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. 2026 Jun 27;26:1590. doi: 10.1186/s12870-026-09365-1

The RcMYB21 transcription factor activates flavonol biosynthesis by targeting RcFLS in Chinese raspberry

Ting Lei 1,2,#, Jun Huang 1,3,#, Wei Qian 1, Qi Zhou 1, Haixiang Ruan 1, Zhou Fang 1, Yunsheng Wang 1,3,✉
PMCID: PMC13576136  PMID: 42374227

Abstract

Background

Chinese raspberry (Rubus chingii Hu) is a medicinal and edible plant belonging to genus Rubus in traditional Chinese medicine. Flavonoids are key bioactive compounds found in abundance in raspberry. However, the regulatory mechanism underlying flavonoid biosynthesis in raspberry remains poorly understood.

Results

Here, we identified RcMYB21, a novel R2R3-MYB transcription factor derived from R. chingii. RT-qPCR analysis revealed a co-expression pattern between RcMYB21 and the flavonol synthase gene RcFLS1. Subcellular localization and transactivation assays confirmed that RcMYB21 localizes to the nucleus and exhibits transcriptional activation activity, primarily mediated by its C-terminal domain. Further mechanistic studies (via Y1H and dual-luciferase assays) demonstrated that RcMYB21 directly binds to the promoters of key flavonoid biosynthesis genes, thereby activating RcFLS expression. Transient overexpression of RcMYB21 in raspberry leaves significantly upregulated RcFLS, while stable overexpression in tobacco (N. tabacum) resulted in elevated flavonol accumulation and suppressed anthocyanin levels in flowers, consistent with its role as a flavonoid pathway regulator. Notably, RcMYB21 also conferred stress adaptability. Under salt stress, RcMYB21-OE Arabidopsis exhibited enhanced lateral root development and improved tolerance, implicating its dual function in secondary metabolism and abiotic stress response.

Conclusions

We identified the RcMYB21 transcription factor in R. chingii. Our study indicated that the RcMYB21 transcription factor specifically regulates flavonoid biosynthesis and accumulation, and enhances salt stress tolerance through flavonol accumulation. These findings provide significant insights into the molecular mechanisms underlying flavonoid regulation in R. chingii and highlight the dual functional role of RcMYB21 in both secondary metabolism and stress response.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09365-1.

Keywords: Chinese raspberry, MYB transcription factor, Flavonoid biosynthesis, Flavonol, Secondary metabolism

Introduction

Chinese raspberry (Rubus chingii Hu) is a globally popular fruit crop known for its unique flavor and numerous health benefits. Both the immature dried fruits and ripe fresh fruits are highly valued for their medicinal and nutritional properties. The immature dried fruits of Rubus chingii Hu exhibit a wide range of bioactive properties, including anti-cancer, anti-bacterial, and neuroprotective effects, among others [1–5]. The ripe fresh fruits are rich in polyphenols, sugars, and nutrients, and are consumed as fresh berries, or processed into jams, a common approach to overcome the limited storability of fresh fruit [6]. Raspberry contains various secondary metabolites with significant biological activities, such as flavonoids, phenolic acids, and terpenoids [7–9]. Flavonoids are important organic compounds produced during raspberry metabolism and are widely recognized for their strong antioxidant activity. Among them, flavonols are the predominant type. In raspberry, flavonols primarily exist as flavonol glycosides, which are formed through the conjugation of flavonol aglycones with sugar molecules [2]. Notably, dozens of flavonols and their derivatives have been identified in raspberry, with tiliroside and kaempferol-3-O-rutinoside serving as key markers for its identification and quality control in the Chinese Pharmacopoeia [10, 11].

Gene expression in the flavonoid biosynthesis pathway is crucial for flavonoid accumulation. For example, chalcone synthase (CHS), the initial rate-limiting enzyme in this pathway [12], plays an essential role in flavonoid production; RNA interference of CHS significantly reduces total flavonoids content in tomato [13]. Flavanone 3’-hydroxylase (F3’H) is a key gene determining flavonoid composition in many plants; overexpression of Malus pumila MpF3’H enhances quercetin and cyanidin levels in Arabidopsis and tobacco [14]. Flavonol synthase (FLS) is both a key enzyme and a rate-limiting factor in flavonol biosynthesis [15]. In our previous study, overexpression of raspberry RcFLS1 resulted in reduced floral pigmentation, whereas overexpression of RcDFR enhanced anthocyanin accumulation, leading to deeper flower coloration in tobacco [16]. Additionally, anthocyanidin synthase (ANS) catalyzes the conversion of leucoanthocyanidins into anthocyanins [17], and its overexpression in strawberry enhances anthocyanin content [18]. However, the regulatory network underlying the biosynthesis and tissue-specific accumulation of flavonoids across diverse plant species remains unclear. According to classical theories, transcription factors such as MYB, bHLH, and WD40 [19] regulate the expression of one or more key structural genes (e.g., CHS, FLS, and DFR) either individually or as a complex (MYB-bHLH-WD40), thereby participating in the transcriptional regulation of flavonoid biosynthesis [20, 21]. Among these, MYB transcription factors exhibit significant diversity.

