Unraveling GhMYB102: A Dual-Function of the Gh_A01G069800 Gene in Promoting Anthocyanin Biosynthesis Inhibition, and Drought Tolerance in Cotton

Unraveling GhMYB102: A Dual-Function of the Gh_A01G069800 Gene in Promoting Anthocyanin Biosynthesis Inhibition, and Drought Tolerance in Cotton | 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 Unraveling GhMYB102: A Dual-Function of the Gh_A01G069800 Gene in Promoting Anthocyanin Biosynthesis Inhibition, and Drought Tolerance in Cotton Dong Wenhao, Richard Odongo Magwanga, Yanchao Xu, Joy Nyangasi Kirungu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7187536/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Cotton plants display significant genetic diversity in petal color, which is essential for resilience against pests and diseases, UV radiation mitigation, and pollinator attraction. The MYB gene family regulates anthocyanin biosynthesis, but the specific functions of the 4th subfamily remain poorly understood in floral color formation. Methods This study focuses on the MYB gene Gh_A01G069800, evaluating its impact on floral pigmentation using virus-induced gene silencing (VIGS). Results The results showed that VIGS plants exhibited higher pigment intensity across four cotton varieties, with the highest anthocyanin levels recorded in Zhongyihong (5.5 nmol/g) and Y52 (5.7 nmol/g), surpassing their wild types. In contrast, overexpression (OE) of Gh_A01G069800 resulted in lower pigmentation (3.0 nmol/g) compared to wild types and mutants, which had anthocyanin levels of 4.0 and 7.5 nmol/g, respectively. Gene expression analysis revealed that while chalcone synthase (CHS) and flavonoid glycosyltransferase (UFGT) were highly upregulated in wild types, genes like chalcone isomerase (CHI), flavonoid-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), and anthocyanin synthase (ANS) showed elevated expression in VIGS plants, suggesting Gh_A01G069800's inhibitory role in anthocyanin biosynthesis. Furthermore, GhMYB102 was shown to directly inhibit CHI, ANS, and F3'5'H expression, affecting anthocyanin synthesis. Overall, Gh_A01G069800 appears to play a crucial role in regulating floral color and enhancing drought stress tolerance in cotton plants. Semi-wild cotton GhMYB102 Petal color Anthocyanins Transcriptional regulation drought stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Cotton belongs to the mallow family, mainly a herb or perennial shrub consisting of more than 50 diploid and tetraploid species [ 1 ]. Cotton species have a wide range of flower colors, with wild diploid and tetraploid cotton species having vibrant petal colors and basal spots [ 2 , 3 ]. Moreover, the floral color do undergo transitional changes post anthesis, with diverse array of petal colors with yellow, cream and red colors being dominant [ 3 ]. The diversity in the floral color is natures gift, and it serves as a critical tool for plant geneticists, ecologists and biochemists in understanding the evolution, diversity, and presence of volatile compounds in plants [ 4 ]. The major pigments which constitutes the floral colors include carotenoids, anthocyanins, and betalains, furthermore, chlorophylls also provide the main flower pigments, but only in selected plant species [ 5 ]. Floral color is determined by both endogenous and exogenous factors, moreover, exogenous factors are predominantly influenced by the environment, for instance, humidity, temperature, and light could also play a role in determining floral pigmentation and scent [ 6 ]. In addition to the major pigments, other conditions are key, for instance the vacuolar pH influence the occurrence of some colors, for blue flowers to emerge, a vacuolar pH of more than 5.5 is critical [ 7 ]. The floral color is also a powerful trait used by plants taxonomist, as a general phenotypic trait, but also confer several biological roles, such as protection against UV-light and attraction of pollinators [ 8 ].The flavonoids are a large subgroup of phenypropanoids [ 9 ], the color determinants are basically water soluble and are located within the cellular vacuoles [ 10 ]. The main flavonoids pigments are the anthocyanins which is the precursor for the pink, red, orange, blue, purple, and blue-black flower colors, and exist in over 90% of angiosperms [ 11 ]. Apart from flowers, flavonoids are also present in fruits, vegetables, grains, bark, roots, stems, tea, and wine [ 12 ], moreover, the flavonoids also play other significant biological activities for instance, protect the plants against plant feeding insects and herbivores [ 13 ]. The flavonoids creates color and aroma of flowers, and in fruits as pollinators and dispersal agents attractants to improve the pollination efficiency and dispersal, respectively [ 10 ]. The flavonoids biosynthesis is well understood due to its high antioxidant and UV light protection, moreover a number of plant metabolites are induced by light. The main derivative of flavonoids is phenylalanine, the process is catalyzed by phenylalanine ammonia-lyase, the and entire process is mediated by a similar step with flavanone 3-hydroxylase (F3H), chalcone synthase (CHS) and fluxed into anthocyanin biosynthesis by dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose [ 14 , 15 ]. Flavonoids can be divided into 6 categories, flavonoids, flavanols, flavanones, isoflavonoids, flavonols, and anthocyanins. Some of the flavonoids (e.g., flavones, flavanols, and orange ketones) can give flowers a yellow color, while anthocyanins can give flowers a red, pink, blue, or purple color. Among them, anthocyanins are the largest subgroup of pigment substances, and the differences in the coloration of plant petals among different species and varieties are mainly caused by the differences in the types of anthocyanins. Hydroxylation, glycosylation, methylation, and acylation modifications of the anthocyanin glycosides B ring, the anthocyanins can be modified into 6 categories, which include the geranins, delphinidins, petunidins, mallowins, paeonidins and the cornflowerins. Moreover, the hydroxylation of the glycosides B ring plays an important role in enhancing stability, and hindering oxidation [ 16 ]. Moreover, anthocyanins glycosylation promotes hypsochomic shift in the absorption maxima of the spectra, and in turn boosts the anthocyanin stability and effective storage at the cellular vacuole [ 17 ]. The unique floral color of Gentiana lutea var. aurantiaca and transgenic petunia ( Petunia× hybrida ) and other such as the orange family (yellow to orange-red) plant petals is due to the presence of the large amount of geraniolins, the Iris tectorum and blue moonflower ( Rosa chinensis Jacq) and other blue family (blue to purple) petals contain large amounts of delphinidin, petunidin, or mallowin, and the red (pink to red) petals of Chrysanthemum morifolium , Lilium spp ., and petunias usually contain large amounts of cornflowerin, and paeonidin. So far, there are more than 600 anthocyanins found in nature [ 18 ]. Cotton germplasms are polymorphic, which the color of the flower, has been used to explore their modern genetic studies [ 19 ]. The color of the cotton flower is due to the effects of both flavonols and anthocyanin [ 3 ]. The flower color could also be attributed to the varietal type, ecological condition, and fertilization [ 3 ], However, the pigment of the cotton flower is less affected by environmental factors than that of its leaf [ 7 ] and thus a stable inheritance [ 20 ]. Few studies have investigated the molecular basis that underpins the formation and accumulation of anthocyanins in wild and cultivated cotton progenitors. The whole genome sequencing of AD [ 21 ], D [ 22 ], and A [ 23 ] genomes provide a perfect platform to explore the diversity of cotton flower colors and therefore, in this research, four cotton germplasms were evaluated, two G. hirsutum races Marie Galante 1139 [ 24 ], punctatum187 [ 25 ], and two upland cotton varieties, Zhongyihong2 and Y52 to explore their floral color. A novel gene, MyB102 was further validated through overexpression, gene knock, and yeast hybridization to further understands its putative role in the determination of the color of the flower, and possible influence in promoting plants tolerance to drought stress. The results therein provide valuable information for breeders to further explore MyB102 in modulating flower color, and breeding for diverse plants varieties with higher adaptability and production efficiency in the face of ever-changing climatic conditions. 2 Materials and methods 2.1 Plant material The wild cotton of Gossypium hirsutum races Marie-galante 1139 (cream colored corolla), punctatum 187 (yellowish corolla), the upland cotton cultivars, Y52 (pink corolla), and Zhongyihong 2 (pink corolla), the model plant, Columbia-type A. thaliana (Col-0), and Ben's Tobacco tabacum were used in this study. The wild cotton germplasms were obtained from the wild cotton germplasm research unit of the Institute of Cotton Research, Anyang, while the land genetic materials Y52 and Zhongyihong 2 were provided by the National Intermediate Cotton Germplasm Resource Bank. The target gene Arabidopsis mutant (N867168) was purchased from the AraShare Arabidopsis mutant germplasm platform [ https://www.arashare.cn/ ]. The seeds for the four cotton germplasms were delinted, and germinated in pots. The plants were grown in the greenhouse, and at the flowering stage, the petals were collected at 10.00 hrs, frozen in liquid nitrogen, and stored at -80 0 C for RNA extraction. 2.2 RNA extraction and qRT-PCR analysis The petal tissues were harvested from Marie-galante 1139 (cream colored corolla), punctatum 187 (yellowish corolla), the land races, Y52 (pink corolla), and Zhongyihong 2 (pink corolla), in liquid nitrogen, then transferred to -80 0 C for temporary storage. The total RNA of the petal tissue was extracted using an RNA extraction kit, and during the extraction process, it was necessary to ensure that all the consumables used were enzyme-free. The concentration of RNA was determined by a NanoDrop 2000 spectrophotometer, and the quality of the RNA was examined by agarose gel electrophoresis. Only those RNA samples with an OD260/280 of about 1.8 were used. The extracted RNA samples were reverse transcribed to synthesize the first strand of cDNA. For RT = qPCR analysis, ABI 7500 Real-Time PCR System (Applied Biosystems, USA) was used. The cotton Ghactin2 was used as an internal reference gene, based on the 2 −ΔΔT method to calculate the relative expression of different genes. Three biological replicates and three technical replicates were set for each sample. 2.3 Cloning and sequence analysis of GhMYB102 gene The full-length CDS of GhMYB102 was obtained by designing gene-specific primers based on the published sequence ( Gh_A01G069800 ). Protein sequences of other R2R3-MYB transcription factors from different species were downloaded from the National Center for Biotechnology Information (NCBI) [ www.NCBI.nlm.nih.gov/BLAST/ ]. The amino acid sequences of GhMYB102 and other crops were compared using SnapGene 6.0.2 software. The flavonoid synthesis-related R2R3-MYB protein sequences of A. thaliana , cotton, maize, apple, grape, and strawberry species were downloaded from the TAIR database [ https://www.arabidopsis.org/index.jsp ] and NCBI, respectively, and the whole protein sequences of GhMYB102 and flavonoid-related R2R3-MYB transcription factors were used to construct the phylogenetic tree by using MEGA 11.0 with the neighbor-joining method (Bootstrap value was set at1000 times). 2.4 Virus-induced gene silencing (VIGS) The CLCrV:MYB102 vector was constructed by using the CCM12 cDNA as a template, and the recombinant primer pairs were designed according to the coding sequence (CDS) of GhMYB102, and the target fragment with a size of 216 bp, then amplified using SnapGene 6.0.2. The recombinant CLCrV:GhMYB102 was transformed into Agrobacterium LBA4404 . Agrobacterium sap containing pCLCrVB and pCLCrV: GhMYB102 was mixed in a 1:1 ratio. The bacterial solution was injected into the abaxial surface of the cotyledons of cotton seedlings using a needleless syringe. The injected plants were grown under a light culture chamber at 23°C with a light/dark cycle of 16h/8h until flowering. Injected empty vector ( pCLCrVA and pCLCrVB ) and positive control PDS ( pCLCrV:Su and pCLCrVB ) plants were used as mock-treated and technical controls, respectively. Sulfur encodes a component of the magnesium chelatase complex and the silencing of Su in cotton results in yellow leaves. Petal RNA from the basal spot-free region of GhMYB102 -silenced and blank plants was extracted to detect the silencing effect of the GhMYB102 gene. 2.5 Genetic transformation and overexpressing of homolog GhMYB102 , gene in A. thaliana GhMYB102 was transferred to tobacco under the control of 35S promoter by using Agrobacterium tumefaciens to infest wild-type A. thaliana leaves by flower dipping method, harvested T0 generation A. thaliana seeds were sterilized, inoculated, and cultured on 0.5 MS medium containing antibiotic Kana, screened for positive seedlings that could grow normally, and continued to be cultured until the seeds were harvested, the T1 were used to generate the T2. The T2 generations were cultured on 0.5 MS medium containing antibiotic Kana, screened for positive seedlings, and then transferred to nutrient pots for growth, leaf DNA was extracted, and the GhMYB102 gene fragment was amplified by specific primers. Wild-type A. thaliana DNA was extracted for amplification as a negative control, the recombinant plasmid was used as a positive control, and the positive pseudo-seedlings were removed by molecular characterization, and cultured until the seeds of T2 generation were harvested. The positive seedlings of T2 generation were continued to be screened and then transferred into the nutrient pot, and the qRT-PCR was used for detecting the expression level of GhMYB102 in the positive plants, and the plants with higher expression were selected to harvest the seeds of T3 generation, and then the subsequent experiments were carried out. 2.6 Extraction and quantitative analysis of Anthocyanin Extraction and quantification of the total anthocyanin from cotton flower petals were performed as previously described [ 26 ]. Each flower was considered as an individual biological replica. From each flower, 0.1 g of petals were ground into powder in liquid nitrogen and at least three biological replicates were used for each flower color. Powders were extracted in the dark with 1 mL of acidic methanol (1% hydrochloric acid, w/v) for 24 h at 4°C. After centrifugation at 12,000 rpm for 10 min at 4°C, 1 mL of supernatant was transferred to 4 mL of acidic methanol. The mixture was used to measure absorbance at 530, 620, and 650 nm using a UV/VIS spectrophotometer. The anthocyanin content was calculated according to the formula Q = ODλ/ε × V/m×106, ODλ=(A530-A620)-0.1×༈A650-A620, where V (mL) is the total volume extracted, m (g) is the fresh weight of the sample, and ε is 4.62 × 10 6 representing the molar extinction coefficient of anthocyanins. 