed the interaction between ScJAZ13 and ScTT8 by using the biomolecular f luorescence complementation (BiFC) assay in tobacco (Fig. 3b).Additionally, the yeast two-hybrid (Y2H) assays revealed an interaction between ScJAZ13 and ScTT8 in yeast (Fig. 3c).In summary, these findings indicated an interaction between ScJAZ13 and ScTT8.

ScFLS expression
Studies have found that functional genes involved in f lavonol biosynthesis were regulated by MYB TFs or the MBW complex [48,49].MYB12 TF independently activated the expression of genes associated with f lavonol biosynthesis in apple [50] and Arabidopsis [31].In this study, transcriptome analysis revealed that the expression of ScMYB12, an ortholog of AtMYB12 in jojoba, was significantly upregulated during cold acclimation (Fig. S7, see online supplementary material).Thus, we conducted yeast one-hybrid (Y1H) assays to determine the regulatory relationship between the ScMYB12 and functional genes associated with f lavonol biosynthesis.We found that ScMYB12 could bind to the ScFLS promoter but not to the ScPAL, ScCHS, or ScCFI promoters (Fig. 4a).The luciferase activity assay demonstrated that ScMYB12 activated the expression of ScFLS (Fig. 4b).
We were interested in whether there was an interaction among ScTT8, ScJAZ13, and ScMYB12 and whether ScTT8 and ScJAZ13 could participate in regulating the expression of functional genes associated with f lavonol biosynthesis through ScMYB12 in jojoba.The LCA showed that ScMYB12 interacted with ScTT8 but not with ScJAZ13 (Fig. 4c), and the BiFC and Y2H assays also confirmed the interaction between ScMYB12 and ScTT8 (Fig. 4d and e).Next, we used a dual luciferase reporter gene controlled by the 35S and ScFLS promoters to investigate the effects of ScTT8, ScJAZ13, and ScMYB12 on the transcriptional activity of ScFLS.When proScFLS::LUC alone penetrated tobacco leaves, we observed a detectable level of luciferase activity, indicating that endogenous MYB TFs in tobacco activated the expression of proScFLS::LUC.Upon co-transfection with ScMYB12 and proScFLS::LUC, there was a substantial increase in luciferase activity, suggesting that ScMYB12 enhanced ScFLS expression.Additionally, when ScMYB12, ScTT8, and proScFLS::LUC were cotransfected, luciferase expression in tobacco leaves was further elevated, indicating that the interaction between ScMYB12 and ScTT8 enhanced the expression of ScFLS.Nevertheless, upon cotransfection with ScJAZ13, ScTT8, ScMYB12, and proScFLS::LUC, there was a marked decrease in luciferase expression in tobacco (Fig. 4f), indicating that ScJAZ13 suppressed the transcriptional activation of the ScTT8 and ScMYB12 complex to ScFLS.

