VvMYB44–VvERF045 complex is involved in abscisic acid-induced sugar accumulation by activating VvSPS4 expression in grapes
Boyang Liu, Jiajia Li, Min Zhou, Ziqin Yu, Zishu Wu, Lei Wang, Shiping Wang, Songtao Jiu
TL;DR
This study identifies a protein complex in grapes that helps increase sugar levels during ripening, offering new insights into fruit quality regulation.
Contribution
The discovery of the VvMYB44–VvERF045 complex's role in ABA-induced sugar accumulation in grapes is novel.
Findings
ABA treatment increases grape berry ripening and soluble sugar levels.
The VvMYB44–VvERF045 complex activates VvSPS4 to influence sucrose metabolism.
Overexpression of VvERF045 in tomatoes also increases soluble sugar levels.
Abstract
Phytohormones play a crucial role in regulating fruit ripening and quality, particularly in soluble sugar accumulation. Despite this, the molecular mechanisms behind hormone-induced sugar accumulation in grapes are not well understood. Our study shows that abscisic acid (ABA) enhances grape berry ripening and soluble sugar levels. We generated a transcriptome dataset from grape berries subjected to hormone treatment and constructed a potential regulatory network related to sugar accumulation using weighted gene co-expression network analysis (WGCNA). Furthermore, we identified five structural genes (SGs) and 44 transcription factors (TFs) responsive to ABA that potentially regulate sugar accumulation in grape berries. Notably, VvMYB44 was emphasized due to its highest expression in ABA-treated mature fruits among these TFs. It binds to the promoter of VvSPS4 and activates its…
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Figure 7- —China Agriculture Research System
- —Key Research and Development Program of Shaanxi Province
- —National Natural Science Foundation of China10.13039/501100001809
- —Fujian Province Science and Technology Plan Project
- —Ningbo Science and Technology Development Special Fund
- —Postdoctoral Fellowship Program of CPSF
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TopicsPlant Molecular Biology Research · Horticultural and Viticultural Research · Plant nutrient uptake and metabolism
Introduction
Sugars, the primary products of photosynthesis, serve as essential energy sources and metabolic substrates for growth and development, while also acting as key signaling molecules in response to environmental changes in plants [1, 2]. In fruit crops, sugars are a major component of soluble solids and play a crucial role in determining fruit flavor quality. The accumulation of soluble sugars in the vacuole during the ripening stage significantly affects both fruit sweetness and commercial value [3]. Sugar content is synergistically regulated by a series of enzymes and transporters, which are intricately controlled at transcriptional, post-transcriptional, and post-translational levels [4, 5]. Understanding the mechanisms that regulate sugar accumulation is vital for improving flavor quality in fleshy fruits.
Sucrose, a major form of photosynthate, is synthesized in the source organs (leaves) and subsequently transported over long distances through the phloem system, ultimately being unloaded into sink organs such as fruits, roots, and flowers [6]. The allocation of sugars from source to sink involves several transmembrane steps at the subcellular level and requires various essential enzymes and sugar transporters [7]. In fruit crops, key enzymes in the sucrose metabolic cycle—a pivotal process in sugar metabolism—include invertase (INV), sucrose synthase (SS), hexokinase (HXK), fructokinase (FRK), phosphoglucose isomerase (GPI), sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), and among others [5, 8]. Additionally, the main sugar transporter families include sucrose carrier/transporter (SUT/SUC), monosaccharide sugar transporter (MST, which encompasses sugar transporter protein (STP), hexose transporter (HT), tonoplast monosaccharide transporter (TMT), early response to dehydration 6-like (ERD6L), vacuolar glucose transporter (vGT), plastidic glucose transporter (pGlcT), polyol monosaccharide transporter (PMT), and inositol transporter (INT)), as well as sugars will eventually be exported transporter (SWEET). These transporters function coordinately in fruits [9–11]. The intensity and speed of sugar unloading are largely dependent on the abundance and activity of these enzymes and sugar transporters, which ultimately determine the sugar content of the fruit.
Phytohormones are crucial regulators of fruit ripening and quality, modulating gene expression and protein abundance that influence various quality attributes. Sugar metabolism and transport intersect with different hormone pathways, including ABA, ethylene, auxin, and others [12, 13]. Ethylene and ABA serve as predominant regulators, playing crucial roles in the ripening and quality formation of climacteric and non-climacteric fruits, respectively. For instance, ethylene treatment enhances sugar accumulation during ripening in banana, apple, and kiwifruit [14]. Silencing of FaNCED1, a key ABA biosynthetic gene, leads to a substantial decrease in sugar content in strawberry fruit, a phenomenon that can be rescued with exogenous ABA treatment [15]. Moreover, brassinosteroids (BRs) are important for facilitating fruit ripening. The heterologous expression of a BR biosynthetic gene, GhDWF4 from cotton, has been shown to promote fruit ripening and soluble sugar accumulation in tomatoes [16]. In contrast, the biosynthesis of auxins, gibberellins (GAs) and cytokinins (CTKs) generally decreases as fruit matures. The antagonistic relationship between auxin and ABA exerts regulatory control over fruit sugar homeostasis, with auxin-mediated downregulation of key transporters and metabolic enzymes significantly impeding the accumulation of soluble solids during ripening transitions [17]. Similarly, lower hexose accumulation has been observed in grape berries treated with exogenous CTKs and GAs during veraison [18]. Additionally, jasmonic acid (JA) delays fruit ripening by inhibiting ethylene biosynthesis in pears [19]. Thus, there is a strong association between sugars and phytohormones during fruit ripening, which warrants thorough investigation in specific contexts.
