MEVALONATE KINASE represses anthocyanin biosynthesis via sucrose transporters and gibberellin synthesis pathways in Arabidopsis

MEVALONATE KINASE represses anthocyanin biosynthesis via sucrose transporters and gibberellin synthesis pathways in Arabidopsis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MEVALONATE KINASE represses anthocyanin biosynthesis via sucrose transporters and gibberellin synthesis pathways in Arabidopsis Jinku Kang, Sua Cho, Kiyoon Kang, Daewon Kim, Sang-Il Bae, Eunji Shin, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8124382/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anthocyanins, a class of flavonoid pigments, function as crucial modulators of plant responses to environmental stressors by mitigating oxidative damage and facilitating cellular adaptation. Anthocyanin biosynthesis is tightly regulated by transcriptional networks that respond to developmental cues and external stimuli. Here, we identify MEVALONATE KINASE (MVK), a key enzyme of the cytosolic isoprenoid biosynthesis pathway, as a repressor of sucrose-induced anthocyanin production in Arabidopsis. Loss-of-function mvk mutants show increased anthocyanin levels compared to wild-type (WT) plants under high sucrose conditions. The expression of anthocyanin biosynthetic and regulatory genes, such as CHS , DFR , and MYB75/PAP1 , is increased in mvk-1 mutants grown in the presence of high sucrose. mvk-1 mutants exhibited elevated sucrose accumulation through upregulation of sucrose transporters compared to WT under high sucrose conditions. Furthermore, reduced levels of gibberellins in mvk-1 mutants resulted in the stabilization of DELLA proteins, which are known repressors of gibberellin signaling, thereby facilitating sucrose-induced anthocyanin accumulation. Our findings demonstrate that MVK negatively regulates sucrose-induced anthocyanin biosynthesis by modulating sucrose transport and gibberellin homeostasis in Arabidopsis. Anthocyanin Arabidopsis thaliana Gibberellic acid Mevalonate pathway MVK SUC1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key Message Arabidopsis MVK negatively regulates sucrose-induced anthocyanin accumulation by modulating SUC1-mediated sucrose transport and gibberellin homeostasis. Introduction Anthocyanins, a pivotal class of water-soluble flavonoids, are responsible for pigmentation of vegetative (leaves, stems, and roots) and reproductive (flowers and fruits) organs in plants. These pigments are not only aesthetic, but also serve crucial ecological functions, such as attracting pollinators and seed dispersers, which are essential for reproduction and survival (Harborne and Williams 2000 ). Beyond their ecological roles, anthocyanins are widely recognized for the role in abiotic stress responses, particularly through antioxidant properties that mitigate oxidative damage caused by reactive oxygen species (ROS) (Grotewold 2006 ; Xu et al. 2017 ). Additionally, anthocyanins offer nutritional benefits to humans, making anthocyanin-rich plants increasingly important research targets (Khoo et al. 2017 ). The biosynthesis of anthocyanins is regulated by a combination of developmental cues and environmental signals, including light, temperature, and several endogenous molecules (LaFountain and Yuan 2021 ). Among them, sucrose is known to promote anthocyanin biosynthesis (Solfanelli et al. 2006 ). In addition, phytohormones, including ethylene, jasmonic acid (JA), gibberellic acid (GA), abscisic acid (ABA), and cytokinin (CK), interact with sucrose signaling pathways, collectively modulating anthocyanin production (Das et al. 2012 ; Loreti et al. 2008 ). Anthocyanins are synthesized in the cytosol, then modified into various derivatives and subsequently transported into vacuoles. In Arabidopsis ( Arabidopsis thaliana ), structural genes involved in anthocyanin biosynthesis are classified into early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) (Deroles 2009). Briefly, the pathway starts with condensation of one molecule of 4-coumaroyl-coenzyme A and three molecules of malonyl-CoA leading to the formation of naringenin chalcone. EBGs such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol 3-hydroxylase (F3H), and flavonol 3-hydroxylase (F3′H) further metabolize naringenin chalcone leading to the production of flavonols. Subsequently, LBGs, including dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin reductase (ANR), and UDP-Glc:flavonoid 3-O-glucosyltransferase (UF3GT) facilitate the final steps that produce anthocyanins and proanthocyanidins (Holton and Cornish 1995 ). Anthocyanin biosynthesis is tightly regulated at the transcriptional level by the MBW (MYB-bHLH-WD40) complex, which consists of R2R3-MYB, bHLH, and WD40-repeat proteins (Hichri et al. 2011 ). In Arabidopsis, the MBW complex includes R2R3-MYBs (MYB75/PAP1, MYB90/PAP2, MYB113, and MYB114), bHLH transcription factors [TRANSPARENT TESTA 8 (TT8), GLABRA 3 (GL3), and ENHANCER OF GLABRA 3 (EGL3)], and the WD40-repeat protein TRANSPARENT TESTA GLABRA 1 (TTG1) (Broun 2005 ; Gonzalez et al. 2008 ). This complex orchestrates anthocyanin biosynthesis by activating the expression of structural genes such as Phenylalanine ammonia-lyase ( PAL ), CHS , CHI , F3H , DFR , ANS , and UF3GT , ultimately promoting anthocyanin accumulation in vegetative and reproductive tissues (Das et al. 2012 ). Recent studies in Arabidopsis showed that GLK1 promotes anthocyanin accumulation by directly interacting with MYB75, MYB90, and MYB113, thereby boosting their transcriptional activity (Li et al. 2023 ). In addition, energy deficiency suppresses anthocyanin accumulation through the action of SnRK1, a master metabolic regulator. SnRK1 destabilizes MYB75, thus repressing MBW-mediated transcription and anthocyanin production under low energy conditions (Broucke et al. 2023 ). Homologs such as FvMYB10, FvMYB41, and FvMYB105 interact with bHLH partners like FvbHLH33 and FvMYC1 to regulate the synthesis of stage-specific anthocyanin and proanthocyanidin during fruit ripening in woodland strawberry (Xu et al. 2021 ). Importantly, the MBW activity is subject to modulation by internal and external signals, such as hormones and sugar. DELLA proteins, mostly acting as repressors of gibberellin (GA) signaling, promote anthocyanin biosynthesis by sequestering JAZ and MYBL2 repressors, thereby enabling MBW activation (Xie et al. 2016 ). In addition, sucrose enhances anthocyanin biosynthesis by stabilizing DELLA proteins and inducing expression of MBW-regulated genes such as MYB75/PAP1 , CHS , and DFR (Li et al. 2014 ). Given its dual role as both a carbon source and a signaling molecule, sucrose not only provides metabolic substrates but also functions as a key regulator that integrates hormonal and transcriptional networks to promote anthocyanin accumulation (Loreti et al. 2008 ; Sakr et al. 2018 ). Sucrose content in plants may increase due to alterations in sucrose metabolism or the activity of sucrose transporters (Julius et al. 2017 ). Sucrose transporters (SUCs) are sucrose-proton symporters involved in sucrose translocation (Braun 2022 ). Nine putative SUC genes have been identified in Arabidopsis, and some are directly linked to anthocyanin accumulation. For example, SUC1 plays a critical role in sucrose-induced anthocyanin accumulation, and sucrose-induced anthocyanin accumulation is inhibited in SUC1 knockout mutants (Sivitz et al. 2008 ). In addition, the Arabidopsis pho3 mutants, which is defective in SUC2 function, exhibits enhanced anthocyanin accumulation (Lloyd and Zakhleniuk 2004 ). Among SUC genes, SUC5 is expressed specifically in the endosperm, SUC6 and SUC7 encode aberrant proteins, and SUC8 and SUC9 are predominantly expressed in floral tissues (Baud et al. 2005 ; Meyer et al. 2000 ; Sauer et al. 2004 ). The mevalonate (MVA) pathway, functioning in the cytosol of plant cells, plays a crucial role in primary metabolism by producing isoprenoids, sterols, and other key metabolites (Pulido et al. 2012 ). It is conserved across plants, fungi, and animals and supports diverse physiological processes (Miziorko 2011 ; Ruiz-Sola et al. 2016 ; Yang et al. 2021 ). Within this pathway, mevalonate kinase (MVK) is a key enzyme that phosphorylates mevalonic acid to produce mevalonate 5-phosphate (Riou et al., 1994 ). A recent study showed that Arabidopsis MVK is a direct phosphorylation target of P2K1, leading to activation of the MVA pathway in response to extracellular ATP (eATP) elicitation (Cho et al. 2022 ). The relationship between the MVA pathway and anthocyanin production has been studied in apple trees ( Malus domestica Borkh). This pathway produces isoprenoids and sterols and was shown to influence anthocyanin accumulation by positively regulating IAA and ABA synthesis while inhibiting GA synthesis (Flores-Perez et al. 2010; Li et al. 2018 ). However, the mechanism of MVA-mediated anthocyanin regulation in plants remains to be elucidated. In this study, we aimed to elucidate the role of MVK, a core enzyme of the cytosolic isoprenoid biosynthesis pathway, in the regulation of sucrose-induced anthocyanin biosynthesis in Arabidopsis. Although the MVA pathway was previously associated with various metabolic and hormonal signaling events, its connection to anthocyanin production in response to sucrose remains poorly understood. Our findings reveal that mvk-1 mutants accumulate more anthocyanins than WT under high sucrose conditions. This phenotype is accompanied by elevated gene expression of anthocyanin biosynthesis such as CHS and DFR , and transcriptional regulators such as MYB75/PAP1 . Furthermore, we demonstrate that MVK negatively regulates anthocyanin accumulation through two distinct mechanisms. First, MVK inhibits the expression of SUC1 . Genetic analysis of mvk-1 suc1-5 double mutants further revealed that this regulation is mediated by a SUC1-dependent regulatory pathway. Second, mutation of MVK reduces gibberellin levels, thereby promoting stabilization of DELLA protein. Collectively, our results uncover a previously uncharacterized function of MVK as a negative regulator of sucrose-induced anthocyanin biosynthesis, integrating sugar transport and hormonal signaling into a coordinated regulatory network. Materials and methods Plant material and growth conditions Wild-type aequorin-expressing transgenic Arabidopsis ColQ (Col-0 background) plants were provided by Marc Knight (Knight et al. 1996 ). The mvk-1 mutant (ColQ background) has been described previously (Cho et al. 2022 ). Arabidopsis seeds were surface-sterilized and sown on half-strength Murashige and Skoog (MS) medium supplemented with 0% (w/v) sucrose, 0.5% (w/v) agar (MB Gellan Gum, Cat No. MB-G4367), and 0.05% (w/v) MES (pH 5.7). Following a 3-day cold stratification at 4°C, the plates were positioned vertically in a growth chamber set to a 16 h light/8 h dark photoperiod at 22°C, and 100 µmole m⁻² s⁻¹ light intensity. Generating CRISPR-Cas9 Generating CRISPR-Cas9 The suc1-5 mutant was generated via CRISPR/Cas9-mediated genome editing, with a single guide RNA (sgRNA, 5`-CTCGATCCCTGGGACATTCCTGG-3`) targeting the SUC1 coding region being designed using the CRISPR direct program ( http://crispr.dbcls.jp/ ) (Naito et al. 2015 ). The tRNA–gRNA–Cas9 fragment was inserted into the pRGEB32 vector (Xie et al. 2015 ). This binary vector was transformed into the Agrobacterium tumefaciens strain GV3101, which was then used to transform Arabidopsis plants via the floral dip method (Clough and Bent 1998 ). The homozygous lines were screened based on hygromycin resistance. Confirmation of this selection was achieved by directly sequencing PCR-amplified genomic products, which were amplified with the use of primers targeting the specific region listed in Supplementary Table 1. Anthocyanin assay Anthocyanins were extracted using a modified version of a previously described method (Nakata et al. 2013 ). Three-day-old Arabidopsis seedlings were transferred to half-strength MS medium supplemented with 1% or 3% (w/v) sucrose and grown for another three days. Samples were then extracted in 45% methanol and 5% acetic acid (v/v). The relative anthocyanin content was determined spectrophotometrically by measuring the absorbance at 520 nm and 657 nm, and the relative values were calculated accordingly. RNA extraction and RT-qPCR analyses Total RNA was extracted from Arabidopsis plants using GeneAll Hybrid-R (GeneAll Biotechnology, Republic of Korea) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 2 µg total RNA using M-MLV reverse transcriptase (Promega, Madison, USA). For RT-qPCR, GoTaq PCR Mix (Promega, Madison, USA) was used according to the manufacturer's instructions. qPCR was performed using a LightCycler 2.0 system (Roche Diagnostics, Mannheim, Germany). Transcript levels were normalized to the expression of the UBQ gene. The primers used for RT-qPCR analysis are provided in Supplementary Table 1. Sucrose measurements For sucrose extraction, 20 mg (fresh weight) of Arabidopsis rosette leaves was ground in liquid nitrogen and extracted using 80% (v/v) ethanol. The extracted samples were centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was filtered before analysis. Soluble sucrose content was quantified by high-performance liquid chromatography (HPLC) on a Dionex Ultimate 3000 system (Thermo Fisher, Sunnyvale, USA) equipped with a Shodex RI-101 refractive index detector (Shoko, Tokyo, Japan) at the Seoul National University NICEM. Separation was performed on a Sugar-Pak column (Waters, 300 mm × 6.5 mm) at 70°C. The mobile phase consisted of ultrapure water (Milli-Q grade) at a flow rate of 0.5 mL/min. The injection volume was set at 10 µL for each sample. Chromeleon ver. 6 software was used for data acquisition and processing. Calibration was carried out with sucrose standard (Sigma, 99.5% purity). Quantification was performed by comparing sample peak areas to those of the standard curve generated from known sucrose concentrations. DELLA protein stability For the DELLA protein stability analysis, five-day-old seedlings were grown in 6-well plates with 1 mL liquid half-strength MS medium supplemented with 0% (w/v) sucrose (pH 5.7) under long-day conditions (16 h light/8 h dark, 21°C). After 3 days in LD conditions, 5% (w/v) Suc was added for an additional 3 days. Then, these seedlings were treated with 10 µM GA for 2 h, and total protein was extracted using extraction buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 0.5% Triton-X 100, 10% glycerol, 1 mM DTT, 0.2 mM PMSF, and 1× Pierce protease inhibitor (Thermo Fisher, Rockford, USA). The extracted proteins were mixed with 5× Laemmli loading buffer containing 10% SDS, 50% glycerol, 0.01% bromophenol blue, 10% beta-mercaptoethanol, and 0.3 M Tris-HCl (pH 6.8), and boiled at 95°C for 5 min. Total extracted proteins were separated by 10% SDS-PAGE gel electrophoresis, and proteins were transferred to a PVDF membrane (Immobilon®-P, Millipore) with a semi-dry transfer system (Trans-Blot® SD, Bio-Rad, Hercules, USA). After blocking with 5% skim milk, the membrane was incubated with RGA/DELLA antibody (Agrisera, Cat No. AS11-1630, dilution 1:1000) in 5% skim milk