ScMYB12 overexpression promoted expression of enzyme genes in flavanol biosynthesis pathway and conferred cold tolerance in jojoba
To assess the effects of ScMYB12 overexpression on f lavonol biosynthesis in jojoba, we constructed a genetic transformation system and obtained ScMYB12-overexpressed hairy roots (Fig. S8, see online supplementary material).The rol B gene in the Ri plasmid of Agrobacterium rhizogenes was detected in OE1, OE2, OE4, OE5, and OE9 (Fig. 5a), indicating that these lines were positive hairy roots.The expression level of ScMYB12 in these lines was significantly higher than that of the wild type (WT) (Fig. 5b).The expression levels of two key TFs closely associated with f lavonoid biosynthesis, ScTT2 and ScTT8, were significantly upregulated in the hairy roots overexpressing ScMYB12 (Fig. S9, see online supplementary material).Additionally, the f lavonoid content in hairy roots overexpressing ScMYB12 was significantly increased (Fig. 5c).Expression profiling revealed a significant upregulation of enzyme genes in the f lavonol synthesis pathway, such as ScPAL, ScCFI, and ScFLS gene (Fig. 5d).These results indicated that ScMYB12 overexpression promoted the accumulation of f lavonols in jojoba by activating the expression of enzyme genes in the f lavonol biosynthetic pathway.To further analyse the effect of overexpressing ScMYB12 on the cold tolerance of jojoba, we measured the MDA content in hairy roots after 24 h of cold stress at 4 • C. The results showed the MDA content in the hairy roots overexpressing ScMYB12 was significantly less than that of the WT hairy roots (Fig. 5e, f and g).This finding indicated that ScMYB12 was a positive regulator of cold stress in jojoba.
The biochemical evidence above indicated that ScJAZ13 inhibited the transcriptional activity of the ScMYB12/ScTT8 complex to ScFLS.To confirm the regulatory role of the ScJAZ-MYB module in cold acclimation of jojoba, both WT and ScMYB12 overexpressing plants were subjected to exogenous MeJA and cold stress treatments.Following treatment with 100 μM MeJA, it was found that the increase of f lavonoids content in the ScMYB12overexpressing hairy roots was significantly greater than that in WT, and the activation of ScFLS was stronger in overexpressing plants (Fig. 5h and i).Similar phenomena were observed after 24 h of cold treatment at 4 • C, where the accumulation of f lavonoids was significantly higher in the ScMYB12-overexpressing hairy roots than in WT hairy roots, and the increase in ScFLS expression levels was also more pronounced (Fig. 5j and k).These results indicated that ScJAZ-MYB module positively regulated the induction of f lavonols by cold stress.

