RsWRKY44 participated in anthocyanin biosynthesis regulation in radish through interaction with RsMYB1a

RsWRKY44 participated in anthocyanin biosynthesis regulation in radish through interaction with RsMYB1a | 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 RsWRKY44 participated in anthocyanin biosynthesis regulation in radish through interaction with RsMYB1a Biao Lai, Chenxi Gao, Li Jiang, Li Wen, Xushuo Zhang, Wei Shen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5445538/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2025 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract The regulation of anthocyanin biosynthesis in radish is primarily controlled by RsMYB1a and RsbHLH4, while the involvement of other factors in this process is not well understood. This study identified a WRKY transcription factor, RsWRKY44, as a key player in anthocyanin biosynthesis regulation. The expression of RsWRKY44 showed a strong correlation with anthocyanin content across different radish cultivars. RsWRKY44 was found to be expressed in the nuclei and exhibit transactivation activity. It was observed that only when RsWRKY44 was co-expressed with RsMYB1a, anthocyanin accumulation was induced in tobacco leaves, while RsWRKY44 alone did not. Additionally, RsWRKY44, along with RsMYB1a, activated the expression of tobacco endogenous anthocyanin biosynthesis regulatory genes NtAN1a and NtAN1b , as well as the structural genes NtCHS , NtCHI , NtDFR , NtF3H , NtANS , NtUFGT in transgenic tobacco. BiFC, FLC and DLA assays confirmed the interaction between RsWRKY44 and RsMYB1a leading to the activation of radish genes RsCHI and RsUFGT , promoting anthocyanin biosynthesis. This study sheds light on a new molecular mechanism of RsWRKY44 involved in anthocyanin biosynthesis regulation in radish. Radish anthocyanin RsWRKY44 RsMYB1a Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Message RsWRKY44 transcription factor, associated with anthocyanin biosynthesis in different radish cultivars, highly facilitates the activation of RsCHI and RsUFGT genes through its interaction with RsMYB1a, thereby promoting anthocyanin production. Introduction Radishes are globally recognized and widely grown as a horticultural crop that has high nutritional and economic value. Radish is low in calories and rich in calcium, magnesium, copper, manganese, potassium, vitamin B6, vitamin C, folate, fat and fatty related compounds, flavonoids, non-flavonoids polyphenols et al. (Gamba, et al. 2021 ). Radishes exhibit a diverse color palette, ranging from stark white to vibrant green, and extending to vivid red, deep purple, and even black. The rich red, purple, and black hues observed in radishes are attributed to the presence of anthocyanin pigments. Anthocyanins are a type of flavonoid, a class of water-soluble compounds with antioxidant effects. Anthocyanins, the main pigments in plants, originate from the phenylpropanoid pathway. They are synthesized through a series of reactions catalyzed by enzymes such as CHS, CHI, F3H, DFR, ANS, and UFGT. These enzymes are encoded by structural genes, leading to the production of various anthocyanins(Jaakola 2013 ). Research shows that the regulation of anthocyanin biosynthesis in plants primarily occurs at the transcriptional level. Three main classes of transcription factors are involved in regulating anthocyanin biosynthesis, namely MYB, basic Helix-Loop-Helix (bHLH), and WD40 proteins. These three types of transcription factors interact to form complexes that collectively regulate the expression of structural genes involved in anthocyanin biosynthesis, thereby affecting the activity of enzymes and ultimately impacting the accumulation of anthocyanins (Jaakola 2013 ; Petroni and Tonelli 2011 ). In the model plant Arabidopsis , PAP1 (MYB) and PAP2 (MYB) can interact with R/B-like bHLH proteins to activate the promoter of the anthocyanin biosynthetic structural gene DFR(Zimmermann, et al. 2004 ). AtPAP1 and AtPAP2 interact with the distinct bHLH transcription factors AtEGL3 and AtGL3 to regulate diverse anthocyanin biosynthesis pathway genes in Arabidopsis (Gonzalez, et al. 2008 ; Zhang, et al. 2003 ). In apple, MdMYB1 and AtEGL3 can cooperate to activate the promoters of MdDFR and MdUFGT (Takos, et al. 2006 ). In bayberry, when MrMYB1 and AtbHLH are co-expressed, the activity of the AtDFR promoter is significantly increased. MrMYB1 can interact with MrbHLH1 to regulate most of the structural genes in the anthocyanin biosynthetic pathway(Liu, et al. 2013 ; Niu, et al. 2010 ). Our previous research suggested that RsMYB1 can interact with RsbHLH4, which in turn regulates the accumulation of anthocyanin in radish taproots. (Lai, et al. 2019 ). MBW (MYB-bHLH-WD40) complex plays a crucial role in regulating anthocyanin accumulation in plants, and all three components are indispensable. In addition to the MBW transcription factor complex, studies have also found that other types of transcription factors such as NAC, MADS, bZIP, and SPL, especially WRKY, are extensively involved in the transcriptional regulation of plant anthocyanin biosynthesis(Ma, et al. 2021 ). PyWRKY26 can form a complex with PybHLH3, directly binding to the key anthocyanin regulatory gene PyMYB114 promoter to promote the transcription of PyMYB114, leading to anthocyanin accumulation in red-skinned pear fruit(Li, et al. 2020 ). PbWRKY75 in pear promotes anthocyanin accumulation by binding to the PbDFR , PbUFGT , and PbMYB10b promoters to enhance their transcription (Cong, et al. 2021 ). In this study, we identified a WRKY transcription factor named RsWRKY44 was highly corelated with anthocyanin content in different radish skin and flesh. RsWRKY44 interacted with RsMYB1a, a key promoter of anthocyanin synthesis enhanced anthocyanin accumulation when transient expressed in tabaco leaf. Altogether, our findings provide novel insights into the functions of RsWRKY44 in anthocyanin synthesis in radish taproot. Materials and methods Plant materials Twelve radish cultivars, including ‘Chuanxin’, ‘Yanzhi’, ‘Shawo’, ‘Xiari’, and ‘Chunbulao’, ‘Zimeiren', ‘Sichuan Hongpi’, ‘Bingqilin’, and ‘Chongqing Hongpi’, ‘Xinlimei’, ‘Hongxin 1–1’ and ‘Shaguan 1’ were grown in the experimental field of Yangtze Normal University. The radish samples were all carefully divided into skin and flesh components, then chopped into small sections. Following this, they were promptly frozen using liquid nitrogen. Subsequently, the samples were stored at a temperature of -80 degrees for further study. Determination of total anthocyanin contents The contents of anthocyanins in the radish tissues were determined as previously described by our group (Wei et al. 2011 ). RNA and DNA extraction RNA and DNA extraction The isolation of the total RNA from the tobacco leaves, as well as from the radish skin and flesh, was performed utilizing a HiPure Total RNA Mini Kit (Magen, China). The isolated RNA subsequently underwent gel electrophoresis for integrity verification. The quantification of the RNA concentration was executed using an UV5Nano spectrophotometer (Mettler Toledo, USA). DNA from the radish leaves was extracted using a DNA isolation kit (Aidlab, China) and subsequently utilized for the isolation of promoter sequences. qRT‑PCR The first-strand complementary DNA (cDNA) was synthesized from 2 µg of total RNA utilizing oligo (dT) primers using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). The synthesized cDNA was subsequently employed as a template for gene amplification and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The qRT-PCR assays were carried out using the QuantStudio 3 system (Applied Biosystems, USA) as previously established methodology (the primers are detailed in Supplementary Table S1 ) (Lai, et al. 2021 ). Transient infiltration assay The complete coding sequences of RsWRKY44 complementary DNA (cDNA) were amplified from the 'Hongxin 1' flesh cDNA utilizing a primer with incorporated attB sites (refer to Supplementary Table S1 for primer sequences). The purified DNA fragments were subsequently recombined with the pDONR/Zeo vector (Invitrogen, USA) via the BP Clonase™ II Enzyme mix (Invitrogen). This process generated the entry clones pDONR/Zeo-RsWRKY44. The transient expression vectors pEAQ-RsWRKY44 were constructed by recombining the entry clones with pEAQ-HT-DEST1 utilizing the LR Clonase™ II Enzyme mix (Invitrogen). The constructs pEAQ-RsMYB1a and pEAQ-RsbHLH4 were previously established (Lai, et al. 2021 ). These transient expression vectors were subsequently introduced into the Agrobacterium tumefaciens strain GV3101. A. tumefaciens harboring pEAQ-RsMYB1a, pEAQ-RsbHLH4, and pEAQ-RsWRKY44 were infiltrated into tobacco leaves either individually or collectively. The leaves infiltrated with a pEAQ-HT construct served as the control group. Photographs were captured five days post-infiltration. Bimolecular fluorescence complementation (BiFC) assay pEarleyGate202-RsWRKY44-YN were constructed by the recombination of the entry clone with pEarleyGate202-YN using an LR Clonase™ II Enzyme mix (Invitrogen). A. tumefaciens harboring both pEarleyGate201-RsMYB1a-YC and pEarleyGate202-RsbHLH4-YN, or maintained separately with an empty vector, were infiltrated into Nicotiana benthamiana plant leaves. The YFP signal was observed 2 days after infiltration using a fluorescence microscope (OLYMPUS, BX43, Japan). Firefly Luciferase Complementation Imaging Assay (FLC) assay pEAQ-RsWRKY44-cLUC and pEAQ-nLUC-RsMYB1a were constructed by the recombination of the entry clone with pEAQ-cLUC-GW and pEAQ-nLUC-GW using an LR Clonase™ II Enzyme mix (Invitrogen). A. tumefaciens harboring both pEAQ-RsWRKY44-cLUC and pEAQ-nLUC-RsMYB1a, or maintained separately with an empty vector, were infiltrated into N. benthamiana plant leaves. The luciferase signal was observed 2 days after infiltration using a cold CCD camara (Biotanon, Tanon 5200, China). Dual luciferase assay Specific primers with recombination sites were designed to amplify the RsCHI and RsUFGT promoter based on the radish genome sequence. The primers are shown in Supplementary Table S1 . The amplified sequences were then inserted into the pGreenII 0800-LUC vector at the 5′ end of a LUC gene using the Gibson assembling method. Infiltration, transient expression analysis, and the determination of the enzyme activities of LUC and REN were conducted as described by Hellens et al. using 6-week-old N. benthamiana leaves (Hellens, et al. 2005 ). Results The identification of RsWRKY44 in radish Through co-expression analysis across different radish cultivars (not published), we identified a WRKY transcription factor, designated as RsWRKY44, whose expression was highly correlated with radish color. The open reading frame of RsWRKY44 spans 1236 base pairs, encoding a 411-residue polypeptide. The protein features two conserved WRKY domains (WRKYGQK) and two CX 4 CX 23 HXH zinc finger domains, which are characteristic of group I WRKY superfamily members, including RsWRKY44 (Fig. 1 B). Multiple sequence alignment revealed that RsWRKY44 shares 73% and 75% similarity at the amino acid level with Arabidopsis AtTTG2 and Brassica napus BnTTG2, respectively. These proteins are known to be involved in the regulation of proanthocyanidin biosynthesis in Brassica napus and trichome patterning in Arabidopsis (Johnson, et al. 2002 ; Qu, et al. 2013 ). Phylogenetic tree analysis indicates that RsWRKY44 is closely related to BnTTG2 and other members associated with anthocyanin biosynthesis, such as AcWRKY44 and VvWRKY26 (Fig. 1 A) (Amato, et al. 2017 ; Peng, et al. 2020 ). The subcellular localization of RsWRKY44 was assessed by engineering a fusion construct comprised of the RsWRKY44 coding sequence and the gene encoding green fluorescent protein (GFP). This construct was transiently expressed within the leaves of N. benthamiana . Fluorescence microscopy, facilitated by GFP detection, manifested the exclusive presence of RsWRKY44–GFP within the nuclei, contrarily to the ubiquitous distribution of lone GFP throughout the entire cell (Fig. 1 C). This evidence strongly substantiates the classification of RsWRKY44 as a nuclear protein. A transcription activation assay was conducted in yeast to identify if RsWRKY44 has transcriptional activity. pGBKT7-RsWRKY44 with the empty pGADT7 vector were co-transformed into yeast strain Y2H gold. All transformants survived on SD/-Trp/-Leu/-His media with 200 ng/mL AbA and the growth was completely inhibited with 300 ng/mL AbA (Fig. 1 D). These outcomes suggest that RsWRKY44 has the capacity to activate transcription in yeast cells. The expression of RsWRKY44 was highly correlated with anthocyanin accumulation in radish To gain further insights into the expression pattern of RsWRKY44, real-time PCR analysis was conducted on the skin and flesh tissues of 10 distinct radish cultivars. As depicted in Fig. 2 , ‘Chuanxin’ exhibited the highest anthocyanin content in the skin, while both the skin and flesh of ‘Yanzhi’ displayed elevated levels of anthocyanin. Conversely, ‘Shawo’, ‘Xiari’, and ‘Chunbulao’ exhibited no anthocyanin accumulation in either the skin or flesh. Remarkably, ‘Zimeiren' and 'Yanzi’ demonstrated substantial anthocyanin accumulation in both skin and flesh tissues. Anthocyanins were primarily localized in the skin of ‘Sichuan Hongpi’, ‘Bingqilin’, ‘Chuanxin’, and ‘Chongqing Hongpi’, with little to no presence in the flesh. Notably, ‘Xinlimei’ only exhibited anthocyanin accumulation in the flesh, while the skin remained devoid of anthocyanins. Additionally, we analyzed the expression levels of key genes involved in the anthocyanin biosynthesis pathway, including RsCHS , RsCHI , RsF3H , RsANS , RsDFR , and RsUFGT , in these tissue samples. As depicted in Fig. 3 , the expression of six anthocyanin biosynthesis structural genes is only evident in the skin and flesh of radishes where anthocyanin has accumulated. The expression of RsWRKY44 shown the same expression pattern with two key regulatory genes RsMYB1a and RsbHLH4 , indicating that RsWRKY44 may related to anthocyanin biosynthesis regulation in radish taproot. Co-expression of RsWRKY44 with RsMYB1a induced anthocyanin accumulation in tobacco leaf To further investigate the function of RsWRKY44 , the pSAK277-RsWRKY44 vector was transiently infiltrated into tobacco leaves using Agrobacterium . However, no anthocyanin was detected in tobacco leaves when RsWRKY44 was expressed alone. Based on previous studies indicating the importance of RsMYB1a as a key regulatory gene in radish, we co-transformed RsWRKY44 with RsMYB1a in tobacco leaves. While individual expression of RsMYB1a did not induce anthocyanin synthesis, co-expression of RsMYB1a with RsWRKY44 significantly promoted anthocyanin synthesis. This induction was lower compared to the combined expression of RsMYB1a with RsbHLH4 (Fig. 4 ). The expression of genes involved in anthocyanin biosynthesis and regulation in tobacco was further examined. It was observed that NtAN2 , which encodes a MYB transcription factor, was not expressed in tobacco leaves (data not shown). NtAN1a and NtAN1b , encoding bHLH transcription factors, exhibited upregulation only when RsMYB1a was co-expressed with RsWRKY44 , not when expressed alone (Fig. 4 A and B). Moreover, the structural genes involved in anthocyanin biosynthesis, such as NtCHS , NtCHI , NtF3H , NtDFR , NtANS and NtUFGT , were all upregulated when RsMYB1a was transformed with RsWRKY44 . The control experiment, involving RsMYB1a with RsbHLH4 , significantly induced anthocyanin accumulation and the expression of related structural and regulatory genes (Fig. 4 C). These findings suggest that RsWRKY44 may regulate anthocyanin biosynthesis by interacting with RsMYB1a, subsequently activating bHLH and structural genes. RsWRKY44 interact RsMYB1a activate anthocyanin biosynthesis pathway genes Considering the observed correlation between the expression patterns of RsWRKY44 and RsMYB1a in different radish cultivars, alongside the demonstrated promotion of anthocyanin accumulation in tobacco leaves through their co-expression, we hypothesize that RsWRKY44 and RsMYB1 proteins could potentially interact to form a complex. BiFC (Bimolecular fluorescence complementation) technology was used to analyze whether RsWRKY44 and RsMYB1 could interact with each other. We fused RsWRKY44 with NYFP and RsMYB1 with CYFP, and then injected them into the leaves of N. benthamiana through Agrobacterium -mediated transient expression. The results showed that when RsWRKY44-NYFP and RsMYB1-CYFP were co-injected, yellow fluorescent protein could be observed in the nuclei of N. benthamiana leaf cells. This indicates that RsWRKY44 and RsMYB1 can interact with each other in plant cells. The promoter of radish anthocyanin biosynthesis pathway gene RsCHI and RsUFGT were drive LUC reporter gene. As shown in Fig. 6 , when RsMYB1a and RsWRKY44 were expressed independently, the RsCHI promoter was not activated. However, a combination of RsMYB1a and RsWRKY44 significantly activated the RsCHI promoter. Similarly, RsMYB1a on its own could activate the RsUFGT promoter, but RsWRKY44 could not. The RsUFGT promoter was highly activated when RsMYB1a and RsWRKY44 were combined. RsMYB1a is not the target of RsWRKY44 ‘Hongxin 1–1’ and ‘Shaguan 1’ are natural radish varieties with dark red skin and white flesh (pictures can be found at Lai et al, 2019 ). The expression of RsMYB1a and RsWRKY44 was examined in ‘Hongxin 1–1’ and ‘Shaguan 1’ shin and flesh. RsMYB1a was found to be expressed normally in both skin and flesh, however, no expression of RsWRKY44 was detected in the white flesh of ‘Hongxin 1–1’ and ‘Shaguan 1’. These results suggested that the transcription of RsMYB1a may not regulated by RsWRKY44. Discussion RsWRKY44: a non-light-dependent regulator of anthocyanin biosynthesis in radish Anthocyanins are a group of secondary metabolites that give plants their vibrant red, blue, and purple colors. They also have antioxidant properties and play a role in defense against pathogens and environmental stresses. WRKY family transcription factors were known to play a crucial role in the regulation of plant defense responses, development, and metabolism, including anthocyanin biosynthesis(Cappellini, et al. 2021; Jiang, et al. 2017 ). In our transcriptome data (not published), we found a WRKY transcription factor named RsWRKY44 was highly related anthocyanin content in different radish cultivars. RsWRKY44 has two conserved WRKY domains (WRKYGQK) and two CX4CX23HXH zinc finger domains which belongs to group I WRKY superfamily. Phylogenetic tree analysis shows that RsWRKY44 is close to AtTTG2 which is involved in the regulation of proanthocyanidin and mucilage biosynthesis in the seed coat, trichome differentiation, and root hair patterning (Johnson, et al. 2002 ; Pesch, et al. 2014 ). The PhPH3 gene in petunia has a similar function to its homologous gene AtTTG2 in Arabidopsis. However, PhPH3 plays an important role in petunias by regulating vacuolar acidification in the cells of the petal, thereby influencing the color of the flower (Verweij, et al. 2016 ). In this study, the expression of RsWRKY44 was analyzed in the shin and flesh