for 2 h. Subsequenctly, the membrane was washed 3 times and incubated with secondary goat anti-rabbit-HRP (Santa Cruz, Cat No. sc-2004, dilution 1:10000) for 2 h. Subsequently, the membrane was washed 5 times in TBST (50 mM Tris, 150 mM NaCl, 0,05% Tween 20), incubated with Pierce SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific, Cat No. 34578) for 1 min and exposed to film. Gibberellins quantification GA quantification was analyzed by the previous method (Xin et al. 2020 ). Briefly, 1 g of 10-day-old seedlings were ground to a find powder in liquid nitrogen, and the samples were freeze-dried for 3 days. 1 mL of solution containing 80% (v/v) methanol was added to each sample and incubated for 12 h at 4°C. The samples were centrifuged at 12,000 x g for 15 min at 4°C. The solvent was then dried down using a Speed Vac concentrator at room temperature (25°C). The dried pellets were resuspended in 100 µL of solution containing 80% (v/v) methanol and resuspended samples were immediately subjected to liquid chromatography-mass spectrometry (LC-MS) hormonal analysis (Seoul National University NICEM, Republic of Korea). Results mvk-1 mutants exhibit enhanced sucrose-induced anthocyanin accumulation The MVA pathway plays a crucial role in the biosynthesis of a wide range of isoprenoids, including phytohormones (Pulido et al. 2012 ). It was reported that CK, GA, and ABA regulate the induction of anthocyanin biosynthesis by sugar in Arabidopsis (Loreti et al. 2008 ). This led us to examine whether mutation of MVK affects anthocyanin levels. We first investigated anthocyanin accumulation in mvk-1 mutants. To determine the role of MVK in anthocyanin accumulation, 10-day-old WT and mvk-1 plants were grown on half-strength Murashige and Skoog (MS) medium in the absence or presence of 3% sucrose for 3 days. The leaves and shoot apical meristem region of mvk-1 mutants showed intense purple coloration after being grown on 3% sucrose (Fig. 1 a). Furthermore, a pronounced anthocyanin accumulation phenotype was observed across the abaxial surface of 10-day-old mvk-1 mutants grown on 5% sucrose medium. (Supplementary Fig. S1). There was no difference in anthocyanin content under mock conditions, whereas mvk-1 mutants exhibited an almost 3-fold increase in anthocyanin content compared to WT under 3% sucrose conditions (Fig. 1 b). Next, to identify whether these phenotypes were due to upregulation of anthocyanin biosynthetic genes at the transcriptional level, we examined the relative expression of genes involved in the anthocyanin biosynthetic pathway ( CHS, CHI, F3H, F3’H, DFR, ANS / LDOX and UF3GT ). Under 3% sucrose conditions, mvk-1 mutants exhibited higher expression levels of anthocyanin biosynthetic genes compared with WT (Fig. 1 c-i). We also measured the relative expression level of MYB75 , which is a transcription factor composing the MBW complex, involved in the transcriptional activation of anthocyanin biosynthetic genes (Teng et al., 2005 ). Interestingly, the expression level of MYB75 was also upregulated in mvk-1 mutants compared to WT under 3% sucrose conditions (Fig. 1 j). Taken together, these findings indicate that a loss-of-function of MVK results in enhanced anthocyanin accumulation, likely through transcriptional activation of the MBW complex and the subsequent upregulation of key genes in the anthocyanin biosynthetic pathway. High sucrose accumulation in the leaves of mvk-1 mutants Sucrose is widely recognized as a signaling molecule that induces anthocyanin biosynthesis (Yoon et al. 2021 ). As shown in Fig. 1 , mvk-1 mutants exhibited increased anthocyanin accumulation under high sucrose conditions. We hypothesized that this phenotype is associated with elevated sucrose levels. To confirm this hypothesis, we collected leaves from WT and mvk-1 mutants grown under the same conditions and subjected them to the treatments with or without 5% sucrose. Sucrose content was measured using HPLC. Under mock conditions, no difference was observed in sucrose levels (i.e., peak at ~ 7.2 mins) between WT and mvk-1 mutants. However, mvk-1 mutants showed higher internal sucrose levels than WT in the presence of 5% sucrose (Fig. 2 a, b). The standard sucrose was detected at ~ 7.2 mins (Fig. 2 b). These results suggest that Arabidopsis MVK influences sucrose accumulation, potentially contributing to an enhanced anthocyanin phenotype observed in mvk-1 mutants. Expression patterns of SUCs in mvk-1 mutants Sucrose transporters are known to play a central role in regulating sucrose levels in plants (Julius et al. 2017 ). Since mvk-1 mutants exhibited increased sucrose accumulation under high exogenous sucrose treatments (Fig. 2 c), we investigated whether this phenotype was associated with altered expression of sucrose transporter genes. In order to measure expression of SUC genes, we compared their transcript levels between WT and mvk-1 seedlings under high-sucrose conditions. The expression of SUC1 was increased in mvk-1 mutants under high-sucrose conditions (Fig. 3 a), whereas that of SUC2 , SUC3 , and SUC4 showed no significant change regardless of sucrose concentrations (Fig. 3 b-d). In addition to SUC genes, we also examined the SWEET ( Sugars Will Eventually be Exported Transporters ) genes encoding sugar transporters, which function as unidirectional uniporters mediating sucrose efflux across the plasma membrane and tonoplast (Ji et al. 2022 ). Among the 17 Arabidopsis SWEET genes, SWEET11 , SWEET12 , SWEET13 , and SWEET14 are known to participate in sucrose transport. However, no significant differences in the expression of these genes were detected between WT and mvk-1 mutants under high-sucrose conditions (Fig. 3 e-h). Taken together, these results indicated that the mutation of MVK specifically alters the expression of SUC1 , while other sucrose transporters ( SUC2 , SUC3 , SUC4 , and SWEET11-14) remain unaffected. MVK genetically affects SUC1-mediated anthocyanin accumulation Arabidopsis SUC1 is plasma membrane-localized sucrose/H⁺ symporters with distinct expression patterns, where SUC1 mediates local sucrose uptake in roots, trichomes, and pollen (Sauer and Stolz 1994 ; Sivitz et al. 2008 ; Stadler and Sauer 2019 ). Since exogenous sucrose strongly induces anthocyanin biosynthesis and SUC1 primarily functions in sucrose uptake in roots, we hypothesized that SUC1 expression is a downstream target of MVK. To investigate whether MVK regulates anthocyanin accumulation through SUC1 , we generated the CRISPR/Cas9-mediated suc1-5 mutants in WT background, harboring a single base insertion that resulted in a premature stop codon in the SUC1 coding region (Supplementary Fig. S2). Additionally, the mvk-1 suc1-5 double mutants were created by introducing the SUC1 CRISPR/Cas9 construct into the mvk-1 background to examine their genetic interaction. Sequencing analysis confirmed a single base insertion in SUC1 , which is identical to the suc1-5 mutant allele (Supplementary Fig. S2b). Consistent with previous studies (Sivitz et al. 2008 ), suc1-5 mutants exhibited less anthocyanin accumulation under 3% sucrose treatment compared to WT (Fig. 4 a, b). Interestingly, mvk-1 suc1-5 double mutants showed intermediate anthocyanin levels, higher than suc1-5 but lower than mvk-1 mutants (Fig. 4 a, b). At the transcriptional level, anthocyanin biosynthetic genes were markedly downregulated in suc1-5 mutants, but significantly upregulated in mvk-1 suc1-5 double mutants compared with WT under sucrose treatment (Fig. 4 c-i). Similarly, MYB75 expression level was reduced in suc1-5 mutants, whereas it was induced in mvk-1 suc1-5 double mutants (Fig. 4 j). Together, these results suggested that MVK regulates anthocyanin accumulation partially by downregulating SUC1 expression, while also acting through additional SUC1-independent pathways. MVK regulates DELLA stability via GA biosynthesis Interestingly, mvk - 1 suc1-5 double mutants showed significantly higher anthocyanin accumulation compared to suc1-5 mutants under sucrose treatment (Fig. 4 a, b). Since the MVA and MEP pathway synthesizes isoprenoid precursors essential for phytohormones, such as brassinosteroids, CK, GA, and ABA. (Pulido et al. 2012 ), and GA suppresses sucrose-induced anthocyanin accumulation (Loreti et al. 2008 ), we hypothesized that MVK may regulate anthocyanin accumulation not only via a SUC1-mediated pathway but also through GA biosynthesis. To confirm this, we compared the anthocyanin accumulation of WT and mvk-1 mutants under 5% sucrose with or without 50 µM GA treatments. As shown in Fig. 5 a, the accumulation of anthocyanins in mvk-1 mutants under 5% sucrose treatment was attenuated by GA application. Since GA represses sucrose signaling by promoting degradation of DELLA proteins (Li et al. 2014 ), we measured the level of DELLA proteins in WT and mvk-1 mutants under sucrose treatment with or without GA treatments. Remarkably, mvk-1 mutants showed higher levels of DELLA proteins compared to WT, regardless of GA treatment under each sucrose conditions (Fig. 5 b). Since the mutants exhibited elevated levels of DELLA protein, we investigated whether this was associated with altered GA content. Analysis by LC-MS revealed that GA 1 levels in the mvk-1 mutants were significantly lower than those in WT (Fig. 5 c). Previous studies demonstrated that GA biosynthetic genes Gibberellin 3-oxidase 1 ( GA3ox1 ) and Gibberellin 20-oxidase 1 ( GA20ox1 ) exhibit increased expression under GA-deficient conditions, consistent with feedback regulation mechanisms (Fukazawa et al. 2014 ). Similarly, our results revealed significant upregulation of GA3ox1 and GA20ox1 expression in mvk-1 mutants, whereas their expression was downregulation in suc1-5 mutants compared to WT (Supplementary Fig. S4). Notably, mvk-1 suc1-5 mutants showed moderate expression levels, significantly higher than suc1-5 mutants but lower than mvk-1 mutants. Taken together, our results suggest that reduced GA biosynthesis in mvk-1 mutants impedes the degradation of DELLA proteins, resulting in higher anthocyanin accumulation compared to WT. Discussion Loss of MVK enhances sucrose-specific induction of anthocyanin biosynthetic pathway Anthocyanin accumulation is closely linked to the availability of sucrose, since sucrose transporters such as SUCs and SWEETs import extracellular sucrose, thereby inducing the anthocyanin biosynthesis. It was previously reported that several kinases regulate sucrose transporters. For example, Sucrose-Induced Receptor Kinase 1 (SIRK1) phosphorylates and thereby activates several membrane proteins including SWEET11 under sucrose-specific osmotic response (Wu et al. 2013 ). Furthermore, Wall-Associated Kinase Like 8 (WAKL8) phosphorylates SUC2, thereby increasing its transport activity (Xu et al. 2020 ). However, although kinase-mediated regulation of sucrose transporters has been demonstrated, no studies have yet reported a mechanism by which such kinase-dependent modulation of sucrose transporters directly influences anthocyanin biosynthesis. In this study, we showed that the knockout mutation of MVK enhances sucrose-specific anthocyanin accumulation (Fig. 1 ). The mvk-1 mutants showed increased expression of anthocyanin biosynthetic genes, along with higher transcript levels of MYB75 , a key transcriptional regulator of anthocyanin biosynthetic genes (Fig. 1 j). Measurement of sucrose contents in leaves revealed that mvk-1 mutants accumulate higher levels of sucrose under high-sucrose conditions compared to WT (Fig. 2 ). Consistently, mvk-1 mutant plants showed significantly increased expression of SUC1 , indicating that MVK negatively regulates SUC1 expression. Furthermore, mvk-1 suc1-5 double mutants showed significantly higher anthocyanin accumulation than the suc1-5 mutants, indicating that the enhanced anthocyanin phenotype of mvk-1 mutants is at least partially dependent on SUC1 . DELLA proteins levels remained higher in mvk-1 mutants than WT under sucrose treatment. LC-MS analysis also indicated that mvk-1 mutants had lower GA 1 levels compared to WT. Taken together, these results strongly suggest that MVK regulates anthocyanin accumulation in plants by modulating SUC1 expression. In addition, MVK controls GA levels, and the absence of MVK activity leads to reduced GA 1 content and increased DELLA protein stability, which further promotes the expression of anthocyanin biosynthetic genes. These regulatory mechanisms contribute to enhanced anthocyanin accumulation observed in mvk-1 mutants (Fig. 6 ). The MVA pathway regulates anthocyanin accumulation via GA biosynthesis Anthocyanin accumulation is tightly controlled by the interplay between sucrose and phytohormones. Auxin and cytokinin promote anthocyanin accumulation through transcriptional activation and antioxidant regulation, whereas ethylene exerts context-dependent effects (Bhaskar et al. 2021 ; Chandler 2016 ; Ni et al. 2021 ). Abscisic acid strongly induces anthocyanin biosynthesis under stress conditions, while GA consistently acts as a negative regulator (Karppinen et al. 2018 ; Loreti et al. 2008 ). Moreover, sucrose signaling was shown to interact with several hormones, including IAA, ABA, MeJA, and SA, but is antagonized by GA (Li et al. 2014 ). Our results expand this framework by demonstrating that MVK, a key enzyme in the MVA pathway, influences anthocyanin accumulation through GA biosynthesis. The mvk-1 suc1-5 double mutants accumulated more anthocyanin than suc1-5 mutants (Fig. 4 a, b). Moreover, the double mutants also exhibited reduced root length (Supplementary Fig. S3). These observations suggest the existence of another regulatory pathway compensating for the loss of SUC1-mediated sucrose signaling. Notably, the dwarfism observed in both mvk-1 and mvk-1 suc1-5 mutants (Supplementary Figs. S1, S3) aligns with established roles of the MVA pathway in isoprenoid biosynthesis, which supplies precursors for GA synthesis (Cho et al. 2022 ). In mvk - 1 mutants, reduced GA 1 levels and elevated DELLA protein accumulation (Fig. 5 c) alleviate GA-mediated repression of sucrose signaling, thereby enabling enhanced anthocyanin accumulation in mvk-1 suc1-5 mutants even in the absence of SUC1 (Fig. 4 ). Furthermore, exogenous GA treatment rescued the hyperaccumulation of anthocyanin phenotype in mvk-1 mutants (Fig. 5 a). Disruption of MVK reduces the amount of GGPP-derived GA precursors, thereby leading to the stabilization of DELLA proteins. These findings highlight how GA hormonal signals linked to the MVA pathway are intertwined in fine-tuning plant secondary metabolism, such as anthocyanin biosynthesis. Our study uncovers a previously unrecognized role of MVK in repressing the expression of SUC1 (Fig. 3 a). This transcriptional repression connects the MVA-GA signaling module to sucrose transport, suggesting that MVK serves as an integrative regulator bridging hormonal and metabolic cues. Our findings therefore propose a broader role for the MVK-SUC1 regulatory axis in anthocyanin biosynthesis. Since anthocyanin accumulation is tightly controlled by sucrose availability and stress-induced signaling pathways, the MVK-SUC1 connection may represent a critical node that coordinates primary metabolism, signaling networks, and secondary metabolism. This integration could enable plants to balance growth and stress adaptation by modulating anthocyanin levels. Declarations Author contributions Jinku Kang, Sua Cho, Eunji Shin, and Sung-Hwan Cho performed most of the experiments. Kiyoon Kang analyzed the data; Sang-Il Bae performed GA measurement. Jinku Kang, Sua Cho, Daewon Kim, So-Yon Park, Gary Stacey, Nam-Chon Paek, and Sung-Hwan Cho wrote the manuscript. Kiyoon Kang, Daewon Kim, So-Yon Park, Gary Stacey, Nam-Chon Paek, and Sung-Hwan Cho acquired funding. Nam-Chon Paek, and Sung-Hwan Cho designed and supervised the projects and wrote the paper. Funding We thank Dr Katalin Toth (INARI company) for valuable advice and helpful comments. This study was supported by the Brain Pool Program funded by the Ministry of Science and Information and Communication Technology through the National Research Foundation of Korea (Grant Nos. 2022H1D3A2A01096185 and RS-2024-00410063 to N.C.P. and S.H.C.), the Basic Science Research Program through the National Research Foundation of Korea (Grant No. RS-2023-00247376 to S.H.C.), the Chungbuk National University National University Development Project (NUDP) program (2025, to S.H.C.), the National Research Foundation of Korea(NRF) (Grant No. RS-2025-02216304 to N.C.P.), the National Research Foundation of Korea (RS-2024-00452677 to K.K.), the Learning & Academic research institution for Master’s, PhD students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2023-00301974 to D.K.), the US Department of Agriculture’s National Institute of Food and Agriculture (Grant No. USDA-AFRI-2023-67013-39896 to S.Y.P.), and the National Science Foundation (Grant No. IOS-PGRP-2348319 to S.Y.P.), the US National Science Foundation Plant Genome Program (Grant No. IOS-2048410 to G.S.), and the US National Institute of General Medical Sciences of the National Institutes of Health (Grant No. R01GM121445 to G.S.). Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations We declare we have no competing interests. References Baud S, Wuillème S, Lemoine R et al (2005) The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis. Plant J 43:824–836. https://doi.org/10.1111/j.1365-313X.2005.02496.x Bhaskar A, Paul LK, Sharma E (2021) OsRR6, a type-A response regulator in rice, mediates cytokinin, light and stress responses when over-expressed in Arabidopsis. Plant Physiol Biochem 161:98–112. https://doi.org/10.1016/j.plaphy.2021.01.047 Braun DM (2022) Phloem Loading and Unloading of Sucrose: What a Long, Strange Trip from Source to Sink. Annu Rev Plant Biol 73:553–584. https://doi.org/10.1146/annurev-arplant-070721-083240 Broucke E, Dang TTV, Li Y et al (2023) SnRK1 inhibits anthocyanin biosynthesis through both transcriptional regulation and direct phosphorylation and dissociation of the MYB / bHLH / TTG1 MBW complex. Plant J 115:1193–1213. https://doi.org/10.1111/tpj.16312 Broun P (2005) Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr Opin Plant Biol 8:272–279. https://doi.org/10.1016/j.pbi.2005.03.006 Chandler JW (2016) Auxin response factors. Plant Cell Environ 39:1014–1028. https://doi.org/10.1111/pce.12662 Cho S-H, Tóth K, Kim D et al (2022) Activation of the plant mevalonate pathway by extracellular ATP. Nat Commun 13:450. https://doi.org/10.1038/s41467-022-28150-w Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant J 16:735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x Das PK, Shin DH, Choi S-B, Park Y-I (2012) Sugar-Hormone Cross-Talk in Anthocyanin Biosynthesis. Mol Cells 34:501–508. https://doi.org/10.1007/s10059-012-0151-x Deroles S (2008) Anthocyanin Biosynthesis in Plant Cell Cultures: A Potential Source of Natural Colourants. In: Winefield C, Davies K, Gould K (eds) Anthocyanins. Springer New York, New York, pp 108–167 Flores-Pérez Ú, Pérez-Gil J, Closa M et al (2010) PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) Integrates the Regulation of Sugar Responses with Isoprenoid Metabolism in Arabidopsis. Mol Plant 3:101–112. https://doi.org/10.1093/mp/ssp100 Fukazawa J, Teramura H, Murakoshi S et al (2014) DELLAs Function as Coactivators of GAI-ASSOCIATED FACTOR1 in Regulation of Gibberellin Homeostasis and Signaling in Arabidopsis . Plant Cell 26:2920–2938. https://doi.org/10.1105/tpc.114.125690 Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53:814–827. https://doi.org/10.1111/j.1365-313X.2007.03373.x Grotewold E, THE GENETICS AND BIOCHEMISTRY OF FLORAL PIGMENTS (2006) Annu Rev Plant Biol 57:761–780. https://doi.org/10.1146/annurev.arplant.57.032905.105248 Harborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504. https://doi.org/10.1016/S0031-9422(00)00235-1 Hichri I, Barrieu F, Bogs J et al (2011) Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot 62:2465–2483. https://doi.org/10.1093/jxb/erq442 Holton TA, Cornish EC (1995) Genetics and Biochemistry of Anthocyanin Biosynthesis. Plant Cell 1071–1083. https://doi.org/10.1105/tpc.7.7.1071 Ji J, Yang L, Fang Z et al (2022) Plant SWEET Family of Sugar Transporters: Structure, Evolution and Biological Functions. Biomolecules 12:205. https://doi.org/10.3390/biom12020205 Julius BT, Leach KA, Tran TM et al (2017) Sugar Transporters in Plants: New Insights and Discoveries. Plant Cell Physiol 58:1442–1460. https://doi.org/10.1093/pcp/pcx090 Karppinen K, Tegelberg P, Häggman H, Jaakola L (2018) Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits. Front Plant Sci 9:1259. https://doi.org/10.3389/fpls.2018.01259 Khoo HE, Azlan A, Tang ST, Lim SM (2017) Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food Nutr Res 61:1361779. https://doi.org/10.1080/16546628.2017.1361779 Knight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8:489–503. https://doi.org/10.1105/tpc.8.3.489 LaFountain AM, Yuan Y (2021) Repressors of anthocyanin biosynthesis. New Phytol 231:933–949. https://doi.org/10.1111/nph.17397 Li W-F, Mao J, Yang S-J et al (2018) Anthocyanin accumulation correlates with hormones in the fruit skin of ‘Red Delicious’ and its four generation bud sport mutants. BMC Plant Biol 18:363. https://doi.org/10.1186/s12870-018-1595-8 Li Y, Lei W, Zhou Z et al (2023) Transcription factor GLK1 promotes anthocyanin biosynthesis via an MBW complex-dependent pathway in Arabidopsis thaliana . JIPB 65:1521–1535. https://doi.org/10.1111/jipb.13471 Li Y, Van Den Ende W, Rolland F (2014) Sucrose Induction of Anthocyanin Biosynthesis Is Mediated by DELLA. Mol Plant 7:570–572. https://doi.org/10.1093/mp/sst161 Lloyd JC, Zakhleniuk OV (2004) Responses of primary and secondary metabolism to sugar accumulation revealed by microarray expression analysis of the Arabidopsis mutant, pho3. J Exp Bot 55:1221–1230. https://doi.org/10.1093/jxb/erh143 Loreti E, Povero G, Novi G et al (2008) Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis . New Phytol 179:1004–1016. https://doi.org/10.1111/j.1469-8137.2008.02511.x Meyer S, Melzer M, Truernit E et al (2000) AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer. Plant J 24:869–882. https://doi.org/10.1046/j.1365-313x.2000.00934.x Miziorko HM (2011) Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys 505:131–143. https://doi.org/10.1016/j.abb.2010.09.028 Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123. https://doi.org/10.1093/bioinformatics/btu743 Nakata M, Mitsuda N, Herde M A bHLH-Type Transcription Factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED et al (2013) MYC2-LIKE1, Acts as a Repressor to Negatively Regulate Jasmonate Signaling in Arabidopsis . Plant Cell 25:1641–1656. https://doi.org/10.1105/tpc.113.111112 Ni J, Premathilake AT, Gao Y et al (2021) Ethylene-activated PpERF105 induces the expression of the repressor‐type R2R3‐MYB gene PpMYB140 to inhibit anthocyanin biosynthesis in red pear fruit. Plant J 105:167–181. https://doi.org/10.1111/tpj.15049 Pulido P, Perello C, Rodriguez-Concepcion M (2012) New Insights into Plant Isoprenoid Metabolism. Mol Plant 5:964–967. https://doi.org/10.1093/mp/sss088 Riou C, Tourte Y, Lacroute F, Karst F (1994) Isolation and characterization of a cDNA encoding Arabidopsis thaliana mevalonate kinase by genetic complementation in yeast. Gene 148:293–297. https://doi.org/10.1016/0378-1119(94)90701-3 Ruiz-Sola MÁ, Coman D, Beck G et al (2016) Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis‐related isoprenoids. New Phytol 209:252–264. https://doi.org/10.1111/nph.13580 Sakr S, Wang M, Dédaldéchamp F et al (2018) The Sugar-Signaling Hub: Overview of Regulators and Interaction with the Hormonal and Metabolic Network. IJMS 19:2506. https://doi.org/10.3390/ijms19092506 Sauer N, Ludwig A, Knoblauch A et al (2004) AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes. Plant J 40:120–130. https://doi.org/10.1111/j.1365-313X.2004.02196.x Sauer N, Stolz J (1994) SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana ; expression and characterization in baker’s yeast and identification of the histidine-tagged protein. Plant J 6:67–77. https://doi.org/10.1046/j.1365-313X.1994.6010067.x Sivitz AB, Reinders A, Ward JM (2008) Arabidopsis Sucrose Transporter AtSUC1 Is Important for Pollen Germination and Sucrose-Induced Anthocyanin Accumulation. Plant Physiol 147:92–100. https://doi.org/10.1104/pp.108.118992 Solfanelli C, Poggi A, Loreti E et al (2006) Sucrose-Specific Induction of the Anthocyanin Biosynthetic Pathway in Arabidopsis. Plant Physiol 140:637–646. https://doi.org/10.1104/pp.105.072579 Stadler R, Sauer N (2019) The AtSUC2 Promoter: A Powerful Tool to Study Phloem Physiology and Development. In: Liesche J (ed) Phloem. Springer New York, New York, pp 267–287 Teng S, Keurentjes J, Bentsink L et al (2005) Sucrose-Specific Induction of Anthocyanin Biosynthesis in Arabidopsis Requires the MYB75/PAP1 Gene. Plant Physiol 139:1840–1852. https://doi.org/10.1104/pp.105.066688 Wu XN, Sanchez Rodriguez C, Pertl-Obermeyer H et al (2013) Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis. Mol Cell Proteom 12:2856–2873. https://doi.org/10.1074/mcp.M113.029579 Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–3575. https://doi.org/10.1073/pnas.1420294112 Xie Y, Tan H, Ma Z, Huang J (2016) DELLA Proteins Promote Anthocyanin Biosynthesis via Sequestering MYBL2 and JAZ Suppressors of the MYB/bHLH/WD40 Complex in Arabidopsis thaliana. Mol Plant 9:711–721. https://doi.org/10.1016/j.molp.2016.01.014 Xin P, Guo Q, Li B et al (2020) A Tailored High-Efficiency Sample Pretreatment Method for Simultaneous Quantification of 10 Classes of Known Endogenous Phytohormones. Plant Commun 1:100047. https://doi.org/10.1016/j.xplc.2020.100047 Xu P, Wu L, Cao M et al (2021) Identification of MBW Complex Components Implicated in the Biosynthesis of Flavonoids in Woodland Strawberry. Front Plant Sci 12:774943. https://doi.org/10.3389/fpls.2021.774943 Xu Q, Yin S, Ma Y et al (2020) Carbon export from leaves is controlled via ubiquitination and phosphorylation of sucrose transporter SUC2. Proc Natl Acad Sci USA 117:6223–6230. https://doi.org/10.1073/pnas.1912754117 Xu Z, Mahmood K, Rothstein SJ (2017) ROS Induces Anthocyanin Production Via Late Biosynthetic Genes and Anthocyanin Deficiency Confers the Hypersensitivity to ROS-Generating Stresses in Arabidopsis. Plant Cell Physiol 58:1364–1377. https://doi.org/10.1093/pcp/pcx073 Yang X, Jiang X, Yan W et al (2021) The Mevalonate Pathway Is Important for Growth, Spore Production, and the Virulence of Phytophthora sojae. Front Microbiol 12:772994. https://doi.org/10.3389/fmicb.2021.772994 Yoon J, Cho L-H, Tun W et al (2021) Sucrose signaling in higher plants. Plant Sci 302:110703. https://doi.org/10.1016/j.plantsci.2020.110703 Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8124382","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":566949399,"identity":"98572347-c0a0-4774-af1b-c827816f9089","order_by":0,"name":"Jinku Kang","email":"","orcid":"","institution":"Seoul National University Agriculture and Life Sciences Library: Seoul National University College of Agriculture and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jinku","middleName":"","lastName":"Kang","suffix":""},{"id":566949400,"identity":"9c255967-fe50-45f3-ab0e-574a44868e19","order_by":1,"name":"Sua Cho","email":"","orcid":"","institution":"Seoul National University Agriculture and Life Sciences Library: Seoul National University College of Agriculture and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sua","middleName":"","lastName":"Cho","suffix":""},{"id":566949401,"identity":"5c93dd62-da9e-4f5c-b756-00e501594c82","order_by":2,"name":"Kiyoon Kang","email":"","orcid":"","institution":"Incheon National University","correspondingAuthor":false,"prefix":"","firstName":"Kiyoon","middleName":"","lastName":"Kang","suffix":""},{"id":566949402,"identity":"b233facd-80c8-4632-bcc7-040b127ba93c","order_by":3,"name":"Daewon Kim","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Daewon","middleName":"","lastName":"Kim","suffix":""},{"id":566949403,"identity":"a318ce54-147c-4f8a-99a1-dfe1248dbffa","order_by":4,"name":"Sang-Il Bae","email":"","orcid":"","institution":"Seoul National University Agriculture and Life Sciences Library: Seoul National University College of Agriculture and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sang-Il","middleName":"","lastName":"Bae","suffix":""},{"id":566949404,"identity":"e77b81da-8a34-48ae-a775-cbcebeff495e","order_by":5,"name":"Eunji Shin","email":"","orcid":"","institution":"Seoul National University College of Agriculture and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Eunji","middleName":"","lastName":"Shin","suffix":""},{"id":566949405,"identity":"95cde86d-11aa-412f-9e26-b874c2a475b2","order_by":6,"name":"So-Yon Park","email":"","orcid":"","institution":"University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"So-Yon","middleName":"","lastName":"Park","suffix":""},{"id":566949406,"identity":"e069a115-03c2-4d11-b69e-c7e1b7ce31b0","order_by":7,"name":"Gary Stacey","email":"","orcid":"","institution":"University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Gary","middleName":"","lastName":"Stacey","suffix":""},{"id":566949407,"identity":"9c6a2876-2074-4193-8635-5563a0cc83c1","order_by":8,"name":"Nam-Chon Paek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYNCCAzYwVgLRWtJI13KYBC3m7GcPfvhw5ry9uUQC44cfDGn5BLVY9uQlS864cTtx54wEZskehhzLBkJaDG7wGEjzfLidYHAjgUGagaHCgKAtQC3Gv/98OGcP1ML8m1gtZtIMNw4wbriRwAa0JYcILWfy0ix7ziQnbjjzsM2yxyCNCC3Hzx6+8eOYnb3B8WQgoyKZsBYGBh4Yg7EBaAIRGpC0jIJRMApGwSjAAQBMKj0tQRUaVAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9827-2287","institution":"Seoul National University College of Agriculture and Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Nam-Chon","middleName":"","lastName":"Paek","suffix":""},{"id":566949408,"identity":"4679ea52-8ea9-470a-bdd5-6e727565fd30","order_by":9,"name":"Sung-Hwan Cho","email":"","orcid":"","institution":"Chungbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Sung-Hwan","middleName":"","lastName":"Cho","suffix":""}],"badges":[],"createdAt":"2025-11-15 22:33:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8124382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8124382/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99283809,"identity":"e4cab7db-1cfc-4074-8179-c10f7a0319be","added_by":"auto","created_at":"2025-12-31 09:02:58","extension":"xml","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12367,"visible":true,"origin":"","legend":"","description":"","filename":"pcrePCRED2501387.xml","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/6d13c428c464dfc5b960ef23.xml"},{"id":99320793,"identity":"ab9a1732-5ea4-4832-977e-be703eb82457","added_by":"auto","created_at":"2025-12-31 16:38:54","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":939,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED250138720393.