Discussion
Jojoba is recognized as one of the three most crucial non-food oil plants globally and is economically crucial.In arid regions, cultivating jojoba increases incomes and helps maintain fragile ecosystems.However, the sensitivity of jojoba to low temperatures severely impedes its introduction to temperate regions.Studying gene expression networks and metabolic pathways associated with the cold tolerance of jojoba and identifying regulatory proteins that play crucial roles in cold acclimation would promote genetic breeding studies aimed at enhancing the cold resistance of jojoba.
Flavonoids, primarily anthocyanins, f lavonols, and f lavanols, are polyphenolic secondary metabolites in plants.In this study, metabolomic analysis showed a substantial elevation in the levels of diverse f lavonoids in jojoba leaves after cold acclimation, with the highest upregulation observed in f lavonol biosynthesis, particularly in quercetin, spiraeoside, and hyperoxide (Table S3, see online supplementary material).Transcriptomic analysis revealed substantial upregulation of several crucial genes associated with f lavonol biosynthesis in jojoba during cold acclimation, including PAL, CHS, CYP75B1, and GT (Fig. 1e).Several abiotic stresses, such as salt [51], drought [52], and cold stress [53], have been reported to lead to the accumulation of f lavonols in plants.Flavonoid synthesis was a determining factor for Arabidopsis to acquire freezing and cold tolerance via cold acclimation [54].In our study, the exogenous addition of f lavonols, such as quercetin, spiraeoside, and hyperoside, enhanced cold resistance in tobacco and yeast (Fig. 1f and j).Studies have reported that f lavonols possessed significant antioxidant activity [55].Several f lavonols have been found in the extracts of Bauhinia galpinii and mulberry leaves, and the addition of these extracts significantly enhanced the ability of animal cells to scavenge free radicals [56,57].Therefore, we speculated that the accumulation of f lavonols after cold acclimation helped jojoba manage the oxidative damage caused by cold stress.
There was a small overlap between the DEGs at different time points during the cold acclimation of jojoba (Fig. S3d, see online supplementary material), indicating that different biological events may occur at different time points.Consistent with this speculation, GO enrichment analysis identified several time point-specific and stage-specific-enriched GO biological process terms during cold acclimation.Notably, terms such as response to stimulus and response to abiotic stress showed significant enrichment at 6 h, suggesting that early biological processes in jojoba cold acclimation were primarily related to environmental stress responses.GO terms such as RNA metabolic process, gene expression, translation, protein folding, protein degradation, mitochondrial RNA modification, and chloroplast mRNA processing were mainly enriched at 24 h and 48 h, indicating that at this stage, leaf cells began to regulate the function of mitochondria and chloroplasts through gene expression and protein synthesis, rebuilding homeostasis under prolonged short-term cold acclimation.The GO terms f lavonoid metabolic process and f lavonoid biosynthetic process were enriched at 48 h, suggesting that the f lavonoid synthesis pathway was activated at this time (Fig. 1c).Despite the occurrence of diverse biological events at different time points, some biological processes associated with hormone signaling pathways, such as plant hormone signaling and response to hormones, were enriched throughout nearly all stages of cold acclimation.This underscored the essential role of plant hormones in providing tolerance to cold stress induced by acclimation in jojoba plants.The significant positive correlation module identified by WGCNA at the late stage of cold acclimation of jojoba contained some genes associated with plant hormone signaling (Fig. 2a), further emphasizing the importance of hormones in the cold acclimation of jojoba.The hairy roots overexpressing ScMYB12 were obtained by CDB delivery system, and the hairy roots growing for one month were collected for each measurement.Each experiment contained at least three independent biological replicates.Graphpad Prism 9 was used for data analysis and plotting.Each value was the mean ± SD.All data were subjected to ANOVA followed by an LSD test.* P < 0.05.* * P < 0.01.
JAs are a class of important signaling molecules that govern multiple biological processes related to plant growth and development and mediate plant responses to environmental stress [58][59][60].Both transcriptome and WGCNA analyses indicated a crucial regulatory role for the JA signaling pathway in the cold acclimation of jojoba.The JA content of jojoba leaves increased significantly after cold acclimation.Simultaneously, the majority of genes responsible for synthesizing enzymes in the JA pathway, including AOC and AOS, along with genes encoding components of the JA signaling pathway, such JAR1 and COI1, were upregulated (Fig. 2d, e, and f).These findings indicated that JA biosynthesis was triggered by cold acclimation.In our study, jojoba seedlings treated with exogenous MeJA had lower REL and MDA levels and higher delayed f luorescence intensity under cold stress than the control group did (Fig. 2g, h, and i), suggesting that MeJA improved the cold tolerance of jojoba by reducing damage to cell membranes and chloroplasts caused by cold stress.Improved cold resistance through exogenous MeJA application has been reported in other plants, such as grapes and tomatoes [61,62].These results showed that JA played a crucial role in the response to cold acclimation in jojoba.