2.7 Transcriptome sequencing materials To better understand the color of cotton petals during flower development, the floral development was stratified into six stages, designated S1 to S6. The cotton floral development stages were done though with modification as compared to that of the model plant, Arabidopsis thaliana [ 27 ]. In the S1 stage, the bracts were lime green, with a bud size of 1.5 ± 0.2mm, in the S2 and S3 where bracts emerged with a bud size of 2 ± 0.2mm and 2.5 ± 0.2mm, respectively, in S4 just a day before flowering with bud size of 3 ± 0.2mm, while S5 and S6 are stages immediately after flowering. The samples were corded as per the development stage of the flower and the mode of treatment, from wild types (WT): WTS1 to WTS5), the VIGS-plants: VGS1 to VGS5; Sample materials were ground, total RNA isolated and cDNA libraries constructed. Fifteen cDNA libraries were sequenced on the Illumina HiSeq 2000 platform. Raw data were transformed, filtered, and mapped to the reference genome of G. arboreum using Hisat2. Expression levels were calculated using transcripts per kilobase per million mapped reads (TPM) for exon modeling. Count files were used for differential expression analysis using the R package DESeq2 with a fold change threshold of 1 and a p-value of 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R package ClusterProfiler. Raw data can be found in the NCBI Sequence Read Archive database under accession number PRJNA904836. 2.8 Yeast single hybridization validation Target fragments of cotton chalcone synthesis (GhCHS), chalcone isomerase (GhCHI), anthocyanin synthase (GhANS), and flavonoids − 3’,5’-hydroxylase (GhF3'5'H) promoter sequences containing MYB cis-acting elements of 400–600 bp in size were amplified by specific primers. The amplified promoter fragments were ligated into pAbAi vector as Bait. the amplified product of the complete coding sequence region of GhMYB102 gene was ligated into pGADT7 vector as Pray. 4 µg of pAbAi recombinant plasmid was digested using BstB I, and 1 µg was linearized into pAbAi-proGhCHS , pAbAi-proGhCHI , pAbAi-proGhANS , pAbAi-proGhF3'5'H into Y1HGold strain, coated with SD/-Ura solid-deficient medium and incubated at 30°C for 3–5 days. Single clones were picked from SD/-Ura plates, inoculated in 1mL SD/-Ura liquid medium, incubated at 30°C for 24h, and used as a template for PCR identification. The positive bait clones were resuspended in 0.9% NaCl solution, making the OD600 0.002; 100 µL were coated in SD/-Ura with AbA (0, 100, 300, 500 ng/mL) solid-deficient medium and incubated at 30°C for 3–5 days. The lowest AbA concentration that can inhibit the growth of yeast colonies was selected according to the colony growth. The empty vector pGADT7 and prey vector pGADT7-GhMYB102 were transfected into Y1HGold receptor cells containing the bait plasmid by LiAc/PEG method, respectively, and coated with 150 µL of solid-deficient medium with SD/-Leu and SD/-Leu with optimal AbA, and incubated at 30°C for 3–5 days. Negative control and blank control were also set up. 2.9 Dual luciferase assay The coding regions of GhCHI, GhDFR, GhANS, and GhF3'5'H promoter fragment sequences were cloned onto the pGreen II 0800-LUC vector. The GhMYB102 gene was ligated to the pGreenII 62 –SK . Equal amounts of Agrobacterium cultures containing CLuc and NLuc constructs were mixed and then co-infiltrated into tobacco leaves. The injected tobacco was incubated at 25 ℃ under darkness and shade for 24h, and then placed in a 25 ℃ light culture room with a light/dark cycle of 16h/8h for 24h. Before observation, tobacco leaves were picked and coated with 1 mmol/L D-worm fluorescein potassium salt in the injection area, and placed in a dark environment for 10 minutes for the fluorescence assay. The leaves were observed under a fluorescence microscope. All experiments were repeated at least three times for each plasmid combination. 2.10 Subcellular localization of the GFP fusion expression vector construct The p2300 plasmid with green fluorescent protein tag was used as the backbon to, clone the GhMYB102 gene fragment, and to construct the green fluorescent protein transient fusion expression vector p2300-eGFP-Flag-MYB102 . The transformed Agrobacterium sensu lato GV3101 , and the monoclonal colony was inoculated into the LB liquid medium containing Cannae and rifampicin resistance and cultivated for 12h; then transferred to 0.5 mL of the bacterial solution to 50 mL of liquid LB, and incubated until the OD600 was about 1.0, the bacterium, was then suspended and adjusted the OD600 to about 0.5, then left under room temperature for 3.5h. The p2300-eGFP-Flag vector was used as a control, and p2300-eGFP-Flag-MYB102 , were both injected into the leaves of the tobacco and observed under the fluorescence signals of the injected tobacco leaves under a laser confocal microscope after 48h to determine the subcellular localization of GhMYB102. 2.11 Data analysis All experimental data were analyzed; the mean values were obtained for at least three independent biological replicates. Data were analyzed using SPSS software, and statistical comparisons of differences were analyzed for significance of multiple data using ANOVA statistics, and plotted using GraphPad Prism 9.0. 3 Results 3.1 Cloning and sequence analysis of the GhMYB102 gene Gene annotation information indicated that the candidate gene Gh_A01G069800 CDS sequence was 984 bp in full length, encoding 327 amino acids, and the gene ID was named GhMYB102. The conserved sequence of the GhMYB102 protein was compared with the conserved structural domains of the homologous gene sequences of other species using MEGA11 software, which showed that GhMYB102 was composed of conserved R2 and R3 DNA-binding structural domains at the N-end which was consistent of the conserved R2 and R3 DNA-binding domains. The R3 DNA-binding domain contained the bHLH -binding motif [D/E] Lx2[R/K]x3Lx6Lx3R, and the conserved motif "DNEI" was also found. The conserved motifs downstream of the C-terminus indicated a high degree of divergence (Figure. 2a), which is typical of the R2R3-MYB transcription factors [ 28 – 30 ]. The reported R2R3-MYB sequences of multiple species involved in the regulation of flavonoid metabolism were downloaded from the NCBI and Arabidopsis tair databases, and the phylogenetic tree was constructed by comparing the GhMYB102 with other genes. The results showed that all the genes had a similarity index of 70.77%, and both were MYB transcription factors involved in the inhibition of the anthocyanin accumulation. Gene similarity index of 70% and above is found to be a true type, moreover, in sequence analysis, 30–70% sequence similarity are always retained [ 31 ]. Genetic evolutionary tree analysis showed that GhMYB102 were phylogenetically classified together with Arabidopsis AtMYB4, AtMYB32, AtMYB7, and strawberry FaMYB1, belonging to the subfamily 4 of the R2R3-MYB transcription factors (Fig. 1 b), whereas the majority of the members of subfamily 4 were negative regulators in the anthocyanin synthesis pathway, i.e., involved in the inhibiting phytocannabinoid synthesis [ 28 ], this implied that the transcription factor GhMYB102 was likely to be involved in the negative regulation of the anthocyanin synthesis. To determine the regulatory effect of the GhMYB102 gene on cotton petal color, the expression of the GhMYB102 gene was downregulated in Marie-galante 1139 (creamy white flowers), punctatum 187 (yellowish flowers), Y52, and Zhongyihong 2 (pink flowers), respectively, by virus-induced gene silencing (VIGS) mechanism. The petals of the silenced plants were harvested for RNA extraction. The gene silencing efficiency was assessed through RT-qPCR, and the results showed that the expression of GhMYB102 was significantly low in the VIGS-plants ( CLCrV: MYB102 silenced plants) compared to the WT and null-loaded plants (Fig. 2 a). The downregulation of the silenced gene showed that the novel gene was effectively silenced. Petal phenotypes of the wild-type and CLCrV :MYB102 silent plants, Marie-galante 1139 (cream flower), punctatum 187 (yellowish flower), Y52 (pink flower), and Zhongyihong 2 (pink flower) were monitored, and there was no morphological variation in the flower pattern between the petals of wild-type and CLCrV:MYB102 silenced plants, however in quantifying the anthocyanin content revealed that significantly higher anthocyanin concentrations in the silenced plants compared to the WT and the positive controlled ones. (Fig. 2 b). Furthermore, a significant observation was made between the WT and the VIGS-plants, in Zhongyihong and Y52 with pink flowers, the pigmentation intensity was significantly higher in VIGS-plant compared to WT. The anthocyanin content in petals of the VIGS-plants was significantly higher than that in the WT and positive controlled plants, which was about three times higher than that of wild-type petals (Fig. 2 c). Based on these results, we concluded that the knockdown of GhMYB102 gene significantly increases the accumulation of anthocyanin content in cotton petals and that GhMYB102 may be playing an inhibitory role in the anthocyanin biosynthesis pathway. The results obtained were in agreement with previous findings, in which anthocyanins synthesis in plants is influenced by transcriptional regulatory factors, majorly the MYB, bHLH, and WD40 [ 32 ], which constitutes anthocyanin biosynthesis regulatory system by binding to structural gene promoters. 3.2 GhMYB102 induces Drought resistance and maintenance of leave pigmentation The GhMYB102 homolog was overexpressed (OE) in A. thaliana , a mutant form, and the wild types were obtained, the three were subjected to drought treatment, and the results showed that the OE plants exhibited higher tolerance to drought stress, leaves were green, and no sign of wilting, while the mutant and the wild type, were significantly affected by drought. Moreover, in the mutant forms, 9 out of the 12 planted cups suffered severe drought and dried. A unique observation however was noted among the wild type, the leaves became more chlorotic, and the survival level was significantly higher compared to the mutant forms (Fig. 3 a). Phenotypic evaluation of the leaves of the OE, WT, and the positive control under drought, all the plants exhibited normal leave shape and color under well-watered conditions, however, OE plants only experienced mild wilting, and the leaf color was still bright green; WT plants had extensive leaf curling, and the leaf color shifted to dark green compared with that of the control group with normal watering; and while the mutant plants had severe wilting and even died, and the leaf color changed to dark purple (Fig. 3bi). A larger group of the MyBs promotes anthocyanin accumulation, for instance, overexpression of SlMYB75 gene enhanced anthocyanin accumulation in both vegetative and reproductive tissues in tomatoes, even though expression levels of SlMYB75 were found to be relatively low in wilt types [ 33 ]. Moreover, quantification of the anthocyanin levels among the OE, WT, and the mutant forms under drought stress conditions, revealed a higher anthocyanin content in the mutant form, closely followed by the WT but significantly low in the OE plants (Fig. 3bii). Anthocyanin accumulation in plants more so in the leaves when plants are under stress plays a survival role, the anthocyanins scavenge on the reactive oxygen species (ROS), thereby protecting plants from oxidative damage and enhancing their sustainability [ 34 ]. 3.3 Subcellular localization To study the expression site of the protein encoded by GhMYB102 gene, p2300-eGFP-Flag-MYB102 fusion expression vector was constructed. The green fluorescence signal was observed under a laser confocal microscope (Leica TCS SP8 DTED) with the help of a tobacco transient expression system. The green fluorescent protein was expressed on both the cell membrane and nucleus of P2300-eGFP-Flag ; while the p2300-eGFP-Flag-MYB102 fusion vector was expressed on the nucleus (Fig. 4 ). 