R2R3-MYB transcription factors are among the largest families in plants [22] and serve as key regulators of flavonoid biosynthesis [23]. The R2R3-MYB transcription factors across the ten subgroups (SGs) exhibit structural and functional diversity [24]. Different subgroups of MYB transcription factors regulate the synthesis of various flavonoid compounds. For example, the expression pattern of MsMYB22 in Malus sieversii is directly associated with that of FLS, and MsMYB22 can bind to the promoter of MsFLS to enhance its expression [25]. In pear fruit (Pyrus bretschneideri), the transient expression of PbMYB12b promotes the expression of PbCHSb and PbFLS, thereby enhancing the biosynthesis of quercetin and isoquercitrin [26]. In Daucus carota, DcMYB113 activates the expression of genes such as DcbHLH3, DcCHS1, and DcDFR1, thereby promoting the accumulation of anthocyanins [27]. Additionally, some MYB subfamilies inhibit flavonoid gene expression. For instance, CsMYB4 and CsMYB7 in tea plants (Camellia sinensis) exhibit an expression pattern that is opposite to that of CsMYB12, and they bind to the promoter of CsFLS and to suppress its expression [28]. PhMYB27 in Petunia hybrida significantly inhibits the expression of genes such as PhF3H, PhF3’5’H, PhDFR, and PhANS, leading to a substantial reduction in anthocyanin accumulation; knocking out PhMYB27 results in the opposite phenotype [29].

The Raspberry fruit is an aggregate fruit formed from multiple pistils within a single flower, and pollen fertility and pollination efficiency playing key roles in determining fruit development and quality. Beyond their well-known roles in vegetative tissues, flavonols also participate in critical reproductive processes, including the regulation of male fertility and the attraction of pollinators, thereby influencing reproductive success and fruit yield [30–32]. There may be specific transcription factors in raspberry that regulate the biosynthesis of flavonol in flowers. The Arabidopsis SG7 transcription factors, such as AtMYB11, AtMYB12, and AtMYB111 are thought to regulate flavonol accumulation by activating genes such as CHS, F3H, and FLS in the flavonoid biosynthesis pathway, though their expression is spatially specific [33, 34]. For example, the biosynthesis of flavonols in Arabidopsis roots and cotyledons is regulated by AtMYB12 and AtMYB111, respectively. Similarly, FeMYBF1 in buckwheat, PpMYBF1 in peach, and PbMYB12b in avocado, all belonging to SG7, can regulate the flavonol biosynthesis in plants [26, 35, 36]. Although many studies have reported that R2R3-MYB transcription factors of SG7 are involved in the regulation of flavonol biosynthesis, none of the members are highly expressed in flowers. In addition to SG7, AtMYB21 and its homologs AtMYB24 and AtMYB57, which belong to the R2R3-MYB subgroup 19, are also thought to regulate flavonol biosynthesis [37]. Among them, AtMYB21 directly binds to the GARE element in the AtFLS1 promoter, activating its expression and promoting flavonol accumulation, which further affects stamen development and fertility. Similarly, FhFLS2 is crucial for the biosynthesis of flavonols in the petals, stamens, and pistils of mature flowers, and it can be activated by FhMYB21L2 [38]. Furthermore, CmMYB21 from chrysanthemum, AmMYB305 from snapdragon, and LxSMYB305 from ornamental tobacco all belong to SG19, are highly expressed in flowers, and regulate flavonoid accumulation in plant flowers [39–41]. However, the precise molecular mechanism by which R2R3-MYB transcription factors of the SG19 subgroup orchestrate the regulation of flavonol biosynthesis remains unclear, thus warranting further investigation.

In this study, the functional role of the RcMYB21 transcription factor in raspberry was systematically investigated. The subcellular localization and transcriptional activation activity of RcMYB21 were analyzed using N. benthamiana and yeast expression systems. Subsequently, the binding activity of RcMYB21 to key flavonoid biosynthetic genes and its regulatory mode were initially characterized using yeast one-hybrid (Y1H) and dual-luciferase reporter assays. Furthermore, the biological function of RcMYB21 in modulating flavonoid biosynthesis was validated via transient expression in raspberry leaves and stable heterologous expression in tobacco and Arabidopsis. Collectively, these findings provide mechanistic insights into the regulatory role of SG19 MYB transcription factors in flavonoid biosynthesis in Rubus chingii Hu.

Results

Phylogenetic analysis and tissue-specific expression of RcMYB21

R2R3-MYB transcription factors are crucial regulatory factors in flavonol biosynthesis [37]. The evolutionary relationships of R2R3-MYB transcription factors involved in flavonoid biosynthesis in R. chingii and other plants were investigated via phylogenetic analysis using the Neighbor-Joining method in MEGA7 software (Figure S1A). The MYB transcription factors were classified into five subfamilies according to the differences in their amino acid sequences. The transcription factors in SG7 and SG19 exhibit a closely intertwined evolutionary relationship and are documented to influence flavonol synthesis. The classification of RcMYB21, OsMYB305, and NtMYB305 into SG19, along with AtMYB21, AtMYB24, and AtMYB57, suggests their involvement in flavonol biosynthesis. The critical amino acid residues and motifs associated with RcMYB21 were identified via multiple amino acid sequence alignment using CLUSTALW (Figure S1B). The results showed that all SG19 transcription factors, including RcMYB21, contain conserved R2 and R3 domains and a relatively stable C-terminal motif.