of ten different radish cultivars. The expression of RsWRKY44 was highly correlated with anthocyanin content was confirmed by real-time PCR assay. The expression of anthocyanin biosynthesis structural genes in these radish samples was also examined. The results showed that the transcription levels of RsCHS , RsCHI , RsF3H , RsDFR , RsANS , and RsUFGT were all relatively higher in radish skin or flesh rich in anthocyanin. These results of structural genes exhibit a similar expression pattern to that of RsWRKY44 , indicating RsWRKY44 may played important role in radish anthocyanin accumulation regulation. Weighted Gene Co-expression Network Analysis (WGCNA) identified key anthocyanin regulators StWRKY70 and SmWRKY44 in potato and eggplant, their expression showed high correlation with anthocyanin biosynthetic genes (He, et al. 2021 ; Zhang, et al. 2024a). Despite radish roots growing underground, in certain cultivars such as ‘Yanzhi’ and ‘Zimeiren’, anthocyanin accumulation in both the skin and flesh remains unaffected, and the expression of RsWRKY44 , RsMYB1a , and other structural genes involved in anthocyanin biosynthesis remains active. However, in red-skinned pears, anthocyanin biosynthesis was induced by light, light-induced transcription factor PpWRKY44 promotes light-triggered anthocyanin biosynthesis was regulated by the light signaling component PpBBX18 (Alabd, et al., 2022 ). The observation that Desiree potato tubers require light for anthocyanin accumulation despite growing underground, with StWRKY13 expression also induced by light (Zhang, et al., 2021 ). The RsWRKY44 - RsMYB1a protein complex regulates anthocyanin biosynthesis Our study indicates that transient expression of either RsWRKY44 or RsMYB1a alone in tobacco leaves does not lead to the accumulation of anthocyanin. Interestingly, RsWRKY44 appears to have an effect similar to that of RsbHLH4, it can help RsMYB1a to regulate anthocyanin biosynthesis (Lai, et al. 2020). Co-expression of RsMYB1a and RsWRKY44 upregulated the expression of not only structural genes but also NtAN1a and NtAN1b (two tobacco endogenous bHLH genes). These results suggest that the interaction between RsMYB1a and RsWRKY44 is critical for regulating anthocyanin biosynthesis in radish. This is because RsWRKY44 has the potential to help RsMYB1a enhance the activation of key structural genes, RsCHI and RsUGFT , involved in anthocyanin biosynthesis (Fig. 6 ). VvWRKY5 plays a positive regulatory role in grape wounding induced anthocyanin synthesis by interacting with VvMYBA1 and then enhance the activation of VvUFGT promoter (Zhang, et al. 2024b ). SmWRKY44 could interact with SmMYB1 promote the biosynthesis of anthocyanins in eggplant leaves and activating anthocyanin biosynthesis pathway genes such as SmCHS , SmF3H , SmDFR , and SmANS (He, et al., 2021 ). However, CitWRKY75 identified by WGCNA regulate early stage of blood orange and citrus juvenile tissues anthocyanin accumulation by bind to the promoter of CitRuby1 , a R2R3-MYB gene (Lu, et al. 2024 ). Functional divergence from orthologous WRKY proteins in anthocyanin regulation Studies have suggested that WRKY transcription factors are involved in the regulation of anthocyanin biosynthesis, the exact mechanisms are different and not fully understood in different plants. Several WRKY transcription factors in apples have been found to participate in the regulation of apple fruit anthocyanin biosynthesis. MdWRKY11 can bind to the promoters of MdMYB10 , MdMYB11 and MdUFGT to participate in anthocyanin biosynthesis regulation during red-flesh apple fruit development (Liu, et al. 2019 ; Wang, et al. 2018 ). Interaction between MdWRKY40 and MdMYB1 can promote the transcription of MdMYB1, inducing mechanical damage-induced anthocyanin biosynthesis in apple fruit (An, et al. 2019 ). UV-B-induced MdWRKY72 could bind to the promoter of MdHY5 and MdMYB1 to regulation anthocyanin accumulation when apple fruit under Ultraviolet-B radiation (Hu, et al. 2020 ). Under light induction, MdWRKY1 promotes the expression of the long non-coding RNA MdLNC499, which in turn promotes the transcription of MdERF109 which activates the expression of genes related to anthocyanin biosynthesis in apple (Ma, et al. 2021 ). Notably, in some radish cultivars, RsMYB1a is normally expressed, but RsWRKY44 remains unexpressed (Fig. 7 ), resulting in no anthocyanin accumulation. This suggests that RsWRKY44 may not act as an upstream regulator of RsMYB1a. Moreover, it also indicates that RsWRKY44 plays a crucial role in anthocyanin biosynthesis in radishes. The transient expression of RsWRKY44 alone in tobacco leaves does not induce the expression of anthocyanin biosynthesis and regulatory genes, as illustrated in Fig. 4 . This remains the case even when combined with RsMYB1a, which doesn't activate the key anthocyanin regulatory MYB gene NtAN2 (data not shown). Similarly, in radishes, we found that the transcription of RsMYB1a may not be activated by RsWRKY44, as shown in Fig. 7 . Given that radish taproot is an underground tissue not exposed to light, it's possible that light is unnecessary for anthocyanin biosynthesis. This could potentially explain why RsMYB1a does not require activation by RsWRKY44, which functions independently of light. In radish, RsWRKY44 primarily induces anthocyanin accumulation by interacting with RsMYB1a to form a complex, thereby activating the expression of structural genes involved in anthocyanin biosynthesis, such as RsCHI and RsUFGT . The expression of LhWRKY44 is light dependent which involved in anthocyanin accumulation regulation by interacting with LhMYBSPLATTER and activate its transcription in lily (Bi, et al. 2023 ). These results suggest that WRKYs could participate both directly and indirectly in the regulation of anthocyanin biosynthesis during plant development, under various environmental conditions and stress scenarios. On the other hand, some studies have suggested that certain WRKY proteins may negatively regulate anthocyanin biosynthesis. For instance, mutants of AtWRKY41 in Arabidopsis exhibit increased anthocyanin content in rosette leaves, and the homologous gene BnWRKY41-1 from Brassica napus can restore the phenotype of the AtWRKY41 mutant, indicating that WRKY41 can suppress anthocyanin biosynthesis (Duan, et al. 2018 ). MdWRKY41 forms a complex by interacting with MdMYB16 to inhibit the expression of MdMYB12 , MdLAR , MdUFGT , and MdDFR , thereby suppressing apple anthocyanin and proanthocyanidin synthesis (Mao, et al. 2021 ). AtWRKY33 directly binds to the AtDFR promoter to repress its expression and indirectly affects AtDFR activation by interacting with PAP1 to interfere with the MBW complex, negatively regulating anthocyanin production (Tao, et al., 2024 ). The regulation of anthocyanin biosynthesis by WRKY proteins is complex and may be influenced by various factors such as environmental conditions and developmental stage in different plants. Declarations Author contributions LND designed the research; BL, CXG, LJ, LW, XSZ, WS, YLX, HBY, FBC and PF performed the experiments and analyzed the data; BL and LND wrote the manuscript. All authors read and approved the final manuscript. Funding This study was supported by Chongqing Natural Science Foundation (Grant No. CSTB2022NSCQ-MSX0228), Chongqing Natural Science Foundation (Grant No. CSTC2021JCYJ-MSXMX0083) and Yangtze Normal University (Grant No. 2016KYQD20 and 2016XJQN06). Data availability The data underlying this article are available in the article and in its online supplemental material. Conflict of interest The authors declare that they have no conflict of interest. References Alabd A, Ahmad M, Zhang X, Gao YH, Peng L, Zhang L, Ni JB, Bai SL, Teng YW (2022) Light-responsive transcription factor PpWRKY44 induces anthocyanin accumulation by regulating PpMYB10 expression in pear. 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Plant Cell 33(10):3309–3330 Ma Y, Ma X, Gao X, Wu W, Zhou B (2021) Light induced regulation pathway of anthocyanin biosynthesis in plants. Int J Mol Sci 22(20):11116 Mao Z, Jiang H, Wang S, Wang Y, Yu L, Zou Q, Liu W, Jiang S, Wang N, Zhang Z, Chen X (2021) The MdHY5-MdWRKY41-MdMYB transcription factor cascade regulates the anthocyanin and proanthocyanidin biosynthesis in red-fleshed apple. Plant Sci 306:110848 Niu SS, Xu CJ, Zhang WS, Zhang B, Li X, Lin-Wang K, Ferguson IB, Allan AC, Chen KS (2010) Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry ( Myrica rubra ) fruit by a R2R3 MYB transcription factor. Planta 231(4):887–899 Peng Y, Thrimawithana AH, Cooney JM, Jensen DJ, Espley RV, Allan AC (2020) The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway. Sci Rep 10(1):14161 Pesch M, Dartan B, Birkenbihl R, Somssich IE, Hülskamp M (2014) Arabidopsis TTG2 regulates TRY expression through enhancement of activator complex-triggered activation. Plant Cell 26(10):4067–4083 Petroni K, Tonelli C (2011) Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci 181(3):219–229 Qu C, Fu F, Lu K, Zhang K, Wang R, Xu X, Wang M, Lu J, Wan H, Zhanglin T, Li J (2013) Differential accumulation of phenolic compounds and expression of related genes in black- and yellow-seeded Brassica napus. J Exp Bot 64(10):2885–2898 Takos AM, Jaffé FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol 142(3):1216–1232 Tao H, Gao F, Li LY, He YQ, Zhang XY, Wang MY, Wei J, Zhao Y, Zhang C, Wang QM, Hong GJ (2024) WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation. Plant Commun 5:100821 Verweij W, Spelt CE, Bliek M, de Vries M, Wit N, Faraco M, Koes R, Quattrocchio FM (2016) Functionally similar