MYB TFs, characterized by their well-conserved MYB domain, are widely identified across various species and play crucial roles in numerous biological activities and metabolic processes, including growth, stress response, hormone signal transduction, and secondary metabolism. The MYB family is classified into three subfamilies: R1-MYB, R2R3-MYB, and R1R2R3-MYB; notably, most MYBs in plants belong to the R2R3-MYB category [20]. Several studies have highlighted the functions of R2R3-MYB TFs in either promoting or inhibiting sugar accumulation in fruit crops. For example, CmMYB113 mediates ethylene-dependent sucrose accumulation by transcriptionally activating CmACO1 and CmSPS1, ultimately increasing sweetness in climacteric melon fruit [21]. The MdMYB305–MdbHLH33 complex positively regulates the expression of three sugar-related genes (MdCWI1, MdVGT3, and MdTMT2), thereby elevating sugar content in apple fruit [22]. PuMYB12 binds to the promoter region of *PuSUT4-*like to enhance its expression, thus promoting sugar accumulation in pear fruit [23]. Conversely, FaMYB44.2 negatively regulates the expression of FaSPS3, thereby inhibiting sucrose accumulation in strawberries [24]. Additionally, CmERFI-2 represses CmMYB44, which derepresses CmSPS1 and CmACO1, thereby coordinating the interplay between sugar and ethylene in melon [25]. While these findings enhance our understanding of the role of MYB TFs in sugar accumulation, critical regulatory aspects remain to be fully elucidated. This highlights the necessity for systematic investigations, which will be presented in subsequent analyses.
Grape (Vitis vinifera L.) is one of the most extensively cultivated perennial fruit crops, highly valued in global markets for its flavor, nutritional benefits, and versatility in processing properties, such as wine, juice, raisins, and table grapes [26]. As a typical non-climacteric fruit, grapes offer a compelling experimental system for investigating the relationship between hormones and non-climacteric fruit ripening [27]. In our previous research, we reported an ABA-mediated transcriptional cascade involving VvGRIP55, VvMYB15, and VvSWEET15, which is associated with sugar transport in grape berries [28]. In the current study, we explored the effects of ABA on ripening and soluble sugar content in grape berries. Furthermore, a transcriptome dataset was generated from four stages of fruit samples under hormone treatment, allowing us to construct a potential regulatory network related to soluble sugar accumulation using WGCNA. Our findings identified VvMYB44, a TF with the highest expression in ABA-treated mature fruits among potential candidates, which enhances sucrose biosynthesis by directly binding to the promoter of VvSPS4 and activating its transcription. Moreover, VvMYB44 and VvERF045 synergistically promote the expression of VvSPS4. Notably, overexpressing VvERF045 promoted sugar accumulation in tomato fruits. In general, this study provides new insights into the regulatory mechanisms governing sugar accumulation in grapes.
Results
Effects of ABA treatment on ripening and soluble sugar content in grape berries
To investigate the effects of ABA on fruit ripening and soluble sugar content, pre-veraison ‘Muscat Hamburg’ grape berries were treated with 500 mg L^−1^ ABA. ABA treatment accelerated the progression of ripening, as evidenced by earlier skin coloration and enhanced berry expansion (Fig. 1A). Compared to the control, fruit weight, equatorial diameter (ED), and longitudinal diameter (LD) increased significantly under ABA treatment (Supplementary Data Fig. S1A–C). Notably, the application of ABA resulted in faster and higher total soluble solids (TSS) accumulation compared to the control (Supplementary Data Fig. S1D). Furthermore, the contents of glucose and fructose were higher and accumulated more rapidly in ABA-treated berries. Sucrose content remained low in grape berries and showed no significant change at 36 days after treatment (Fig. 1B–D). Consequently, ABA strongly promoted soluble sugar accumulation in grape berries. To elucidate genome-wide transcriptional responses during ripening, RNA-seq was conducted on ABA-treated ‘Muscat Hamburg’ grape berries. After adapter trimming and quality filtering (Q30 ≥ 91.99%), clean reads (ranging from 5.92 to 10.24 Gb/sample) were aligned to the V. vinifera reference genome, yielding mapping rates between 68.48% and 92.76% (Supplementary Table S1). In total, 26 087 genes were expressed across the 21 samples, demonstrating high correlations among the three biological replicates (Supplementary Table S2).
ABA treatment promotes fruit ripening and soluble sugar accumulation in ‘Muscat Hamburg’ grapes. (A) Phenotypes of grape berries under control and ABA treatment. Scale bars = 1 cm. (B-D) Sucrose (B), glucose (C), and fructose (D) contents of grape berries under control and ABA treatment. Data are presented as means ± SD with n = 3. * indicates values significantly different from the control (P < 0.05), ** indicates values highly significantly different from the control (P < 0.01).
Construction of weighted gene co-expression networks
To gain insights into the regulatory effects of ABA treatment on metabolic changes in grape berries, transcriptome data from 14 samples were utilized for WGCNA. The clustering dendrogram revealed high correlations among biological replicates, with branches indicating samples that exhibited similar expression patterns (Fig. 2A). A total of 10 co-expression modules were identified based on their analogous expression profiles (Fig. 2B; Supplementary Table S3). The heatmap illustrating module-trait correlations demonstrated that transcripts within the brown, blue, magenta, and turquoise modules were negatively correlated with key traits such as the accumulation of soluble sugars, including sucrose, fructose, and glucose, which significantly increase during ripening (Fig. 2C). These results suggest that genes within these four modules are primarily associated with the processes of fruit ripening and soluble sugar accumulation in ‘Muscat Hamburg’ grapes.