The R2R3-MYB transcription factor LG06.275 from R. chingii was identified via a local BLAST search of genomic data and designated as RcMYB21 (GeneBank: PQ628090) due to its high similarity to Arabidopsis AtMYB21. The correlation between RcMYB21 expression patterns and flavonoid accumulation was examined by performing RT-qPCR on various tissue and organ samples from R. chingii (Fig. 1A). RcMYB21 is predominantly expressed in R. chingii flowers, followed by leaves, with significantly higher expression levels compared to other tissues (Fig. 1B).

Fig. 1.

Fig. 1

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Tissue-specific expression of RcMYB21 gene. A Different tissues and fruit stages of R. chingii, including root, stem, leaf, flower, fruitlet, immature fruit, and ripe fruit. B Expression levels of RcMYB21 in different tissues and fruit stages. C Different stages of R. chingii flower development (S1, S2 and S3). D Expression levels of RcMYB21, RcFLS1/2 and RcDFR genes at different flowering stages. The short white lines in figure A and C are 1 cm scale bars. Data represent means ± SD; Tukey’s test, n = 3; P < 0.05

Our previous studies revealed that flavonols predominantly accumulate in the leaves and flowers of R. chingii, with RcFLS1 also highly expressed in these tissues [16], suggesting a potential link between the expression levels of RcMYB21 and RcFLS1 and the pattern of flavonol accumulation. Additionally, RcMYB21 expression levels decrease throughout the developmental stages of R. chingii fruit, consistent with the observed proanthocyanidinsaccumulation pattern in these fruits. Furthermore, R. chingii flowers were categorized into three distinct developmental stages: S1 (small bud stage), S2 (bud stage), and S3 (full bloom stage) (Fig. 1C). The upregulation of RcMYB21 during flowering implies its involvement in R. chingii flower development (Fig. 1D). RcMYB21 and RcFLS1 exhibit coordinated expression across flower developmental stages, showing an increasing expression trend. In contrary, RcFLS2 and RcDFR exhibit high expression levels during the S2 stage; however, both demonstrate a slight decline in expression during the subsequent S3 stage, with no significant difference observed between these two stages.

Nuclear localization and transcriptional activity of RcMYB21

The presence of nuclear localization signals and transcriptional regulatory domains is a characteristic feature of typical transcription factors [42]. To confirm whether RcMYB21 is an active transcription factor in R. chingii, subcellular localization and transcriptional activation assays were performed (Fig. 2A). GFP and the RcMYB21-GFP chimeric protein were transiently expressed in N. benthamiana leaves for subcellular visualization, and nuclei were stained with DAPI. Our results show that RcMYB21 is exclusively localized in the nucleus, confirming its role as a transcription factor.

Fig. 2.

Fig. 2

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Subcellular localization and transcriptional activation activity analysis of RcMYB21. A Subcellular localization of RcMYB21 in epidermic cells of N. benthamiana leaves. B Domain prediction of RcMYB21 protein. C Truncation analysis of RcMYB21 transcriptional activation activity

The protein domains of the RcMYB21 were predicted using the PlantCare database (Fig. 2B). The predicted domain of RcMYB21 included a low-complexity region (LCR), a C-terminal motif, and the highly conserved R2 and R3 domains (Fig. 2B). Since MYB transcription factors have a highly conserved MYB DNA-binding domain and a diverse C-terminal regulatory region [43], we speculated that the C-terminal motif of RcMYB21 is involved in transcriptional activation. The transcriptional activity of RcMYB21 was further confirmed using a yeast-based assay (Fig. 2C). Yeast cells transformed with BD-RcMYB21 (1-209) and various truncated plasmids (1-192, 1-166, and 1-132) grow well on SD medium lacking tryptophan. However, only yeast cells transformed with BD-RcMYB21 (1-209) are able to grow on SD medium without histidine and turn blue in the presence of X-α-Gal (Fig. 2C). These findings indicate that RcMYB21 is a nuclear-localized R2R3-MYB transcription factor with a C-terminal motif responsible for its transcriptional activation activity.

RcMYB21 binds and activates flavonol pathway genes

The ability of RcMYB21 to bind to the promoters of key flavonoid biosynthesis genes was verified by examining its interactions with the promoters of RcFLS1, RcFLS2, RcDFR, and RcANS (Fig. 3). Predicted cis-acting elements related to MYB transcription factor binding were identified in each promoter using the PlantCare website (Fig. 3A). The results showed that each promoter sequence contained cis-acting elements associated with MYB transcription factor binding, including the MYB cis-element (TAACCA), MYB-like cis-element (CAACCA), and MBS cis-element (CAACGT). Yeast one-hybrid assays showed that the yeast cells co-transformed with AD-RcMYB21 and pAbAi-proRcFLS1/FLS2/DFR/ANS can still grow on SD medium lacking leucine but supplemented with aureobasidin A (-L + AbA), whereas no growth was observed in the negative control. These results suggest that RcMYB21 can bind to the promoters of these genes (Fig. 3B). The regulatory effect of RcMYB21 on the aforementioned genes was examined using a dual-luciferase assay (Fig. 3C-J). We co-transformed 35 S: RcMYB21 and proRcFLS1:Luc into N. benthamiana leaves, and observed a significant activation of RcFLS1 expression by RcMYB21, as indicated by a notable increased luciferase activity (Fig. 3C, G). Likewise, RcMYB21 activated RcFLS2 expression but did not have a significant influence on RcDFR and RcANS (Fig. 3D-F, H-J).

Fig. 3.