WRKY proteins regulate vacuolar acidification in petunia and hair development in Arabidopsis . Plant Cell 28(3):786–803 Wang N, Liu W, Zhang T, Jiang S, Xu H, Wang Y, Zhang Z, Wang C, Chen X (2018) Transcriptomic analysis of red-fleshed apples reveals the novel role of MdWRKY11 in flavonoid and anthocyanin biosynthesis. J Agric Food Chem 66(27):7076–7086 Wei Y, Hu F, Hu G, Li X, Huang X, Wang H (2011) Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn. PLoS ONE 6:e19455 Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A (2003) A network of redundant bHLH proteins functions in all TTG1 -dependent pathways of Arabidopsis . Development 130(20):4859–4869 Zhang Y, Pu Y, Zhang Y, Li K, Bai S, Wang J, Xu M, Liu S, Zhou Z, Wu Y, Hu R, Wu Q, Kear P, Du M, Qi J 2024a. Tuber transcriptome analysis reveals a novel WRKY transcription factor StWRKY70 potentially involved in potato pigmentation. Plant Physiol Biochem 213, 108792 Zhang Z, Chen C, Jiang C, Lin H, Zhao Y, Guo Y (2024b) VvWRKY5 positively regulates wounding-induced anthocyanin accumulation in grape by interplaying with VvMYBA1 and promoting jasmonic acid biosynthesis. Hortic Res 11(5):uhae083 Zhang HL, Zhang ZH, Zhao YH, Guo DL, Zhao XJ, Gao W, Zhang JP, Song BT (2021) StWRKY13 promotes anthocyanin biosynthesis in potato ( Solanum tuberosum ) tubers. Funct. Plant Biol 49(1):102–114 Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40(1):22–34 Supplementary Files SupplementaryTableS1.xlsx Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2025 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Accept 01 Apr, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers invited by journal 23 Mar, 2025 Editor assigned by journal 23 Mar, 2025 First submitted to journal 21 Mar, 2025 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. 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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-5445538","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432834516,"identity":"afb27e56-20c8-4b8e-b440-8b4ecda408af","order_by":0,"name":"Biao Lai","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Biao","middleName":"","lastName":"Lai","suffix":""},{"id":432834517,"identity":"fd8e3020-1d2d-4035-af30-b08deb562b3d","order_by":1,"name":"Chenxi Gao","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Gao","suffix":""},{"id":432834518,"identity":"09eb9bb9-0de2-4bb9-9ae3-79fa21a93dd9","order_by":2,"name":"Li Jiang","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Jiang","suffix":""},{"id":432834519,"identity":"da59cb03-694c-482d-973c-bc6bac04f386","order_by":3,"name":"Li Wen","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Wen","suffix":""},{"id":432834520,"identity":"9fcd9075-9991-489c-80e7-52d9bf7a6788","order_by":4,"name":"Xushuo Zhang","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xushuo","middleName":"","lastName":"Zhang","suffix":""},{"id":432834521,"identity":"e9e94be3-53f8-432c-9f9c-e347fd982946","order_by":5,"name":"Wei Shen","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Shen","suffix":""},{"id":432834522,"identity":"8550d58d-48b5-4810-945b-2bf3ce666bda","order_by":6,"name":"Yanling Yu","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yanling","middleName":"","lastName":"Yu","suffix":""},{"id":432834523,"identity":"3c61f11b-5021-4258-b303-0ee1bc20550c","order_by":7,"name":"Hanbing Yang","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hanbing","middleName":"","lastName":"Yang","suffix":""},{"id":432834524,"identity":"6288ed52-2fda-4e57-8c25-3a9e73359beb","order_by":8,"name":"Fabo Chen","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Fabo","middleName":"","lastName":"Chen","suffix":""},{"id":432834525,"identity":"b0a44a3d-5858-4827-b473-9e2cf935f860","order_by":9,"name":"Ping Fang","email":"","orcid":"","institution":"Yangtze Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Fang","suffix":""},{"id":432834526,"identity":"2f367cf0-555e-497d-ad4e-add454034222","order_by":10,"name":"Lina Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYDCCA8wNBxgYJOr5mZkPPyBSCyNIi02CZDtbmgHRWoBkWoLBeR4FCaJ08N1IbDxcUHM4z/gwD4MBQ41NNEEtkjcSGw7POHa42Oww74EHDMfSchsIaTEAaeFhO8y47TBfggFjw2Fitfw7zLi5mcdAgngtvG1piRuYidUieeYhUEufjbHEYWAgJxDjF77jyYc/83yTkOPvP3z4wYcaG8JaUEECacpHwSgYBaNgFOACAApmRapnO4WhAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1504-8571","institution":"Yangtze Normal University","correspondingAuthor":true,"prefix":"","firstName":"Lina","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2024-11-13 09:10:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5445538/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5445538/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-025-03487-w","type":"published","date":"2025-04-21T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79172774,"identity":"1dbf15e6-3efd-4968-8d31-4c7719c1c6a9","added_by":"auto","created_at":"2025-03-25 09:44:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5220644,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the RsWRKY44 transcription factor.\u003c/p\u003e\n\u003cp\u003eA, Phylogenetic analysis of RsWRKY44 and anthocyanin-related WRKY proteins from other plant species, the GenBank accession numbers were followed behind the WRKY protein names. B, Multi-sequence alignment between RsWRKY44 and other WRKY proteins, the underlined part was WRKY domain. C, Subcellular localization of the LhWRKY44-GFP in \u003cem\u003eN. benthamiana\u003c/em\u003e leaf epidermal cells. D, Transcriptional activation activity of RsWRKY44 in Y2H Gold yeast cell.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/d3f3d00375cfbb87313037d3.png"},{"id":79171694,"identity":"ff3a8a1e-d541-40f3-936d-77427625a164","added_by":"auto","created_at":"2025-03-25 09:36:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2803137,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of anthocyanin content in the skin and flesh of different radish cultivars\u003c/p\u003e\n\u003cp\u003eA, Phenotypic images of various radish cultivars. B, Measurements of anthocyanin content in the skin and flesh of these diverse radish cultivars. The error bars show average±SE of 3 biological replicates.\u003c/p\u003e","description":"","filename":"Figure2new.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/3bbafbf45638253a8dbb8fee.png"},{"id":79171698,"identity":"fe78ef0e-339e-49fe-b6c9-250a07fb5519","added_by":"auto","created_at":"2025-03-25 09:36:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":471346,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of anthocyanin biosynthesis structural and regulatory genes in the skin and flesh of different radish cultivars\u003c/p\u003e\n\u003cp\u003eThe radish \u003cem\u003eactin\u003c/em\u003egene was used as the control. The error bars show average±SE of 3 biological replicates.\u003c/p\u003e","description":"","filename":"Figure3new.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/5e1a02a305c73ac6f7f9010f.png"},{"id":79172775,"identity":"ac385fa7-91a0-46f3-9991-5a3cd97c1758","added_by":"auto","created_at":"2025-03-25 09:44:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21166351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransient overexpression of RsWRKY44 and RsMYB1a in tobacco leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Phenotypic imagery of tobacco leaves following the transient overexpression of RsWRKY44, and co-expression of RsWRKY44 with RsMYB1a. B, Anthocyanin content in tobacco leaves post the overexpression of RsWRKY44 and the co-expression of RsWRKY44 with RsMYB1a. C, Expression levels of genes related to anthocyanin accumulation in tobacco leaves, after transient expression of RsWRKY44 and simultaneous expression of RsWRKY44 with RsMYB1a. The error bars show average±SE of 3 biological replicates.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/c1d9bef105aae8a3fda2bc9b.png"},{"id":79172771,"identity":"9dbb6167-c431-4b7e-872a-d998d8df9570","added_by":"auto","created_at":"2025-03-25 09:44:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":841304,"visible":true,"origin":"","legend":"\u003cp\u003einteraction between RsWRKY44 and RsMYB1\u003c/p\u003e\n\u003cp\u003eA, BiFC assays of RsWRKY44 and RsMYB1a in \u003cem\u003eN. benthamiana\u003c/em\u003e leaf epidermal cells. B, FLC assay in \u003cem\u003eN. benthamiana\u003c/em\u003e leaf. LUC signals were imaged using a CCD camera.\u003c/p\u003e","description":"","filename":"Figure5new.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/568f7bcedf30db49b7a7bcd6.png"},{"id":79172773,"identity":"82de1fd2-aa48-4102-9c58-750d923b0e8f","added_by":"auto","created_at":"2025-03-25 09:44:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2597554,"visible":true,"origin":"","legend":"\u003cp\u003eRsWRKY44 with RsMYB1aactivates the promoter activity of \u003cem\u003eRsCHI \u003c/em\u003eand \u003cem\u003eRsUFGT\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA, Schematic diagrams of Effector and reporter plasmids. B, \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves were co-infiltrated with \u003cem\u003eAgrobacterium\u003c/em\u003e strains carrying the indicated constructs. Control (35S:Empty/RsCHSpro:LUC or RsUFGTpro:LUC), co-expression of 35S: RsWRKY44/RsCHSpro:LUC or RsUFGTpro:LUC, co-expression of 35S:RsMYB1a/RsCHSpro:LUC or RsUFGTpro:LUC; co-expression of 35S:RsMYB1a/35S:RsWRKY44/RsCHSpro:LUC or RsUFGTpro:LUC. LUC signals were imaged using a CCD camera. C, Expression values were determined by calculating the ratio of LUC activity to REN activity (LUC/REN). The error bars show average±SE of six biological replicates.