3.4 Transcriptome data enrichment analysis Gene expression is temporally and spatially specific, and this study screened for differentially expressed genes (DEGs) between the comparison groups. The comparison groups were divided into WTS4 vs WTS1, WTS5 vs WTS1, WTS5 vs WTS4 among wild type; VGS5 vs VGS4 among silenced strains; and VGS4 vs WTS4, VGS5 vs WTS5 among wild type and silenced strains, totaling six comparison groups (Fig. 5 a). A comparison of GhMYB102 gene expression in WTS5 vs VGS5 using qRT-PCR analysis confirmed that the gene expression in VGS5 was significantly lower than that in WTS5. To understand the biological functions of the differentially expressed genes, KEGG enrichment was used in this study to classify DEGs for functional enrichment. The expression of genes enriched to flavonoid synthesis pathway genes in all the samples was jointly displayed with some flavonoid metabolism schematics, and the gene enrichment is shown in (Supplementary Fig. 1). Differential gene enrichment analysis of the VGS5 vs WTS5 group, which had significant phenotypic differences in petals, revealed significant enrichment in the flavonoid synthesis pathway, with 19 genes significantly up-regulated and 1 gene significantly down-regulated (Fig. 5 b). The enrichment analysis of up-regulated genes in the VGS5 vs WTS5 group again showed that the flavonoid synthesis pathway was ranked first in significance, and the significantly up-regulated genes included CHS, CHI, F3'5'H, F3'H, F3H, ANS, FLS, and LAR, which are structural genes involved in the synthesis of flavonols and anthocyanins (Table 1 ). The above results suggest that the transcription factor GhMYB102 may directly or indirectly inhibit the expression of structural genes in the flavonoid metabolic pathway, thus affecting the synthesis of flavonoids and anthocyanins. Table 1 Expression levels of the flower pigmentation responsive genes under WT, and VIGS plants. Gene Name Gene ID WTS5 VGS5 VGS5/WTS5 F3'5'H Gh_A07G130200.1 5.242 14.108 2.69 Gh_A07G130000.1 0.4623 1.3443 2.91 LAR Gh_D12G188200.1 3.7419 8.826 2.36 CHI Gh_Contig01002G 001700.1 4.7022 11.12 2.36 F3H Gh_A12G064100.1 18.492 91.319 4.94 Gh_D12G061800.1 5.9692 50.555 8.47 Gh_A11G186200.1 7.4602 18.051 2.42 CHS Gh_A05G312100.1 0.7676 4.0172 5.23 Gh_A09G000900 0.3665 1.2489 3.41 Gh_A02G031300 2.5245 16.911 6.70 Gh_A09G000100 2.3735 8.2105 3.46 Gh_D02G036800 4.4697 23.725 5.31 Gh_D05G322000 0.0516 0.445 8.62 Gh_D09G000100.1 1.7385 8.3984 4.83 Gh_Contig00311G 000400.1 5.0616 14.841 2.93 FLS Gh_A05G400400.1 3.5118 14.597 4.16 Gh_D04G022800.1 0.4526 4.4799 9.90 ANS Gh_A13G262900.1 5.9781 12.991 2.17 F3'H Gh_A12G204700.1 2.1729 8.4589 3.89 3.5 qRT-PCR gene expression analysis of flavonoid synthesis genes To further investigate whether GhMYB102 could regulate key flavonoid genes in cotton petals, we used qRT-PCR to compare the expression of CHS, CHI, F3H, F3'H, F3'5'H, DFR, and ANS in the Y52 material after the interference of WT and VIGS, UFGT eight flavonoid synthesis-related gene expression. The results showed that GhMYB102 strongly affected the transcript levels of genes involved in flavonoid and anthocyanin biosynthesis. When the expression of GhMYB102 decreased, the expression of flavonoid and anthocyanin synthesis-related genes CHI, F3H, F3'H, F3'5'H, DFR, and ANS increased significantly (Fig. 6 ). The results obtained were in agreement with previous findings in which the expression of anthocyanidin synthase, RnANS1 gene was upregulated during fruit maturation, which positively correlated with anthocyanin accumulation [ 35 ]. Furthermore, the levels of the anthocyanin biosynthetic genes are positively correlated to anthocyanin contents at different flower developmental stages, being dihydroflavonol 4-reductase (DFR) is vital for anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonol to leucoanthocyanidin [ 36 ]. The results were consistent with the transcriptome sequencing results, indicating that the transcriptome data were reliable and suggesting that the transcription factor GhMYB102 may directly or indirectly deter the expression of structural genes in the flavonoid metabolic pathway, thus affecting the synthesis of flavonoids and anthocyanins. 3.6 Yeast single hybridization To determine whether there is a direct regulatory relationship between GhMYB102 and the structural genes of flavonoid metabolism, we first analyzed the promoter sequences of eight genes related to flavonoid synthesis, namely, CHS, CHI, F3H, F3'H, F3'5'H, DFR, ANS, and UFGT, in the Plant CARE database. Flavonoid synthesis-related genes were analyzed, and it was found that the 2000bp region upstream of these genes had multiple MYB binding elements. After that, we ligated the complete coding region sequence of GhMYB102 gene into pGADT7 vector as a prey protein, and amplified the 2000bp upstream sequences of GhCHS , GhCHI , GhANS , and GhF3'5'H genes, and ligated them into pAbAi vector as bait proteins. Self-activation assay was performed on Bait, and the results showed that yeast cells transformed with the bait plasmid grew normally on SD/-Ura solid-deficient medium. Observation of yeast cell growth under 100, 300, and 500 ng/mL of antibiotics showed that there had been essentially no growth on SD/-Ura/AbA (100 ng/mL) solid-deficient medium, indicating that pAbAi-proGhCHS , pAbAi-proGhCHI , pAbAi-proGhANS , pAbAi- proGhF3'5'H had a self-activating response, but 100 ng/mL gold tanshin suppressed the background expression of the AbAr reporter gene in the transformed strain (Fig. 7 a). Positive clones on SD/-Ura solid-deficient medium were picked to make Y1H Gold yeast receptor cells. The yeast cells were transfected into a plasmid containing yeast prey recombinant vector pGADT7-GhMYB102 and empty pGADT7 (negative control), respectively, and the growth of yeast cells was observed on the corresponding solid-deficient medium. The results showed that all yeast strains could grow normally on SD/-Leu solid-deficient medium, but except for the controlled group pGADT7 + pAbAi-proGhCHS that showed strain growth, only the yeast strain transformed with the pGADT7-GhMYB102 plasmid could grow normally on the SD/-Leu solid-deficient medium containing 150 ng/mL AbA medium for normal growth (Fig. 7 b). This indicated that GhMYB102 was able to bind cis-acting elements in the GhCHI, GhANS, and GhF3'5'H promoters and activate expression. 3.7 Dual luciferase validation To detect whether the transcription factor GhMYB102 regulates the promoters of ANS , F3'5'H , DFR , and CHI genes, a dual luciferase assay experiment was performed. A 2000 bp fragment upstream of the four promoters was ligated into the plant expression vector pGreenII 0800-LUC via SalI and KpnI restriction endonucleases to constitute the Reporter, while the full-field sequences of the coding regions of the genes of GhMYB102 were ligated into the plant expression vector pGreenII 62-SK via XbaI and KpnI restriction endonucleases to form Effector. Prepare bacterial solution for transient transformation of tobacco leaves, the leaves were divided into four equal parts during transformation, the upper left area is the combination of 62-SK empty and 0800-promoter, the lower left area is the combination of 62SK-MYB102 and 0800 empty, the lower right is the combination of 62-SK empty and 0800 empty, and the above serves as the control group ; upper right is the combination of 62SK-MYB102 and 0800-promoter, which served as the experimental group. Each combination was set up with at least 3 technical replicates (Fig. 8 a). Fluorescence imaging results showed that the combination of pGreenII 62-SK and 0800-promoter on the upper left had a higher fluorescence intensity (Fig. 8 b), indicating that the ANS promoter can self-activate, and the combination of 62SK-MYB102 and 0800-promoter on the upper right had a fainter fluorescence intensity, indicating that the transcription factor GhMYB102 represses the ANS promoter's expression. Similarly, GhMYB102 also repressed the expression of F3'5'H, the CHI promoter, and had almost no effect on the CHS promoter. 4 Discussion Petal colors in nature are varied and diverse, which are mainly influenced by the external environment such as light, temperature, and soil pH, as well as internal color-developing pigments [ 37 ]. The main determinant of floral pigmentation is the flavonoids, which their metabolic pathways are very conserved in plants, and it is an enzyme-catalyzed reaction encoded by a series of structural genes. One of the largest plant transcriptome factors with a greater role in primary and secondary metabolic reactions are the MyB transcription factors [ 38 ]. The R2R3-MYB transcription factors have the highest percentage in the MYB family [ 39 ]. Studies have shown that the MYBs do regulate petal color in multiple species, such as lily, moonflower, daffodil, and gerbera [ 40 ]. Moreover, in cotton, the GhTT2 of the MYB family promotes proanthocyanidin biosynthesis [ 40 ]. Additionally, overexpression of GhMYB1a leads to increased accumulation of flavonols [ 28 ]; GhMYB113 controls the formation of purple spots at the base of petals in tetraploid sea-island cotton [ 41 ]; and dominant genes in Asian cotton. Furthermore, a loss of function of GaPC in the mutant forms, results in a phenotype in which GaPC masks other color genes and exhibits recessive epistasis, whereas petals of GaPC -silenced sea and land cotton exhibit albinism. Furthermore, infiltration of a gene that is identical to that of GbBM homologous to R2R3-MYB gene fragment GhPF in land cotton showed a rare pink color [ 42 ]. It has also been reported that the change of corolla color from pink to white may also be due to a promoter variant of a GhTT19 gene in the region of the infiltrated fragment [ 43 ]. In this study, a new transcription factor GhMYB102 belonging to R2R3-MYB, was identified from a wild cotton germline of terrestrial cotton. Phylogenetically, this gene was found to be a member of the 4th subfamily, known to have the inhibitory role of anthocyanin biosynthesis, moreover, a single nucleotide mutation in the gene resulted in a higher pigmentation intensity in the VIGS-plants, an indication that the suppression of its expression has a positive effect on the anthocyanin biosynthesis pathways. The results were in agreement with previous studies in which TgMYB4 repression negatively regulated the Anthocyanin Biosynthesis in Tulipa gesneriana L [ 44 ]. The results were further validated by overexpressing the gene in A. thaliana , the mutant form exhibited higher anthocyanin content and purplish leave color compared to the OE plants, which further revealed the inhibitory role of the identified gene. However, it is important to note that the MYB genes are critical in anthocyanin biosynthesis pathways and have been explored in several crops, for instance, SlMYB75, an MYB-type transcription factor has been found to have a positive regulatory role in anthocyanin biosynthesis and also promote the volatile compound synthesis in tomatoes [ 33 ]. Thus the negative role of the identified gene, clearly indicates that the gene is a member of the 4th subfamily. Even though the mutant recorded the highest concentration levels of the anthocyanin, the performance under drought stress conditions revealed a negative adaptation towards drought stress, the OE plants maintained green leaves, and with significantly low phenotypic attributes of wilting compared to the mutant and the wild type. The mutant and WT suffered severe effects of drought stress, in plants when plants are exposed to either drought or salt stress, the amount of reactive oxygen species (ROS) released increases, and thus the high induction levels of the anthocyanins would help in reducing the ROS levels, however this was not observed. MYB transcription factors have been found to enhance drought stress tolerance [ 45 ]. The ZmRL6 gene a member of the MYBs has been found to have a positive effect on enhancing drought tolerance in maize [ 46 ]. Furthermore, overexpression of MYB12 and MYB75 genes has been found to increase flavonoid accumulation with significantly enhanced antioxidative activities [ 47 ]. Thus the OE plant’s response to drought stress could have been due to the oxidative properties of the anthocyanin thereby reducing the oxidative stress, minimizing the cell damage, and preventing massive cell death. Moreover, the drought effect wasn’t so severe, which points to the possible role of the anthocyanin to scavenge on the reactive oxygen species (ROS). Based on transcriptome sequencing, secondary metabolite pathway exploration, and functional gene mining are carried out to study the molecular mechanism of cotton flower color regulation. To further investigate the molecular mechanism of GhMYB102 regulating anthocyanin synthesis, we performed transcriptome analysis using wild-type S1, S4, and S5 period petals of land cotton genetic material "Y52" (control) and S4 and S5 period petals of CLCrV: MYB102 (VIGS). We analyzed 74,474 expressed genes for KEGG categorical enrichment, and a total of 40 genes were enriched in the flavonoid metabolic pathway. Enrichment analysis of up-regulated genes in the VGS5 vs WTS5 group, which had significant phenotypic differences in petals, ranked first in flavonoid synthesis pathway significance, and the 19 genes that were significantly up-regulated included CHS, CHI, F3'5'H, F3'H, F3H, ANS, FLS, LAR, these genes are involved in the synthesis of flavonols, anthocyanins. The above results suggest that the transcription factor GhMYB102 may directly or indirectly deter the expression of structural genes in the flavonoid metabolic pathway, thus affecting the synthesis of flavonoids and anthocyanins. Based on the analysis of the transcriptional regulatory network of GhMYB102, we further verified the expression of flavonoid structural genes CHS, CHI, F3'5'H, F3'H, F3H, ANS, DFR, UFGT using qRT-PCR, which was consistent with the results of transcriptome sequencing. transcriptome sequencing results were consistent. To determine whether there is a direct regulatory relationship between GhMYB102 and the structural genes of flavonoid metabolism, we carried out yeast one-hybrid (Y1H) and dual-luciferase (dual-LUC) experiments on the structural genes of flavonoids analyzed by qRT-PCR, to elucidate the molecular mechanism of GhMYB102 in regulating phytochromes. The results indicated that the transcription factor GhMYB102 can directly repress the expression activity of GhCHI, GhF3'5'H, and GhANS promoters. 