Fig. 3

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Analysis of RcMYB21 binding and interaction with promoters of key genes for flavonoid biosynthesis. A Prediction of cis-acting elements in promoters of key flavonoid biosynthesis genes. B-J Yeast one-hybrid and dual luciferase assays to verify the binding and interaction of RcMYB21 with the promoters of key genes in flavonoid biosynthesis. Data represent means ± SD; Tukey’s test, n = 3; *, P < 0.05; **, P < 0.01

RcMYB21 promotes the expression of flavonol synthesis genes in R. chingii

The function of RcMYB21 was investigated by transiently overexpressing it in detached raspberry leaves using Agrobacterium (Fig. 4). Young raspberry leaves were bisected along the midvein, covered with filter paper, and continuously infected with an appropriate amount of fresh Agrobacterium resuspension (Fig. 4A). The filter paper was removed after infection, and the leaves were subsequently exposed to normal light. RT-qPCR was performed to assess the expression levels of the related genes after 48 h. The relative expression level of RcMYB21 in infected raspberry leaves was significantly higher than that in the control, indicating the successful overexpression of RcMYB21 (Fig. 4B). In addition, the expression levels of RcFLS1 and RcFLS2 were significantly higher in raspberry leaves overexpressing RcMYB21 higher than in the control (Fig. 4C-D), indicating that RcMYB21 plays a pivotal role in activating both RcFLS1 and RcFLS2. However, the expression levels of RcDFR and RcANS showed no significant difference compared to the control (Fig. 4E-F).

Fig. 4.

Fig. 4

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Transient overexpression of RcMYB21 in R. chingii leaves. A Schematic diagram of the infection for detached R. chingii leaves. B Relative expression of RcMYB21 in R. chingii leaves after infection; (C-F) Expression levels of RcFLS1, RcFLS2, RcDFR and RcANS genes in R. chingii leaves after infection. Data represent means ± SD; Tukey’s test, n = 3; *, P < 0.05; **, P < 0.01

RcMYB21 regulates flavonoid profiles in N. tabacum

The function of RcMYB21 was investigated using transgenic N. tabacum overexpressing RcMYB21 (Fig. 5). Compared with the pink petals of control plants, transgenic N. tabacum line RcMYB21-3 (with high RcMYB21 expression) exhibits significantly lighter floral color (Fig. 5A), whereas transgenic lines RcMYB21-1 and RcMYB21-2 (with low RcMYB21 expression) show no significant color change (Fig. 5A). These findings suggest a potential correlation between flower color and RcMYB21 expression level in N. tabacum. RT-qPCR results confirmed that the RcMYB21 expression level in each transgenic N. tabacum line is consistent with that of semi-quantitative PCR (Fig. 5A, B). Anthocyanin quantification in RcMYB21-OE N. tabacum flowers was conducted using spectrophotometry (Fig. 5C). The results show a significant decrease in anthocyanin content in RcMYB21-3 flowers compared to the control.

Fig. 5.

Fig. 5

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Changes of flavonoid content and gene expression in overexpressing RcMYB21 N. tabacum flowers. A Phenotype of overexpressing RcMYB21 N. tabacum flowers. B Relative expression levels of RcMYB21 in N. tabacum flowers. C The relative content of anthocyanins in overexpressing RcMYB21 N. tabacum flowers (colorimetric method). D Relative expression levels of endogenous flavonoid biosynthesis genes in N. tabacum flowers. E-G The relative contents of kaempferol, quercetin and anthocyanin in overexpressing RcMYB21 N. tabacum flowers. K3G, Kaempferol-3-O-glucoside; K3RG, Kaempferol-3-O-rhamnosylglucoside; K3GG, Kaempferol-3-O-glucosylglucoside; K3RG7G, Kaempferol-3-O-rhamnosylglucoside-7-O-glucoside; Q3G, Quercetin-3-O-glucoside; Q3,7R, Quercetin-3,7-di-O-rhamnoside; Q3RG, Quercetin-3-O-rhamnosylglucoside; Q3RG7G, Quercetin-3-O-rhamnosylglucoside-7-O-glucoside; C3Ru, Cyanidin-3-O-rutinoside; C3G, Cyanidin-3-O-glucoside, P3G, Pelargonidin-3-O-glucoside. Data represent means ± SD; Tukey’s test, n = 3; *, P < 0.05; **, P < 0.01

To verify the impact of RcMYB21 on the regulation of genes involved in flavonoid biosynthesis, RT-qPCR was performed to evaluate the expression of endogenous flavonoid biosynthesis genes in N. tabacum flowers. NtFLS expression is significantly upregulated in all RcMYB21-OE lines, with the highest level in RcMYB21-3 flowers (Fig. 5D). In addition, the expression levels of NtCHS, NtCHI, NtF3H, and NtF3’H are significantly upregulated in RcMYB21-OE N. tabacum flowers. However, no significant change was observed in the expression levels of NtDFR, NtANS, NtANR, and NtLAR compared to the control (Fig. 5D).