\u003c/p\u003e","description":"","filename":"Figure6new.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/86df2a26f543ed4c61a14570.png"},{"id":79172770,"identity":"07ab63e5-1750-4a49-b2ba-1c241c6a7330","added_by":"auto","created_at":"2025-03-25 09:44:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":83078,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of \u003cem\u003eRsMYB1a\u003c/em\u003e and \u003cem\u003eRsWRKY44\u003c/em\u003e in ‘Hongxin 1-1’ and ‘Shaguan 1’ skin and flesh\u003c/p\u003e\n\u003cp\u003eA, Anthocyanin content in the skin and flesh of ‘Hongxin 1-1’ and ‘Shaguan 1’ radish. B and C, The expression of \u003cem\u003eRsMYB1a\u003c/em\u003e and \u003cem\u003eRsWRKY44\u003c/em\u003e in ‘Hongxin 1-1’ and ‘Shaguan 1’ skin and flesh. The error bars show average±SE of 3 biological replicates.\u003c/p\u003e","description":"","filename":"Figure7new.png","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/949b2eb76f7723de729f41b3.png"},{"id":81569529,"identity":"81515eb7-3448-45f5-b2d0-8fa914aeae8b","added_by":"auto","created_at":"2025-04-28 16:05:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":63735772,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/8eebd32c-3721-4361-8390-62dc87cc3dbf.pdf"},{"id":79172776,"identity":"340c1511-bdd2-451c-93f2-dc9820d0614a","added_by":"auto","created_at":"2025-03-25 09:44:08","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":10844,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5445538/v1/f82c84c242b5cdcdbeaefed0.xlsx"}],"financialInterests":"","formattedTitle":"RsWRKY44 participated in anthocyanin biosynthesis regulation in radish through interaction with RsMYB1a","fulltext":[{"header":"Key Message","content":"\u003cp\u003eRsWRKY44 transcription factor, associated with anthocyanin biosynthesis in different radish cultivars, highly facilitates the activation of \u003cem\u003eRsCHI\u003c/em\u003e and \u003cem\u003eRsUFGT\u003c/em\u003e genes through its interaction with RsMYB1a, thereby promoting anthocyanin production.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eRadishes are globally recognized and widely grown as a horticultural crop that has high nutritional and economic value. Radish is low in calories and rich in calcium, magnesium, copper, manganese, potassium, vitamin B6, vitamin C, folate, fat and fatty related compounds, flavonoids, non-flavonoids polyphenols et al. (Gamba, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Radishes exhibit a diverse color palette, ranging from stark white to vibrant green, and extending to vivid red, deep purple, and even black. The rich red, purple, and black hues observed in radishes are attributed to the presence of anthocyanin pigments. Anthocyanins are a type of flavonoid, a class of water-soluble compounds with antioxidant effects. Anthocyanins, the main pigments in plants, originate from the phenylpropanoid pathway. They are synthesized through a series of reactions catalyzed by enzymes such as CHS, CHI, F3H, DFR, ANS, and UFGT. These enzymes are encoded by structural genes, leading to the production of various anthocyanins(Jaakola \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch shows that the regulation of anthocyanin biosynthesis in plants primarily occurs at the transcriptional level. Three main classes of transcription factors are involved in regulating anthocyanin biosynthesis, namely MYB, basic Helix-Loop-Helix (bHLH), and WD40 proteins. These three types of transcription factors interact to form complexes that collectively regulate the expression of structural genes involved in anthocyanin biosynthesis, thereby affecting the activity of enzymes and ultimately impacting the accumulation of anthocyanins (Jaakola \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Petroni and Tonelli \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the model plant \u003cem\u003eArabidopsis\u003c/em\u003e, PAP1 (MYB) and PAP2 (MYB) can interact with R/B-like bHLH proteins to activate the promoter of the anthocyanin biosynthetic structural gene DFR(Zimmermann, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). AtPAP1 and AtPAP2 interact with the distinct bHLH transcription factors AtEGL3 and AtGL3 to regulate diverse anthocyanin biosynthesis pathway genes in \u003cem\u003eArabidopsis\u003c/em\u003e(Gonzalez, et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In apple, MdMYB1 and AtEGL3 can cooperate to activate the promoters of \u003cem\u003eMdDFR\u003c/em\u003e and \u003cem\u003eMdUFGT\u003c/em\u003e(Takos, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In bayberry, when MrMYB1 and AtbHLH are co-expressed, the activity of the \u003cem\u003eAtDFR\u003c/em\u003e promoter is significantly increased. MrMYB1 can interact with MrbHLH1 to regulate most of the structural genes in the anthocyanin biosynthetic pathway(Liu, et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Niu, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our previous research suggested that RsMYB1 can interact with RsbHLH4, which in turn regulates the accumulation of anthocyanin in radish taproots. (Lai, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). MBW (MYB-bHLH-WD40) complex plays a crucial role in regulating anthocyanin accumulation in plants, and all three components are indispensable.\u003c/p\u003e \u003cp\u003eIn addition to the MBW transcription factor complex, studies have also found that other types of transcription factors such as NAC, MADS, bZIP, and SPL, especially WRKY, are extensively involved in the transcriptional regulation of plant anthocyanin biosynthesis(Ma, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PyWRKY26 can form a complex with PybHLH3, directly binding to the key anthocyanin regulatory gene PyMYB114 promoter to promote the transcription of PyMYB114, leading to anthocyanin accumulation in red-skinned pear fruit(Li, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PbWRKY75 in pear promotes anthocyanin accumulation by binding to the \u003cem\u003ePbDFR\u003c/em\u003e, \u003cem\u003ePbUFGT\u003c/em\u003e, and \u003cem\u003ePbMYB10b\u003c/em\u003e promoters to enhance their transcription (Cong, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we identified a WRKY transcription factor named RsWRKY44 was highly corelated with anthocyanin content in different radish skin and flesh. RsWRKY44 interacted with RsMYB1a, a key promoter of anthocyanin synthesis enhanced anthocyanin accumulation when transient expressed in tabaco leaf. Altogether, our findings provide novel insights into the functions of RsWRKY44 in anthocyanin synthesis in radish taproot.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eTwelve radish cultivars, including \u0026lsquo;Chuanxin\u0026rsquo;, \u0026lsquo;Yanzhi\u0026rsquo;, \u0026lsquo;Shawo\u0026rsquo;, \u0026lsquo;Xiari\u0026rsquo;, and \u0026lsquo;Chunbulao\u0026rsquo;, \u0026lsquo;Zimeiren', \u0026lsquo;Sichuan Hongpi\u0026rsquo;, \u0026lsquo;Bingqilin\u0026rsquo;, and \u0026lsquo;Chongqing Hongpi\u0026rsquo;, \u0026lsquo;Xinlimei\u0026rsquo;, \u0026lsquo;Hongxin 1\u0026ndash;1\u0026rsquo; and \u0026lsquo;Shaguan 1\u0026rsquo; were grown in the experimental field of Yangtze Normal University. The radish samples were all carefully divided into skin and flesh components, then chopped into small sections. Following this, they were promptly frozen using liquid nitrogen. Subsequently, the samples were stored at a temperature of -80 degrees for further study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of total anthocyanin contents\u003c/h3\u003e\n\u003cp\u003eThe contents of anthocyanins in the radish tissues were determined as previously described by our group (Wei et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eRNA and DNA extraction\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA and DNA extraction\u003c/div\u003e \u003cp\u003eThe isolation of the total RNA from the tobacco leaves, as well as from the radish skin and flesh, was performed utilizing a HiPure Total RNA Mini Kit (Magen, China). The isolated RNA subsequently underwent gel electrophoresis for integrity verification. The quantification of the RNA concentration was executed using an UV5Nano spectrophotometer (Mettler Toledo, USA). DNA from the radish leaves was extracted using a DNA isolation kit (Aidlab, China) and subsequently utilized for the isolation of promoter sequences.\u003c/p\u003e\n\u003ch3\u003eqRT‑PCR\u003c/h3\u003e\n\u003cp\u003eThe first-strand complementary DNA (cDNA) was synthesized from 2 \u0026micro;g of total RNA utilizing oligo (dT) primers using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). The synthesized cDNA was subsequently employed as a template for gene amplification and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The qRT-PCR assays were carried out using the QuantStudio 3 system (Applied Biosystems, USA) as previously established methodology (the primers are detailed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) (Lai, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eTransient infiltration assay\u003c/h3\u003e\n\u003cp\u003eThe complete coding sequences of RsWRKY44 complementary DNA (cDNA) were amplified from the 'Hongxin 1' flesh cDNA utilizing a primer with incorporated attB sites (refer to Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for primer sequences). The purified DNA fragments were subsequently recombined with the pDONR/Zeo vector (Invitrogen, USA) via the BP Clonase\u0026trade; II Enzyme mix (Invitrogen). This process generated the entry clones pDONR/Zeo-RsWRKY44. The transient expression vectors pEAQ-RsWRKY44 were constructed by recombining the entry clones with pEAQ-HT-DEST1 utilizing the LR Clonase\u0026trade; II Enzyme mix (Invitrogen). The constructs pEAQ-RsMYB1a and pEAQ-RsbHLH4 were previously established (Lai, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These transient expression vectors were subsequently introduced into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring pEAQ-RsMYB1a, pEAQ-RsbHLH4, and pEAQ-RsWRKY44 were infiltrated into tobacco leaves either individually or collectively. The leaves infiltrated with a pEAQ-HT construct served as the control group. Photographs were captured five days post-infiltration.