Studies have reported that JA inf luenced f lavonol biosynthesis [63,64].The application of MeJA was reported to significantly elevate f lavonol content in tomato seedlings [65].Supplementing a cell culture suspension of Polygonum multif lorum with JA led to a notable increase in the f lavonol content of the cells [66].Therefore, we hypothesized that there may be an intrinsic connection between JA induced by cold acclimation and f lavonol accumulation in jojoba.JAZ proteins played crucial roles as inhibitory factors in the JA signaling pathway and were believed to impede plant responses to JA regulation by interacting with downstream TFs [67,68].Some studies found that JA may affect f lavonoid biosynthesis through the negative regulatory factor JAZ.In Arabidopsis, JAZ proteins weakened the transcriptional activity of the MBW complex by interacting with bHLH and MYB TFs, repressing JAinduced anthocyanin accumulation [33,69].In tea plant, CsJAZ6 interacted with the MBW components CsEGL3 and CsTTG1, and the induction of JA weakened the inhibitory effect of CsJAZ6 on CsEGL3 and CsTTG1, leading to an increase in the biosynthesis of catechins under heat stress [70].In our study, we found 13 ScJAZ genes in the jojoba genome, five of which were selected for analysis of their interactions with bHLH TF-ScTT8.The LCA, Y2H, and BiFC assays indicated that ScJAZ13 interacted with ScTT8 (Fig. 3).This result implied that JA may inf luence f lavonol biosynthesis in jojoba through ScJAZ13.
The regulatory mechanisms underlying f lavonol biosynthesis have been partially elucidated [71].AtMYB11, AtMYB12, and AtMYB111 have been shown to directly activate the expression of CHS, F3H, and FLS to promote the accumulation of f lavonols [30][31][32]72].In apples, MdMYB22 bound directly to the FLS promoter, promoting f lavonol biosynthesis.MdMYB22 overexpression in apple calli and AtMYB12/11/111 mutants induced f lavonol accumulation [50].In pears, PbMYB12 activation of PbFLS expression promoted the biosynthesis of quercetin and kaempferol glycosides [73].In this study, ScMYB12 expression was significantly upregulated under cold acclimation (Fig. S7, see online supplementary material).The Y1H and luciferase activity assays demonstrated that ScMYB12 enhanced the expression of ScFLS by binding to its promoter (Fig. 4a and b).In jojoba hairy roots overexpressing ScMYB12, the f lavonoid content was substantially higher than that in WT hairy roots (Fig. 5c), and the expression levels of key enzyme genes, such as PAL, CFI, and FLS, were significantly upregulated (Fig. 5d).The MDA content in hairy roots overexpressing ScMYB12 decreased significantly after cold stress (Fig. 5g).These results indicated that ScMYB12 promoted the synthesis of f lavonols with antioxidant capabilities, enhancing the cold tolerance of jojoba.Furthermore, after cold stress and MeJA treatment, the increase of f lavonoids content and the expression of ScFLS in the hairy roots overexpressing ScMYB12 were higher compared to WT hairy roots (Fig. 5h, i, j, and k).These findings suggested that JA might positively regulate the accumulation of cold-induced f lavonols through the JAZ-MYB12 module.
Studies showed that MYB TFs regulated f lavonol synthesis by interacting with the bHLH members of the f lavonoid-regulating complex MBW [74,75].In Arabidopsis, MYB75 enhanced the transcription of dihydrof lavonol-4-reductase (DFR) by interacting with TT8 [33].In apples, MdMYB12 activated the transcription activity of MdFLS through its interaction with bHLH3/bHLH33 [50].In Arabidopsis, TCP3, a bHLH TF, interacted with AtMYB12 and AtMYB111, and plants overexpressing TCP3 exhibited a significant increase in f lavonol content [76].Thus, we hypothesized that ScTT8 interacted with ScMYB12, affecting f lavonol biosynthesis in jojoba.In our study, the LCA, BiFC, and Y2H assays confirmed the interaction between ScMYB12 and ScTT8 (Fig. 4c, d, and e).Co-expression of ScTT8 with ScMYB12 resulted in a substantial increase in the expression of ScFLS in tobacco leaves (Fig. 4f).This suggested that the interaction between ScMYB12 and ScTT8 enhanced f lavonol accumulation by promoting the transcription of ScFLS in jojoba.
JAZs are a family of negative regulatory factors involved in the JA signaling pathway.Studies have reported that in some plants, such as Arabidopsis and tea plants, JAZ proteins interacted with bHLH or MYB TFs to negatively regulate f lavonoid accumulation.In Arabidopsis, JAZ1 interacted with MYB75 and TT8.When MYB75, TT8, and JAZ1 were co-expressed, the transcription levels of enzymes related to f lavonoid biosynthesis, such as DFR, decreased, leading to a reduction in anthocyanin synthesis compared with the expression of MYB75 alone or in combination with TT8 [33].In tea plants, CsJAZ6 weakened the transcription of the anthocyanin synthesis enzyme (ANS) by interacting with the CsEGL3 and CsTTG1, further repressing catechin production [70].In this study, we found that when ScJAZ13, ScTT8, and ScMYB12 were co-expressed, the transcription levels of ScFLS were significantly less than those with the expression of ScMYB12 alone or in combination with ScTT8 (Fig. 4f).This suggested that ScJAZ13, which acted as a negative regulatory factor, impeded the accumulation of f lavonols in jojoba by suppressing the transcriptional activity of the ScMYB12 and ScTT8 complex.
Collectively, our results indicated that JA promoted the accumulation of f lavonol during cold acclimation, ultimately enhancing the cold tolerance of jojoba.The mechanism by which JA regulated the cold response has been partially elucidated in other plants.In Arabidopsis and apples, JA regulated the CBF/DREB1 pathway-mediated cold resistance response through AtICE1 and MdBBX37 [45,77].In tomatoes, JA promoted ABA biosynthesis, endowing tomatoes with cold tolerance [78].Our results indicated that JA enhanced cold tolerance by promoting the biosynthesis of f lavonol with antioxidant capabilities, mediating the response to cold stress in a CBF/DREB1-independent manner.In tea, Arabidopsis, and apple, JA promoted f lavonoid biosynthesis [33,50,70], implying that the mechanism by which JA promoted f lavonol synthesis under cold stress observed in jojoba has a certain degree of conservation.This mechanism held a broad evolutionary advantage in maintaining such pathways to cope with environmental challenges.The discovery of the regulatory function of JAZ-MYB module in cold acclimation of jojoba suggests that plants can regulate the synthesis of f lavonols by integrating environmental temperature and plant hormone signals, thereby adapting to environmental changes.In conclusion, our study reveals the molecular mechanism by which JA positively regulates jojoba's cold tolerance through the JAZ-TT8/MYB module.At the same time, our research broadens the applicability of JAZ proteins and further indicates that the JA pathway is a valuable candidate pathway for the cold breeding and cultivation of jojoba.