5 Conclusion Cotton is an important crop, and the main source of white fiber, which is the primary raw material in the textile industries. Its production has greatly declined due effects of abiotic factors, which have become a limiting factor not only to cotton but other crops, moreover, use of pesticides has also significantly reduced the population of the natural pollinators. The diversity that exist within the cotton gnome provided an excellent platform to understand the role of MyB genes in regulating the floral color. In this work, a novel gene GhMyB was identified from the semi wild cotton germplasms (Data to be published). The gene has been found to play an inhibitory role in the anthocyanin biosynthesis pathways, and has additive effect in enhancing drought tolerance in cotton. Further exploitation is required to understand the antagonistic role, of this gene, being anthocyanin is known to have ROS scavenging capacity, so its inhibitory nature, and the positive effect of the gene in enhancing drought tolerance requires further exploration. Declarations Author contribution statement Conceptualization, DW, ZZ, ROM, LF. and YX.; methodology, DW, ROM, YX, JNK, ZZ.; software, DW and ROM.; validation, DW, ROM, ZZ, JNK, YH, QKL, JH, YX and ZZ.; formal analysis, DW, ROM, JNK, YH.; investigation, DW.; resources, ZZ, XC, YH, YYZ, QKL and JZ.; data curation, JZ, FL, and ZZ.; writing—original draft preparation, DW, and ROM.; writing—review and editing, ROM.; All authors have read and agreed to the published version of the manuscript. Funding This research was funded by National Natural Science Foundation of China (32401824, 32171994, 32272090); Natural Science Youth Fund of Henan Province (242300421595); the National Key R&D Program of China (2024YFD1200300,2022YFD1200345) Data Availability Statement All data that support this publication are fully provided within the text and its supplementary files Acknowledgments We acknowledge the enormous support provided by the entire research group of the wild cotton germplasm resources, of the institute of cotton research (ICR), Anyang, China, the lab technicians and laboratory managers for the support accorded to us during this research work. Conflicts of Interest The authors declare no conflicts of interest. References Yu J, Jung S, Cheng C, Ficklin SP, Lee T, Zheng P, et al. CottonGen : a genomics , genetics and breeding database for cotton research. 2014;42 November 2013:1229–36. Khidirov MT, Ernazarova DK, Rafieva FU, Ernazarova ZA, Toshpulatov AK, Umarov RF, et al. 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Enhancement of oxidative and drought tolerance in Arabidopsis by over accumulation of antioxidant flavonoids. Plant J. 2014; 77:367–79. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviewers agreed at journal 10 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers invited by journal 07 Sep, 2025 Editor invited by journal 24 Jul, 2025 Editor assigned by journal 23 Jul, 2025 Submission checks completed at journal 23 Jul, 2025 First submitted to journal 22 Jul, 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. 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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-7187536","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512843477,"identity":"8357808e-0c76-4a55-8cb4-cbe6b0985be0","order_by":0,"name":"Dong Wenhao","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Wenhao","suffix":""},{"id":512843478,"identity":"ac4c61cf-1700-4677-bf0e-3827fa0625f2","order_by":1,"name":"Richard Odongo Magwanga","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYFADZgY2ECnHwMBDohZjErQwQLQkNhDSott+/JrEzx218ubtzM8e/Nxjnb7h+NmDDz4w2MnpNmDXYnYmp0yy98xxwzmH2cwNe56l5244k5dsOIMh2djsAA4tB3LSbvC2HWOcwczDJsFz4HDuhgM5ZtI8DAcSt+HScv5N2s2/bcfsQVok/xw4nG5w/g0BLTfSj93mbatJBGmRBtqSYHCDkC033rD/lm07kDyDmc1MWuZAuuHMG2+MDWcY4PHL+fTHhm/b6mxn8B9+JvnmgLU83/kcwwcfKuzkcGkBRpwBkDiM4CuAVRrgUg4C7A+ARB2CL9+AT/UoGAWjYBSMRAAAZCxh4elmGAMAAAAASUVORK5CYII=","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":true,"prefix":"","firstName":"Richard","middleName":"Odongo","lastName":"Magwanga","suffix":""},{"id":512843479,"identity":"5566e960-0cf0-4bea-b033-78b1552cf933","order_by":2,"name":"Yanchao Xu","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Yanchao","middleName":"","lastName":"Xu","suffix":""},{"id":512843480,"identity":"900e1014-a706-4abb-bbb9-12d68ac4bbd0","order_by":3,"name":"Joy Nyangasi Kirungu","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Joy","middleName":"Nyangasi","lastName":"Kirungu","suffix":""},{"id":512843482,"identity":"504a6d98-0d7c-4b19-9cf9-ae7fc17dadd3","order_by":4,"name":"Yuqing Hou","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Hou","suffix":""},{"id":512843483,"identity":"c0e5fa7f-fda1-48ba-a21f-261a60fa7a4d","order_by":5,"name":"QianKun Liu","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"QianKun","middleName":"","lastName":"Liu","suffix":""},{"id":512843484,"identity":"f50cf0ff-c3cc-4d66-bff1-262e136e7078","order_by":6,"name":"Jiale Han","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Jiale","middleName":"","lastName":"Han","suffix":""},{"id":512843485,"identity":"c4ba2d69-adfd-4999-948f-4f1ca45f28c5","order_by":7,"name":"Xiaoyan Cai","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Cai","suffix":""},{"id":512843486,"identity":"9f090cde-be4d-43fe-bb6c-4af7cc5f70aa","order_by":8,"name":"Fang Liu","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Liu","suffix":""},{"id":512843487,"identity":"507c5a2c-649c-4460-bb70-a68b1a791fea","order_by":9,"name":"Zhongli Zhou","email":"","orcid":"","institution":"Zhengzhou University, Chinese Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Zhongli","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-07-22 13:23:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7187536/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7187536/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91163021,"identity":"d172d2a5-085d-4142-b21e-77e4af332fed","added_by":"auto","created_at":"2025-09-12 09:44:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2071129,"visible":true,"origin":"","legend":"\u003cp\u003e(a) R2R3-MYB transcription factor protein sequence alignment; (b) Evolutionary tree of R2R3-MYB transcription factors associated with multispecies flavonoid synthesis pathways\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/bfddfc1552d7e7becfd5a956.png"},{"id":91162182,"identity":"ecb103e2-9cf7-4ce9-9f2a-2c2978e3b051","added_by":"auto","created_at":"2025-09-12 09:28:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2826831,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis and anthocyanin quantification, (a i-iv). \u003cem\u003eMyB102 \u003c/em\u003egene expression levels in Maria Galante, punctatum, zhongyihong2 and YS2, respectively. (b) the flower color of the four cotton varieties at WT and VIGS-plants, (c) Quantification of anthocyanin levels on WT, CLCrV and \u003cem\u003eCLCrV: MyB102\u003c/em\u003e plants. The bar indicates the standard error (SE). Different letters indicate the significant differences between the wild type (WT) and VIGS-plants (ANOVA; \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/9841293e5e8034fd5a1f32f0.jpg"},{"id":91162632,"identity":"89e425f1-e946-43cb-b0b7-bfdb2dedba52","added_by":"auto","created_at":"2025-09-12 09:36:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3391840,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Morphological evaluation of overexpressed \u003cem\u003eArabidopsis thaliana\u003c/em\u003e under drought stress conditions. (b) (i)Wilting and color change on leaves excised from overexpressed, wilt types and the mutant. (ii). Anthocyanin quantification as extracted from the leaves of Mutant, WT and OE plants under drought stress conditions. The bar indicates the standard error (SE). Different letters indicate the significant differences between the wild type (WT) and VIGS-plants (ANOVA; \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Figure3.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/d71cc04abf6c4db337052cb3.jpg"},{"id":91162189,"identity":"52504256-e3b6-4e47-a4c3-123686196175","added_by":"auto","created_at":"2025-09-12 09:28:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4273797,"visible":true,"origin":"","legend":"\u003cp\u003eLocalization of GhMYB102 in tobacco, the tobacco epidermal cells transformed with P2300-eGFP-flag and P2300-eGFP-flag-MyB102\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/7fb98fdc5605291c29a9956c.png"},{"id":91162186,"identity":"ee85d04e-a0c9-4eb1-bc03-5686a53894d1","added_by":"auto","created_at":"2025-09-12 09:28:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":621515,"visible":true,"origin":"","legend":"\u003cp\u003e(a) (i)Flower development stages, (ii)Flower color and anthocyanin intensity on the wild type and the VIGS-plants, (iii) Extraction of anthocyanin from the petals of WT and VIGS-plants, (iv) Anthocyanin quantification at different floral development stages from S1 to S6, (b) The expression of flavonoid metabolism structure gene was significantly increased in VGS5 vs WTS5 group. Bar indicates the standard error (SE). Different letters indicate the significant differences (ANOVA; \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Figure5.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/e4c84d4a5f9e1e1d2a6a69f5.jpg"},{"id":91162633,"identity":"dbc305ce-c18f-4315-b32f-704bef34962e","added_by":"auto","created_at":"2025-09-12 09:36:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2459057,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of flavonoid synthesis gene expression in cotton petals of gene interference strain and wild type. WT indicates wild-type Y52 strain petals; GhMYB102 represents VIGS silent strain petals; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; F3′H, flavonoid-3′-hydroxylase; F3′5′H, flavonoid-3′,5′-hydroxylase; DFR, dihydroflavanol-4-reductase; ANS. anthocyanin synthase; UFGT, flavonoid glycosyltransferase. Asterisks represent significant differences from the wild type at the \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e level. Bars indicate the standard error of three biological replicates.\u003c/p\u003e","description":"","filename":"Figure6.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/0872628631d52f12925fd8be.jpg"},{"id":91162631,"identity":"075ef721-a11e-4ebb-8b39-b2484c0e1eb9","added_by":"auto","created_at":"2025-09-12 09:36:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2058165,"visible":true,"origin":"","legend":"\u003cp\u003eGhMYB102 specifically binds to downstream gene promoters\u003c/p\u003e","description":"","filename":"Figure7.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/426b78e11973523aa78118d6.jpg"},{"id":91162190,"identity":"e7de4d44-e318-445f-96b5-c85fd2ca35ed","added_by":"auto","created_at":"2025-09-12 09:28:47","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1397877,"visible":true,"origin":"","legend":"\u003cp\u003e(a). Bacterial injection combination, (b) The expression of downstream gene is inhibited by GhMYB102 in tobacco\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/d9471f5b872df1a3f887ad2f.jpg"},{"id":91163505,"identity":"0a2d0b34-5d5d-48f4-9466-cdb5a9e33214","added_by":"auto","created_at":"2025-09-12 09:52:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19049588,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7187536/v1/3bf7cc56-90e4-4d58-b703-25ee64664ebf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unraveling GhMYB102: A Dual-Function of the Gh_A01G069800 Gene in Promoting Anthocyanin Biosynthesis Inhibition, and Drought Tolerance in Cotton","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCotton belongs to the mallow family, mainly a herb or perennial shrub consisting of more than 50 diploid and tetraploid species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Cotton species have a wide range of flower colors, with wild diploid and tetraploid cotton species having vibrant petal colors and basal spots [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, the floral color do undergo transitional changes post anthesis, with diverse array of petal colors with yellow, cream and red colors being dominant [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The diversity in the floral color is natures gift, and it serves as a critical tool for plant geneticists, ecologists and biochemists in understanding the evolution, diversity, and presence of volatile compounds in plants [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The major pigments which constitutes the floral colors include carotenoids, anthocyanins, and betalains, furthermore, chlorophylls also provide the main flower pigments, but only in selected plant species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Floral color is determined by both endogenous and exogenous factors, moreover, exogenous factors are predominantly influenced by the environment, for instance, humidity, temperature, and light could also play a role in determining floral pigmentation and scent [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition to the major pigments, other conditions are key, for instance the vacuolar pH influence the occurrence of some colors, for blue flowers to emerge, a vacuolar pH of more than 5.5 is critical [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The floral color is also a powerful trait used by plants taxonomist, as a general phenotypic trait, but also confer several biological roles, such as protection against UV-light and attraction of pollinators [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].The flavonoids are a large subgroup of phenypropanoids [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the color determinants are basically water soluble and are located within the cellular vacuoles [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The main flavonoids pigments are the anthocyanins which is the precursor for the pink, red, orange, blue, purple, and blue-black flower colors, and exist in over 90% of angiosperms [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Apart from flowers, flavonoids are also present in fruits, vegetables, grains, bark, roots, stems, tea, and wine [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], moreover, the flavonoids also play other significant biological activities for instance, protect the plants against plant feeding insects and herbivores [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The flavonoids creates color and aroma of flowers, and in fruits as pollinators and dispersal agents attractants to improve the pollination efficiency and dispersal, respectively [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The flavonoids biosynthesis is well understood due to its high antioxidant and UV light protection, moreover a number of plant metabolites are induced by light. The main derivative of flavonoids is phenylalanine, the process is catalyzed by phenylalanine ammonia-lyase, the and entire process is mediated by a similar step with flavanone 3-hydroxylase (F3H), chalcone synthase (CHS) and fluxed into anthocyanin biosynthesis by dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Flavonoids can be divided into 6 categories, flavonoids, flavanols, flavanones, isoflavonoids, flavonols, and anthocyanins. Some of the flavonoids (e.g., flavones, flavanols, and orange ketones) can give flowers a yellow color, while anthocyanins can give flowers a red, pink, blue, or purple color. Among them, anthocyanins are the largest subgroup of pigment substances, and the differences in the coloration of plant petals among different species and varieties are mainly caused by the differences in the types of anthocyanins.