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation (BiFC) assay\u003c/h2\u003e \u003cp\u003epEarleyGate202-RsWRKY44-YN were constructed by the recombination of the entry clone with pEarleyGate202-YN using an LR Clonase\u0026trade; II Enzyme mix (Invitrogen). \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring both pEarleyGate201-RsMYB1a-YC and pEarleyGate202-RsbHLH4-YN, or maintained separately with an empty vector, were infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plant leaves. The YFP signal was observed 2 days after infiltration using a fluorescence microscope (OLYMPUS, BX43, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFirefly Luciferase Complementation Imaging Assay (FLC) assay\u003c/h3\u003e\n\u003cp\u003epEAQ-RsWRKY44-cLUC and pEAQ-nLUC-RsMYB1a were constructed by the recombination of the entry clone with pEAQ-cLUC-GW and pEAQ-nLUC-GW using an LR Clonase\u0026trade; II Enzyme mix (Invitrogen). \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring both pEAQ-RsWRKY44-cLUC and pEAQ-nLUC-RsMYB1a, or maintained separately with an empty vector, were infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e plant leaves. The luciferase signal was observed 2 days after infiltration using a cold CCD camara (Biotanon, Tanon 5200, China).\u003c/p\u003e\n\u003ch3\u003eDual luciferase assay\u003c/h3\u003e\n\u003cp\u003eSpecific primers with recombination sites were designed to amplify the \u003cem\u003eRsCHI\u003c/em\u003e and \u003cem\u003eRsUFGT\u003c/em\u003e promoter based on the radish genome sequence. The primers are shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The amplified sequences were then inserted into the pGreenII 0800-LUC vector at the 5\u0026prime; end of a LUC gene using the Gibson assembling method. Infiltration, transient expression analysis, and the determination of the enzyme activities of LUC and REN were conducted as described by Hellens et al. using 6-week-old \u003cem\u003eN. benthamiana\u003c/em\u003e leaves (Hellens, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eThe identification of RsWRKY44 in radish\u003c/h2\u003e\n\u003cp\u003eThrough co-expression analysis across different radish cultivars (not published), we identified a WRKY transcription factor, designated as RsWRKY44, whose expression was highly correlated with radish color. The open reading frame of RsWRKY44 spans 1236 base pairs, encoding a 411-residue polypeptide. The protein features two conserved WRKY domains (WRKYGQK) and two CX\u003csub\u003e4\u003c/sub\u003eCX\u003csub\u003e23\u003c/sub\u003eHXH zinc finger domains, which are characteristic of group I WRKY superfamily members, including RsWRKY44 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Multiple sequence alignment revealed that RsWRKY44 shares 73% and 75% similarity at the amino acid level with \u003cem\u003eArabidopsis\u003c/em\u003e AtTTG2 and \u003cem\u003eBrassica napus\u003c/em\u003e BnTTG2, respectively. These proteins are known to be involved in the regulation of proanthocyanidin biosynthesis in \u003cem\u003eBrassica napus\u003c/em\u003e and trichome patterning in \u003cem\u003eArabidopsis\u003c/em\u003e(Johnson, et al. \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e; Qu, et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Phylogenetic tree analysis indicates that RsWRKY44 is closely related to BnTTG2 and other members associated with anthocyanin biosynthesis, such as AcWRKY44 and VvWRKY26 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) (Amato, et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Peng, et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe subcellular localization of RsWRKY44 was assessed by engineering a fusion construct comprised of the RsWRKY44 coding sequence and the gene encoding green fluorescent protein (GFP). This construct was transiently expressed within the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e. Fluorescence microscopy, facilitated by GFP detection, manifested the exclusive presence of RsWRKY44\u0026ndash;GFP within the nuclei, contrarily to the ubiquitous distribution of lone GFP throughout the entire cell (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). This evidence strongly substantiates the classification of RsWRKY44 as a nuclear protein.\u003c/p\u003e\n\u003cp\u003eA transcription activation assay was conducted in yeast to identify if RsWRKY44 has transcriptional activity. pGBKT7-RsWRKY44 with the empty pGADT7 vector were co-transformed into yeast strain Y2H gold. All transformants survived on SD/-Trp/-Leu/-His media with 200 ng/mL AbA and the growth was completely inhibited with 300 ng/mL AbA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). These outcomes suggest that RsWRKY44 has the capacity to activate transcription in yeast cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eThe expression of RsWRKY44 was highly correlated with anthocyanin accumulation in radish\u003c/h2\u003e\n\u003cp\u003eTo gain further insights into the expression pattern of RsWRKY44, real-time PCR analysis was conducted on the skin and flesh tissues of 10 distinct radish cultivars. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, \u0026lsquo;Chuanxin\u0026rsquo; exhibited the highest anthocyanin content in the skin, while both the skin and flesh of \u0026lsquo;Yanzhi\u0026rsquo; displayed elevated levels of anthocyanin. Conversely, \u0026lsquo;Shawo\u0026rsquo;, \u0026lsquo;Xiari\u0026rsquo;, and \u0026lsquo;Chunbulao\u0026rsquo; exhibited no anthocyanin accumulation in either the skin or flesh. Remarkably, \u0026lsquo;Zimeiren' and 'Yanzi\u0026rsquo; demonstrated substantial anthocyanin accumulation in both skin and flesh tissues. Anthocyanins were primarily localized in the skin of \u0026lsquo;Sichuan Hongpi\u0026rsquo;, \u0026lsquo;Bingqilin\u0026rsquo;, \u0026lsquo;Chuanxin\u0026rsquo;, and \u0026lsquo;Chongqing Hongpi\u0026rsquo;, with little to no presence in the flesh. Notably, \u0026lsquo;Xinlimei\u0026rsquo; only exhibited anthocyanin accumulation in the flesh, while the skin remained devoid of anthocyanins. Additionally, we analyzed the expression levels of key genes involved in the anthocyanin biosynthesis pathway, including \u003cem\u003eRsCHS\u003c/em\u003e, \u003cem\u003eRsCHI\u003c/em\u003e, \u003cem\u003eRsF3H\u003c/em\u003e, \u003cem\u003eRsANS\u003c/em\u003e, \u003cem\u003eRsDFR\u003c/em\u003e, and \u003cem\u003eRsUFGT\u003c/em\u003e, in these tissue samples. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the expression of six anthocyanin biosynthesis structural genes is only evident in the skin and flesh of radishes where anthocyanin has accumulated. The expression of \u003cem\u003eRsWRKY44\u003c/em\u003e shown the same expression pattern with two key regulatory genes \u003cem\u003eRsMYB1a\u003c/em\u003e and \u003cem\u003eRsbHLH4\u003c/em\u003e, indicating that \u003cem\u003eRsWRKY44\u003c/em\u003e may related to anthocyanin biosynthesis regulation in radish taproot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-expression of\u003c/strong\u003e \u003cstrong\u003eRsWRKY44\u003c/strong\u003e \u003cstrong\u003ewith\u003c/strong\u003e \u003cstrong\u003eRsMYB1a\u003c/strong\u003e \u003cstrong\u003einduced anthocyanin accumulation in tobacco leaf\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the function of \u003cem\u003eRsWRKY44\u003c/em\u003e, the pSAK277-RsWRKY44 vector was transiently infiltrated into tobacco leaves using \u003cem\u003eAgrobacterium\u003c/em\u003e. However, no anthocyanin was detected in tobacco leaves when \u003cem\u003eRsWRKY44\u003c/em\u003e was expressed alone. Based on previous studies indicating the importance of \u003cem\u003eRsMYB1a\u003c/em\u003e as a key regulatory gene in radish, we co-transformed \u003cem\u003eRsWRKY44\u003c/em\u003e with \u003cem\u003eRsMYB1a\u003c/em\u003e in tobacco leaves. While individual expression of \u003cem\u003eRsMYB1a\u003c/em\u003e did not induce anthocyanin synthesis, co-expression of \u003cem\u003eRsMYB1a\u003c/em\u003e with \u003cem\u003eRsWRKY44\u003c/em\u003e significantly promoted anthocyanin synthesis. This induction was lower compared to the combined expression of \u003cem\u003eRsMYB1a\u003c/em\u003e with \u003cem\u003eRsbHLH4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe expression of genes involved in anthocyanin biosynthesis and regulation in tobacco was further examined. It was observed that \u003cem\u003eNtAN2\u003c/em\u003e, which encodes a MYB transcription factor, was not expressed in tobacco leaves (data not shown). \u003cem\u003eNtAN1a\u003c/em\u003e and \u003cem\u003eNtAN1b\u003c/em\u003e, encoding bHLH transcription factors, exhibited upregulation only when \u003cem\u003eRsMYB1a\u003c/em\u003e was co-expressed with \u003cem\u003eRsWRKY44\u003c/em\u003e, not when expressed alone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Moreover, the structural genes involved in anthocyanin biosynthesis, such as \u003cem\u003eNtCHS\u003c/em\u003e, \u003cem\u003eNtCHI\u003c/em\u003e, \u003cem\u003eNtF3H\u003c/em\u003e, \u003cem\u003eNtDFR\u003c/em\u003e, \u003cem\u003eNtANS\u003c/em\u003e and \u003cem\u003eNtUFGT\u003c/em\u003e, were all upregulated when \u003cem\u003eRsMYB1a\u003c/em\u003e was transformed with \u003cem\u003eRsWRKY44\u003c/em\u003e. The control experiment, involving \u003cem\u003eRsMYB1a\u003c/em\u003e with \u003cem\u003eRsbHLH4\u003c/em\u003e, significantly induced anthocyanin accumulation and the expression of related structural and regulatory genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings suggest that RsWRKY44 may regulate anthocyanin biosynthesis by interacting with RsMYB1a, subsequently activating \u003cem\u003ebHLH\u003c/em\u003e and structural genes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eRsWRKY44 interact RsMYB1a activate anthocyanin biosynthesis pathway genes\u003c/h2\u003e\n\u003cp\u003eConsidering the observed correlation between the expression patterns of \u003cem\u003eRsWRKY44\u003c/em\u003e and \u003cem\u003eRsMYB1a\u003c/em\u003e in different radish cultivars, alongside the demonstrated promotion of anthocyanin accumulation in tobacco leaves through their co-expression, we hypothesize that RsWRKY44 and RsMYB1 proteins could potentially interact to form a complex.