LC-MS was used to detect flavonoid contents in the flowers of each N. tabacum line (Fig. 5, Figure S2). The content of kaempferol-3-O-rhamnosylglucoside (K3RG) in RcMYB21-2 flowers is significantly higher than that in the control. Similarly, RcMYB21-1 and RcMYB21-3 N. tabacum flowers show a higher K3RG content, although the difference is not statistically significant (Fig. 5E). The contents of kaempferol-3-O-glucoside (K3G) and kaempferol-3-O-glucosylglucoside (K3GG) are significantly increased in all RcMYB21-OE N. tabacum flowers, whereas the content of kaempferol-3-O-rhamnosylglucoside-7-O-glucoside (K3RG7G) shows no significant difference among the flowers of different N. tabacum lines (Fig. 5E). Additionally, no significant differences were detected in the levels of quercetin derivatives among the flowers of different N. tabacum lines (Fig. 5F). However, compared to the control group, RcMYB21-2 and RcMYB21-3 N. tabacum flowers exhibit a significant reduction in cyanidin-3-O-rutinoside (C3Ru) content (Fig. 5G). Overexpression of RcMYB21 in tobacco leads to upregulation of NtFLS, increased flavonol accumulation, and reduced anthocyanin levels, consistent with its branch-specific role in diverting metabolic flux toward flavonol biosynthesis.

RcMYB21 enhances salt stress tolerance via Flavonol accumulation in Arabidopsis

The function of RcMYB21 was further investigated using transgenic Arabidopsis plants overexpressing RcMYB21 (Fig. 6).

Fig. 6.

Fig. 6

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Abiotic stress treatment in overexpressing RcMYB21 A. thaliana. A The contents of flavonols and proanthocyanidins in green pods of overexpressing RcMYB21 A. thaliana. B The relative expression of RcMYB21 and A. thaliana endogenous flavonoid biosynthesis genes in green pods. C, F Drought and salt stress treatment in overexpressing RcMYB21 A. thaliana. D, G Phenotypic parameters of each plant under drought and salt stress treatment. E, H The relative contents of flavonols and anthocyanins in each plant under drought and salt stress treatment. The short white lines in C and F are 0.5 cm scale bars. Data represent means ± SD; Tukey’s test, n = 3; *, P < 0.05; **, P < 0.01

The phenotype of RcMYB21-OE Arabidopsis does not differ significantly from that of the control; however, a notable increase in flavonol and proanthocyanidin contents was observed in their green pods (Fig. 6A). To clarify the regulatory effect of RcMYB21 on flavonoid biosynthesis genes in Arabidopsis, RT-qPCR was performed to measure the expression levels of related genes (Fig. 6B). The results indicated a significant upregulation of AtFLS expression in all RcMYB21-OE lines. Similarly, the expression levels of AtCHS, AtCHI, AtF3H, and AtF3’H are notably increased in the green pods of RcMYB21-OE Arabidopsis. In contrast, the expression levels of AtDFR, AtANS, and AtBAN remain statistically unchanged compared to the control. These findings suggest that RcMYB21 is involved in regulating flavonoid biosynthesis in Arabidopsis (Fig. 6B).

To further investigate the physiological functions of RcMYB21, abiotic stress treatments were performed on RcMYB21-OE Arabidopsis. The results show differences in the growth status of RcMYB21-OE Arabidopsis compared to the control (Fig. 6C, F).

Under mannitol-simulated drought stress, the growth of all Arabidopsis plants is inhibited, characterized by slow growth, leaf senescence, and anthocyanin accumulation (Fig. 6C). The growth status of RcMYB21-OE Arabidopsis is similar to that of the control, with no significant differences observed in primary root length and lateral root number (Fig. 6D). However, quantitative detection using spectrophotometry shows that flavonol content in RcMYB21-OE Arabidopsis is significantly higher than that in the control after drought stress, whereas anthocyanin content is significantly lower (Fig. 6E). Under salt stress, RcMYB21-OE Arabidopsis exhibits enhanced root system development and reduced anthocyanin accumulation in leaves compared to the control (Fig. 6F). Under salt stress, the primary root length of RcMYB21-OE Arabidopsis is significantly shorter than that of the control, whereas both the length and number of lateral roots are significantly increased (Fig. 6G). In addition, after salt stress, the flavonol content in RcMYB21-OE Arabidopsis is significantly higher than that in the control, whereas anthocyanin content is significantly lower (Fig. 6H), suggesting that RcMYB21-OE Arabidopsis plants may exhibit stronger stress resistance. The elevated flavonol content and enhanced root growth in RcMYB21-OE Arabidopsis under salt stress suggest a role for flavonols in mitigating oxidative damage and promoting adaptive root architecture.

Discussion

RcMYB21 regulates the biosynthesis of flavonol

Flavonoid compounds represent significant secondary metabolites in plants, and their biosynthesis is regulated by a diverse array of genes. Notably, MYB transcription factors exert a crucial influence on this process.

SbMYB3 can activate the promoter of SbFNSII-2, which encodes a rate-limiting enzyme in isoflavone synthesis in Scutellaria baicalensis. RNA interference (RNAi) or gene knockout of SbMYB3 decreases the contents of baicalin and oroxindin, whereas overexpression of SbMYB3 significantly increases the accumulation of these compounds [44]. In Paeonia suffruticosa, PsMYBPA2 activates the expression of PsF3H, providing sufficient precursor substances for anthocyanin biosynthesis, whereas PsMYB21 activates the expression of both PsF3H and PsFLS, thereby promoting flavonol biosynthesis. PsMYB21 is also significantly overexpressed in the non-spotted areas of peony petals, where it competes for substrates involved in anthocyanin synthesis, thereby inhibiting spots formation [45]. However, the regulatory mechanism underlying flavonoid biosynthesis in raspberry remains unclear.