\u003c/p\u003e\u003cp\u003eHydroxylation, glycosylation, methylation, and acylation modifications of the anthocyanin glycosides B ring, the anthocyanins can be modified into 6 categories, which include the geranins, delphinidins, petunidins, mallowins, paeonidins and the cornflowerins. Moreover, the hydroxylation of the glycosides B ring plays an important role in enhancing stability, and hindering oxidation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, anthocyanins glycosylation promotes hypsochomic shift in the absorption maxima of the spectra, and in turn boosts the anthocyanin stability and effective storage at the cellular vacuole [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The unique floral color of \u003cem\u003eGentiana lutea\u003c/em\u003e var. aurantiaca and transgenic petunia (\u003cem\u003ePetunia\u0026times; hybrida\u003c/em\u003e) and other such as the orange family (yellow to orange-red) plant petals is due to the presence of the large amount of geraniolins, the \u003cem\u003eIris tectorum\u003c/em\u003e and blue moonflower (\u003cem\u003eRosa chinensis\u003c/em\u003e Jacq) and other blue family (blue to purple) petals contain large amounts of delphinidin, petunidin, or mallowin, and the red (pink to red) petals of \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e, Lilium \u003cem\u003espp\u003c/em\u003e., and petunias usually contain large amounts of cornflowerin, and paeonidin. So far, there are more than 600 anthocyanins found in nature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCotton germplasms are polymorphic, which the color of the flower, has been used to explore their modern genetic studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The color of the cotton flower is due to the effects of both flavonols and anthocyanin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The flower color could also be attributed to the varietal type, ecological condition, and fertilization [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], However, the pigment of the cotton flower is less affected by environmental factors than that of its leaf [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and thus a stable inheritance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Few studies have investigated the molecular basis that underpins the formation and accumulation of anthocyanins in wild and cultivated cotton progenitors. The whole genome sequencing of AD [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], D [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and A [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] genomes provide a perfect platform to explore the diversity of cotton flower colors and therefore, in this research, four cotton germplasms were evaluated, two \u003cem\u003eG. hirsutum\u003c/em\u003e races Marie Galante 1139 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], punctatum187 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and two upland cotton varieties, Zhongyihong2 and Y52 to explore their floral color. A novel gene, \u003cem\u003eMyB102\u003c/em\u003e was further validated through overexpression, gene knock, and yeast hybridization to further understands its putative role in the determination of the color of the flower, and possible influence in promoting plants tolerance to drought stress. The results therein provide valuable information for breeders to further explore \u003cem\u003eMyB102\u003c/em\u003e in modulating flower color, and breeding for diverse plants varieties with higher adaptability and production efficiency in the face of ever-changing climatic conditions.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plant material\u003c/h2\u003e\u003cp\u003eThe wild cotton of \u003cem\u003eGossypium hirsutum\u003c/em\u003e races Marie-galante 1139 (cream colored corolla), punctatum 187 (yellowish corolla), the upland cotton cultivars, Y52 (pink corolla), and Zhongyihong 2 (pink corolla), the model plant, Columbia-type \u003cem\u003eA. thaliana\u003c/em\u003e (Col-0), and Ben's \u003cem\u003eTobacco tabacum\u003c/em\u003e were used in this study. The wild cotton germplasms were obtained from the wild cotton germplasm research unit of the Institute of Cotton Research, Anyang, while the land genetic materials Y52 and Zhongyihong 2 were provided by the National Intermediate Cotton Germplasm Resource Bank. The target gene Arabidopsis mutant (N867168) was purchased from the AraShare Arabidopsis mutant germplasm platform [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arashare.cn/\u003c/span\u003e\u003cspan address=\"https://www.arashare.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. The seeds for the four cotton germplasms were delinted, and germinated in pots. The plants were grown in the greenhouse, and at the flowering stage, the petals were collected at 10.00 hrs, frozen in liquid nitrogen, and stored at -80\u003csup\u003e0\u003c/sup\u003eC for RNA extraction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 RNA extraction and qRT-PCR analysis\u003c/h2\u003e\u003cp\u003eThe petal tissues were harvested from Marie-galante 1139 (cream colored corolla), punctatum 187 (yellowish corolla), the land races, Y52 (pink corolla), and Zhongyihong 2 (pink corolla), in liquid nitrogen, then transferred to -80\u003csup\u003e0\u003c/sup\u003eC for temporary storage. The total RNA of the petal tissue was extracted using an RNA extraction kit, and during the extraction process, it was necessary to ensure that all the consumables used were enzyme-free. The concentration of RNA was determined by a NanoDrop 2000 spectrophotometer, and the quality of the RNA was examined by agarose gel electrophoresis. Only those RNA samples with an OD260/280 of about 1.8 were used. The extracted RNA samples were reverse transcribed to synthesize the first strand of cDNA. For RT\u0026thinsp;=\u0026thinsp;qPCR analysis, ABI 7500 Real-Time PCR System (Applied Biosystems, USA) was used. The cotton \u003cem\u003eGhactin2\u003c/em\u003e was used as an internal reference gene, based on the 2\u003csup\u003e\u0026minus;ΔΔT\u003c/sup\u003e method to calculate the relative expression of different genes. Three biological replicates and three technical replicates were set for each sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cloning and sequence analysis of \u003cem\u003eGhMYB102\u003c/em\u003e gene\u003c/h2\u003e\u003cp\u003eThe full-length CDS of GhMYB102 was obtained by designing gene-specific primers based on the published sequence (\u003cem\u003eGh_A01G069800\u003c/em\u003e). Protein sequences of other R2R3-MYB transcription factors from different species were downloaded from the National Center for Biotechnology Information (NCBI) [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.NCBI.nlm.nih.gov/BLAST/\u003c/span\u003e\u003cspan address=\"http://www.NCBI.nlm.nih.gov/BLAST/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. The amino acid sequences of GhMYB102 and other crops were compared using SnapGene 6.0.2 software.\u003c/p\u003e\u003cp\u003eThe flavonoid synthesis-related R2R3-MYB protein sequences of \u003cem\u003eA. thaliana\u003c/em\u003e, cotton, maize, apple, grape, and strawberry species were downloaded from the TAIR database [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e] and NCBI, respectively, and the whole protein sequences of GhMYB102 and flavonoid-related R2R3-MYB transcription factors were used to construct the phylogenetic tree by using MEGA 11.0 with the neighbor-joining method (Bootstrap value was set at1000 times).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Virus-induced gene silencing (VIGS)\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eCLCrV:MYB102\u003c/em\u003e vector was constructed by using the CCM12 cDNA as a template, and the recombinant primer pairs were designed according to the coding sequence (CDS) of GhMYB102, and the target fragment with a size of 216 bp, then amplified using SnapGene 6.0.2. The recombinant \u003cem\u003eCLCrV:GhMYB102\u003c/em\u003e was transformed into Agrobacterium \u003cem\u003eLBA4404\u003c/em\u003e. Agrobacterium sap containing \u003cem\u003epCLCrVB\u003c/em\u003e and \u003cem\u003epCLCrV: GhMYB102\u003c/em\u003e was mixed in a 1:1 ratio. The bacterial solution was injected into the abaxial surface of the cotyledons of cotton seedlings using a needleless syringe. The injected plants were grown under a light culture chamber at 23\u0026deg;C with a light/dark cycle of 16h/8h until flowering. Injected empty vector (\u003cem\u003epCLCrVA\u003c/em\u003e and \u003cem\u003epCLCrVB\u003c/em\u003e) and positive control PDS (\u003cem\u003epCLCrV:Su\u003c/em\u003e and \u003cem\u003epCLCrVB\u003c/em\u003e) plants were used as mock-treated and technical controls, respectively. Sulfur encodes a component of the magnesium chelatase complex and the silencing of Su in cotton results in yellow leaves. Petal RNA from the basal spot-free region of \u003cem\u003eGhMYB102\u003c/em\u003e-silenced and blank plants was extracted to detect the silencing effect of the \u003cem\u003eGhMYB102\u003c/em\u003e gene.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Genetic transformation and overexpressing of homolog \u003cem\u003eGhMYB102\u003c/em\u003e, gene in \u003cem\u003eA. thaliana\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eGhMYB102 was transferred to tobacco under the control of 35S promoter by using Agrobacterium tumefaciens to infest wild-type \u003cem\u003eA. thaliana\u003c/em\u003e leaves by flower dipping method, harvested T0 generation \u003cem\u003eA. thaliana\u003c/em\u003e seeds were sterilized, inoculated, and cultured on 0.5 MS medium containing antibiotic Kana, screened for positive seedlings that could grow normally, and continued to be cultured until the seeds were harvested, the T1 were used to generate the T2. The T2 generations were cultured on 0.5 MS medium containing antibiotic Kana, screened for positive seedlings, and then transferred to nutrient pots for growth, leaf DNA was extracted, and the \u003cem\u003eGhMYB102\u003c/em\u003e gene fragment was amplified by specific primers. Wild-type \u003cem\u003eA. thaliana\u003c/em\u003e DNA was extracted for amplification as a negative control, the recombinant plasmid was used as a positive control, and the positive pseudo-seedlings were removed by molecular characterization, and cultured until the seeds of T2 generation were harvested. The positive seedlings of T2 generation were continued to be screened and then transferred into the nutrient pot, and the qRT-PCR was used for detecting the expression level of GhMYB102 in the positive plants, and the plants with higher expression were selected to harvest the seeds of T3 generation, and then the subsequent experiments were carried out.