\u003c/p\u003e\n\u003cp\u003eBiFC (Bimolecular fluorescence complementation) technology was used to analyze whether RsWRKY44 and RsMYB1 could interact with each other. We fused RsWRKY44 with NYFP and RsMYB1 with CYFP, and then injected them into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e through \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transient expression. The results showed that when RsWRKY44-NYFP and RsMYB1-CYFP were co-injected, yellow fluorescent protein could be observed in the nuclei of \u003cem\u003eN. benthamiana\u003c/em\u003e leaf cells. This indicates that RsWRKY44 and RsMYB1 can interact with each other in plant cells.\u003c/p\u003e\n\u003cp\u003eThe promoter of radish anthocyanin biosynthesis pathway gene \u003cem\u003eRsCHI\u003c/em\u003e and \u003cem\u003eRsUFGT\u003c/em\u003e were drive LUC reporter gene. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, when RsMYB1a and RsWRKY44 were expressed independently, the \u003cem\u003eRsCHI\u003c/em\u003e promoter was not activated. However, a combination of RsMYB1a and RsWRKY44 significantly activated the \u003cem\u003eRsCHI\u003c/em\u003e promoter. Similarly, RsMYB1a on its own could activate the \u003cem\u003eRsUFGT\u003c/em\u003e promoter, but RsWRKY44 could not. The \u003cem\u003eRsUFGT\u003c/em\u003e promoter was highly activated when RsMYB1a and RsWRKY44 were combined.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eRsMYB1a is not the target of RsWRKY44\u003c/h2\u003e\n\u003cp\u003e\u0026lsquo;Hongxin 1\u0026ndash;1\u0026rsquo; and \u0026lsquo;Shaguan 1\u0026rsquo; are natural radish varieties with dark red skin and white flesh (pictures can be found at Lai et al, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The expression of \u003cem\u003eRsMYB1a\u003c/em\u003e and \u003cem\u003eRsWRKY44\u003c/em\u003e was examined in \u0026lsquo;Hongxin 1\u0026ndash;1\u0026rsquo; and \u0026lsquo;Shaguan 1\u0026rsquo; shin and flesh. \u003cem\u003eRsMYB1a\u003c/em\u003e was found to be expressed normally in both skin and flesh, however, no expression of \u003cem\u003eRsWRKY44\u003c/em\u003e was detected in the white flesh of \u0026lsquo;Hongxin 1\u0026ndash;1\u0026rsquo; and \u0026lsquo;Shaguan 1\u0026rsquo;. These results suggested that the transcription of \u003cem\u003eRsMYB1a\u003c/em\u003e may not regulated by RsWRKY44.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRsWRKY44: a non-light-dependent regulator of anthocyanin biosynthesis in radish\u003c/h2\u003e \u003cp\u003eAnthocyanins are a group of secondary metabolites that give plants their vibrant red, blue, and purple colors. They also have antioxidant properties and play a role in defense against pathogens and environmental stresses. WRKY family transcription factors were known to play a crucial role in the regulation of plant defense responses, development, and metabolism, including anthocyanin biosynthesis(Cappellini, et al. 2021; Jiang, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In our transcriptome data (not published), we found a WRKY transcription factor named RsWRKY44 was highly related anthocyanin content in different radish cultivars. RsWRKY44 has two conserved WRKY domains (WRKYGQK) and two CX4CX23HXH zinc finger domains which belongs to group I WRKY superfamily. Phylogenetic tree analysis shows that RsWRKY44 is close to AtTTG2 which is involved in the regulation of proanthocyanidin and mucilage biosynthesis in the seed coat, trichome differentiation, and root hair patterning (Johnson, et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Pesch, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The PhPH3 gene in petunia has a similar function to its homologous gene AtTTG2 in Arabidopsis. However, PhPH3 plays an important role in petunias by regulating vacuolar acidification in the cells of the petal, thereby influencing the color of the flower (Verweij, et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the expression of \u003cem\u003eRsWRKY44\u003c/em\u003e was analyzed in the shin and flesh of ten different radish cultivars. The expression of \u003cem\u003eRsWRKY44\u003c/em\u003e was highly correlated with anthocyanin content was confirmed by real-time PCR assay. The expression of anthocyanin biosynthesis structural genes in these radish samples was also examined. The results showed that the transcription levels of \u003cem\u003eRsCHS\u003c/em\u003e, \u003cem\u003eRsCHI\u003c/em\u003e, \u003cem\u003eRsF3H\u003c/em\u003e, \u003cem\u003eRsDFR\u003c/em\u003e, \u003cem\u003eRsANS\u003c/em\u003e, and \u003cem\u003eRsUFGT\u003c/em\u003e were all relatively higher in radish skin or flesh rich in anthocyanin. These results of structural genes exhibit a similar expression pattern to that of \u003cem\u003eRsWRKY44\u003c/em\u003e, indicating \u003cem\u003eRsWRKY44\u003c/em\u003e may played important role in radish anthocyanin accumulation regulation. Weighted Gene Co-expression Network Analysis (WGCNA) identified key anthocyanin regulators StWRKY70 and SmWRKY44 in potato and eggplant, their expression showed high correlation with anthocyanin biosynthetic genes (He, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang, et al. 2024a). Despite radish roots growing underground, in certain cultivars such as \u0026lsquo;Yanzhi\u0026rsquo; and \u0026lsquo;Zimeiren\u0026rsquo;, anthocyanin accumulation in both the skin and flesh remains unaffected, and the expression of \u003cem\u003eRsWRKY44\u003c/em\u003e, \u003cem\u003eRsMYB1a\u003c/em\u003e, and other structural genes involved in anthocyanin biosynthesis remains active. However, in red-skinned pears, anthocyanin biosynthesis was induced by light, light-induced transcription factor PpWRKY44 promotes light-triggered anthocyanin biosynthesis was regulated by the light signaling component PpBBX18 (Alabd, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The observation that Desiree potato tubers require light for anthocyanin accumulation despite growing underground, with \u003cem\u003eStWRKY13\u003c/em\u003e expression also induced by light (Zhang, et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe RsWRKY44 - RsMYB1a protein complex regulates anthocyanin biosynthesis\u003c/h2\u003e \u003cp\u003eOur study indicates that transient expression of either RsWRKY44 or RsMYB1a alone in tobacco leaves does not lead to the accumulation of anthocyanin. Interestingly, RsWRKY44 appears to have an effect similar to that of RsbHLH4, it can help RsMYB1a to regulate anthocyanin biosynthesis (Lai, et al. 2020). Co-expression of RsMYB1a and RsWRKY44 upregulated the expression of not only structural genes but also \u003cem\u003eNtAN1a\u003c/em\u003e and \u003cem\u003eNtAN1b\u003c/em\u003e (two tobacco endogenous bHLH genes). These results suggest that the interaction between RsMYB1a and RsWRKY44 is critical for regulating anthocyanin biosynthesis in radish. This is because RsWRKY44 has the potential to help RsMYB1a enhance the activation of key structural genes, \u003cem\u003eRsCHI\u003c/em\u003e and \u003cem\u003eRsUGFT\u003c/em\u003e, involved in anthocyanin biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). VvWRKY5 plays a positive regulatory role in grape wounding induced anthocyanin synthesis by interacting with VvMYBA1 and then enhance the activation of \u003cem\u003eVvUFGT\u003c/em\u003e promoter (Zhang, et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). SmWRKY44 could interact with SmMYB1 promote the biosynthesis of anthocyanins in eggplant leaves and activating anthocyanin biosynthesis pathway genes such as \u003cem\u003eSmCHS\u003c/em\u003e, \u003cem\u003eSmF3H\u003c/em\u003e, \u003cem\u003eSmDFR\u003c/em\u003e, and \u003cem\u003eSmANS\u003c/em\u003e (He, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, CitWRKY75 identified by WGCNA regulate early stage of blood orange and citrus juvenile tissues anthocyanin accumulation by bind to the promoter of \u003cem\u003eCitRuby1\u003c/em\u003e, a R2R3-MYB gene (Lu, et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFunctional divergence from orthologous WRKY proteins in anthocyanin regulation\u003c/h2\u003e \u003cp\u003eStudies have suggested that WRKY transcription factors are involved in the regulation of anthocyanin biosynthesis, the exact mechanisms are different and not fully understood in different plants. Several WRKY transcription factors in apples have