Plant materials and stress treatment
Jojoba seeds (KH-1201) were collected from Kehe Town, Sichuan Province, China.Seed sterilization and germination were performed as previously described [21].After germination, the seedlings were transported to a greenhouse with a temperature of 28 • C for cultivation.One-month-old seedlings were transferred to growth chambers (Percival Scientific, Perry, IA, USA) for cold acclimation, with conditions set at 15 • C during the day and 10 • C at night.Leaves from the uppermost part of each jojoba seedling were harvested at 0 (control, CK), 6, 24, 48, 72 h, and one week (cold acclimation, CA).All leaf samples were collected between 09:00 a.m. and 11:00 a.m.The gathered samples were promptly immersed in liquid nitrogen and preserved at −80 • C for subsequent experiments.Leaf samples from the six time points were subjected to transcriptome analysis, and the CK and CA groups were subjected to metabolomic analysis.Detailed sampling time points are shown in Fig. S1 (see online supplementary material).Samples were collected from a minimum of three distinct jojoba seedlings, and each group was subjected to three independent biological replicates.

Transcriptome analysis
RNA samples were obtained from jojoba leaves using the procedure of Geng et al. [79].The purity, concentration, and integrity of RNA were assessed using a method described previously [21].RNA sequencing and data pre-processing were performed at Beijing Boyun Huakang Gene Technology Co., Ltd (Beijing, China).Transcriptome data were analysed as described previously [21].In brief, DEseq2 was employed for the detection of DEGs, with the criteria for DEG identification comprising (i) FDR < 0.05 and (ii) a fold change ≥2 or ≤ 0.5.GO and enrichment analyses were conducted as described previously [21].Enriched metabolic pathways were screened based on Q-values.WGCNA was performed on the BMKCloud platform by using the default parameters (www.biocloud.net).

qRT-PCR analysis
The synthesis of cDNA and the reaction for qRT-PCR were performed using a method previously described [21].All primers were designed using Primer Premier 5.0.A comprehensive list of all primers is provided in Table S6 (see online supplementary material).18S rRNA (NCBI accession number: AF094562) was used as an internal control.

Metabolomic analysis
Metabolite extraction and detection were conducted by referring to a method previously described [21].PCA and PLS-DA of all metabolites were conducted using MetaboAnalyst 5.0 [80].Criteria for identifying DAMs were as follows: (i) P. adjust (P value after BH adjustment) <0.05 and (ii) a fold change ≥1.5 or ≤0.67.KEGG enrichment analyses were conducted using methods described previously [21].Enriched metabolic pathways were screened based on Q-values.

Effects of exogenous flavonols on cold tolerance of yeast
The cold tolerance of yeast strain W303-1A was analysed using a previously described method [81].Yeast cells were cultured in YPA medium to the exponential phase (OD 600 between 0.6 ∼ 0.8) and then resuspended in YPA medium with 0.1 mg/mL concentrations of quercetin, spiraeoside, hyperin, and DMSO, respectively.After incubating at 30 • C for 30 min, yeast cells were transferred to −20 • C for 36 h and cultured on YPD medium at 30 • C for 2∼3 d to observe the growth of colonies.