Construction of weighted gene co-expression networks. (A) Cluster dendrogram of transcriptomic samples of ‘Muscat Hamburg’ grapes. (B) Cluster dendrogram of co-expression modules identified by WGCNA. The major tree branches constitute 10 modules. (C) Heatmap of module-trait correlations, where each row corresponds to a module colored distinctly and each column corresponds to a trait. Yellow indicates a positive correlation, whereas purple denotes a negative correlation.
Generation of soluble sugar metabolic regulatory networks
To further explore the potential transcriptional regulatory mechanisms underlying soluble sugar metabolism, we screened for SGs involved in soluble sugar metabolism and transport, as well as TFs from the brown, blue, magenta, and turquoise modules based on the aforementioned transcriptome dataset of control and ABA-treated grape berries, using an eigengene connectivity value threshold (|KME| ≥ 0.8; Supplementary Table S4). This process identified a total of 19 SGs, including INVs, SSs, SPSs, SUCs, HTs, and SWEETs, along with 135 TFs, such as MYBs, WRKYs, ERFs, ARFs, NACs, bZIPs, and bHLHs (Supplementary Table S5). Subsequently, we refined our selection to 12 SGs whose expression levels were highly correlated with the accumulation patterns of the three soluble sugars. We also identified 128 TFs that showed strong correlation with these 12 SGs. This gene set was then visualized as a correlation network (Fig. 3A; Supplementary Table S6). Furthermore, we extracted the transcription levels of the 140 genes. Among them, five SGs and 44 TFs exhibited differential expression between the ABA treatment and control groups (Fig. 3B; Supplementary Table S7). These findings suggest that the 49 genes identified within this regulatory network are likely responsive to ABA signaling and play critical roles in regulating soluble sugar metabolism in grape berries.
Regulatory network of soluble sugars and expression heatmap of candidate genes in ‘Muscat Hamburg’ grapes. (A) Regulatory network of sucrose, fructose, and glucose. Hexagons represent the three soluble sugars, octagons represent structural genes involved in their metabolism and transport, and circles represent TFs whose transcript levels correlate with the expression of SGs. (B) Expression heatmap of differentially expressed candidate genes in RNA-seq data. C0, C1, C2, and C3 correspond to berries from the control group at 0-, 7-, 15-, and 36-day post-treatment; A1, A2, and A3 correspond to berries from the ABA-treated group at 7-, 15-, and 36-day post-treatment.
VvMYB44 regulates soluble sugar accumulation in grape calli and tomato
To assess the role of genes within the regulatory networks, we focused on the VvMYB44 gene, with highest expression in ABA-treated mature fruits among aforementioned 44 TFs (Supplementary Table S7). We successfully isolated the full-length coding sequence (CDS) of VvMYB44 from V. vinifera cv. ‘Muscat Hamburg’. Phylogenetic analysis indicated that VvMYB44 belongs to the R2R3-MYB family and shares homology with Arabidopsis AtMYB44, AtMYB70, AtMYB73, and AtMYB77 (Fig. 4A). The VvMYB44-YFP fusion protein was transiently expressed in tobacco leaves alongside a nucleus marker. As expected, complete colocalization of VvMYB44-YFP and NLS-mCherry was observed, confirming that VvMYB44 is located in the nucleus (Fig. 4B). We then assessed the expression pattern of VvMYB44 throughout developmental stages in ‘Muscat Hamburg’ grape berries. The VvMYB44 transcript showed high abundance from 60 to 110 days post anthesis (DPA) (Fig. 4C). To investigate the functional role of VvMYB44 in regulating target genes and influencing sugar accumulation, we generated three lines of grape calli overexpressing VvMYB44 (Lines 2, 4, and 5) (Fig. 4D). The effectiveness of the genetic transformation was validated via qRT-PCR analysis (Fig. 4G). Notably, the contents of glucose, fructose, and sucrose were significantly higher in the three overexpression lines compared to wild-type (WT) (Fig. 4E). Analysis of the regulatory network revealed a strong correlation between VvMYB44 and 10 SGs involved in sugar metabolism, including INVs, SSs, SPSs, and SWEETs (Fig. 4F). We examined the expression of some important target genes, finding VvNINV3, VvSS2, VvSPS4, and VvERD6L8 were up-regulated in VvMYB44 overexpression calli (Fig. 4G). Given these correlations, VvNINV3, VvSS2, and VvSPS4 were identified as candidate target genes for VvMYB44. In addition, we generated three overexpression lines of VvMYB44 in tomato (Lines 1, 3, and 7) (Fig. 4H–J). Consistent with our findings in grape calli, soluble sugar contents were significantly elevated in the overexpression fruit (Fig. 4K). We further analyzed the expression levels of the homologs of candidate target genes in the overexpressed tomato fruit, discovering significant up-regulation of SlLIN7, SlLIN8, SlNINV, SlVINV, SlSS4, SlSPS, SlSPS4, and SlSPSB compared to WT (Supplementary Data Fig. S2A). Additionally, other genes associated with sugar accumulation in tomato, such as SlSUT1, SlSUT4, SlTST1, and SlTST2, were also significantly induced in the VvMYB44 overexpression lines (Supplementary Data Fig. S2B). Overall, these results suggest that VvMYB44 plays a crucial role in soluble sugar accumulation by modulating the expression of VvNINV3, VvSS2, and VvSPS4 in grape berries.