been found to participate in the regulation of apple fruit anthocyanin biosynthesis. MdWRKY11 can bind to the promoters of \u003cem\u003eMdMYB10\u003c/em\u003e, \u003cem\u003eMdMYB11\u003c/em\u003e and \u003cem\u003eMdUFGT\u003c/em\u003e to participate in anthocyanin biosynthesis regulation during red-flesh apple fruit development (Liu, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Interaction between MdWRKY40 and MdMYB1 can promote the transcription of MdMYB1, inducing mechanical damage-induced anthocyanin biosynthesis in apple fruit (An, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). UV-B-induced MdWRKY72 could bind to the promoter of MdHY5 and MdMYB1 to regulation anthocyanin accumulation when apple fruit under Ultraviolet-B radiation (Hu, et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Under light induction, MdWRKY1 promotes the expression of the long non-coding RNA MdLNC499, which in turn promotes the transcription of MdERF109 which activates the expression of genes related to anthocyanin biosynthesis in apple (Ma, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, in some radish cultivars, \u003cem\u003eRsMYB1a\u003c/em\u003e is normally expressed, but \u003cem\u003eRsWRKY44\u003c/em\u003e remains unexpressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), resulting in no anthocyanin accumulation. This suggests that RsWRKY44 may not act as an upstream regulator of RsMYB1a. Moreover, it also indicates that RsWRKY44 plays a crucial role in anthocyanin biosynthesis in radishes.\u003c/p\u003e \u003cp\u003eThe transient expression of \u003cem\u003eRsWRKY44\u003c/em\u003e alone in tobacco leaves does not induce the expression of anthocyanin biosynthesis and regulatory genes, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. This remains the case even when combined with RsMYB1a, which doesn't activate the key anthocyanin regulatory MYB gene \u003cem\u003eNtAN2\u003c/em\u003e (data not shown). Similarly, in radishes, we found that the transcription of \u003cem\u003eRsMYB1a\u003c/em\u003e may not be activated by RsWRKY44, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Given that radish taproot is an underground tissue not exposed to light, it's possible that light is unnecessary for anthocyanin biosynthesis. This could potentially explain why RsMYB1a does not require activation by RsWRKY44, which functions independently of light. In radish, RsWRKY44 primarily induces anthocyanin accumulation by interacting with RsMYB1a to form a complex, thereby activating the expression of structural genes involved in anthocyanin biosynthesis, such as \u003cem\u003eRsCHI\u003c/em\u003e and \u003cem\u003eRsUFGT\u003c/em\u003e. The expression of LhWRKY44 is light dependent which involved in anthocyanin accumulation regulation by interacting with LhMYBSPLATTER and activate its transcription in lily (Bi, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These results suggest that WRKYs could participate both directly and indirectly in the regulation of anthocyanin biosynthesis during plant development, under various environmental conditions and stress scenarios.\u003c/p\u003e \u003cp\u003eOn the other hand, some studies have suggested that certain WRKY proteins may negatively regulate anthocyanin biosynthesis. For instance, mutants of \u003cem\u003eAtWRKY41\u003c/em\u003e in Arabidopsis exhibit increased anthocyanin content in rosette leaves, and the homologous gene \u003cem\u003eBnWRKY41-1\u003c/em\u003e from \u003cem\u003eBrassica napus\u003c/em\u003e can restore the phenotype of the AtWRKY41 mutant, indicating that WRKY41 can suppress anthocyanin biosynthesis (Duan, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). MdWRKY41 forms a complex by interacting with MdMYB16 to inhibit the expression of \u003cem\u003eMdMYB12\u003c/em\u003e, \u003cem\u003eMdLAR\u003c/em\u003e, \u003cem\u003eMdUFGT\u003c/em\u003e, and \u003cem\u003eMdDFR\u003c/em\u003e, thereby suppressing apple anthocyanin and proanthocyanidin synthesis (Mao, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). AtWRKY33 directly binds to the \u003cem\u003eAtDFR\u003c/em\u003e promoter to repress its expression and indirectly affects \u003cem\u003eAtDFR\u003c/em\u003e activation by interacting with PAP1 to interfere with the MBW complex, negatively regulating anthocyanin production (Tao, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The regulation of anthocyanin biosynthesis by WRKY proteins is complex and may be influenced by various factors such as environmental conditions and developmental stage in different plants.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e LND designed the research; BL, CXG, LJ, LW, XSZ, WS, YLX, HBY, FBC and PF performed the experiments and analyzed the data; BL and LND wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was supported by Chongqing Natural Science Foundation (Grant No. CSTB2022NSCQ-MSX0228), Chongqing Natural Science Foundation (Grant No. CSTC2021JCYJ-MSXMX0083) and Yangtze Normal University (Grant No. 2016KYQD20 and 2016XJQN06).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The data underlying this article are available in the article and in its online supplemental material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlabd A, Ahmad M, Zhang X, Gao YH, Peng L, Zhang L, Ni JB, Bai SL, Teng YW (2022) Light-responsive transcription factor PpWRKY44 induces anthocyanin accumulation by regulating \u003cem\u003ePpMYB10\u003c/em\u003e expression in pear. 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Development 130(20):4859\u0026ndash;4869\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Pu Y, Zhang Y, Li K, Bai S, Wang J, Xu M, Liu S, Zhou Z, Wu Y, Hu R, Wu Q, Kear P, Du M, Qi J 2024a. Tuber transcriptome analysis reveals a novel WRKY transcription factor StWRKY70 potentially involved in potato pigmentation. Plant Physiol Biochem 213, 108792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Chen C, Jiang C, Lin H, Zhao Y, Guo Y (2024b) \u003cem\u003eVvWRKY5\u003c/em\u003e positively regulates wounding-induced anthocyanin accumulation in grape by interplaying with \u003cem\u003eVvMYBA1\u003c/em\u003e and promoting jasmonic acid biosynthesis. Hortic Res 11(5):uhae083\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang HL, Zhang ZH, Zhao YH, Guo DL, Zhao XJ, Gao W, Zhang JP, Song BT (2021) StWRKY13 promotes anthocyanin biosynthesis in potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e) tubers. Funct. Plant Biol 49(1):102\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40(1):22\u0026ndash;34\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Radish, anthocyanin, RsWRKY44, RsMYB1a","lastPublishedDoi":"10.21203/rs.3.rs-5445538/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5445538/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe regulation of anthocyanin biosynthesis in radish is primarily controlled by RsMYB1a and RsbHLH4, while the involvement of other factors in this process is not well understood. This study identified a WRKY transcription factor, RsWRKY44, as a key player in anthocyanin biosynthesis regulation. The expression of \u003cem\u003eRsWRKY44\u003c/em\u003e showed a strong correlation with anthocyanin content across different radish cultivars. RsWRKY44 was found to be expressed in the nuclei and exhibit transactivation activity. It was observed that only when RsWRKY44 was co-expressed with RsMYB1a, anthocyanin accumulation was induced in tobacco leaves, while RsWRKY44 alone did not. Additionally, RsWRKY44, along with RsMYB1a, activated the expression of tobacco endogenous anthocyanin biosynthesis regulatory genes \u003cem\u003eNtAN1a\u003c/em\u003e and \u003cem\u003eNtAN1b\u003c/em\u003e, as well as the structural genes \u003cem\u003eNtCHS\u003c/em\u003e, \u003cem\u003eNtCHI\u003c/em\u003e, \u003cem\u003eNtDFR\u003c/em\u003e, \u003cem\u003eNtF3H\u003c/em\u003e, \u003cem\u003eNtANS\u003c/em\u003e, \u003cem\u003eNtUFGT\u003c/em\u003e in transgenic tobacco. BiFC, FLC and DLA assays confirmed the interaction between RsWRKY44 and RsMYB1a leading to the activation of radish genes \u003cem\u003eRsCHI\u003c/em\u003e and \u003cem\u003eRsUFGT\u003c/em\u003e, promoting anthocyanin biosynthesis. This study sheds light on a new molecular mechanism of RsWRKY44 involved in anthocyanin biosynthesis regulation in radish.\u003c/p\u003e","manuscriptTitle":"RsWRKY44 participated in anthocyanin biosynthesis regulation in radish through interaction with RsMYB1a","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 09:36:03","doi":"10.21203/rs.3.rs-5445538/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-04-01T12:38:41+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-24T01:06:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-24T01:04:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-23T13:56:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-03-21T08:37:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"00093063-f51a-4f05-bf50-bf509e86f96b","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T15:58:56+00:00","versionOfRecord":{"articleIdentity":"rs-5445538","link":"https://doi.org/10.1007/s00299-025-03487-w","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2025-04-21 15:56:58","publishedOnDateReadable":"April 21st, 2025"},"versionCreatedAt":"2025-03-25 09:36:03","video":"","vorDoi":"10.1007/s00299-025-03487-w","vorDoiUrl":"https://doi.org/10.1007/s00299-025-03487-w","workflowStages":[]},"version":"v1","identity":"rs-5445538","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5445538","identity":"rs-5445538","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}