Effects of exogenous flavonols on cold tolerance of tobacco
Forty tobacco seedlings with identical growth statuses were randomly allocated into four groups and subjected to spray treatment with 1 mM quercetin, spiraoside, hyperin, and H 2 O.Each tobacco seedling was sprayed with 10 mL of the solution.After 3 d, the tobacco seedlings were transferred to growth chambers for cold treatment at 4 • C for 24 h, immediately followed by phenotypic observation and delayed f luorescence imaging.Delayed f luorescence was a noninvasive approach for assessing the impact of abiotic stress on the functionality and performance of the photosynthetic apparatus [82].Delayed f luorescence was imaged using a NightSGADE LB 985 (Berthold, Bad Wildbad, Germany) following the previously described method [83].

Detection of plant endogenous hormone content
The JA content in jojoba leaves after cold acclimation was assessed by Nanjing Ruiyuan Biotechnology Co., Ltd.(Nanjing, China), which used the method of Zhang et al. [84].

Effects of exogenous MeJA on cold tolerance of jojoba
Sixty one-month-old jojoba seedlings with the same growth status were randomly allocated into three groups and watered with 0, 10, or 100 μM MeJA every 3 d.Six days later, half of the plants in each group were transferred to growth chambers for 24 h of cold stress at 4 • C; the remaining seedlings were designated as the control.REL and MDA content were determined, and delayed f luorescence imaging of the leaves was performed.The MDA content and REL were determined using a previously described method [85].

LCA
ScTT8 was fused to the plasmid pCAMBIA1300-nLUC to obtain the target construct ScTT8-nLUC, and ScJAZ1, ScJAZ6, ScJAZ7, ScJAZ8, ScJAZ13, and ScMYB12 were fused to the plasmid pCAMBIA1300-cLUC to obtain different constructs.The Agrobacterium tumefaciens GV3101 harboring the recombinant plasmid was co-transformed into tobacco leaves in different combinations.After 2∼3 d of culture, the luminescence intensity was detected using a Tanon-4800 Chemiluminescent Imaging System (Tanon, China) following dark treatment for 10 min, according to a previously described method [86].Each experiment consisted of a minimum of five independent biological replicates.

BiFC assay
ScTT8 was fused to plasmid pSPYNE-35S to obtain the target construct ScTT8-nYFP, and ScJAZ13 and ScMYB12 were fused to plasmid pSPYCE-35S to obtain the target constructs ScJAZ13-cYFP and ScMYB12-cYFP.The A. tumefaciens GV3101 harboring the recombinant plasmid was co-transformed into tobacco leaves in different combinations.After 2∼3 d, YFP f luorescence was observed using a confocal microscope (Leica, Mannheim, Germany).

Y2H assay
ScTT8 was fused into the pGBKT7 (BD) plasmid to generate the target construct BD-ScTT8, and ScJAZ13 and ScMYB12 were fused into the pGADT7 (AD) plasmid to generate the target constructs AD-ScJAZ13 and AD-ScMYB12, respectively.Different combinations were transformed into the yeast strain Y2H-Gold (Coolaber, Beijing, China).The yeast cells were cultured at 28 • C on SD/−Trp/−Leu (−T-L) plates for 48∼96 h.Subsequently, the colonies were transferred to SD/−Trp/−Leu/-His/−Ade (−T-L-A-H) plates with the addition of Aureobasidin A (AbA) and X-α-gal for growth.

Y1H assays
The promoter sequences (1500 bp upstream of the start codon) of ScPAL, ScCHS, ScCFI, and ScFLS were cloned into the pAbAi yeast-integrating vector to obtain different constructs.Different promoter constructs were transformed into yeast strain Y1H-Gold (Coolaber, Beijing, China).Yeast cells were cultured at 28 • C on SD/-Ura plates.Subsequently, the optimal concentration of AbA required to inhibit the self-activation of the promoter was screened on SD/-Ura plates.The target construct, AD-ScMYB12, was obtained by inserting full-length ScMYB12 into AD plasmids.The obtained AD-ScMYB12 was transformed into the yeast strain Y1H-Gold cells harboring different promoter fragments.The colonies were cultured on SD/−Leu plates at the optimal concentration of AbA.