VvMYB44 modulates sugar accumulation and expression of related genes in grape calli and tomato fruit. (A) Phylogenetic analysis of VvMYB44 and its homologs from grape and Arabidopsis, constructed using the neighbor-joining (NJ) method with 1000 bootstrap tests. (B) Subcellular localization of VvMYB44 in tobacco leaf epidermal cells, co-expressed with nuclear localization marker (NLS-mCherry) and VvMYB44-YFP. Scale bars = 10 μm. (C) Transcript abundance of VvMYB44 during fruit development and ripening. (D) Phenotypes of WT and VvMYB44-overexpressing grape calli. Scale bar = 1 cm. (E) Contents of sucrose, fructose, and glucose in WT and VvMYB44-overexpressing grape calli. (F) Transcriptional regulatory network of VvMYB44; hexagons represent sugars, diamonds represent VvMYB44, and circles represent correlated SGs. Red lines indicate positive correlation; blue lines indicate negative correlation. (G) Relative expression levels of VvMYB44 and target genes from the regulatory network in VvMYB44 overexpression calli. (H) Fruit phenotype of VvMYB44 overexpression lines compared to WT. Scale bars = 1 cm. (I, J) Transgenic identification of VvMYB44 overexpression lines. (K) Soluble sugar contents in fruits from VvMYB44 overexpression lines and WT. Data presented are means ± SD (n = 3). * indicates significantly different from WT plants (P < 0.05), ** indicates highly significantly different from WT plants (P < 0.01).
VvMYB44 binds to the promoter region of VvSPS4 to enhance its expression
To investigate whether VvMYB44 mediates soluble sugar accumulation through the transcriptional activation, we first scanned the promoter regions of the three candidate target genes. This analysis identified a binding motif for AtMYB44 exclusively in the promoter of VvSPS4. We conducted a yeast one-hybrid (Y1H) assay to confirm that VvMYB44 binds to the VvSPS4 promoter. The VvSPS4 promoter was inserted into the pLacZi vector, while the CDS of VvMYB44 was inserted into the pB42AD vector. Yeast cells of strain EGY48 harboring both the proVvSPS4-pLacZi and VvMYB44-pB42AD constructs turned blue on SD/-Trp/-Ura medium with X-gal. In contrast, yeast cells harboring proVvSPS4-pLacZi with an empty pB42AD plasmid did not show blue coloration (Fig. 5A). Additionally, given the presence of the MBS motif in the VvSPS4 promoter, we performed an electrophoretic mobility shift assay (EMSA) using the VvMYB44-His fusion protein. The results demonstrated that VvMYB44-His could directly bind to the MBS motif. Importantly, competitive cold probes significantly reduced the signal associated with the binding of VvMYB44 to the VvSPS4 promoter, while a mutated probe disrupted binding (Fig. 5B). These findings confirmed the interaction between VvMYB44 and the VvSPS4 promoter in vitro. Subsequently, we performed dual-luciferase reporter (DLR) assays in tobacco leaves. VvMYB44 was cloned into an effector vector, and the VvSPS4 promoter was cloned into the pGREENII0800 reporter vector (Fig. 5C). Leaves simultaneously co-expressing 35S::VvMYB44 and proVvSPS4-LUC exhibited stronger luminescence intensity than those expressing the empty vector alongside proVvSPS4-LUC (Fig. 5D). Collectively, these results indicate that VvMYB44 binds to the promoter region of VvSPS4, enhancing its expression both in vivo and in vitro.
VvMYB44 directly binds to the VvSPS4 promoter and activates its expression. (A) Y1H assay showing that VvMYB44 binds to the VvSPS4 promoter in vitro. (B) EMSA demonstrating that VvMYB44 binds to cis-acting elements within the VvSPS4 promoter in vitro. ‘+’ and ‘−’ indicate the presence and absence of the corresponding probe or recombinant protein, respectively. (C) Schematic diagrams of the reporter and effector constructs used in the DLR assay. (D) DLR assay showing that VvMYB44 activates VvSPS4 promoter activity, with luminescence image of tobacco leaves infiltrated with the constructs (upper panel) and quantitative analysis of relative LUC/REN activity (lower panel). REN activity served as an internal control. Data are presented as means ± SD (n = 5). * indicates a significant difference compared with the EV group (P < 0.05), ** indicates a highly significant different (P < 0.01).
VvMYB44 and VvERF045 synergistically activate the expression of VvSPS4 and enhance soluble sugar accumulation
To further investigate how VvMYB44 regulates soluble sugar accumulation, a yeast two-hybrid (Y2H) assay was performed to evaluate the interaction between VvMYB44 and VvERF045, a critical integrator of ethylene, BR and ABA pathways [58]. As shown in Fig. 6A, yeast cells harboring VvMYB44-pGBKT7 and VvERF045-pGADT7 grew robustly and turned blue on SD/–Trp/–Leu/–Ade/–His medium containing X-α-Gal. We then performed pull-down assay using resin coated with GST-antibody. The results showed that VvMYB44-GST directly bound to VvERF045-His, while GST alone did not show binding (Fig. 6B). To substantiate the interaction in vivo, a BiFC assay was performed in tobacco leaves. Co-expression with the pair of VvMYB44-nYFP and VvERF045-cYFP led to the reconstitution of functional YFP in the nucleus, whereas no YFP signals were observed in control combinations (Fig. 6C). Furthermore, a Co-IP assay demonstrated that VvERF045-Myc was co-immunoprecipitated using anti-GFP in the presence of VvMYB44-GFP, while no Flag signal was detected in the negative control (Fig. 6D). These findings collectively confirm that VvERF045 interacts with VvMYB44 in vitro and in vivo. To evaluate whether this interaction affects the transcriptional regulation of VvSPS4, DLR assays were performed in tobacco leaves using the aforementioned reporters and effectors, along with additional 35S::VvERF045 effector (Fig. 6E). Compared with VvMYB44 alone, co-expression of VvMYB44 and VvERF045 further enhanced luminescence and LUC activity driven by VvSPS4 promoter (Fig. 6F and G). To investigate whether VvERF045 affects soluble sugar accumulation, we generated three tomato lines overexpressing VvERF045 (Lines 2, 3, and 5) with successful transformation validation (Fig. 6H–J). Notably, glucose, fructose, and sucrose levels were significantly higher in all three overexpression lines compared to WT (Fig. 6K). Overall, these results indicate that VvMYB44 and VvERF045 synergistically activate the expression of VvSPS4 and enhance soluble sugar accumulation.