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Extraction and quantitative analysis of Anthocyanin\u003c/h2\u003e\u003cp\u003eExtraction and quantification of the total anthocyanin from cotton flower petals were performed as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Each flower was considered as an individual biological replica. From each flower, 0.1 g of petals were ground into powder in liquid nitrogen and at least three biological replicates were used for each flower color. Powders were extracted in the dark with 1 mL of acidic methanol (1% hydrochloric acid, w/v) for 24 h at 4\u0026deg;C. After centrifugation at 12,000 rpm for 10 min at 4\u0026deg;C, 1 mL of supernatant was transferred to 4 mL of acidic methanol. The mixture was used to measure absorbance at 530, 620, and 650 nm using a UV/VIS spectrophotometer. The anthocyanin content was calculated according to the formula Q\u0026thinsp;=\u0026thinsp;ODλ/ε\u0026thinsp;\u0026times;\u0026thinsp;V/m\u0026times;106, ODλ=(A530-A620)-0.1\u0026times;༈A650-A620, where V (mL) is the total volume extracted, m (g) is the fresh weight of the sample, and ε is 4.62 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e representing the molar extinction coefficient of anthocyanins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Transcriptome sequencing materials\u003c/h2\u003e\u003cp\u003eTo better understand the color of cotton petals during flower development, the floral development was stratified into six stages, designated S1 to S6. The cotton floral development stages were done though with modification as compared to that of the model plant, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the S1 stage, the bracts were lime green, with a bud size of 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2mm, in the S2 and S3 where bracts emerged with a bud size of 2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2mm and 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2mm, respectively, in S4 just a day before flowering with bud size of 3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2mm, while S5 and S6 are stages immediately after flowering. The samples were corded as per the development stage of the flower and the mode of treatment, from wild types (WT): WTS1 to WTS5), the VIGS-plants: VGS1 to VGS5;\u003c/p\u003e\u003cp\u003eSample materials were ground, total RNA isolated and cDNA libraries constructed. Fifteen cDNA libraries were sequenced on the Illumina HiSeq 2000 platform. Raw data were transformed, filtered, and mapped to the reference genome of \u003cem\u003eG. arboreum\u003c/em\u003e using Hisat2. Expression levels were calculated using transcripts per kilobase per million mapped reads (TPM) for exon modeling. Count files were used for differential expression analysis using the R package DESeq2 with a fold change threshold of 1 and a p-value of 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R package ClusterProfiler. Raw data can be found in the NCBI Sequence Read Archive database under accession number PRJNA904836.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Yeast single hybridization validation\u003c/h2\u003e\u003cp\u003eTarget fragments of cotton chalcone synthesis (GhCHS), chalcone isomerase (GhCHI), anthocyanin synthase (GhANS), and flavonoids \u0026minus;\u0026thinsp;3\u0026rsquo;,5\u0026rsquo;-hydroxylase (GhF3'5'H) promoter sequences containing MYB cis-acting elements of 400\u0026ndash;600 bp in size were amplified by specific primers. The amplified promoter fragments were ligated into \u003cem\u003epAbAi\u003c/em\u003e vector as Bait. the amplified product of the complete coding sequence region of \u003cem\u003eGhMYB102\u003c/em\u003e gene was ligated into \u003cem\u003epGADT7\u003c/em\u003e vector as Pray. 4 \u0026micro;g of \u003cem\u003epAbAi\u003c/em\u003e recombinant plasmid was digested using BstB I, and 1 \u0026micro;g was linearized into \u003cem\u003epAbAi-proGhCHS\u003c/em\u003e, \u003cem\u003epAbAi-proGhCHI\u003c/em\u003e, \u003cem\u003epAbAi-proGhANS\u003c/em\u003e, \u003cem\u003epAbAi-proGhF3'5'H\u003c/em\u003e into Y1HGold strain, coated with SD/-Ura solid-deficient medium and incubated at 30\u0026deg;C for 3\u0026ndash;5 days. Single clones were picked from SD/-Ura plates, inoculated in 1mL SD/-Ura liquid medium, incubated at 30\u0026deg;C for 24h, and used as a template for PCR identification. The positive bait clones were resuspended in 0.9% NaCl solution, making the OD600 0.002; 100 \u0026micro;L were coated in SD/-Ura with AbA (0, 100, 300, 500 ng/mL) solid-deficient medium and incubated at 30\u0026deg;C for 3\u0026ndash;5 days. The lowest AbA concentration that can inhibit the growth of yeast colonies was selected according to the colony growth.\u003c/p\u003e\u003cp\u003eThe empty vector \u003cem\u003epGADT7\u003c/em\u003e and prey vector \u003cem\u003epGADT7-GhMYB102\u003c/em\u003e were transfected into Y1HGold receptor cells containing the bait plasmid by LiAc/PEG method, respectively, and coated with 150 \u0026micro;L of solid-deficient medium with SD/-Leu and SD/-Leu with optimal AbA, and incubated at 30\u0026deg;C for 3\u0026ndash;5 days. Negative control and blank control were also set up.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Dual luciferase assay\u003c/h2\u003e\u003cp\u003eThe coding regions of GhCHI, GhDFR, GhANS, and GhF3'5'H promoter fragment sequences were cloned onto the \u003cem\u003epGreen II 0800-LUC\u003c/em\u003e vector. The \u003cem\u003eGhMYB102\u003c/em\u003e gene was ligated to the \u003cem\u003epGreenII 62 \u0026ndash;SK\u003c/em\u003e. Equal amounts of Agrobacterium cultures containing \u003cem\u003eCLuc\u003c/em\u003e and \u003cem\u003eNLuc\u003c/em\u003e constructs were mixed and then co-infiltrated into tobacco leaves. The injected tobacco was incubated at 25 ℃ under darkness and shade for 24h, and then placed in a 25 ℃ light culture room with a light/dark cycle of 16h/8h for 24h. Before observation, tobacco leaves were picked and coated with 1 mmol/L D-worm fluorescein potassium salt in the injection area, and placed in a dark environment for 10 minutes for the fluorescence assay. The leaves were observed under a fluorescence microscope. All experiments were repeated at least three times for each plasmid combination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Subcellular localization of the GFP fusion expression vector construct\u003c/h2\u003e\u003cp\u003eThe p2300 plasmid with green fluorescent protein tag was used as the backbon to, clone the \u003cem\u003eGhMYB102\u003c/em\u003e gene fragment, and to construct the green fluorescent protein transient fusion expression vector \u003cem\u003ep2300-eGFP-Flag-MYB102\u003c/em\u003e. The transformed Agrobacterium sensu lato \u003cem\u003eGV3101\u003c/em\u003e, and the monoclonal colony was inoculated into the LB liquid medium containing Cannae and rifampicin resistance and cultivated for 12h; then transferred to 0.5 mL of the bacterial solution to 50 mL of liquid LB, and incubated until the OD600 was about 1.0, the bacterium, was then suspended and adjusted the OD600 to about 0.5, then left under room temperature for 3.5h. The \u003cem\u003ep2300-eGFP-Flag\u003c/em\u003e vector was used as a control, and \u003cem\u003ep2300-eGFP-Flag-MYB102\u003c/em\u003e, were both injected into the leaves of the tobacco and observed under the fluorescence signals of the injected tobacco leaves under a laser confocal microscope after 48h to determine the subcellular localization of GhMYB102.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Data analysis\u003c/h2\u003e\u003cp\u003eAll experimental data were analyzed; the mean values were obtained for at least three independent biological replicates. Data were analyzed using SPSS software, and statistical comparisons of differences were analyzed for significance of multiple data using ANOVA statistics, and plotted using GraphPad Prism 9.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Cloning and sequence analysis of the \u003cem\u003eGhMYB102\u003c/em\u003e gene\u003c/h2\u003e\u003cp\u003eGene annotation information indicated that the candidate gene Gh_A01G069800 CDS sequence was 984 bp in full length, encoding 327 amino acids, and the gene ID was named GhMYB102. The conserved sequence of the GhMYB102 protein was compared with the conserved structural domains of the homologous gene sequences of other species using MEGA11 software, which showed that GhMYB102 was composed of conserved R2 and R3 DNA-binding structural domains at the N-end which was consistent of the conserved R2 and R3 DNA-binding domains. The R3 DNA-binding domain contained the \u003cem\u003ebHLH\u003c/em\u003e-binding motif [D/E] Lx2[R/K]x3Lx6Lx3R, and the conserved motif \"DNEI\" was also found. The conserved motifs downstream of the C-terminus indicated a high degree of divergence (Figure. 2a), which is typical of the \u003cem\u003eR2R3-MYB\u003c/em\u003e transcription factors [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe reported R2R3-MYB sequences of multiple species involved in the regulation of flavonoid metabolism were downloaded from the NCBI and Arabidopsis tair databases, and the phylogenetic tree was constructed by comparing the GhMYB102 with other genes. The results showed that all the genes had a similarity index of 70.77%, and both were MYB transcription factors involved in the inhibition of the anthocyanin accumulation. Gene similarity index of 70% and above is found to be a true type, moreover, in sequence analysis, 30\u0026ndash;70% sequence similarity are always retained [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Genetic evolutionary tree analysis showed that GhMYB102 were phylogenetically classified together with Arabidopsis AtMYB4, AtMYB32, AtMYB7, and strawberry FaMYB1, belonging to the subfamily 4 of the R2R3-MYB transcription factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), whereas the majority of the members of subfamily 4 were negative regulators in the anthocyanin synthesis pathway, i.e., involved in the inhibiting phytocannabinoid synthesis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], this implied that the transcription factor GhMYB102 was likely to be involved in the negative regulation of the anthocyanin synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the regulatory effect of the \u003cem\u003eGhMYB102\u003c/em\u003e gene on cotton petal color, the expression of the \u003cem\u003eGhMYB102\u003c/em\u003e gene was downregulated in Marie-galante 1139 (creamy white flowers), punctatum 187 (yellowish flowers), Y52, and Zhongyihong 2 (pink flowers), respectively, by virus-induced gene silencing (VIGS) mechanism. The petals of the silenced plants were harvested for RNA extraction. The gene silencing efficiency was assessed through RT-qPCR, and the results showed that the expression of GhMYB102 was significantly low in the VIGS-plants (\u003cem\u003eCLCrV: MYB102\u003c/em\u003e silenced plants) compared to the WT and null-loaded plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The downregulation of the silenced gene showed that the novel gene was effectively silenced.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePetal phenotypes of the wild-type and CLCrV :MYB102 silent plants, Marie-galante 1139 (cream flower), punctatum 187 (yellowish flower), Y52 (pink flower), and Zhongyihong 2 (pink flower) were monitored, and there was no morphological variation in the flower pattern between the petals of wild-type and CLCrV:MYB102 silenced plants, however in quantifying the anthocyanin content revealed that significantly higher anthocyanin concentrations in the silenced plants compared to the WT and the positive controlled ones. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Furthermore, a significant observation was made between the WT and the VIGS-plants, in Zhongyihong and Y52 with pink flowers, the pigmentation intensity was significantly higher in VIGS-plant compared to WT. The anthocyanin content in petals of the VIGS-plants was significantly higher than that in the WT and positive controlled plants, which was about three times higher than that of wild-type petals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Based on these results, we concluded that the knockdown of \u003cem\u003eGhMYB102\u003c/em\u003e gene significantly increases the accumulation of anthocyanin content in cotton petals and that GhMYB102 may be playing an inhibitory role in the anthocyanin biosynthesis pathway. The results obtained were in agreement with previous findings, in which anthocyanins synthesis in plants is influenced by transcriptional regulatory factors, majorly the MYB, bHLH, and WD40 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], which constitutes anthocyanin biosynthesis regulatory system by binding to structural gene promoters.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 \u003cem\u003eGhMYB102\u003c/em\u003e induces Drought resistance and maintenance of leave pigmentation\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eGhMYB102\u003c/em\u003e homolog was overexpressed (OE) in \u003cem\u003eA. thaliana\u003c/em\u003e, a mutant form, and the wild types were obtained, the three were subjected to drought treatment, and the results showed that the OE plants exhibited higher tolerance to drought stress, leaves were green, and no sign of wilting, while the mutant and the wild type, were significantly affected by drought. Moreover, in the mutant forms, 9 out of the 12 planted cups suffered severe drought and dried. A unique observation however was noted among the wild type, the leaves became more chlorotic, and the survival level was significantly higher compared to the mutant forms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Phenotypic evaluation of the leaves of the OE, WT, and the positive control under drought, all the plants exhibited normal leave shape and color under well-watered conditions, however, OE plants only experienced mild wilting, and the leaf color was still bright green; WT plants had extensive leaf curling, and the leaf color shifted to dark green compared with that of the control group with normal watering; and while the mutant plants had severe wilting and even died, and the leaf color changed to dark purple (Fig.\u0026nbsp;3bi). A larger group of the MyBs promotes anthocyanin accumulation, for instance, overexpression of \u003cem\u003eSlMYB75\u003c/em\u003e gene enhanced anthocyanin accumulation in both vegetative and reproductive tissues in tomatoes, even though expression levels of SlMYB75 were found to be relatively low in wilt types [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, quantification of the anthocyanin levels among the OE, WT, and the mutant forms under drought stress conditions, revealed a higher anthocyanin content in the mutant form, closely followed by the WT but significantly low in the OE plants (Fig.