Luciferase activity assay
The ScMYB12 and proScFLS sequences were inserted into pRI-GFP and pCAMBIA1302-LUC using homologous recombination to obtain the target constructs, pRI-ScMYB12 and pCAMBIA1302-proScFLS.The A. tumefaciens GV3101 harboring the recombinant plasmid was co-transformed into tobacco leaves in different combinations.After 2∼3 d, the luciferase activity in tobacco leaves was analysed using the Tanon-4800 Chemiluminescent Imaging System (Tanon, China) with the procedure outlined by Cheng et al. [87].

Dual luciferase activity assay
The ScFLS promoter and ScMYB12, ScTT8, and ScJAZ13 were inserted into the pGreenII 0800-LUC and pGreenII 62-SK vectors to generate the reporter and effector constructs, respectively.The A. tumefaciens GV3101 harboring the recombinant plasmid was co-transformed into tobacco leaves in different combinations.After 2∼3 d, luciferase activity was assessed using a dual luciferase reporter assay kit (Vazyme, Nanjing, China).

Transformation of hairy roots of jojoba
The genetic transformation system for jojoba was constructed according to a cut-dip-budding (CDB) delivery system by using a previously described method [88].ScMYB12 was cloned into the pCAMBIA1305 vector to generate the 35S:ScMYB12 construct.Hairy roots of jojoba overexpressing ScMYB12 were obtained using a CDB delivery system, and PCR amplification of rol B from A. rhizogenes was performed to confirm the success of the transgenic process [89].The total f lavonoid content in the hairy roots of different lines was determined using a plant f lavonoid assay kit (Solarbio, Beijing, China).The ScMYB12-overexpressing plants were watered every three days with 100 μM MeJA.After six days, the f lavonoid content and gene expression levels in the hairy roots were quantified.

Figure 1 .
Figure 1.Cold acclimation stimulated f lavonoid production in jojoba.a Classification of the f lavonoids in all DAMs.b KEGG analysis of the DAM.c, d GO and KEGG analysis of all DEGs.e Metabolites transformation and the corresponding enzyme gene changes in f lavonoid metabolism pathways under cold acclimation.The dots and rectangle, respectively, indicated metabolites and the genes encoding the corresponding enzymes.Red and blue represented the increase and decrease of metabolites/genes after cold acclimation, respectively.f The morphology changes of the treatment group (spiraeoside, hyperin, and quercetin) and the control group (H 2 O) after cold stress.g The delayed f luorescence imaging of the treatment groups and the control group.h MDA content of control group and treatment group.i REL measurements.Statistical analysis of the data was conducted using Duncan's multiple range test.j Exogenous addition of spiraeoside, hyperin, and quercetin enhanced the cold tolerance of yeast strain W303-1A.

Figure 2 .
Figure 2. Analysis of WGCNA and synthesis and signal transduction pathways of JA. a Top 10 GO terms of biological process in GO enrichment analysis in the MEbrown module.b KEGG analysis of MEbrown module.c Co-expression network analysis of the MEbrown module.The gene in the middle denoted the hub gene.d The alterations in JA content in jojoba leaves pre-and post-cold acclimation.e Trend analysis of gene expression levels in JA signaling pathway.The heatmap was generated using the log2FC values of the corresponding genes.f The qRT-PCR analysis of crucial genes in synthesis and signal transduction pathways of JA under cold acclimation in jojoba.Each error bar showed the mean ± SD.All data were compared by an ANOVA followed by a Fisher's least significant difference (LSD) test.* P < 0.05, * * P < 0.01.g MDA content measurement of control and MeJA treatment group.h REL analysis of control and MeJA treatment group.i Delayed f luorescence imaging of control and MeJA treatment group.Lowercase letters indicate the significance analysis of different groups in the control group, and uppercase letters denote the significance analysis of different groups in the stressed group.Multiple comparisons were performed using the R package (Duncan's multiple range test, P < 0.05).