VvMYB44 physically interacts with VvERF045 to further activate the expression of VvSPS4. (A) Y2H assay showing the interaction between VvMYB44 and VvERF045 in vitro. SD/−TL denotes SD/−Trp/−Leu medium, and SD/-TLHA + X denotes SD/−Trp/−Leu/-His/−Ade/+ X-α-Gal medium. (B) Pull-down assay showing that VvMYB44 interacts with VvERF045 in vitro. ‘+’ and ‘−’ indicate the presence and absence of the corresponding protein. (C) BiFC assay showing the interaction between VvMYB44 and VvERF045 in tobacco leaves. Unfused nYFP and cYFP served as controls. (D) Co-IP assay confirming the interaction between VvMYB44 and VvERF045 in vivo. ‘+’ and ‘−’ indicate the presence and absence of the corresponding protein. (E) Schematic diagrams of the reporter and effector constructs used in the DLR assay. (F and G) DLR assay showing the effect of VvMYB44–VvERF045 interaction on the transcriptional activation of VvSPS4, with luminescence images of tobacco leaves (F) and quantitative analysis of relative LUC/REN activity (G). REN activity served as an internal control. Data presented are means ± SD (n = 5). (H) Fruit phenotypes of VvERF045 overexpression lines compared to WT. Scale bars = 1 cm. (I and J) Identification of VvERF045-overexpressing lines. (K) Three sugar contents in fruits of VvERF045-overexpressing lines compared to WT. Data presented are means ± SD (n = 3). * indicates significantly different from WT plants (P < 0.05), ** indicates highly significantly different from WT plants (P < 0.01).
Discussion
The study of fruit ripening has been notably constrained by the limited availability of extensive mutant collections in fruit crops. Consequently, most research relies on the application of various hormones or compounds during different stages of fruit development. This is typically followed by extensive sampling and subsequent transcriptomic and metabolomic analyses to unravel their effects on transcript abundance and metabolite content [27]. ABA functions as a crucial regulatory molecule governing the ripening processes of both climacteric and non-climacteric fruits. During the ripening of most fleshy fruits, including strawberry, grape, and tomato, endogenous ABA levels typically increase [29–31]. Exogenous ABA application also promotes the fruit sweetness, coloration, and flavor. For instance, ‘Summer Black’ grapes harvested after ABA treatment matured 15 days earlier than untreated grapes, showing significantly higher levels of soluble sugars and anthocyanins [32]. ABA treatment also significantly accelerated the softening of sweet cherry fruits [33] and enhanced carotenoid, phenolic, and aromatic volatile accumulation in tomato [34]. Furthermore, both application of ABA and fluridone (an ABA inhibitor) markedly influenced sugar accumulation in apples, promoting and inhibiting it, respectively [35]. In our study, ABA treatment promoted grape berry ripening, specifically, enhanced coloration and increased soluble sugar content (Fig. 1). Overall, these findings underscore ABA as a common and essential regulator of ripening across diverse fruit species.
Numerous TFs have been identified as key modulators of fruit ripening and sugar accumulation by regulating the expression of genes involved in sugar metabolism, transport, and phytohormone synthesis. For example, ClNAC68 positively regulates sugar accumulation in watermelon by repressing ClINV [36]. MaMADS36 binds to the promoter of MaBAM9b, activating its transcription and promoting starch degradation during banana fruit ripening [37]. MdERDL6-mediated glucose circulation activates the SnRK2.3-AREB1-TST1/2 cascade module, resulting in increased sugar levels in both apple and tomato [38]. In grapes, VvMSA that induced by sucrose and ABA signals positively regulates sugar accumulation by activating VvHT1 [39]. VvERF105 and VvNAC72 regulate the spatial and temporal expression of VvSWEET15 in opposing manners, thereby influencing sugar accumulation during grape berry ripening [40]. In this study, by constructing a soluble sugar–associated regulatory network, we identified 44 TFs, including MYBs, WRKYs, ERFs, NACs, and bHLHs, that may respond to ABA signaling to modulate soluble sugar metabolism in grape berries (Fig. 3). We further characterized the positive role of VvMYB44 in regulating soluble sugar accumulation in grape calli and tomato (Fig. 4). Notably, a recent report highlighted the role of VvMYB44 in high-temperature-induced inhibition of anthocyanin biosynthesis in grape berries [41]. Our findings demonstrate that VvMYB44 transcriptionally activates the expression of VvSPS4, thereby promoting sucrose metabolism in grapes (Fig. 5). Collectively, these results underscore the important and diverse functions of VvMYB44 in fruit ripening and soluble sugar accumulation.