\u0026nbsp;3bii). Anthocyanin accumulation in plants more so in the leaves when plants are under stress plays a survival role, the anthocyanins scavenge on the reactive oxygen species (ROS), thereby protecting plants from oxidative damage and enhancing their sustainability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Subcellular localization\u003c/h2\u003e\u003cp\u003eTo study the expression site of the protein encoded by \u003cem\u003eGhMYB102\u003c/em\u003e gene, \u003cem\u003ep2300-eGFP-Flag-MYB102\u003c/em\u003e fusion expression vector was constructed. The green fluorescence signal was observed under a laser confocal microscope (Leica TCS SP8 DTED) with the help of a tobacco transient expression system. The green fluorescent protein was expressed on both the cell membrane and nucleus of \u003cem\u003eP2300-eGFP-Flag\u003c/em\u003e; while the \u003cem\u003ep2300-eGFP-Flag-MYB102\u003c/em\u003e fusion vector was expressed on the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Transcriptome data enrichment analysis\u003c/h2\u003e\u003cp\u003eGene expression is temporally and spatially specific, and this study screened for differentially expressed genes (DEGs) between the comparison groups. The comparison groups were divided into WTS4 vs WTS1, WTS5 vs WTS1, WTS5 vs WTS4 among wild type; VGS5 vs VGS4 among silenced strains; and VGS4 vs WTS4, VGS5 vs WTS5 among wild type and silenced strains, totaling six comparison groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA comparison of \u003cem\u003eGhMYB102\u003c/em\u003e gene expression in WTS5 vs VGS5 using qRT-PCR analysis confirmed that the gene expression in VGS5 was significantly lower than that in WTS5. To understand the biological functions of the differentially expressed genes, KEGG enrichment was used in this study to classify DEGs for functional enrichment. The expression of genes enriched to flavonoid synthesis pathway genes in all the samples was jointly displayed with some flavonoid metabolism schematics, and the gene enrichment is shown in (Supplementary Fig.\u0026nbsp;1). Differential gene enrichment analysis of the VGS5 vs WTS5 group, which had significant phenotypic differences in petals, revealed significant enrichment in the flavonoid synthesis pathway, with 19 genes significantly up-regulated and 1 gene significantly down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The enrichment analysis of up-regulated genes in the VGS5 vs WTS5 group again showed that the flavonoid synthesis pathway was ranked first in significance, and the significantly up-regulated genes included CHS, CHI, F3'5'H, F3'H, F3H, ANS, FLS, and LAR, which are structural genes involved in the synthesis of flavonols and anthocyanins (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The above results suggest that the transcription factor GhMYB102 may directly or indirectly inhibit the expression of structural genes in the flavonoid metabolic pathway, thus affecting the synthesis of flavonoids and anthocyanins.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExpression levels of the flower pigmentation responsive genes under WT, and VIGS plants.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWTS5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVGS5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eVGS5/WTS5\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF3'5'H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A07G130200.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.242\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A07G130000.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.4623\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.3443\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_D12G188200.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.7419\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_Contig01002G\u003c/p\u003e\u003cp\u003e001700.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.7022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eF3H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A12G064100.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18.492\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e91.319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_D12G061800.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.9692\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e50.555\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A11G186200.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.4602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18.051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eCHS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A05G312100.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7676\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.0172\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A09G000900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.3665\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.2489\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A02G031300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.5245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16.911\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A09G000100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.3735\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.2105\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_D02G036800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.4697\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_D05G322000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.445\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_D09G000100.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.7385\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.3984\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_Contig00311G\u003c/p\u003e\u003cp\u003e000400.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.0616\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.841\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFLS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A05G400400.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.5118\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.597\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_D04G022800.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.4526\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.4799\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eANS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A13G262900.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.9781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.991\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3'H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGh_A12G204700.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.1729\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.4589\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 qRT-PCR gene expression analysis of flavonoid synthesis genes\u003c/h2\u003e\u003cp\u003eTo further investigate whether GhMYB102 could regulate key flavonoid genes in cotton petals, we used qRT-PCR to compare the expression of CHS, CHI, F3H, F3'H, F3'5'H, DFR, and ANS in the Y52 material after the interference of WT and VIGS, UFGT eight flavonoid synthesis-related gene expression. The results showed that GhMYB102 strongly affected the transcript levels of genes involved in flavonoid and anthocyanin biosynthesis. When the expression of GhMYB102 decreased, the expression of flavonoid and anthocyanin synthesis-related genes CHI, F3H, F3'H, F3'5'H, DFR, and ANS increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results obtained were in agreement with previous findings in which the expression of anthocyanidin synthase, \u003cem\u003eRnANS1\u003c/em\u003e gene was upregulated during fruit maturation, which positively correlated with anthocyanin accumulation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, the levels of the anthocyanin biosynthetic genes are positively correlated to anthocyanin contents at different flower developmental stages, being dihydroflavonol 4-reductase (DFR) is vital for anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonol to leucoanthocyanidin [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The results were consistent with the transcriptome sequencing results, indicating that the transcriptome data were reliable and suggesting that the transcription factor GhMYB102 may directly or indirectly deter the expression of structural genes in the flavonoid metabolic pathway, thus affecting the synthesis of flavonoids and anthocyanins.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Yeast single hybridization\u003c/h2\u003e\u003cp\u003eTo determine whether there is a direct regulatory relationship between GhMYB102 and the structural genes of flavonoid metabolism, we first analyzed the promoter sequences of eight genes related to flavonoid synthesis, namely, CHS, CHI, F3H, F3'H, F3'5'H, DFR, ANS, and UFGT, in the Plant CARE database. Flavonoid synthesis-related genes were analyzed, and it was found that the 2000bp region upstream of these genes had multiple MYB binding elements. After that, we ligated the complete coding region sequence of \u003cem\u003eGhMYB102\u003c/em\u003e gene into pGADT7 vector as a prey protein, and amplified the 2000bp upstream sequences of \u003cem\u003eGhCHS\u003c/em\u003e, \u003cem\u003eGhCHI\u003c/em\u003e, \u003cem\u003eGhANS\u003c/em\u003e, and \u003cem\u003eGhF3'5'H\u003c/em\u003e genes, and ligated them into pAbAi vector as bait proteins. Self-activation assay was performed on Bait, and the results showed that yeast cells transformed with the bait plasmid grew normally on SD/-Ura solid-deficient medium. Observation of yeast cell growth under 100, 300, and 500 ng/mL of antibiotics showed that there had been essentially no growth on \u003cem\u003eSD/-Ura/AbA\u003c/em\u003e (100 ng/mL) solid-deficient medium, indicating that \u003cem\u003epAbAi-proGhCHS\u003c/em\u003e, \u003cem\u003epAbAi-proGhCHI\u003c/em\u003e, \u003cem\u003epAbAi-proGhANS\u003c/em\u003e, \u003cem\u003epAbAi- proGhF3'5'H\u003c/em\u003e had a self-activating response, but 100 ng/mL gold tanshin suppressed the background expression of the \u003cem\u003eAbAr\u003c/em\u003e reporter gene in the transformed strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Positive clones on \u003cem\u003eSD/-Ura\u003c/em\u003e solid-deficient medium were picked to make Y1H Gold yeast receptor cells. The yeast cells were transfected into a plasmid containing yeast prey recombinant vector \u003cem\u003epGADT7-GhMYB102\u003c/em\u003e and empty \u003cem\u003epGADT7\u003c/em\u003e (negative control), respectively, and the growth of yeast cells was observed on the corresponding solid-deficient medium. The results showed that all yeast strains could grow normally on SD/-Leu solid-deficient medium, but except for the controlled group \u003cem\u003epGADT7\u0026thinsp;+\u0026thinsp;pAbAi-proGhCHS\u003c/em\u003e that showed strain growth, only the yeast strain transformed with the \u003cem\u003epGADT7-GhMYB102\u003c/em\u003e plasmid could grow normally on the SD/-Leu solid-deficient medium containing 150 ng/mL AbA medium for normal growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). This indicated that GhMYB102 was able to bind cis-acting elements in the GhCHI, GhANS, and GhF3'5'H promoters and activate expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Dual luciferase validation\u003c/h2\u003e\u003cp\u003eTo detect whether the transcription factor GhMYB102 regulates the promoters of \u003cem\u003eANS\u003c/em\u003e, \u003cem\u003eF3'5'H\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, and \u003cem\u003eCHI\u003c/em\u003e genes, a dual luciferase assay experiment was performed. A 2000 bp fragment upstream of the four promoters was ligated into the plant expression vector \u003cem\u003epGreenII 0800-LUC\u003c/em\u003e via \u003cem\u003eSalI\u003c/em\u003e and \u003cem\u003eKpnI\u003c/em\u003e restriction endonucleases to constitute the Reporter, while the full-field sequences of the coding regions of the genes of GhMYB102 were ligated into the plant expression vector \u003cem\u003epGreenII 62-SK\u003c/em\u003e via \u003cem\u003eXbaI\u003c/em\u003e and \u003cem\u003eKpnI\u003c/em\u003e restriction endonucleases to form Effector. Prepare bacterial solution for transient transformation of tobacco leaves, the leaves were divided into four equal parts during transformation, the upper left area is the combination of \u003cem\u003e62-SK\u003c/em\u003e empty and 0800-promoter, the lower left area is the combination of \u003cem\u003e62SK-MYB102\u003c/em\u003e and 0800 empty, the lower right is the combination of \u003cem\u003e62-SK\u003c/em\u003e empty and 0800 empty, and the above serves as the control group ; upper right is the combination of \u003cem\u003e62SK-MYB102\u003c/em\u003e and 0800-promoter, which served as the experimental group. Each combination was set up with at least 3 technical replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFluorescence imaging results showed that the combination of \u003cem\u003epGreenII 62-SK\u003c/em\u003e and 0800-promoter on the upper left had a higher fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb), indicating that the ANS promoter can self-activate, and the combination of 62SK-MYB102 and 0800-promoter on the upper right had a fainter fluorescence intensity, indicating that the transcription factor GhMYB102 represses the ANS promoter's expression. Similarly, GhMYB102 also repressed the expression of F3'5'H, the CHI promoter, and had almost no effect on the CHS promoter.