Figure 3 .
Figure 3. ScJAZ13 interacted with ScTT8.a LCA for testing the interactions of the ScTT8 with ScJAZ1, ScJAZ6, ScJAZ7, ScJAZ8, and ScJAZ13.The ScTT8 was fused with nLUC, and these JAZs were fused with cLUC.The luminescence intensity was detected by Tanon-4800 Chemiluminescent Imaging System.b BiFC assay of ScTT8 and ScJAZ13.The ScTT8 was fused with nYFP, and these JAZs were fused with cYFP.The scale bars denoted 50 μm.c Y2H assay for detecting the interactions of ScTT8 (fused with BD) with ScJAZ13 (fused with AD).Positive control, pGADT7-T and pGBKT7-53.Negative control, pGADT7-T and pGBKT7-lam.

Figure 4 .
Figure 4. ScMYB12 interacted with ScTT8 and promoted ScFLS expression.a Y1H assay for detecting the regulatory relationship between ScMYB12 and the promoter of ScPAL, ScCHS, ScCFI, and ScFLS.The promoter fragments of ScPAL, ScCHS, ScCFI, and ScFLS were cloned into pAbAi plasmids, and the optimal concentration of AbA to inhibit the self-activation of the promoter was screened on SD/-Ura medium.pAbAi-P53 and pGADT7-53 was used as positive controls.b Luciferase activity assay in tobacco leaves.The ScMYB12 was fused with pRI-GFP, and the proScFLS was fused with pCAMBIA1302-LUC.The luminescence intensity was detected by Tanon-4800 Chemiluminescent Imaging System.c LCA of ScMYB12 (fused with cLUC) and ScTT8 and ScJAZ13 (fused with nLUC) in tobacco leaves.d BiFC assay of ScMYB12 and ScTT8 in tobacco leaves.The ScTT8 was fused with nYFP, and ScMYB12 was fused with cYFP.The scale bars denoted 50 μm.e Y2H assay for detecting the interactions of ScTT8 (fused with BD) with ScMYB12 (fused with AD).Positive control, pGADT7-T, and pGBKT7-53.Negative control, pGADT7-T, and pGBKT7-lam.f Dual luciferase activity assay.The effectors ScMYB12, ScTT8, and ScJAZ13 were ligated in pGreenII 62-SK vectors, and the reporter proScFLS was ligated in pGreenII 0800-LUC vectors.Statistical analysis of the data was conducted using Duncan's multiple range test.

Figure 5 .
Figure 5.Effect of ScMYB12 overexpression on gene expression of f lavonol synthesis-related enzymes and cold stress tolerance.a Screening of ScMYB12-overexpressing hairy roots.M represented the DNA ladder DL2000, WT indicated the hairy roots of jojoba without Agrobacterium rhizogenes infection, and OE1-OE10 denoted different infection lines.b qRT-PCR analysis of hairy roots overexpressing ScMYB12.c Flavonoid content assay of hairy roots overexpressing ScMYB12.d The expression levels of enzyme genes associated with the f lavonol biosynthesis were analysed in ScMYB12-overexpressing hairy root lines OE1 and OE5. e Screening of ScMYB12-overexpressing hairy roots.f qRT-PCR analysis of hairy roots overexpressing ScMYB12.g MDA content measurement.h Analysis of f lavonoids content after MeJA treatment.i Analysis of ScFLS expression levels after MeJA treatment.j Analysis of f lavonoids content after cold stress.k Analysis of ScFLS expression levels after cold stress.The hairy roots overexpressing ScMYB12 were obtained by CDB delivery system, and the hairy roots growing for one month were collected for each measurement.Each experiment contained at least three independent biological replicates.Graphpad Prism 9 was used for data analysis and plotting.Each value was the mean ± SD.All data were subjected to ANOVA followed by an LSD test.* P < 0.05.* * P < 0.01.