Sucrose plays a critical role in carbohydrate metabolism, serving as the primary form for transporting leaf photosynthates to fruits [6]. SPS is the rate-limiting enzyme in sucrose synthesis, catalyzing the conversion of fructose-6-phosphate and uridine diphosphate glucose into sucrose-6-phosphate, which is subsequently hydrolyzed by SPP to produce sucrose [42]. Since the first SPS gene was isolated from maize, homologous genes have been identified across various species, including Arabidopsis, rice, wheat, tomato, and apple [43–47]. SPS genes play a crucial role in providing energy during vegetative and reproductive growth. AtSPSA1 is involved in photosynthetic sucrose synthesis in Arabidopsis leaves [44]. Notably, sps1 mutants exhibit sterile pollen in rice [48]. Overexpressing SoSPS1 has been shown to enhance sucrose levels in sugarcane [49]. A strong correlation exists between SPS enzyme activity and sucrose content in fruit crops. For instance, SPS activity significantly increases alongside rapid sucrose accumulation during banana fruit ripening [50]. CmSPS1 contributes to ethylene-induced sucrose accumulation in postharvest climacteric melon fruit [21]. Overexpression of FaSPS3 raises sucrose levels in strawberry fruit [24]. Additionally, the expression of MdSPSA2.3 also correlates positively with sucrose content during the ripening of ‘Golden Delicious’ apples; with silencing MdSPSA2.3 leading to a significant inhibition of sucrose accumulation [47]. Here, we found that VvSPS4 as a function target of VvMYB44 that promoting soluble sugar accumulation in grapes (Fig. 5). Similarly, MaNAC19 has been shown to directly activate the MaSPS1 to promote sucrose synthesis in bananas [51]. CitSAR binds directly to the GCC-box of the CitSPS4 promoter, activating its expression and sucrose metabolism in citrus fruits [52]. Therefore, understanding the molecular regulation of SPS genes is essential for improving fruit flavor quality.
Transcription factors often function cooperatively through protein–protein interactions to co-regulate the expression of target genes. The MYB-bHLH-WD40 complex is involved in anthocyanin biosynthesis in various species [53]. MdMYB63 interacts with MdERF106 to amplify the transcriptional activity of the target MdSOS1, ultimately enhancing the salt tolerance of apple [54]. VqWRKY53 positively regulates stilbene biosynthesis and Pst DC3000 resistance in wild grape by interacting with VqMYB14 and VqMYB15 [55]. TF oligomers also participate in controlling soluble sugar accumulation. ABA signaling regulators MdbZIP23 and MdbZIP46 interact with MdWRKY9, enhancing its regulatory impact on MdSWEET9b, which ultimately promotes sugar accumulation in apple fruits [35]. PpNAP4, in conjunction with PpNAP6, serves as a key regulator of sucrose synthesis by influencing the expression of PpSUS1 and PpSPS2 [56]. The FvNAC073–FvCMB1L complex competitively bind to the promoters of FvSPS1 and FvSUS2, thereby antagonistically regulating the sucrose accumulation during the ripening of strawberry fruit [57]. Here, VvERF045 interacts directly with VvMYB44 to further activates the VvSPS4 expression. Overexpressing VvERF045 also boosted the soluble sugar levels in tomato fruits (Fig. 6). In our recent research, VvERF045 was identified as a critical integrator mediating the crosstalk among ethylene, BR, and ABA pathways, regulating anthocyanin, BR and ABA biosynthesis [58]. Consequently, the synergistic effect of multiple TFs is a crucial system to regulate soluble sugar accumulation in plants.
Conclusion
In this study, we highlighted the positive impacts of ABA on grape ripening and soluble sugar accumulation. Utilizing a transcriptome dataset from ‘Muscat Hamburg’ grape berries subjected to exogenous hormone treatment, we constructed a potential regulatory network for soluble sugar accumulation. From this analysis, we identified five SGs and 44 TFs that may respond to ABA signaling to regulate soluble sugar metabolism in grape berries. Notably, we characterized the VvMYB44 TF, which is highly expressed during the veraison and ripening stages in grape berries. VvMYB44 appears to influence sucrose metabolism by binding to the promoter of VvSPS4, thus activating its expression. Moreover, VvERF045 was found to interact with VvMYB44, further enhancing its transcriptional activation of VvSPS4 (Fig. 7). These findings provide new insights into the molecular mechanisms underlying soluble sugar accumulation in grapes.
Proposed model depicting the role of VvMYB44 in promoting soluble sugar accumulation in response to ABA in grapes.
Materials and methods
Plant materials and growth conditions
The grape cultivar ‘Muscat Hamburg’ (V. vinifera) was grown in the greenhouse at Shanghai Jiao Tong University (31°11′ N, 121°29′ E). Eight individual plants were cultivated under natural light and irrigated weekly throughout the growing season. One fruit cluster was retained per branch, ensuring that the total number of grape berries and leaf area for each plant were approximately equal. The ‘Gamay’ calli used for stable transformation were cultured on B5 medium, maintained in the dark at 24°C, and subcultured once a month. Nicotiana benthamiana, the wild-type tomato ‘Micro-Tom’, and various transgenic lines were grown in controlled-environment chambers at 22°C, with a long-day cycle and a photosynthetically active radiation (PAR) level of 600 μmol m^−2^·s^−1^.