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/03dd1240eb5f68269337cf21.xml"},{"id":99283815,"identity":"de5b60ff-9960-4738-a5cb-9a83f058ae33","added_by":"auto","created_at":"2025-12-31 09:02:58","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":819,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED2501387Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/c1bf26e9938f0e37425eb3dd.xml"},{"id":99283819,"identity":"2bd79001-1788-48e2-8edb-b77d1880a788","added_by":"auto","created_at":"2025-12-31 09:02:58","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148123,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25013870enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/92d5b02cc9869cc3ade357ba.xml"},{"id":99283821,"identity":"6d8f4132-ba13-4e39-adbc-775ae1f20da5","added_by":"auto","created_at":"2025-12-31 09:02:58","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107327,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/52e8b997d00cf05283dcd92a.jpeg"},{"id":99321015,"identity":"a3285c7d-e01e-40e4-af5e-9d54fabfb359","added_by":"auto","created_at":"2025-12-31 16:39:03","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97223,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/19108d5a840bc11cab7d2915.jpeg"},{"id":99319583,"identity":"9eee46c0-0a20-47e2-a6a4-03819bee0dbe","added_by":"auto","created_at":"2025-12-31 16:37:31","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178505,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/3f82c793a61ad06920320aef.jpeg"},{"id":99283825,"identity":"827f0578-3c1b-47f3-b60d-c8d0d47ad2a6","added_by":"auto","created_at":"2025-12-31 09:02:58","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152420,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/bf4ecd87a8ffa6e574804b19.jpeg"},{"id":99321209,"identity":"3f5689d5-6987-4366-8bc5-6e64cd86e02b","added_by":"auto","created_at":"2025-12-31 16:39:16","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117145,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/6bec488602451cc148fa17aa.png"},{"id":99320919,"identity":"64fe3ead-0f16-4c5e-a1c3-4907e2457d21","added_by":"auto","created_at":"2025-12-31 16:38:57","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21285,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/43b9c9356d9962a3b5bfc90d.png"},{"id":99320901,"identity":"baf54cef-c211-4cc0-a87d-72c7dffa70dd","added_by":"auto","created_at":"2025-12-31 16:38:56","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124479,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/6bc81954be8c8764e1425ac0.png"},{"id":99319483,"identity":"17a7a3e8-2a94-4bdb-a8c7-d80c1a1ccb75","added_by":"auto","created_at":"2025-12-31 16:37:21","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91247,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/7731339ab1d96445a9fc289b.png"},{"id":99283833,"identity":"74a7c0ee-0565-4e82-8259-5bde8ec139d5","added_by":"auto","created_at":"2025-12-31 09:02:59","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125137,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/ff7598ef15c936907edc728d.png"},{"id":99320440,"identity":"14b7868e-ddfd-436c-9b36-38b476573c32","added_by":"auto","created_at":"2025-12-31 16:38:35","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90418,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/f8106edfc88689d9c0e52e94.png"},{"id":99319497,"identity":"b4b278b5-8ed5-4f15-b55b-8df7f5829419","added_by":"auto","created_at":"2025-12-31 16:37:24","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107884,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/37e7d4bbee61429534bc741f.png"},{"id":99319072,"identity":"83325e0b-b8af-4a1d-bf7b-d3037329d0f9","added_by":"auto","created_at":"2025-12-31 16:36:13","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40847,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/d6238bc8eb6df524d6d39f0d.png"},{"id":99321223,"identity":"7ddc9d32-aed8-42d6-916f-3c3e56b99565","added_by":"auto","created_at":"2025-12-31 16:39:17","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":56561,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/fd92dfbe5b3bb447c2fff788.png"},{"id":99320893,"identity":"c7515063-e4cf-4a70-91c0-4566a16dbae7","added_by":"auto","created_at":"2025-12-31 16:38:56","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94891,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/5c6534d102c9b5fbf510a840.png"},{"id":99283829,"identity":"f9bd8375-7c49-49b4-89aa-eb1fd2c55005","added_by":"auto","created_at":"2025-12-31 09:02:59","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146501,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25013870structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/8c743840ace16bf785cb30f8.xml"},{"id":99283835,"identity":"6031957f-bbf7-449f-be23-02fcaf3dd50a","added_by":"auto","created_at":"2025-12-31 09:02:59","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163491,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/c63d2cc7abe193c5a8f01d6d.html"},{"id":99283812,"identity":"117c05b8-ac19-49d6-900f-6e75860494a9","added_by":"auto","created_at":"2025-12-31 09:02:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":624751,"visible":true,"origin":"","legend":"\u003cp\u003eMVK is involved in sucrose-induced anthocyanin biosynthesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eThe \u003cem\u003emvk-1\u003c/em\u003e mutants accumulate more anthocyanins in response to sucrose treatment. 10-day-old seedlings of WT and \u003cem\u003emvk-1\u003c/em\u003e mutants grown in half strength Murashige and Skoog (MS) medium were treated with or without 3% (w/v) exogenous sucrose for 3 days. Scale bar represents 1 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003eAnthocyanin content in WT and \u003cem\u003emvk-1\u003c/em\u003e mutants. Anthocyanin contents were determined by measuring the absorbance of plant extracts at 520 nm, subtracting 0.25 times the absorbance at 657 nm, and expressing the result per gram fresh weight (FW). The mean and SD were obtained from three biological replicates. Asterisks indicate significantly different values according to Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003eExpression patterns of anthocyanin biosynthetic and regulatory genes. The relative expression levels of (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eCHS\u003c/em\u003e, (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eCHI\u003c/em\u003e, (\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eF3H\u003c/em\u003e, (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eF3’H\u003c/em\u003e, (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eDFR\u003c/em\u003e, (\u003cstrong\u003eh\u003c/strong\u003e) \u003cem\u003eLDOX\u003c/em\u003e, (\u003cstrong\u003ei\u003c/strong\u003e) \u003cem\u003eUF3GT\u003c/em\u003e, and (\u003cstrong\u003ej\u003c/strong\u003e) \u003cem\u003eMYB75\u003c/em\u003e in WT and \u003cem\u003emvk-1\u003c/em\u003eseedlings. 10-day-old seedlings grown in half-strength MS medium were treated with or without 3% (w/v) sucrose for 3 days. White bars represent WT, blue bars represent \u003cem\u003emvk-1\u003c/em\u003e mutants. Expression levels were determined by RT-qPCR and normalized to the expression of \u003cem\u003eUBQ5\u003c/em\u003e reference gene. The mean and SD were obtained from four biological replicates. Asterisks indicate significantly different values according to Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/14ae9f65031007d65eaa0d17.jpeg"},{"id":99320797,"identity":"568a5a04-54a9-4467-a6d7-7f1e4b7ff16c","added_by":"auto","created_at":"2025-12-31 16:38:54","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":593528,"visible":true,"origin":"","legend":"\u003cp\u003eSucrose content in the leaves of WT and \u003cem\u003emvk-1 \u003c/em\u003emutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eSucrose contents were measured from the leaves of 10-day-old WT and \u003cem\u003emvk-1\u003c/em\u003eseedlings treated with or without 5% (w/v) sucrose for 3 days. Leaf extracts were analyzed by HPLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003eHPLC chromatogram showing the standard sucrose peak. The retention time of sucrose was 7.192 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003eSucrose contents in WT and \u003cem\u003emvk-1\u003c/em\u003emutants. White bars represent WT, blue bars represent \u003cem\u003emvk-1\u003c/em\u003emutants. The mean and SD were obtained from more than four biological replicates. Asterisks indicate significantly different values according to Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/c5a21b9f54ab37e94c877a30.jpeg"},{"id":99319551,"identity":"5edda8f9-9c3e-44ed-a495-ab3a30f90e89","added_by":"auto","created_at":"2025-12-31 16:37:28","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":453342,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression levels of sucrose transporter genes in WT and \u003cem\u003emvk-1 \u003c/em\u003eseedlings.\u003c/p\u003e\n\u003cp\u003eThe relative expression levels of (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eSUC1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eSUC2\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eSUC3\u003c/em\u003e, (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eSUC4\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eSWEET11\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eSWEET12\u003c/em\u003e,\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eSWEET13\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand (\u003cstrong\u003eh\u003c/strong\u003e) \u003cem\u003eSWEET14 \u003c/em\u003ein 10-day-old seedlings grown in half-strength MS medium were treated with or without 3% (w/v) sucrose for 1 day. White bars represent WT, blue bars represent \u003cem\u003emvk-1\u003c/em\u003e mutants. Transcript levels were determined by RT-qPCR and normalized to the expression of \u003cem\u003eUBQ5\u003c/em\u003e reference gene. The mean and SD were obtained from four biological replicates. Asterisks indicate significantly different values according to Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/e58d5a142c9cc5a7a73861a6.jpeg"},{"id":99321000,"identity":"9616754e-71e8-443d-83aa-6fe390285ad2","added_by":"auto","created_at":"2025-12-31 16:39:01","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":720991,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of anthocyanin biosynthetic and regulatory genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eAnthocyanin accumulation with or without 3% (w/v) sucrose treatment in WT, \u003cem\u003emvk-1\u003c/em\u003e, \u003cem\u003esuc1-5\u003c/em\u003e, and \u003cem\u003emvk-1 suc1-5 \u003c/em\u003emutants. 10-day-old seedlings of WT, \u003cem\u003emvk-1\u003c/em\u003e, \u003cem\u003esuc1-5\u003c/em\u003e, and \u003cem\u003emvk-1 suc1-5 \u003c/em\u003emutants grown in half strength MS medium were treated with or without 3% (w/v) sucrose for 3 days. Scale bar represents 0.1 cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003eAnthocyanin contents in WT, \u003cem\u003emvk-1\u003c/em\u003e, \u003cem\u003esuc1-5\u003c/em\u003e, and \u003cem\u003emvk-1 suc1-5 \u003c/em\u003emutants. Anthocyanin contents were determined by measuring the absorbance of plant extracts at 520 nm, subtracting 0.25 times the absorbance at 657 nm, and expressing the result per gram fresh weight (FW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ej\u003c/strong\u003eThe relative expression levels of (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eCHS\u003c/em\u003e, (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eCHI\u003c/em\u003e, (\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eF3H\u003c/em\u003e, (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eF3’H\u003c/em\u003e, (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eDFR\u003c/em\u003e, (\u003cstrong\u003eh\u003c/strong\u003e) \u003cem\u003eLDOX\u003c/em\u003e, (\u003cstrong\u003ei\u003c/strong\u003e) \u003cem\u003eUF3GT\u003c/em\u003e, and (\u003cstrong\u003ej\u003c/strong\u003e) \u003cem\u003eMYB75\u003c/em\u003e in WT, \u003cem\u003emvk-1, suc1-5\u003c/em\u003e, and \u003cem\u003emvk-1 suc1-5 \u003c/em\u003eseedlings. 10-day-old whole seedlings grown in half-strength MS medium were treated with or without 3% (w/v) sucrose for 3 days. The white, blue, red, and green bars represent WT, \u003cem\u003emvk-1\u003c/em\u003e, \u003cem\u003esuc1-5\u003c/em\u003e, and \u003cem\u003emvk-1 suc1-5\u003c/em\u003e mutants, respectively\u003cem\u003e. \u003c/em\u003eExpression levels were determined by RT-qPCR and normalized to the expression of \u003cem\u003eUBQ5\u003c/em\u003e reference gene. The mean and SD were obtained from four biological replicates. Different letters indicate significantly different values according to a one-way ANOVA followed by Duncan’s least significant range test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/d0051032b7b3159fdebe18f7.jpeg"},{"id":99320355,"identity":"778661aa-1833-40f9-afb0-21d8083d16dc","added_by":"auto","created_at":"2025-12-31 16:38:32","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":427644,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003emvk-1\u003c/em\u003e mutants accumulate lower levels of gibberellins (GA\u003csub\u003e1\u003c/sub\u003e) than WT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eAnthocyanin accumulation under 5% (w/v) sucrose with or without 50 μM GA treatments in WT and \u003cem\u003emvk-1 \u003c/em\u003emutants. Scale bar represents 0.3 cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Measurement of DELLA protein levels. WT and \u003cem\u003emvk-1\u003c/em\u003e mutants were grown under with or without 3% (w/v) sucrose treatments with or without GA. Total DELLA proteins were detected by immunoblotting with an anti-RGA/DELLA antibody. Protein loading was visualized by Coomassie brilliant blue (CBB) staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e GA\u003csub\u003e1\u003c/sub\u003e quantification by LC-MS. 14-day-old whole seedlings of WT and \u003cem\u003emvk-1\u003c/em\u003e mutants grown in half-strength MS medium were treated with or without 3% (w/v) sucrose for 3 days. The mean and SD were obtained from more than four biological replicates. Asterisks indicate significantly different values according to Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/82f016770f587c16a611591c.jpeg"},{"id":99320608,"identity":"b66c7c2c-5055-434d-ac1f-5f794a2353fc","added_by":"auto","created_at":"2025-12-31 16:38:48","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":486538,"visible":true,"origin":"","legend":"\u003cp\u003eMVK regulates anthocyanin synthesis by modulating \u003cem\u003eSUC1\u003c/em\u003e expression and GA biosynthesis.