Flavonoid biosynthesis is primarily regulated by R2R3-MYB transcription factors from the subgroups 4 (SG4), 5 (SG5), 6 (SG6), and 7 (SG7) groups [46]. Research has also demonstrated that AtMYB21 stimulates flavonol accumulation in Arabidopsis stamens by positively regulating the expression of AtFLS1 [37]. In this study, an R2R3-MYB transcription factor (RcMYB21), which shares homology with AtMYB21 from the subfamily 19 of A. thaliana, was identified and named in the genome of raspberry. The expression pattern of RcMYB21 in raspberry exhibits a strong correlation with that of RcFLS1 (Fig. 1D). Subsequent investigations revealed that RcMYB21 is localized in the nucleus and possesses transcriptional activation activity via its C-terminal motif (Fig. 2). RcMYB21 can bind to the promoters of RcFLS1, RcFLS2, RcDFR, and RcANS (Fig. 3A, B). However, dual-luciferase assays revealed that RcMYB21 primarily activates the expression of RcFLS1 and RcFLS2, with no significant effect on the activation of RcDFR and RcANS (Fig. 3C-J). Similarly, AtMYB21 and AtMYB24 can directly bind to the promoter region of AtFLS1, activating the expression of genes containing the MYBCORE, MYB1AT, and MYBPZM motifs. Additionally, FhMYB21L2 can also directly regulate the FhFLS2 promoter [38]. Furthermore, transient overexpression of RcMYB21 in detached raspberry leaves upregulates the expression levels of RcFLS1 and RcFLS2 (Fig. 4C, D), indicating that RcMYB21 plays a key role in regulating flavonol biosynthesis in raspberry (Fig. 7).

Fig. 7.

Fig. 7

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RcMYB21 transcription factor involved in flavonols biosynthesis of R. chingii

RcMYB21 transcription factor promotes the accumulation of flavonols in R. chingii by activating the expression of RcFLS1. Examples outside the circle show that MYB transcription factors are involved in regulating responses to various stresses.

Allogenic expression of RcMYB21 in N. tabacum leads to alterations in petal color, suggesting that RcMYB21 may also regulate anthocyanin accumulation. Further studies show that RcMYB21 significantly upregulates the expression of NtCHS, NtCHI, NtF3H, NtF3’H, and NtFLS in N. tabacum flowers, but had no significant effect on the expression of anthocyanin/proanthocyanidin-related genes (such as NtDFR, NtANS, and NtLAR). It can be postulated that the flavonol metabolic pathways in RcMYB21-OE N. tabacum plants are predominantly activated, resulting in a reduction of anthocyanin metabolic flux due to competitive inhibition.

The expression patterns of flavonoid biosynthesis genes in A. thaliana mirrored those observed in N. tabacum. Similarly, overexpression of the SG19 transcription factor AtMYB21 in A. thaliana led to the upregulation of CHS, CHI, F3H, F3’H and FLS1 genes [37]. Moreover, the SG7 transcription factor AtMYB12 in A. thaliana is capable of activating the expression of genes involved in flavonol biosynthesis pathway such as CHS, CHI, F3H and FLS, thereby regulating flavonol accumulation in A. thaliana [47]. Phylogenetic analysis revealed a close clade between SG19 and SG7, suggesting their potential functional similarity. The molecular mechanism underlying the regulation of flavonoid biosynthesis (especially flavonols) by SG19 transcription factors require further investigation.

RcMYB21 performs a variety of physiological functions

Flavonol biosynthesis in A. thaliana is regulated by SG7 transcription factors AtMYB11, AtMYB12, and AtMYB111; flavonol accumulation is significantly reduced in the myb11/myb12/myb111 triple mutant [33]. However, the flavonol content in pollen grains of this mutant is comparable to that in the wild type, suggesting that additional transcription factors may be involved in regulating FLS1 expression in Arabidopsis stamens [48]. Subsequent research has revealed that flavonol biosynthesis in Arabidopsis flowers is directly regulated by SG19 transcription factors AtMYB21 and AtMYB24, which also affect Arabidopsis fertility [37, 38]. As a member of SG19, RcMYB21 exhibits high expression level in raspberry flowers (Fig. 1), indicating its involvement in flavonol biosynthesis, as well as flower organ development and fruit formation. RcMYB21 expression is also relatively high in raspberry leaves, second only to flowers. Transient overexpression of RcMYB21 results in significant upregulation of RcFLS1 and RcFLS2 compared to the control (Fig. 4), suggesting its potential role in regulating flavonol biosynthesis in raspberry leaves. Moreover, the pod of RcMYB21-OE Arabidopsis exhibited significantly elevated flavonol and proanthocyanidin contents compared to the control group (Fig. 6), suggesting that RcMYB21 can enhance overall flavonoid metabolic flux by activating upstream pathway genes such as CHS, CHI, and F3H.

In addition, MYB transcription factors are essential not only for plant growth and development but also for mediating responses to biotic and abiotic stresses. In cotton, the R2R3-MYB transcription factor GhODO1 interacts with the promoters of Gh4CL1 and GhCAD3, thereby activating lignin biosynthesis and enhancing resistance against Verticillium dahliae [49]. Medicago sativa MsMYB12 can bind to the MBS motif in the MsFLS13 promoter to promote its expression. Overexpression of MsFLS13 enhances flavonoid accumulation and inhibits the excessive accumulation of reactive oxygen species (ROS) induced by combined stress, thereby improving resistance to low-temperature and saline-alkali stress [50]. Overexpression of VcMYB4a in blueberry increases the susceptibility of calli to drought and salt stress [51]. Similarly, IbMYB308 from sweet potato (Ipomoea batatas) enhances salt tolerance in transgenic tobacco plants [52]. However, high temperature leads to the direct binding of Chrysanthemum morifolium CmMYB012 to the promoters of CmFNS, CmCHS, CmDFR, CmANS, and CmUFGT, inhibiting their expression and reducing flavonoid and anthocyanin levels, thereby weakening the plant’s ability to cope with high-temperature stress [53].