ABA treatment
The eight plants were divided into two groups, with four plants assigned to each group, each containing 20 fruit clusters. The first group was sprayed with water as the control; the second group received a treatment of 500 mg L^−1^ ABA [59]. Hormonal applications were performed one week before veraison (approximately 60 DPA), and to minimize rapid evaporation, ABA was applied at sunset. Grape berries were collected at 0, 7, 15, and 36 days after treatment. Each group contained three biological replicates, with each replicate involving the random collection of one hundred berries. All samples were immediately frozen in liquid nitrogen and stored at −80°C for subsequent assays.
Measurements of soluble sugar content
Soluble sugar components from grape berries, tomato fruits, and grape calli were extracted according to previously established methods [60]. Levels of soluble sugars were determined using a high-performance liquid chromatography (HPLC) system (LC3000, Shanghai, China) equipped with a XBridge NH_2_ column (Waters, MA, USA). Each sample group included three biological replicates.
Quantification of ABA
The ABA content in grape berries was quantified using a method previously described [61]. ABA levels were determined using an HPLC system (LC100, Shanghai, China) fitted with a XBridge C18 column (Waters, MA, USA). Three biological replicates were measured for each group.
RNA sequencing and data analysis
Total RNA was extracted from grape berries subjected to treatment for 0, 7, 15, and 36 days across different groups using TRIzol® Reagent (Magen, China). Paired-end libraries were prepared using an ABclonal mRNA-seq Library Preparation Kit (ABclonal, China). Sequencing was performed on an Illumina NovaSeq 6000 platform. Raw FASTQ files were processed with in-house Perl scripts to filter out adapter sequences and low-quality reads. These clean reads were aligned to grape reference genome GCF_030704535.1 using HISAT2 software. The differential expression analysis was performed using DESeq2, applying a threshold of log_2_ (Fold Change) > 1 and P < 0.05 to identify significantly differentially expressed genes. Each sample included three biological replicates, with ten berries pooled per replicate. Sample names were designated as follows: C01, C02, and C03 represent control group berries at 0 days post-treatment; C11, C12, and C13 denote control group berries at 7 days post-treatment; C21, C22, and C23 indicate control group berries at 15 days post-treatment; and C31, C32, and C33 depict control group berries at 36 days post-treatment. Similarly, A11, A12, and A13 correspond to berries from the ABA-treated group at 7 days post-treatment.
Weighted gene co-expression network analysis and visualization
Co-expression network modules between metabolites/traits and genes were generated using the R-plugin WGCNA shiny within TBtools v2.056 software [62]. The co-expression modules were obtained using default parameters, with the exception of the soft threshold power set to 26 and a minimum module size of 150. Initially, the clusters were merged based on eigengenes, and eigengenes values of each module were calculated to examine associations with metabolites/traits. Evaluating the Pearson correlation coefficient (PCC > 0.8) between SGs and TFs within the same module to establish the transcriptional regulatory networks. These networks were visualized using Cytoscape (v3.7.2, USA) software [63].
Genes expression analysis
Total RNA from grape calli and tomato samples was extracted using the RNAprep Pure Plant Plus kit (TIANGEN, Beijing, China) according to the manufacturer’s guidelines. First-strand cDNA was synthesized from 200 ng of total RNA using the HiScript IV RT SuperMix (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) assays were performed on a Bio-Rad real-time PCR system (Bio-Rad, CA, USA). Transcripts VvACTIN (XM_002282480.4) and SlUBI (NM_001346406.1) served as reference controls for grape and tomato, respectively. The relative expression levels of the genes were calculated using the 2^−ΔΔCT^ method [64]. The primers utilized in this study are listed in Supplementary Table S8.
Subcellular localization
The full-length CDS (lacking the stop codon) of VvMYB44 was cloned into the pHB-YFP vector. The recombinant constructs were transiently transformed into tobacco leaves. The nucleus was marked using NLS-mCherry. After 72 h, YFP and mCherry fluorescence signals were captured using a Zeiss LSM 780 confocal microscope (Zeiss, Jena, Germany). The primers utilized in this study are listed in Supplementary Table S8.
Genetic transformation of grape calli and tomato
The VvMYB44-pRI101, VvMYB44-pHB, and VvERF045-pHB overexpression vectors were constructed to generate transgenic grape calli and tomato lines. Stable transgenic grape calli and tomato lines were developed using Agrobacterium-mediated genetic transformation, as previously described [65]. The resulting kanamycin-resistant grape calli lines and hygromycin-resistant tomato lines were further verified through PCR and RT-qPCR analyses. Positive transgenic materials were maintained for subsequent experiments. The primers utilized in this study are listed in Supplementary Table S8.
Yeast one-hybrid assay
Y1H assays were performed according to established protocols [66]. The promoter fragment of VvSPS4 was inserted into the pLacZi vector. Additionally, the recombinant pB42AD plasmid containing the full-length CDS of VvMYB44 was constructed. These recombinant plasmids were co-transformed into competent EGY48 yeast strain. Aliquots (10 μl) of three yeast clone suspensions were then plated on SD/-Trp/-Ura/+X-Gal medium and incubated for an additional 3 to 5 days at 30°C. Blue plaques indicated successful binding between DNA and protein, whereas non-blue plaques suggested no interaction. The primers utilized in this study are listed in Supplementary Table S8.
Electrophoretic mobility shift assay
The CDS of VvMYB44 was cloned into the pET32a vector to express the VvMYB44-His fusion protein in E. coli strain Rosetta (DE3). The EMSA was conducted using a Chemiluminescent EMSA Kit (Beyotime, China). The sequences of biotin-labeled probes, cold probes, and mutant probes are listed in Supplementary Table S8.