\u003c/p\u003e\n\u003cp\u003eSchematic model shows the role of MVK in regulating anthocyanin biosynthesis. MVK represses \u003cem\u003eSUC1 \u003c/em\u003eexpression, which in turn downregulates sucrose levels, leading to altered GA levels, both directly and indirectly. Reduced GA levels stabilize DELLA proteins, thereby activating the MBW complex and enhancing anthocyanin accumulation in Arabidopsis.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/c227eabadefd0edd9f51d44d.jpeg"},{"id":99788375,"identity":"70809a41-ed84-4084-9026-baea39e25f1d","added_by":"auto","created_at":"2026-01-08 12:46:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4238466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/c117dd71-a14f-4c3b-a58f-a6822c3c04ea.pdf"},{"id":99320563,"identity":"f5d1a800-81fb-4d48-ab4e-76d067f72801","added_by":"auto","created_at":"2025-12-31 16:38:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":557831,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8124382/v1/e78b2a40d622a5e600af6b46.docx"}],"financialInterests":"","formattedTitle":"MEVALONATE KINASE represses anthocyanin biosynthesis via sucrose transporters and gibberellin synthesis pathways in Arabidopsis","fulltext":[{"header":"Key Message","content":"\u003cp\u003eArabidopsis MVK negatively regulates sucrose-induced anthocyanin accumulation by modulating SUC1-mediated sucrose transport and gibberellin homeostasis.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAnthocyanins, a pivotal class of water-soluble flavonoids, are responsible for pigmentation of vegetative (leaves, stems, and roots) and reproductive (flowers and fruits) organs in plants. These pigments are not only aesthetic, but also serve crucial ecological functions, such as attracting pollinators and seed dispersers, which are essential for reproduction and survival (Harborne and Williams \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Beyond their ecological roles, anthocyanins are widely recognized for the role in abiotic stress responses, particularly through antioxidant properties that mitigate oxidative damage caused by reactive oxygen species (ROS) (Grotewold \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, anthocyanins offer nutritional benefits to humans, making anthocyanin-rich plants increasingly important research targets (Khoo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe biosynthesis of anthocyanins is regulated by a combination of developmental cues and environmental signals, including light, temperature, and several endogenous molecules (LaFountain and Yuan \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among them, sucrose is known to promote anthocyanin biosynthesis (Solfanelli et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In addition, phytohormones, including ethylene, jasmonic acid (JA), gibberellic acid (GA), abscisic acid (ABA), and cytokinin (CK), interact with sucrose signaling pathways, collectively modulating anthocyanin production (Das et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Loreti et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnthocyanins are synthesized in the cytosol, then modified into various derivatives and subsequently transported into vacuoles. In Arabidopsis (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e), structural genes involved in anthocyanin biosynthesis are classified into early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) (Deroles 2009). Briefly, the pathway starts with condensation of one molecule of 4-coumaroyl-coenzyme A and three molecules of malonyl-CoA leading to the formation of naringenin chalcone. EBGs such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol 3-hydroxylase (F3H), and flavonol 3-hydroxylase (F3\u0026prime;H) further metabolize naringenin chalcone leading to the production of flavonols. Subsequently, LBGs, including dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin reductase (ANR), and UDP-Glc:flavonoid 3-O-glucosyltransferase (UF3GT) facilitate the final steps that produce anthocyanins and proanthocyanidins (Holton and Cornish \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnthocyanin biosynthesis is tightly regulated at the transcriptional level by the MBW (MYB-bHLH-WD40) complex, which consists of R2R3-MYB, bHLH, and WD40-repeat proteins (Hichri et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In Arabidopsis, the MBW complex includes R2R3-MYBs (MYB75/PAP1, MYB90/PAP2, MYB113, and MYB114), bHLH transcription factors [TRANSPARENT TESTA 8 (TT8), GLABRA 3 (GL3), and ENHANCER OF GLABRA 3 (EGL3)], and the WD40-repeat protein TRANSPARENT TESTA GLABRA 1 (TTG1) (Broun \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gonzalez et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This complex orchestrates anthocyanin biosynthesis by activating the expression of structural genes such as \u003cem\u003ePhenylalanine ammonia-lyase\u003c/em\u003e (\u003cem\u003ePAL\u003c/em\u003e), \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eANS\u003c/em\u003e, and \u003cem\u003eUF3GT\u003c/em\u003e, ultimately promoting anthocyanin accumulation in vegetative and reproductive tissues (Das et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Recent studies in Arabidopsis showed that GLK1 promotes anthocyanin accumulation by directly interacting with MYB75, MYB90, and MYB113, thereby boosting their transcriptional activity (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, energy deficiency suppresses anthocyanin accumulation through the action of SnRK1, a master metabolic regulator. SnRK1 destabilizes MYB75, thus repressing MBW-mediated transcription and anthocyanin production under low energy conditions (Broucke et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Homologs such as FvMYB10, FvMYB41, and FvMYB105 interact with bHLH partners like FvbHLH33 and FvMYC1 to regulate the synthesis of stage-specific anthocyanin and proanthocyanidin during fruit ripening in woodland strawberry (Xu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Importantly, the MBW activity is subject to modulation by internal and external signals, such as hormones and sugar. DELLA proteins, mostly acting as repressors of gibberellin (GA) signaling, promote anthocyanin biosynthesis by sequestering JAZ and MYBL2 repressors, thereby enabling MBW activation (Xie et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, sucrose enhances anthocyanin biosynthesis by stabilizing DELLA proteins and inducing expression of MBW-regulated genes such as \u003cem\u003eMYB75/PAP1\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, and \u003cem\u003eDFR\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven its dual role as both a carbon source and a signaling molecule, sucrose not only provides metabolic substrates but also functions as a key regulator that integrates hormonal and transcriptional networks to promote anthocyanin accumulation (Loreti et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Sakr et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Sucrose content in plants may increase due to alterations in sucrose metabolism or the activity of sucrose transporters (Julius et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Sucrose transporters (SUCs) are sucrose-proton symporters involved in sucrose translocation (Braun \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nine putative \u003cem\u003eSUC\u003c/em\u003e genes have been identified in Arabidopsis, and some are directly linked to anthocyanin accumulation. For example, SUC1 plays a critical role in sucrose-induced anthocyanin accumulation, and sucrose-induced anthocyanin accumulation is inhibited in \u003cem\u003eSUC1\u003c/em\u003e knockout mutants (Sivitz et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In addition, the Arabidopsis \u003cem\u003epho3\u003c/em\u003e mutants, which is defective in SUC2 function, exhibits enhanced anthocyanin accumulation (Lloyd and Zakhleniuk \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Among \u003cem\u003eSUC\u003c/em\u003e genes, \u003cem\u003eSUC5\u003c/em\u003e is expressed specifically in the endosperm, \u003cem\u003eSUC6\u003c/em\u003e and \u003cem\u003eSUC7\u003c/em\u003e encode aberrant proteins, and \u003cem\u003eSUC8\u003c/em\u003e and \u003cem\u003eSUC9\u003c/em\u003e are predominantly expressed in floral tissues (Baud et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Meyer et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sauer et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe mevalonate (MVA) pathway, functioning in the cytosol of plant cells, plays a crucial role in primary metabolism by producing isoprenoids, sterols, and other key metabolites (Pulido et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It is conserved across plants, fungi, and animals and supports diverse physiological processes (Miziorko \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ruiz-Sola et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Within this pathway, mevalonate kinase (MVK) is a key enzyme that phosphorylates mevalonic acid to produce mevalonate 5-phosphate (Riou et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). A recent study showed that Arabidopsis MVK is a direct phosphorylation target of P2K1, leading to activation of the MVA pathway in response to extracellular ATP (eATP) elicitation (Cho et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The relationship between the MVA pathway and anthocyanin production has been studied in apple trees (\u003cem\u003eMalus domestica\u003c/em\u003e Borkh). This pathway produces isoprenoids and sterols and was shown to influence anthocyanin accumulation by positively regulating IAA and ABA synthesis while inhibiting GA synthesis (Flores-Perez et al. 2010; Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the mechanism of MVA-mediated anthocyanin regulation in plants remains to be elucidated.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to elucidate the role of MVK, a core enzyme of the cytosolic isoprenoid biosynthesis pathway, in the regulation of sucrose-induced anthocyanin biosynthesis in Arabidopsis. Although the MVA pathway was previously associated with various metabolic and hormonal signaling events, its connection to anthocyanin production in response to sucrose remains poorly understood. Our findings reveal that \u003cem\u003emvk-1\u003c/em\u003e mutants accumulate more anthocyanins than WT under high sucrose conditions. This phenotype is accompanied by elevated gene expression of anthocyanin biosynthesis such as \u003cem\u003eCHS\u003c/em\u003e and \u003cem\u003eDFR\u003c/em\u003e, and transcriptional regulators such as \u003cem\u003eMYB75/PAP1\u003c/em\u003e. Furthermore, we demonstrate that MVK negatively regulates anthocyanin accumulation through two distinct mechanisms. First, MVK inhibits the expression of \u003cem\u003eSUC1\u003c/em\u003e. Genetic analysis of \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants further revealed that this regulation is mediated by a SUC1-dependent regulatory pathway. Second, mutation of \u003cem\u003eMVK\u003c/em\u003e reduces gibberellin levels, thereby promoting stabilization of DELLA protein. Collectively, our results uncover a previously uncharacterized function of MVK as a negative regulator of sucrose-induced anthocyanin biosynthesis, integrating sugar transport and hormonal signaling into a coordinated regulatory network.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant material and growth conditions\u003c/h2\u003e\u003cp\u003eWild-type aequorin-expressing transgenic Arabidopsis ColQ (Col-0 background) plants were provided by Marc Knight (Knight et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The \u003cem\u003emvk-1\u003c/em\u003e mutant (ColQ background) has been described previously (Cho et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Arabidopsis seeds were surface-sterilized and sown on half-strength Murashige and Skoog (MS) medium supplemented with 0% (w/v) sucrose, 0.5% (w/v) agar (MB Gellan Gum, Cat No. MB-G4367), and 0.05% (w/v) MES (pH 5.7). Following a 3-day cold stratification at 4\u0026deg;C, the plates were positioned vertically in a growth chamber set to a 16 h light/8 h dark photoperiod at 22\u0026deg;C, and 100 \u0026micro;mole m⁻\u0026sup2; s⁻\u0026sup1; light intensity.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenerating CRISPR-Cas9\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eGenerating CRISPR-Cas9\u003c/div\u003e\u003cp\u003eThe \u003cem\u003esuc1-5\u003c/em\u003e mutant was generated via CRISPR/Cas9-mediated genome editing, with a single guide RNA (sgRNA, 5`-CTCGATCCCTGGGACATTCCTGG-3`) targeting the \u003cem\u003eSUC1\u003c/em\u003e coding region being designed using the CRISPR direct program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.dbcls.jp/\u003c/span\u003e\u003cspan address=\"http://crispr.dbcls.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Naito et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The tRNA\u0026ndash;gRNA\u0026ndash;Cas9 fragment was inserted into the \u003cem\u003epRGEB32\u003c/em\u003e vector (Xie et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This binary vector was transformed into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101, which was then used to transform Arabidopsis plants via the floral dip method (Clough and Bent \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The homozygous lines were screened based on hygromycin resistance. Confirmation of this selection was achieved by directly sequencing PCR-amplified genomic products, which were amplified with the use of primers targeting the specific region listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eAnthocyanin assay\u003c/h3\u003e\n\u003cp\u003eAnthocyanins were extracted using a modified version of a previously described method (Nakata et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Three-day-old Arabidopsis seedlings were transferred to half-strength MS medium supplemented with 1% or 3% (w/v) sucrose and grown for another three days. Samples were then extracted in 45% methanol and 5% acetic acid (v/v). The relative anthocyanin content was determined spectrophotometrically by measuring the absorbance at 520 nm and 657 nm, and the relative values were calculated accordingly.