Under salt stress, the taproot length of RcMYB21-OE Arabidopsis is significantly reduced, whereas the length and number of lateral roots were significantly increased (Fig. 6). This observation suggests that RcMYB21 may play a regulatory role in modulating root system architecture in A. thaliana under saline conditions. It has been reported that flavonols can act as endogenous polar auxin transport inhibitors to regulate plant stem and root growth [30, 54]. The A. thaliana myb12 mutant partially restores the short-root phenotype of ga1-3, indicating that reduced flavonol biosynthesis can promote plant root growth [55]. In conclusion, RcMYB21 may regulate plant root development by modulating flavonol biosynthesis.

In addition, under low-nitrogen conditions, rice plants overexpressing OsMYB305 exhibit significantly increased tiller number, above-ground dry weight, and total nitrogen concentration; however, under high-nitrogen conditions, their root length is significantly decreased, suggesting that OsMYB305 is a potential molecular target for improving nitrogen use efficiency in rice [56]. Additional research is required to further elucidate the physiological role of RcMYB21.

Conclusions

We identified the RcMYB21 transcription factor in R. chingii. Our study indicated that the RcMYB21 transcription factor specifically regulates flavonoid biosynthesis and accumulation, and enhances salt stress tolerance through flavonol accumulation. These findings provide significant insights into the molecular mechanisms underlying flavonoid regulation in R. chingii and highlight the dual functional role of RcMYB21 in both secondary metabolism and stress response.

Materials and methods

Plant Materials

R. chingii plant was collected from Jing County, Anhui Province, China (N: 30°50′6″, E: 117°49′16″) in December 2017. The R. chingii resources were taxonomically authenticated by Yunsheng Wang from the Botany Teaching and Research Department, Anhui Agricultural University and subsequently cultivated at the Agricultural Industrial Park of Anhui Agricultural University (N: 31°55′51″, E: 117°12′12″). The plant specimens are stored in the Plant Specimen Room of the College of Life Sciences at Anhui Agricultural University (Collection Number: AAUB-202606-R01). The 35 S: RcMYB21/Col-0 and 35 S: RcMYB21/G28 seeds were generated by our laboratory, and all vectors used in the experiment were also constructed by our laboratory. A. thaliana used in this experiment was grown in a climate chamber at 22 ± 1℃, while tobacco (N. tabacum and N. benthamiana) plants were at 25 ± 1℃; all plants were planted under a 16 h light/8 h dark (16 h-L/8 h-D) photoperiod.

Total RNA extraction and RT-qPCR analysis

Total RNA was extracted from R. chingii using the RNA Isolation Kit (Vazyme, China), and from tobacco and Arabidopsis thaliana using TRIzol Reagent (Invitrogen, USA). Reverse transcription was performed using the MonScript™ RTIII-PCR Kit (Monad, China). The cDNA templates were diluted with RNA-free water to a final concentration of 100 ng/µL. Thereaction mixtures were prepared on ice as described in the MonAmp™ ChemoHS qPCR Mix kit (Monad, China). The qPCR protocol followed the two-step method provided with the kit: 95℃, 10 min; 95℃, 10 s; 60℃, 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene for each tissue sample, and three biological replicates were performed for each assay. The primers used in this assay are listed in Table S1.

Bioinformatics analysis

Phylogenetic analysis of the amino acid sequences was conducted using MEGA7 software (v1.0) [57], employing the Neighbor-Joining (NJ) method; parameters were set as previously reported [58]. Amino acid sequence alignment was performed using CLUSTALW (https://www.genome.jp/tools-bin/clustalw) with default parameters. After alignment, data visualization was performed using ESPript (v3.0) [59]. Prediction of cis-acting elements was performed using the PlantCare website [60]. After sorting and filtering the prediction results, cis-acting elements were visualized using TBtools software (v2.114) [61].

Subcellular localization and transcriptional activity assay

For subcellular localization assays, the full-length coding sequence (CDS) of RcMYB21 was inserted into the pCAMBIA1305 vector, which contains a green fluorescent protein (GFP) tag.

Agrobacterium tumefaciens strain GV3101, harboring different constructs, was used to infiltrate N. benthamiana leaves. After staining nuclear with 4’,6-diamidino-2-phenylindole (DAPI), the cells were observed using a confocal microscope (Olympus, Japan).

Transcriptional activity was assessed using a yeast-based assay. RcMYB21 was cloned into the pGBKT7 (BD) vector, which was then transformed into the yeast strain Y2H Gold. The transformed yeast cells were cultured on SD/-Trp medium and SD/-His medium supplemented with 20 mg/mL X-α-Gal at 30 °C for 3 additional days.