Yeast two-hybrid assay
Y2H assay was carried out using the Matchmaker™ Gold Yeast Two-Hybrid System according to the user manual. The CDSs of VvMYB44 and VvERF045 were inserted into pGBKT7 and pGADT7 vectors, respectively. These recombinant plasmids were co-transformed into competent Y2HGold yeast strain. Aliquots (10 μl) of three yeast clone suspensions were plated on SD/−Trp/−Leu medium and SD/−Trp/−Leu/−His/−Ade/+ X-α-Gal medium, followed by incubation for an additional 3 to 5 days at 30°C. Blue plaques indicated positive interaction, whereas non-growth plaques suggested a lack of interaction. The primers utilized in this study are listed in Supplementary Table S8.
Pull-down assay
The CDSs of VvMYB44 and VvERF045 were cloned into the pGEX-4T-2 and pET32a vectors to express the VvMYB44-GST and VvERF045-His fusion protein in E. coli strain Rosetta (DE3), respectively. The pull-down assay was conducted using a GST Protein Pull Down Kit (ACE, China). The primers utilized in this study are listed in Supplementary Table S8.
Bimolecular fluorescence complementation assay
The full-length CDS (lacking the stop codon) of VvERF045 was cloned into the pXY104-cYFP vector, while the full-length CDS of VvMYB44 was ligated into the pXY106-nYFP vector. The recombinant constructs were transiently transformed into tobacco leaves. The nucleus was marked using NLS-mCherry. After 72 h, YFP and mCherry fluorescence signals were captured using a Zeiss LSM 780 confocal microscope (Zeiss, Jena, Germany). The primers utilized in this study are listed in Supplementary Table S8.
Co-immunoprecipitation assay
Co-IP assay was performed following previously reported methods [67]. The full-length CDS (lacking the stop codon) of VvERF045 and that of VvMYB44 were cloned into pHB-GFP and pCAMBIA1300-Myc vectors, respectively. The recombinant constructs were transiently transformed into tobacco leaves. After 48 h, the infiltrated tobacco leaves were collected. The primers utilized in this study are listed in Supplementary Table S8.
Dual-luciferase reporter assay
DLR assays were performed following previously reported methods [28]. The promoter fragments of VvSPS4 were inserted into the pGREENII0800 reporter vector, while the full-length CDS of VvMYB44 and VvERF045 were inserted into an effector vector under the control of the constitutive CaMV35S promoter. The combinations of the reporter and effector were transiently co-infiltrated into tobacco leaves. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Gene Assay Kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. Visual LUC signals were observed using a vivo imaging system (Tanon, Shanghai, China) following D-Luciferin potassium salt treatment (Yeasen, Shanghai, China). Each assay included five biological replicates. The primers utilized in this study are listed in Supplementary Table S8.
Statistical analysis
Data are presented as mean ± standard deviation (SD) from at least three independent replicates. Statistical significance was assessed using Student’s t-test (^*^P < 0.05, ^**^P < 0.01) or one-way analysis of variance followed by Tukey’s multiple range test at the P < 0.05 level. Linear regression analysis was conducted using Microsoft Excel (Microsoft, WA, USA). All data were analyzed using SPSS v22.0 (IBM SPSS Statistics, IL, USA) and graphed using SigmaPlot v14.0 software (Systat Software Inc., CA, USA).
Supplementary Material
Web_Material_uhaf318
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Smeekens S, Ma J, Hanson J. et al. Sugar signals and molecular networks controlling plant growth. Curr Opin Plant Biol. 2010;13:273–810.1016/j.pbi.2009.12.00220056477 · doi ↗ · pubmed ↗
- 2Ruan YL . Sucrose metabolism: gateway to diverse carbon use and sugar signaling. Annu Rev Plant Biol. 2014;65:33–6724579990 10.1146/annurev-arplant-050213-040251 · doi ↗ · pubmed ↗
- 3Dai Z, Wu H, Baldazzi V. et al. Inter-species comparative analysis of components of soluble sugar concentration in fleshy fruits. Front Plant Sci. 2016;7:64927242850 10.3389/fpls.2016.00649 PMC 4872523 · doi ↗ · pubmed ↗
- 4Wipf D, Pfister C, Mounier A. et al. Identification of putative interactors of Arabidopsis sugar transporters. Trends Plant Sci. 2021;26:13–2233071187 10.1016/j.tplants.2020.09.009 · doi ↗ · pubmed ↗
- 5Chen T, Zhang Z, Li B. et al. Molecular basis for optimizing sugar metabolism and transport during fruit development. a BIOTECH. 2021;2:330–4036303881 10.1007/s 42994-021-00061-2PMC 9590571 · doi ↗ · pubmed ↗
- 6Wan H, Wu L, Yang Y. et al. Evolution of sucrose metabolism: the dichotomy of invertases and beyond. Trends Plant Sci. 2018;23:163–7729183781 10.1016/j.tplants.2017.11.001 · doi ↗ · pubmed ↗
- 7Braun DM . Phloem loading and unloading of sucrose: what a long, strange trip from source to sink. Annu Rev Plant Biol. 2022;73:553–8435171647 10.1146/annurev-arplant-070721-083240 · doi ↗ · pubmed ↗
- 8Braun DM, Wang L, Ruan YL. Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signaling to enhance crop yield and food security. J Exp Bot. 2014;65:1713–3524347463 10.1093/jxb/ert 416 · doi ↗ · pubmed ↗