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and RT-qPCR analyses\u003c/h3\u003e\n\u003cp\u003e Total RNA was extracted from Arabidopsis plants using GeneAll Hybrid-R (GeneAll Biotechnology, Republic of Korea) according to the manufacturer\u0026rsquo;s instructions. First-strand cDNA was synthesized from 2 \u0026micro;g total RNA using M-MLV reverse transcriptase (Promega, Madison, USA). For RT-qPCR, GoTaq PCR Mix (Promega, Madison, USA) was used according to the manufacturer's instructions. qPCR was performed using a LightCycler 2.0 system (Roche Diagnostics, Mannheim, Germany). Transcript levels were normalized to the expression of the \u003cem\u003eUBQ\u003c/em\u003e gene. The primers used for RT-qPCR analysis are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eSucrose measurements\u003c/h3\u003e\n\u003cp\u003eFor sucrose extraction, 20 mg (fresh weight) of Arabidopsis rosette leaves was ground in liquid nitrogen and extracted using 80% (v/v) ethanol. The extracted samples were centrifuged at 12,000 \u0026times; g for 10 minutes at 4\u0026deg;C, and the supernatant was filtered before analysis. Soluble sucrose content was quantified by high-performance liquid chromatography (HPLC) on a Dionex Ultimate 3000 system (Thermo Fisher, Sunnyvale, USA) equipped with a Shodex RI-101 refractive index detector (Shoko, Tokyo, Japan) at the Seoul National University NICEM. Separation was performed on a Sugar-Pak column (Waters, 300 mm \u0026times; 6.5 mm) at 70\u0026deg;C. The mobile phase consisted of ultrapure water (Milli-Q grade) at a flow rate of 0.5 mL/min. The injection volume was set at 10 \u0026micro;L for each sample. Chromeleon ver. 6 software was used for data acquisition and processing. Calibration was carried out with sucrose standard (Sigma, 99.5% purity). Quantification was performed by comparing sample peak areas to those of the standard curve generated from known sucrose concentrations.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDELLA protein stability\u003c/h2\u003e\u003cp\u003eFor the DELLA protein stability analysis, five-day-old seedlings were grown in 6-well plates with 1 mL liquid half-strength MS medium supplemented with 0% (w/v) sucrose (pH 5.7) under long-day conditions (16 h light/8 h dark, 21\u0026deg;C). After 3 days in LD conditions, 5% (w/v) Suc was added for an additional 3 days. Then, these seedlings were treated with 10 \u0026micro;M GA for 2 h, and total protein was extracted using extraction buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EDTA, 0.5% Triton-X 100, 10% glycerol, 1 mM DTT, 0.2 mM PMSF, and 1\u0026times; Pierce protease inhibitor (Thermo Fisher, Rockford, USA). The extracted proteins were mixed with 5\u0026times; Laemmli loading buffer containing 10% SDS, 50% glycerol, 0.01% bromophenol blue, 10% beta-mercaptoethanol, and 0.3 M Tris-HCl (pH 6.8), and boiled at 95\u0026deg;C for 5 min. Total extracted proteins were separated by 10% SDS-PAGE gel electrophoresis, and proteins were transferred to a PVDF membrane (Immobilon\u0026reg;-P, Millipore) with a semi-dry transfer system (Trans-Blot\u0026reg; SD, Bio-Rad, Hercules, USA). After blocking with 5% skim milk, the membrane was incubated with RGA/DELLA antibody (Agrisera, Cat No. AS11-1630, dilution 1:1000) in 5% skim milk for 2 h. Subsequenctly, the membrane was washed 3 times and incubated with secondary goat anti-rabbit-HRP (Santa Cruz, Cat No. sc-2004, dilution 1:10000) for 2 h. Subsequently, the membrane was washed 5 times in TBST (50 mM Tris, 150 mM NaCl, 0,05% Tween 20), incubated with Pierce SuperSignal\u0026reg; West Pico chemiluminescent substrate (Thermo Scientific, Cat No. 34578) for 1 min and exposed to film.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGibberellins quantification\u003c/h3\u003e\n\u003cp\u003eGA quantification was analyzed by the previous method (Xin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, 1 g of 10-day-old seedlings were ground to a find powder in liquid nitrogen, and the samples were freeze-dried for 3 days. 1 mL of solution containing 80% (v/v) methanol was added to each sample and incubated for 12 h at 4\u0026deg;C. The samples were centrifuged at 12,000 x g for 15 min at 4\u0026deg;C. The solvent was then dried down using a Speed Vac concentrator at room temperature (25\u0026deg;C). The dried pellets were resuspended in 100 \u0026micro;L of solution containing 80% (v/v) methanol and resuspended samples were immediately subjected to liquid chromatography-mass spectrometry (LC-MS) hormonal analysis (Seoul National University NICEM, Republic of Korea).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003emvk-1\u003c/b\u003e \u003cb\u003emutants exhibit enhanced sucrose-induced anthocyanin accumulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe MVA pathway plays a crucial role in the biosynthesis of a wide range of isoprenoids, including phytohormones (Pulido et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). It was reported that CK, GA, and ABA regulate the induction of anthocyanin biosynthesis by sugar in Arabidopsis (Loreti et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This led us to examine whether mutation of MVK affects anthocyanin levels. We first investigated anthocyanin accumulation in \u003cem\u003emvk-1\u003c/em\u003e mutants. To determine the role of \u003cem\u003eMVK\u003c/em\u003e in anthocyanin accumulation, 10-day-old WT and \u003cem\u003emvk-1\u003c/em\u003e plants were grown on half-strength Murashige and Skoog (MS) medium in the absence or presence of 3% sucrose for 3 days. The leaves and shoot apical meristem region of \u003cem\u003emvk-1\u003c/em\u003e mutants showed intense purple coloration after being grown on 3% sucrose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Furthermore, a pronounced anthocyanin accumulation phenotype was observed across the abaxial surface of 10-day-old \u003cem\u003emvk-1\u003c/em\u003e mutants grown on 5% sucrose medium. (Supplementary Fig. S1). There was no difference in anthocyanin content under mock conditions, whereas \u003cem\u003emvk-1\u003c/em\u003e mutants exhibited an almost 3-fold increase in anthocyanin content compared to WT under 3% sucrose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, to identify whether these phenotypes were due to upregulation of anthocyanin biosynthetic genes at the transcriptional level, we examined the relative expression of genes involved in the anthocyanin biosynthetic pathway (\u003cem\u003eCHS, CHI, F3H, F3\u0026rsquo;H, DFR, ANS\u003c/em\u003e/\u003cem\u003eLDOX and UF3GT\u003c/em\u003e). Under 3% sucrose conditions, \u003cem\u003emvk-1\u003c/em\u003e mutants exhibited higher expression levels of anthocyanin biosynthetic genes compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-i). We also measured the relative expression level of \u003cem\u003eMYB75\u003c/em\u003e, which is a transcription factor composing the MBW complex, involved in the transcriptional activation of anthocyanin biosynthetic genes (Teng et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Interestingly, the expression level of \u003cem\u003eMYB75\u003c/em\u003e was also upregulated in \u003cem\u003emvk-1\u003c/em\u003e mutants compared to WT under 3% sucrose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). Taken together, these findings indicate that a loss-of-function of \u003cem\u003eMVK\u003c/em\u003e results in enhanced anthocyanin accumulation, likely through transcriptional activation of the MBW complex and the subsequent upregulation of key genes in the anthocyanin biosynthetic pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHigh sucrose accumulation in the leaves of\u003c/b\u003e \u003cb\u003emvk-1\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSucrose is widely recognized as a signaling molecule that induces anthocyanin biosynthesis (Yoon et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cem\u003emvk-1\u003c/em\u003e mutants exhibited increased anthocyanin accumulation under high sucrose conditions. We hypothesized that this phenotype is associated with elevated sucrose levels. To confirm this hypothesis, we collected leaves from WT and \u003cem\u003emvk-1\u003c/em\u003e mutants grown under the same conditions and subjected them to the treatments with or without 5% sucrose. Sucrose content was measured using HPLC. Under mock conditions, no difference was observed in sucrose levels (i.e., peak at ~\u0026thinsp;7.2 mins) between WT and \u003cem\u003emvk-1\u003c/em\u003e mutants. However, \u003cem\u003emvk-1\u003c/em\u003e mutants showed higher internal sucrose levels than WT in the presence of 5% sucrose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). The standard sucrose was detected at ~\u0026thinsp;7.2 mins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These results suggest that Arabidopsis MVK influences sucrose accumulation, potentially contributing to an enhanced anthocyanin phenotype observed in \u003cem\u003emvk-1\u003c/em\u003e mutants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression patterns of\u003c/b\u003e \u003cb\u003eSUCs\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003emvk-1\u003c/b\u003e \u003cb\u003emutants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSucrose transporters are known to play a central role in regulating sucrose levels in plants (Julius et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Since \u003cem\u003emvk-1\u003c/em\u003e mutants exhibited increased sucrose accumulation under high exogenous sucrose treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), we investigated whether this phenotype was associated with altered expression of sucrose transporter genes. In order to measure expression of \u003cem\u003eSUC\u003c/em\u003e genes, we compared their transcript levels between WT and \u003cem\u003emvk-1\u003c/em\u003e seedlings under high-sucrose conditions. The expression of \u003cem\u003eSUC1\u003c/em\u003e was increased in \u003cem\u003emvk-1\u003c/em\u003e mutants under high-sucrose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), whereas that of \u003cem\u003eSUC2\u003c/em\u003e, \u003cem\u003eSUC3\u003c/em\u003e, and \u003cem\u003eSUC4\u003c/em\u003e showed no significant change regardless of sucrose concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition to \u003cem\u003eSUC\u003c/em\u003e genes, we also examined the \u003cem\u003eSWEET\u003c/em\u003e (\u003cem\u003eSugars Will Eventually be Exported Transporters\u003c/em\u003e) genes encoding sugar transporters, which function as unidirectional uniporters mediating sucrose efflux across the plasma membrane and tonoplast (Ji et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the 17 Arabidopsis \u003cem\u003eSWEET\u003c/em\u003e genes, \u003cem\u003eSWEET11\u003c/em\u003e, \u003cem\u003eSWEET12\u003c/em\u003e, \u003cem\u003eSWEET13\u003c/em\u003e, and \u003cem\u003eSWEET14\u003c/em\u003e are known to participate in sucrose transport. However, no significant differences in the expression of these genes were detected between WT and \u003cem\u003emvk-1\u003c/em\u003e mutants under high-sucrose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-h). Taken together, these results indicated that the mutation of \u003cem\u003eMVK\u003c/em\u003e specifically alters the expression of \u003cem\u003eSUC1\u003c/em\u003e, while other sucrose transporters (\u003cem\u003eSUC2\u003c/em\u003e, \u003cem\u003eSUC3\u003c/em\u003e, \u003cem\u003eSUC4\u003c/em\u003e, and \u003cem\u003eSWEET11-14)\u003c/em\u003e remain unaffected.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMVK genetically affects SUC1-mediated anthocyanin accumulation\u003c/h2\u003e\u003cp\u003eArabidopsis SUC1 is plasma membrane-localized sucrose/H⁺ symporters with distinct expression patterns, where SUC1 mediates local sucrose uptake in roots, trichomes, and pollen (Sauer and Stolz \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Sivitz et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Stadler and Sauer \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since exogenous sucrose strongly induces anthocyanin biosynthesis and SUC1 primarily functions in sucrose uptake in roots, we hypothesized that \u003cem\u003eSUC1\u003c/em\u003e expression is a downstream target of MVK. To investigate whether MVK regulates anthocyanin accumulation through \u003cem\u003eSUC1\u003c/em\u003e, we generated the CRISPR/Cas9-mediated \u003cem\u003esuc1-5\u003c/em\u003e mutants in WT background, harboring a single base insertion that resulted in a premature stop codon in the \u003cem\u003eSUC1\u003c/em\u003e coding region (Supplementary Fig. S2). Additionally, the \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants were created by introducing the \u003cem\u003eSUC1\u003c/em\u003e CRISPR/Cas9 construct into the \u003cem\u003emvk-1\u003c/em\u003e background to examine their genetic interaction. Sequencing analysis confirmed a single base insertion in \u003cem\u003eSUC1\u003c/em\u003e, which is identical to the \u003cem\u003esuc1-5\u003c/em\u003e mutant allele (Supplementary Fig. S2b).\u003c/p\u003e\u003cp\u003eConsistent with previous studies (Sivitz et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003esuc1-5\u003c/em\u003e mutants exhibited less anthocyanin accumulation under 3% sucrose treatment compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Interestingly, \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants showed intermediate anthocyanin levels, higher than \u003cem\u003esuc1-5\u003c/em\u003e but lower than \u003cem\u003emvk-1\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). At the transcriptional level, anthocyanin biosynthetic genes were markedly downregulated in \u003cem\u003esuc1-5\u003c/em\u003e mutants, but significantly upregulated in \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants compared with WT under sucrose treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-i). Similarly, \u003cem\u003eMYB75\u003c/em\u003e expression level was reduced in \u003cem\u003esuc1-5\u003c/em\u003e mutants, whereas it was induced in \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). Together, these results suggested that MVK regulates anthocyanin accumulation partially by downregulating \u003cem\u003eSUC1\u003c/em\u003e expression, while also acting through additional SUC1-independent pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMVK regulates DELLA stability via GA biosynthesis\u003c/h2\u003e\u003cp\u003eInterestingly, \u003cem\u003emvk\u003c/em\u003e-\u003cem\u003e1 suc1-5\u003c/em\u003e double mutants showed significantly higher anthocyanin accumulation compared to \u003cem\u003esuc1-5\u003c/em\u003e mutants under sucrose treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Since the MVA and MEP pathway synthesizes isoprenoid precursors essential for phytohormones, such as brassinosteroids, CK, GA, and ABA. (Pulido et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and GA suppresses sucrose-induced anthocyanin accumulation (Loreti et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), we hypothesized that MVK may regulate anthocyanin accumulation not only via a SUC1-mediated pathway but also through GA biosynthesis. To confirm this, we compared the anthocyanin accumulation of WT and \u003cem\u003emvk-1\u003c/em\u003e mutants under 5% sucrose with or without 50 \u0026micro;M GA treatments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the accumulation of anthocyanins in \u003cem\u003emvk-1\u003c/em\u003e mutants under 5% sucrose treatment was attenuated by GA application. Since GA represses sucrose signaling by promoting degradation of DELLA proteins (Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), we measured the level of DELLA proteins in WT and \u003cem\u003emvk-1\u003c/em\u003e mutants under sucrose treatment with or without GA treatments. Remarkably, \u003cem\u003emvk-1\u003c/em\u003e mutants showed higher levels of DELLA proteins compared to WT, regardless of GA treatment under each sucrose conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Since the mutants exhibited elevated levels of DELLA protein, we investigated whether this was associated with altered GA content. Analysis by LC-MS revealed that GA\u003csub\u003e1\u003c/sub\u003e levels in the \u003cem\u003emvk-1\u003c/em\u003e mutants were significantly lower than those in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePrevious studies demonstrated that GA biosynthetic genes \u003cem\u003eGibberellin 3-oxidase 1\u003c/em\u003e (\u003cem\u003eGA3ox1\u003c/em\u003e) and \u003cem\u003eGibberellin 20-oxidase 1\u003c/em\u003e (\u003cem\u003eGA20ox1\u003c/em\u003e) exhibit increased expression under GA-deficient conditions, consistent with feedback regulation mechanisms (Fukazawa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarly, our results revealed significant upregulation of \u003cem\u003eGA3ox1\u003c/em\u003e and \u003cem\u003eGA20ox1\u003c/em\u003e expression in \u003cem\u003emvk-1\u003c/em\u003e mutants, whereas their expression was downregulation in \u003cem\u003esuc1-5\u003c/em\u003e mutants compared to WT (Supplementary Fig. S4). Notably, \u003cem\u003emvk-1 suc1-5\u003c/em\u003e mutants showed moderate expression levels, significantly higher than \u003cem\u003esuc1-5\u003c/em\u003e mutants but lower than \u003cem\u003emvk-1\u003c/em\u003e mutants. Taken together, our results suggest that reduced GA biosynthesis in \u003cem\u003emvk-1\u003c/em\u003e mutants impedes the degradation of DELLA proteins, resulting in higher anthocyanin accumulation compared to WT.