Y1H and dual luciferase analysis

The promoters of target genes and the RcMYB21 coding sequence were cloned into the pAbAi and pGADT7 vectors, respectively. Two micrograms (2 µg) of the recombinant pAbAi plasmid was linearized with BstBⅠ restriction endonuclease and integrated into Y1H Gold yeast cells using the PEG/LiAc method. The transformed yeast cells were plated on uracil-deficient SD solid medium (SD-Ura). After incubation for 3 days, single colonies were selected for verification by colony PCR. The PEG/LiAc method was used to transform the corresponding recombinant pGADT7 plasmid and negative control plasmid into the verified yeast strain (Y1H Gold-pAbAi); the transformed cells were then plated on SD solid medium lacking leucine (SD-Leu). After incubation for 3 days, single colonies were resuspended in sterile water, and the optical density at 600 nm (OD600) was adjusted to 0.002. Five microliters (5 µL) of the yeast suspension was pipetted and spotted onto fresh SD-Leu-AbA solid medium, followed by incubation at 30 °C for 2–3 days to monitor yeast growth.

The pGreenII 0800-Luc and pCAMBIA1300-MYC vectors were used for dual-luciferase assay. Three days post-infection, a 150 µg/mL solution of D-fluorescein sodium salt was applied to the surfaces of N. tabacum leaves. Fluorescence signals were then captured using an automated chemiluminescence imaging system after the leaves were left in the dark for five minutes. Two infected leaf discs (10 mm in diameter) were placed into a 2 mL centrifuge tube and ground in liquid nitrogen. Once fully ground, 100 µL of lysis buffer was added, and the mixture was incubated on ice for 5 min. The sample was then centrifuged at 13,000 rpm at 4 °C for 5 min. The supernatant was collected and subjected to fluorescence detection according to the protocol provided with the dual-luciferase reporter assay kit.

Transient expression in vivo by infecting R. chingii

RcMYB21 was inserted into the pCAMBIA1300-MYC vector. One milliliter (1 mL) of an overnight Agrobacterium culture was transferred to 50 mL of liquid LB medium and cultured with shaking at 28 °C. Once the OD600 of the culture reached 0.8, the cells were resuspended in an infection buffer containing 10 mM MgCl2, 200 µM acetosyringone, and 10 mM MES (pH 5.7).

Young R. chingii leaves were excised at the petiole base, and several wounds were evenly made on the leaf surfaces using a scalpel. Each leaf was bisected along the main vein and placed into a Petri dish. The materials were stacked in the following order (from bottom to top): Petri dish bottom, qualitative filter paper, half R. chingii leaf, qualitative filter paper, and Petri dish lid.

Agrobacterium resuspension containing the pCAMBIA1300-RcMYB21-MYC plasmid and the corresponding empty vector control resuspension were added to the two petri dishes, respectively, until the filter paper was fully saturated. After 8 h of incubation in the dark, the upper layer of qualitative filter paper was removed, and the leaves were subsequently incubated under light conditions for approximately 48 h. The transient expression plants were identified by RT-qPCR.

Genetic transformation and quantitative analysis of polyphenols

The recombinant pCAMBIA1300-RcMYB21-MYC vector and empty vector were introduced into Agrobacterium tumefaciens strain GV3101 and subsequently used to transform A. thaliana (Col-0) and N. tabacum (G28). Overexpressing lines were identified using semi-quantitative PCR.

Samples were ground into a fine powder in liquid nitrogen. A 0.1 g aliquot of each sample was mixed with 2 mL of extraction solvent and subjected to ultrasonic extraction at low temperature for 30 min. After centrifugation at 13,000 rpm for 10 min at 4 °C, the supernatant was collected. The residues were re-extracted twice, and the supernatants were combined. Polyphenol composition was qualitatively analyzed using Q-TOF LC/MS as previously described [62]. The DMACA solution was prepared as described earlier, and proanthocyanidin content was determined by measuring the absorbance at OD640 [63]. Total flavonoid content was determined using the aluminum chloride assay as previously reported [64].

Abiotic stress treatment of A. thaliana

A. thaliana seeds were sterilized and sown on MS medium supplemented with the appropriate antibiotics, and cultured for approximately one week. For plate-based stress treatments, two types of MS medium were prepared: one containing 150 mmol/L NaCl (for salt stress) and the other containing 200 mmol/L mannitol (for simulated drought stress). Seedlings at the same growth stage were transferred to the aforementioned MS media for salt stress and simulated drought stress treatments, respectively.

Supplementary Information

Supplementary Material 1. (11.6KB, xlsx)
Supplementary Material 2. (2.1MB, docx)

Acknowledgements

Not applicable.

Authors' contributions

# T.L. and J.H. contributed equally to this study. T.L., and Y.W. designed the experiments; T.L., J.H., W.Q. and Q.Z. performed the experiments; T.L., J.H., and Z.F. analyzed the data; J.H., T.L., and H.R. wrote and revised the manuscript. All authors contributed to the article and approved the final manuscript. The authors declare no conflict of interest.

Funding

This study was supported by the Key Research and Development Program of Anhui Province, China (202004a06020009).

Data availability

All data generated and analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Not applicable. In compliance with China’s regulations on wild plant protection, no special permits were required for the collection of R. chingii resources described in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ting Lei and Jun Huang are contributed equally to this study.

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Associated Data

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Supplementary Materials

Supplementary Material 1. (11.6KB, xlsx)
Supplementary Material 2. (2.1MB, docx)

Data Availability Statement

All data generated and analyzed during this study are included in this published article.

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