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003ePetal colors in nature are varied and diverse, which are mainly influenced by the external environment such as light, temperature, and soil pH, as well as internal color-developing pigments [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The main determinant of floral pigmentation is the flavonoids, which their metabolic pathways are very conserved in plants, and it is an enzyme-catalyzed reaction encoded by a series of structural genes. One of the largest plant transcriptome factors with a greater role in primary and secondary metabolic reactions are the MyB transcription factors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The R2R3-MYB transcription factors have the highest percentage in the MYB family [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Studies have shown that the MYBs do regulate petal color in multiple species, such as lily, moonflower, daffodil, and gerbera [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, in cotton, the GhTT2 of the MYB family promotes proanthocyanidin biosynthesis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, overexpression of GhMYB1a leads to increased accumulation of flavonols [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]; \u003cem\u003eGhMYB113\u003c/em\u003e controls the formation of purple spots at the base of petals in tetraploid sea-island cotton [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]; and dominant genes in Asian cotton. Furthermore, a loss of function of GaPC in the mutant forms, results in a phenotype in which \u003cem\u003eGaPC\u003c/em\u003e masks other color genes and exhibits recessive epistasis, whereas petals of \u003cem\u003eGaPC\u003c/em\u003e-silenced sea and land cotton exhibit albinism. Furthermore, infiltration of a gene that is identical to that of \u003cem\u003eGbBM\u003c/em\u003e homologous to \u003cem\u003eR2R3-MYB\u003c/em\u003e gene fragment GhPF in land cotton showed a rare pink color [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It has also been reported that the change of corolla color from pink to white may also be due to a promoter variant of a \u003cem\u003eGhTT19\u003c/em\u003e gene in the region of the infiltrated fragment [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, a new transcription factor GhMYB102 belonging to R2R3-MYB, was identified from a wild cotton germline of terrestrial cotton. Phylogenetically, this gene was found to be a member of the 4th subfamily, known to have the inhibitory role of anthocyanin biosynthesis, moreover, a single nucleotide mutation in the gene resulted in a higher pigmentation intensity in the VIGS-plants, an indication that the suppression of its expression has a positive effect on the anthocyanin biosynthesis pathways. The results were in agreement with previous studies in which TgMYB4 repression negatively regulated the Anthocyanin Biosynthesis in Tulipa gesneriana L [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The results were further validated by overexpressing the gene in \u003cem\u003eA. thaliana\u003c/em\u003e, the mutant form exhibited higher anthocyanin content and purplish leave color compared to the OE plants, which further revealed the inhibitory role of the identified gene. However, it is important to note that the \u003cem\u003eMYB\u003c/em\u003e genes are critical in anthocyanin biosynthesis pathways and have been explored in several crops, for instance, SlMYB75, an MYB-type transcription factor has been found to have a positive regulatory role in anthocyanin biosynthesis and also promote the volatile compound synthesis in tomatoes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus the negative role of the identified gene, clearly indicates that the gene is a member of the 4th subfamily.\u003c/p\u003e\u003cp\u003eEven though the mutant recorded the highest concentration levels of the anthocyanin, the performance under drought stress conditions revealed a negative adaptation towards drought stress, the OE plants maintained green leaves, and with significantly low phenotypic attributes of wilting compared to the mutant and the wild type. The mutant and WT suffered severe effects of drought stress, in plants when plants are exposed to either drought or salt stress, the amount of reactive oxygen species (ROS) released increases, and thus the high induction levels of the anthocyanins would help in reducing the ROS levels, however this was not observed. MYB transcription factors have been found to enhance drought stress tolerance [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The \u003cem\u003eZmRL6\u003c/em\u003e gene a member of the MYBs has been found to have a positive effect on enhancing drought tolerance in maize [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Furthermore, overexpression of MYB12 and MYB75 genes has been found to increase flavonoid accumulation with significantly enhanced antioxidative activities [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Thus the OE plant\u0026rsquo;s response to drought stress could have been due to the oxidative properties of the anthocyanin thereby reducing the oxidative stress, minimizing the cell damage, and preventing massive cell death. Moreover, the drought effect wasn\u0026rsquo;t so severe, which points to the possible role of the anthocyanin to scavenge on the reactive oxygen species (ROS).\u003c/p\u003e\u003cp\u003eBased on transcriptome sequencing, secondary metabolite pathway exploration, and functional gene mining are carried out to study the molecular mechanism of cotton flower color regulation. To further investigate the molecular mechanism of GhMYB102 regulating anthocyanin synthesis, we performed transcriptome analysis using wild-type S1, S4, and S5 period petals of land cotton genetic material \"Y52\" (control) and S4 and S5 period petals of \u003cem\u003eCLCrV: MYB102\u003c/em\u003e (VIGS). We analyzed 74,474 expressed genes for KEGG categorical enrichment, and a total of 40 genes were enriched in the flavonoid metabolic pathway. Enrichment analysis of up-regulated genes in the VGS5 vs WTS5 group, which had significant phenotypic differences in petals, ranked first in flavonoid synthesis pathway significance, and the 19 genes that were significantly up-regulated included CHS, CHI, F3'5'H, F3'H, F3H, ANS, FLS, LAR, these genes are involved in the synthesis of flavonols, anthocyanins. The above results suggest that the transcription factor GhMYB102 may directly or indirectly deter the expression of structural genes in the flavonoid metabolic pathway, thus affecting the synthesis of flavonoids and anthocyanins. Based on the analysis of the transcriptional regulatory network of GhMYB102, we further verified the expression of flavonoid structural genes CHS, CHI, F3'5'H, F3'H, F3H, ANS, DFR, UFGT using qRT-PCR, which was consistent with the results of transcriptome sequencing. transcriptome sequencing results were consistent. To determine whether there is a direct regulatory relationship between GhMYB102 and the structural genes of flavonoid metabolism, we carried out yeast one-hybrid (Y1H) and dual-luciferase (dual-LUC) experiments on the structural genes of flavonoids analyzed by qRT-PCR, to elucidate the molecular mechanism of GhMYB102 in regulating phytochromes. The results indicated that the transcription factor GhMYB102 can directly repress the expression activity of GhCHI, GhF3'5'H, and GhANS promoters.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eCotton is an important crop, and the main source of white fiber, which is the primary raw material in the textile industries. Its production has greatly declined due effects of abiotic factors, which have become a limiting factor not only to cotton but other crops, moreover, use of pesticides has also significantly reduced the population of the natural pollinators. The diversity that exist within the cotton gnome provided an excellent platform to understand the role of \u003cem\u003eMyB\u003c/em\u003e genes in regulating the floral color. In this work, a novel gene GhMyB was identified from the semi wild cotton germplasms (Data to be published). The gene has been found to play an inhibitory role in the anthocyanin biosynthesis pathways, and has additive effect in enhancing drought tolerance in cotton. Further exploitation is required to understand the antagonistic role, of this gene, being anthocyanin is known to have ROS scavenging capacity, so its inhibitory nature, and the positive effect of the gene in enhancing drought tolerance requires further exploration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contribution statement\u003c/p\u003e\n\u003cp\u003eConceptualization, DW, ZZ, ROM, LF. and YX.; methodology, DW, ROM, YX, JNK, ZZ.; software, DW and ROM.; validation, DW, ROM, ZZ, JNK, YH, QKL, JH, YX and ZZ.; formal analysis, DW, ROM, JNK, YH.; investigation, DW.; resources, ZZ, XC, YH, YYZ, QKL and JZ.; data curation, JZ, FL, and ZZ.; writing\u0026mdash;original draft preparation, DW, and ROM.; writing\u0026mdash;review and editing, ROM.; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was funded\u0026nbsp;by National Natural Science Foundation of China (32401824, 32171994, 32272090); Natural Science Youth Fund of Henan Province (242300421595); the National Key R\u0026amp;D Program of China (2024YFD1200300,2022YFD1200345)\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eAll data that support this publication are fully provided within the text and its supplementary files\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe acknowledge the enormous support provided by the entire research group of the wild cotton germplasm resources, of the institute of cotton research (ICR), Anyang, China, the lab technicians and laboratory managers for the support accorded to us during this research work.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYu J, Jung S, Cheng C, Ficklin SP, Lee T, Zheng P, et al. 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J Hered. 2002;93:55\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eHu X, Liang Z, Sun T, Huang L, Wang Y, Chan Z, et al. The R2R3-MYB Transcriptional Repressor TgMYB4 Negatively Regulates Anthocyanin Biosynthesis in Tulips (Tulipa gesneriana L.). Int J Mol Sci. 2024;25.\u003c/li\u003e\n\u003cli\u003eWang X, Niu Y, Zheng Y. Multiple functions of myb transcription factors in abiotic stress responses. Int J Mol Sci. 2021;22.\u003c/li\u003e\n\u003cli\u003eZhang P, Wang T, Cao L, Jiao Z, Ku L, Dou D, et al. Molecular mechanism analysis of ZmRL6 positively regulating drought stress tolerance in maize. Stress Biol. 2023;3.\u003c/li\u003e\n\u003cli\u003eNakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, et al. Enhancement of oxidative and drought tolerance in Arabidopsis by over accumulation of antioxidant flavonoids. Plant J. 2014; 77:367\u0026ndash;79.\u003c/li\u003e\n\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":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Semi-wild cotton, GhMYB102, Petal color, Anthocyanins, Transcriptional regulation, drought stress","lastPublishedDoi":"10.21203/rs.3.rs-7187536/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7187536/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCotton plants display significant genetic diversity in petal color, which is essential for resilience against pests and diseases, UV radiation mitigation, and pollinator attraction. The MYB gene family regulates anthocyanin biosynthesis, but the specific functions of the 4th subfamily remain poorly understood in floral color formation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study focuses on the MYB gene Gh_A01G069800, evaluating its impact on floral pigmentation using virus-induced gene silencing (VIGS).\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results showed that VIGS plants exhibited higher pigment intensity across four cotton varieties, with the highest anthocyanin levels recorded in Zhongyihong (5.5 nmol/g) and Y52 (5.7 nmol/g), surpassing their wild types. In contrast, overexpression (OE) of Gh_A01G069800 resulted in lower pigmentation (3.0 nmol/g) compared to wild types and mutants, which had anthocyanin levels of 4.0 and 7.5 nmol/g, respectively. Gene expression analysis revealed that while chalcone synthase (CHS) and flavonoid glycosyltransferase (UFGT) were highly upregulated in wild types, genes like chalcone isomerase (CHI), flavonoid-3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), and anthocyanin synthase (ANS) showed elevated expression in VIGS plants, suggesting Gh_A01G069800's inhibitory role in anthocyanin biosynthesis. Furthermore, GhMYB102 was shown to directly inhibit CHI, ANS, and F3'5'H expression, affecting anthocyanin synthesis. Overall, Gh_A01G069800 appears to play a crucial role in regulating floral color and enhancing drought stress tolerance in cotton plants.\u003c/p\u003e","manuscriptTitle":"Unraveling GhMYB102: A Dual-Function of the Gh_A01G069800 Gene in Promoting Anthocyanin Biosynthesis Inhibition, and Drought Tolerance in Cotton","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 09:28:43","doi":"10.21203/rs.3.rs-7187536/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-22T08:54:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T03:18:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T10:55:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T21:51:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190858211083098299604792227932124394684","date":"2025-09-10T05:00:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268646452354903908394250516428446755751","date":"2025-09-10T02:42:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128072161245582796602544473457249632448","date":"2025-09-09T06:23:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-07T05:58:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-24T14:49:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-24T01:20:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T01:19:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-07-22T13:11:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a3f49de-c438-4fac-857c-a4b21205c19b","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T20:38:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 09:28:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7187536","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7187536","identity":"rs-7187536","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}