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLoss of MVK enhances sucrose-specific induction of anthocyanin biosynthetic pathway\u003c/h2\u003e\u003cp\u003eAnthocyanin accumulation is closely linked to the availability of sucrose, since sucrose transporters such as SUCs and SWEETs import extracellular sucrose, thereby inducing the anthocyanin biosynthesis. It was previously reported that several kinases regulate sucrose transporters. For example, Sucrose-Induced Receptor Kinase 1 (SIRK1) phosphorylates and thereby activates several membrane proteins including SWEET11 under sucrose-specific osmotic response (Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, Wall-Associated Kinase Like 8 (WAKL8) phosphorylates SUC2, thereby increasing its transport activity (Xu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, although kinase-mediated regulation of sucrose transporters has been demonstrated, no studies have yet reported a mechanism by which such kinase-dependent modulation of sucrose transporters directly influences anthocyanin biosynthesis.\u003c/p\u003e\u003cp\u003eIn this study, we showed that the knockout mutation of \u003cem\u003eMVK\u003c/em\u003e enhances sucrose-specific anthocyanin accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The \u003cem\u003emvk-1\u003c/em\u003e mutants showed increased expression of anthocyanin biosynthetic genes, along with higher transcript levels of \u003cem\u003eMYB75\u003c/em\u003e, a key transcriptional regulator of anthocyanin biosynthetic genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). Measurement of sucrose contents in leaves revealed that \u003cem\u003emvk-1\u003c/em\u003e mutants accumulate higher levels of sucrose under high-sucrose conditions compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consistently, \u003cem\u003emvk-1\u003c/em\u003e mutant plants showed significantly increased expression of \u003cem\u003eSUC1\u003c/em\u003e, indicating that \u003cem\u003eMVK\u003c/em\u003e negatively regulates \u003cem\u003eSUC1\u003c/em\u003e expression. Furthermore, \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants showed significantly higher anthocyanin accumulation than the \u003cem\u003esuc1-5\u003c/em\u003e mutants, indicating that the enhanced anthocyanin phenotype of \u003cem\u003emvk-1\u003c/em\u003e mutants is at least partially dependent on \u003cem\u003eSUC1\u003c/em\u003e. DELLA proteins levels remained higher in \u003cem\u003emvk-1\u003c/em\u003e mutants than WT under sucrose treatment. LC-MS analysis also indicated that \u003cem\u003emvk-1\u003c/em\u003e mutants had lower GA\u003csub\u003e1\u003c/sub\u003e levels compared to WT.\u003c/p\u003e\u003cp\u003eTaken together, these results strongly suggest that \u003cem\u003eMVK\u003c/em\u003e regulates anthocyanin accumulation in plants by modulating \u003cem\u003eSUC1\u003c/em\u003e expression. In addition, MVK controls GA levels, and the absence of MVK activity leads to reduced GA\u003csub\u003e1\u003c/sub\u003e content and increased DELLA protein stability, which further promotes the expression of anthocyanin biosynthetic genes. These regulatory mechanisms contribute to enhanced anthocyanin accumulation observed in \u003cem\u003emvk-1\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eThe MVA pathway regulates anthocyanin accumulation via GA biosynthesis\u003c/h2\u003e\u003cp\u003eAnthocyanin accumulation is tightly controlled by the interplay between sucrose and phytohormones. Auxin and cytokinin promote anthocyanin accumulation through transcriptional activation and antioxidant regulation, whereas ethylene exerts context-dependent effects (Bhaskar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chandler \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ni et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Abscisic acid strongly induces anthocyanin biosynthesis under stress conditions, while GA consistently acts as a negative regulator (Karppinen et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Loreti et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Moreover, sucrose signaling was shown to interact with several hormones, including IAA, ABA, MeJA, and SA, but is antagonized by GA (Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur results expand this framework by demonstrating that MVK, a key enzyme in the MVA pathway, influences anthocyanin accumulation through GA biosynthesis. The \u003cem\u003emvk-1 suc1-5\u003c/em\u003e double mutants accumulated more anthocyanin than \u003cem\u003esuc1-5\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Moreover, the double mutants also exhibited reduced root length (Supplementary Fig. S3). These observations suggest the existence of another regulatory pathway compensating for the loss of SUC1-mediated sucrose signaling. Notably, the dwarfism observed in both \u003cem\u003emvk-1\u003c/em\u003e and \u003cem\u003emvk-1 suc1-5\u003c/em\u003e mutants (Supplementary Figs. S1, S3) aligns with established roles of the MVA pathway in isoprenoid biosynthesis, which supplies precursors for GA synthesis (Cho et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn \u003cem\u003emvk\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e mutants, reduced GA\u003csub\u003e1\u003c/sub\u003e levels and elevated DELLA protein accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) alleviate GA-mediated repression of sucrose signaling, thereby enabling enhanced anthocyanin accumulation in \u003cem\u003emvk-1 suc1-5\u003c/em\u003e mutants even in the absence of \u003cem\u003eSUC1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, exogenous GA treatment rescued the hyperaccumulation of anthocyanin phenotype in \u003cem\u003emvk-1\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Disruption of MVK reduces the amount of GGPP-derived GA precursors, thereby leading to the stabilization of DELLA proteins. These findings highlight how GA hormonal signals linked to the MVA pathway are intertwined in fine-tuning plant secondary metabolism, such as anthocyanin biosynthesis. Our study uncovers a previously unrecognized role of MVK in repressing the expression of \u003cem\u003eSUC1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This transcriptional repression connects the MVA-GA signaling module to sucrose transport, suggesting that MVK serves as an integrative regulator bridging hormonal and metabolic cues. Our findings therefore propose a broader role for the MVK-SUC1 regulatory axis in anthocyanin biosynthesis. Since anthocyanin accumulation is tightly controlled by sucrose availability and stress-induced signaling pathways, the MVK-SUC1 connection may represent a critical node that coordinates primary metabolism, signaling networks, and secondary metabolism. This integration could enable plants to balance growth and stress adaptation by modulating anthocyanin levels.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinku Kang, Sua Cho, Eunji Shin, and Sung-Hwan Cho performed most of the experiments. Kiyoon Kang analyzed the data; Sang-Il Bae performed GA measurement. Jinku Kang, Sua Cho, Daewon Kim, So-Yon Park, Gary Stacey, Nam-Chon Paek, and Sung-Hwan Cho wrote the manuscript. Kiyoon Kang, Daewon Kim, So-Yon Park, Gary Stacey, Nam-Chon Paek, and Sung-Hwan Cho acquired funding. Nam-Chon Paek, and Sung-Hwan Cho designed and supervised the projects and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr Katalin Toth (INARI company) for valuable advice and helpful comments. This study was supported by the Brain Pool Program funded by the Ministry of Science and Information and Communication Technology through the National Research Foundation of Korea (Grant Nos. 2022H1D3A2A01096185 and RS-2024-00410063 to N.C.P. and S.H.C.), the Basic Science Research Program through the National Research Foundation of Korea (Grant No. RS-2023-00247376 to S.H.C.), the Chungbuk National University National University Development Project (NUDP) program (2025, to S.H.C.), the National Research Foundation of Korea(NRF) (Grant No. RS-2025-02216304 to N.C.P.), the National Research Foundation of Korea (RS-2024-00452677 to K.K.), the Learning \u0026amp; Academic research institution for Master’s, PhD students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2023-00301974 to D.K.), the US Department of Agriculture’s National Institute of Food and Agriculture (Grant No. USDA-AFRI-2023-67013-39896 to S.Y.P.), and the National Science Foundation (Grant No. IOS-PGRP-2348319 to S.Y.P.), the US National Science Foundation Plant Genome Program (Grant No. IOS-2048410 to G.S.), and the US National Institute of General Medical Sciences of the National Institutes of Health (Grant No. R01GM121445 to G.S.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare we have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaud S, Wuill\u0026egrave;me S, Lemoine R et al (2005) The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis. Plant J 43:824\u0026ndash;836. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2005.02496.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2005.02496.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhaskar A, Paul LK, Sharma E (2021) OsRR6, a type-A response regulator in rice, mediates cytokinin, light and stress responses when over-expressed in Arabidopsis. Plant Physiol Biochem 161:98\u0026ndash;112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plaphy.2021.01.047\u003c/span\u003e\u003cspan address=\"10.1016/j.plaphy.2021.01.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBraun DM (2022) Phloem Loading and Unloading of Sucrose: What a Long, Strange Trip from Source to Sink. Annu Rev Plant Biol 73:553\u0026ndash;584. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-arplant-070721-083240\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-070721-083240\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroucke E, Dang TTV, Li Y et al (2023) SnRK1 inhibits anthocyanin biosynthesis through both transcriptional regulation and direct phosphorylation and dissociation of the MYB / bHLH / TTG1 MBW complex. Plant J 115:1193\u0026ndash;1213. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.16312\u003c/span\u003e\u003cspan address=\"10.1111/tpj.16312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroun P (2005) Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr Opin Plant Biol 8:272\u0026ndash;279. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pbi.2005.03.006\u003c/span\u003e\u003cspan address=\"10.1016/j.pbi.2005.03.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandler JW (2016) Auxin response factors. Plant Cell Environ 39:1014\u0026ndash;1028. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pce.12662\u003c/span\u003e\u003cspan address=\"10.1111/pce.12662\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCho S-H, T\u0026oacute;th K, Kim D et al (2022) Activation of the plant mevalonate pathway by extracellular ATP. Nat Commun 13:450. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-022-28150-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-022-28150-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClough SJ, Bent AF (1998) Floral dip: a simplified method for \u003cem\u003eAgrobacterium\u003c/em\u003e -mediated transformation of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant J 16:735\u0026ndash;743. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-313x.1998.00343.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.1998.00343.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDas PK, Shin DH, Choi S-B, Park Y-I (2012) Sugar-Hormone Cross-Talk in Anthocyanin Biosynthesis. Mol Cells 34:501\u0026ndash;508. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10059-012-0151-x\u003c/span\u003e\u003cspan address=\"10.1007/s10059-012-0151-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeroles S (2008) Anthocyanin Biosynthesis in Plant Cell Cultures: A Potential Source of Natural Colourants. In: Winefield C, Davies K, Gould K (eds) Anthocyanins. Springer New York, New York, pp 108\u0026ndash;167\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlores-P\u0026eacute;rez \u0026Uacute;, P\u0026eacute;rez-Gil J, Closa M et al (2010) PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) Integrates the Regulation of Sugar Responses with Isoprenoid Metabolism in Arabidopsis. Mol Plant 3:101\u0026ndash;112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/mp/ssp100\u003c/span\u003e\u003cspan address=\"10.1093/mp/ssp100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFukazawa J, Teramura H, Murakoshi S et al (2014) DELLAs Function as Coactivators of GAI-ASSOCIATED FACTOR1 in Regulation of Gibberellin Homeostasis and Signaling in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Cell 26:2920\u0026ndash;2938. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.114.125690\u003c/span\u003e\u003cspan address=\"10.1105/tpc.114.125690\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53:814\u0026ndash;827. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2007.03373.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2007.03373.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrotewold E, THE GENETICS AND BIOCHEMISTRY OF FLORAL PIGMENTS (2006) Annu Rev Plant Biol 57:761\u0026ndash;780. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.arplant.57.032905.105248\u003c/span\u003e\u003cspan address=\"10.1146/annurev.arplant.57.032905.105248\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0031-9422(00)00235-1\u003c/span\u003e\u003cspan address=\"10.1016/S0031-9422(00)00235-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHichri I, Barrieu F, Bogs J et al (2011) Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot 62:2465\u0026ndash;2483. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/erq442\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erq442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHolton TA, Cornish EC (1995) Genetics and Biochemistry of Anthocyanin Biosynthesis. Plant Cell 1071\u0026ndash;1083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.7.7.1071\u003c/span\u003e\u003cspan address=\"10.1105/tpc.7.7.1071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJi J, Yang L, Fang Z et al (2022) Plant SWEET Family of Sugar Transporters: Structure, Evolution and Biological Functions. Biomolecules 12:205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom12020205\u003c/span\u003e\u003cspan address=\"10.3390/biom12020205\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJulius BT, Leach KA, Tran TM et al (2017) Sugar Transporters in Plants: New Insights and Discoveries. Plant Cell Physiol 58:1442\u0026ndash;1460. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/pcp/pcx090\u003c/span\u003e\u003cspan address=\"10.1093/pcp/pcx090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarppinen K, Tegelberg P, H\u0026auml;ggman H, Jaakola L (2018) Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits. Front Plant Sci 9:1259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2018.01259\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2018.01259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhoo HE, Azlan A, Tang ST, Lim SM (2017) Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food Nutr Res 61:1361779. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/16546628.2017.1361779\u003c/span\u003e\u003cspan address=\"10.1080/16546628.2017.1361779\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKnight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8:489\u0026ndash;503. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.8.3.489\u003c/span\u003e\u003cspan address=\"10.1105/tpc.8.3.489\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLaFountain AM, Yuan Y (2021) Repressors of anthocyanin biosynthesis. New Phytol 231:933\u0026ndash;949. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.17397\u003c/span\u003e\u003cspan address=\"10.1111/nph.17397\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi W-F, Mao J, Yang S-J et al (2018) Anthocyanin accumulation correlates with hormones in the fruit skin of \u0026lsquo;Red Delicious\u0026rsquo; and its four generation bud sport mutants. BMC Plant Biol 18:363. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-018-1595-8\u003c/span\u003e\u003cspan address=\"10.1186/s12870-018-1595-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Lei W, Zhou Z et al (2023) Transcription factor GLK1 promotes anthocyanin biosynthesis via an MBW complex-dependent pathway in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. JIPB 65:1521\u0026ndash;1535. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jipb.13471\u003c/span\u003e\u003cspan address=\"10.1111/jipb.13471\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Van Den Ende W, Rolland F (2014) Sucrose Induction of Anthocyanin Biosynthesis Is Mediated by DELLA. Mol Plant 7:570\u0026ndash;572. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/mp/sst161\u003c/span\u003e\u003cspan address=\"10.1093/mp/sst161\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLloyd JC, Zakhleniuk OV (2004) Responses of primary and secondary metabolism to sugar accumulation revealed by microarray expression analysis of the Arabidopsis mutant, pho3. J Exp Bot 55:1221\u0026ndash;1230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/erh143\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erh143\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoreti E, Povero G, Novi G et al (2008) Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in \u003cem\u003eArabidopsis\u003c/em\u003e. New Phytol 179:1004\u0026ndash;1016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1469-8137.2008.02511.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1469-8137.2008.02511.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeyer S, Melzer M, Truernit E et al (2000) AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer. Plant J 24:869\u0026ndash;882. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-313x.2000.00934.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.2000.00934.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiziorko HM (2011) Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys 505:131\u0026ndash;143. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.abb.2010.09.028\u003c/span\u003e\u003cspan address=\"10.1016/j.abb.2010.09.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120\u0026ndash;1123. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btu743\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btu743\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakata M, Mitsuda N, Herde M A bHLH-Type Transcription Factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED et al (2013) MYC2-LIKE1, Acts as a Repressor to Negatively Regulate Jasmonate Signaling in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Cell 25:1641\u0026ndash;1656. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.113.111112\u003c/span\u003e\u003cspan address=\"10.1105/tpc.113.111112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNi J, Premathilake AT, Gao Y et al (2021) Ethylene-activated PpERF105 induces the expression of the repressor‐type R2R3‐MYB gene \u003cem\u003ePpMYB140\u003c/em\u003e to inhibit anthocyanin biosynthesis in red pear fruit. Plant J 105:167\u0026ndash;181. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.15049\u003c/span\u003e\u003cspan address=\"10.1111/tpj.15049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePulido P, Perello C, Rodriguez-Concepcion M (2012) New Insights into Plant Isoprenoid Metabolism. Mol Plant 5:964\u0026ndash;967. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/mp/sss088\u003c/span\u003e\u003cspan address=\"10.1093/mp/sss088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiou C, Tourte Y, Lacroute F, Karst F (1994) Isolation and characterization of a cDNA encoding Arabidopsis thaliana mevalonate kinase by genetic complementation in yeast. Gene 148:293\u0026ndash;297. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0378-1119(94)90701-3\u003c/span\u003e\u003cspan address=\"10.1016/0378-1119(94)90701-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuiz-Sola M\u0026Aacute;, Coman D, Beck G et al (2016) \u003cem\u003eArabidopsis\u003c/em\u003e GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis‐related isoprenoids. New Phytol 209:252\u0026ndash;264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.13580\u003c/span\u003e\u003cspan address=\"10.1111/nph.13580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSakr S, Wang M, D\u0026eacute;dald\u0026eacute;champ F et al (2018) The Sugar-Signaling Hub: Overview of Regulators and Interaction with the Hormonal and Metabolic Network. IJMS 19:2506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms19092506\u003c/span\u003e\u003cspan address=\"10.3390/ijms19092506\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSauer N, Ludwig A, Knoblauch A et al (2004) \u003cem\u003eAtSUC8\u003c/em\u003e and \u003cem\u003eAtSUC9\u003c/em\u003e encode functional sucrose transporters, but the closely related \u003cem\u003eAtSUC6\u003c/em\u003e and \u003cem\u003eAtSUC7\u003c/em\u003e genes encode aberrant proteins in different \u003cem\u003eArabidopsis\u003c/em\u003e ecotypes. Plant J 40:120\u0026ndash;130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2004.02196.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-313X.2004.02196.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSauer N, Stolz J (1994) SUC1 and SUC2: two sucrose transporters from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; expression and characterization in baker\u0026rsquo;s yeast and identification of the histidine-tagged protein. Plant J 6:67\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-313X.1994.6010067.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313X.1994.6010067.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSivitz AB, Reinders A, Ward JM (2008) Arabidopsis Sucrose Transporter AtSUC1 Is Important for Pollen Germination and Sucrose-Induced Anthocyanin Accumulation. Plant Physiol 147:92\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.108.118992\u003c/span\u003e\u003cspan address=\"10.1104/pp.108.118992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSolfanelli C, Poggi A, Loreti E et al (2006) Sucrose-Specific Induction of the Anthocyanin Biosynthetic Pathway in Arabidopsis. Plant Physiol 140:637\u0026ndash;646. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.105.072579\u003c/span\u003e\u003cspan address=\"10.1104/pp.105.072579\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStadler R, Sauer N (2019) The AtSUC2 Promoter: A Powerful Tool to Study Phloem Physiology and Development. In: Liesche J (ed) Phloem. Springer New York, New York, pp 267\u0026ndash;287\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTeng S, Keurentjes J, Bentsink L et al (2005) Sucrose-Specific Induction of Anthocyanin Biosynthesis in Arabidopsis Requires the MYB75/PAP1 Gene. Plant Physiol 139:1840\u0026ndash;1852. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.105.066688\u003c/span\u003e\u003cspan address=\"10.1104/pp.105.066688\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu XN, Sanchez Rodriguez C, Pertl-Obermeyer H et al (2013) Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis. Mol Cell Proteom 12:2856\u0026ndash;2873. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/mcp.M113.029579\u003c/span\u003e\u003cspan address=\"10.1074/mcp.M113.029579\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570\u0026ndash;3575. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1420294112\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1420294112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie Y, Tan H, Ma Z, Huang J (2016) DELLA Proteins Promote Anthocyanin Biosynthesis via Sequestering MYBL2 and JAZ Suppressors of the MYB/bHLH/WD40 Complex in Arabidopsis thaliana. Mol Plant 9:711\u0026ndash;721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molp.2016.01.014\u003c/span\u003e\u003cspan address=\"10.1016/j.molp.2016.01.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXin P, Guo Q, Li B et al (2020) A Tailored High-Efficiency Sample Pretreatment Method for Simultaneous Quantification of 10 Classes of Known Endogenous Phytohormones. Plant Commun 1:100047. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.xplc.2020.100047\u003c/span\u003e\u003cspan address=\"10.1016/j.xplc.2020.100047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu P, Wu L, Cao M et al (2021) Identification of MBW Complex Components Implicated in the Biosynthesis of Flavonoids in Woodland Strawberry. Front Plant Sci 12:774943. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2021.774943\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2021.774943\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu Q, Yin S, Ma Y et al (2020) Carbon export from leaves is controlled via ubiquitination and phosphorylation of sucrose transporter SUC2. Proc Natl Acad Sci USA 117:6223\u0026ndash;6230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1912754117\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1912754117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu Z, Mahmood K, Rothstein SJ (2017) ROS Induces Anthocyanin Production Via Late Biosynthetic Genes and Anthocyanin Deficiency Confers the Hypersensitivity to ROS-Generating Stresses in Arabidopsis. Plant Cell Physiol 58:1364\u0026ndash;1377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/pcp/pcx073\u003c/span\u003e\u003cspan address=\"10.1093/pcp/pcx073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang X, Jiang X, Yan W et al (2021) The Mevalonate Pathway Is Important for Growth, Spore Production, and the Virulence of Phytophthora sojae. Front Microbiol 12:772994. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2021.772994\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2021.772994\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoon J, Cho L-H, Tun W et al (2021) Sucrose signaling in higher plants. Plant Sci 302:110703. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plantsci.2020.110703\u003c/span\u003e\u003cspan address=\"10.1016/j.plantsci.2020.110703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anthocyanin, Arabidopsis thaliana, Gibberellic acid, Mevalonate pathway, MVK, SUC1","lastPublishedDoi":"10.21203/rs.3.rs-8124382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8124382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnthocyanins, a class of flavonoid pigments, function as crucial modulators of plant responses to environmental stressors by mitigating oxidative damage and facilitating cellular adaptation. Anthocyanin biosynthesis is tightly regulated by transcriptional networks that respond to developmental cues and external stimuli. Here, we identify MEVALONATE KINASE (MVK), a key enzyme of the cytosolic isoprenoid biosynthesis pathway, as a repressor of sucrose-induced anthocyanin production in Arabidopsis. Loss-of-function\u003cem\u003e mvk \u003c/em\u003emutants show increased anthocyanin levels compared to wild-type (WT) plants under high sucrose conditions. The expression of anthocyanin biosynthetic and regulatory genes, such as \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, and \u003cem\u003eMYB75/PAP1\u003c/em\u003e, is increased in \u003cem\u003emvk-1\u003c/em\u003e mutants grown in the presence of high sucrose. \u003cem\u003emvk-1\u003c/em\u003e mutants exhibited elevated sucrose accumulation through upregulation of sucrose transporters compared to WT under high sucrose conditions. Furthermore, reduced levels of gibberellins in \u003cem\u003emvk-1\u003c/em\u003e mutants resulted in the stabilization of DELLA proteins, which are known repressors of gibberellin signaling, thereby facilitating sucrose-induced anthocyanin accumulation. Our findings demonstrate that MVK negatively regulates sucrose-induced anthocyanin biosynthesis by modulating sucrose transport and gibberellin homeostasis in Arabidopsis.\u003c/p\u003e","manuscriptTitle":"MEVALONATE KINASE represses anthocyanin biosynthesis via sucrose transporters and gibberellin synthesis pathways in Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 09:02:54","doi":"10.21203/rs.3.rs-8124382/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f181b26f-0b6c-4209-bacd-3dee53d647a2","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-31T09:02:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 09:02:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8124382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8124382","identity":"rs-8124382","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}