JrATHB-12 mediates JrMYB113 and JrMYB27 to control the anthocyanin levels in different types of red walnut

Red walnut has broad market prospects because it is richer in anthocyanins than ordinary walnut.However,the mechanism driving anthocyanin biosynthesis in red walnut is still unknown.We studied two types of red walnut,called red walnut 1(R1),with a red pericarp and seed coat,and red walnut 2(R2),with a red seed coat only.R1 mostly contained cyanidin-3-O-galactoside,while R2 contained a various amounts of cyanidin-3-Ogalactoside,cyanidin-3-O-arabinoside,and cyanidin-3-O-glucoside.The LDOX-2(LOC109007163)and LDOX-3(LOC109010746)genes,which encode leucoanthocyanidin dioxygenase/anthocyanidin synthase(LDOX/ANS),were preliminarily indicated as the crucial genes for anthocyanin biosynthesis in R1 and R2,respectively.The MYB differential genes analysis showed that MYB27 and MYB113 are specifically expressed in the red parts of R1 and R2,respectively,and they are regarded as candidate regulatory genes.Ectopic expression in Arabidopsis and transient injection in walnut showed that both MYB27 and MYB113 were located in the nucleus and promoted anthocyanin accumulation,while MYB27 promoted the expression of LDOX-2,and MYB113 promoted the expression of LDOX-3and UAGT-3.Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind to the LDOX-2 promoter,while MYB113 could bind to the promoters of both LDOX-3 and UAGT-3.In addition,we also identified an HD-Zip transcription factor,ATHB-12,which is specifically expressed in the pericarp.After silencing the expression of ATHB-12,the R2 pericarp turned red,and MYB113 expression increased.Further experiments showed that ATHB-12 could specifically interact with MYB113 and bind to its promoter.This suggests that MYB27controls R1 coloration by regulating LDOX-2,while MYB113 controls R2 coloration by regulating LDOX-3 and UAGT-3,but ATHB-12 can specifically bind to and inhibit the MYB113 of the R2 pericarp so that it becomes unpigmented.This study reveals the anthocyanin biosynthetic mechanisms in two different types of red walnut and provides a scientific basis for the selection and breeding of red walnut varieties.

ChMYB1-ChbHLH42-ChTTG1 module regulates abscisic acidinduced anthocyanin biosynthesis in Cerasus humilis

Cerasus humilis is a kind of economic fruit tree peculiar to China,which is widely used in the food,landscape,and pharmaceutical industries.Anthocyanins are a phenolic metabolite that plays an essential role in fruit coloration.However,the regulatory network of C.humilis in anthocyanin biosynthesis is still unclear.In this study,the R2R3-MYB transcription factor ChMYB1 was isolated from the full genome of the species.Yeast one-hybrid,dual-luciferase assays,and GUS staining showed that ChMYB1 significantly increased anthocyanin contents in C.humilis fruit by promoting the expression of ChCHS and ChUFGT by binding MBS(MYB-binding elements).ChMYB1 interacted with ChbHLH42and ChTTG1 to form the MBW complex and further enhanced the expression of ChUFGT.In addition,abscisic acid(ABA)treatment promoted the expression of ChMYB1 and anthocyanin accumulation in C.humilis fruit.Interestingly,ABA treatment enhanced the interaction between ChMYB1 and ChbHLH42.Furthermore,ChABI5 inhibited the interaction between ChMYB1 and ChbHLH42.Our data elucidated the primary molecular mechanism of anthocyanin biosynthesis in C.humilis fruit,deepening the understanding of the regulatory network affecting anthocyanin metabolism in edible fruit crops.

CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum.However,the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear,which greatly restricts design of molecular breeding in chrysanthemum.Here,a chrysanthemum SG7 R2R3 MYB transcription factor(TF),CmMYB3-like,was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage.Quantitative real time PCR(qRT-PCR)assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage.Genetic transformation of chrysanthemum and dual-luciferase assays in N.benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly.However,overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols.Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation,but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage.Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

Smart Colorimetric Corn Starch Films Combined with Anthocyanin-Loaded Glutenin-Carboxymethyl Chitosan Nanocomplexes for Freshness Monitoring of Chilled Pork

In this study,intelligent,pH-responsive colorimetric films were prepared by encapsulating anthocyanins in nanocomplexes prepared from glutenin and carboxymethyl chitosan.These nanocomplexes were added to a corn starch matrix and used in the freshness monitoring of chilled pork.The effects of anthocyanin-loaded nanocomplexes on the physical,structural,and functional characteristics of the films were investigated.The addition of anthocyanin-loaded nanocomplexes increased the tensile strength,elongation at break,hydrophobicity,and light transmittance of the films while decreasing their water vapor permeability.This is because new hydrogen bonds are formed between the film components,resulting in a more homogeneous and dense structure.The colorimetric film has a significant color response to pH changes.These films were used in experiments on the freshness of chilled pork,in which the pH changes with changing freshness states.The results show that the colorimetric film can monitor changes in the freshness of chilled pork in real time,where orange,pink,and green represent the fresh,secondary fresh,and putrefied states of pork,respectively.Therefore,the intelligent colorimetric film developed in this study has good application potential in the food industry.

Blueberry anthocyanins extract attenuates oxidative stress and angiogenesis on an in vitro high glucose-induced retinopathy model through the miR-33/GLCCI1 axis

Background:Diabetes retinopathy(DR)is a complication of diabetes that affects patients’vision.Previous studies have found blueberry anthocyanins extract(BAE)can inhibit the progression of DR,but its mechanism is not completely clear.Methods:To study the role of BAE in diabetes retinopathy,we treated human retinal endothelial cells(HRCECs)with 30 mM high glucose to simulate the microenvironment of diabetes retinopathy and used BAE to intervene the in vitro high glucose-induced retinopathy model.HRCEC cell viability and apoptosis rates were examined by Cell Counting Kit 8(CCK-8)assay and flow cytometry assay.The binding sites between miR-33 and glucocorticoid-induced transcript 1(GLCCI1)were assessed by luciferase reporter assay.Retinal neovascularization and oxidative stress contribute to diabetic retinopathy.The tubule formation assay was applied to detect the retinal neovascularization.The oxidative stress in the HRCECs was manifested by the reactive oxygen species(ROS)level,the malondialdehyde(MDA)level,and the superoxide dismutase(SOD)activity.Results:Compared with HRCECs cells cultured under normal conditions,high glucose(HG)can induce oxidative stress in HRCRCs,specifically manifested in the increase of ROS and MDA levels,and the decrease of SOD activity.BAE relieved the tubule formation in n the HRCEC.BAE also relieved the ROS and MDA levels and increased the SOD activity.Luciferase reporter assay revealed that GLCCI1 is a target molecule downstream of miR-33.In HRCEC,BAE significantly inhibited the expression of miR-33 induced by HG.miR-33 mimic inhibited the BAE’s effects on oxidative stress and angiogenesis in an in vitro high glucose-induced retinopathy model.Conclusion:BAE alleviated the oxidative stress and microangiogenesis of HRCEC by regulating the miR-33/GLCCI1 axis.

Transcriptomic Analysis Reveals the Formation Mechanism of Anthocyanins Light-Independent Synthesis in Chrysanthemum

Chrysanthemum×morifolium is a horticultural crop which plays a vital role in theflower industry with signifi-cant economic value and has a cultivation history of over three thousand years in China.The accumulation of anthocyanins is always affected by light.Here,we revealed that anthocyanin accumulation is highly dependent on light in‘2021135’genotype chrysanthemum,while it is light-independent in‘2001402’genotype chrysanthe-mum.However,no literature has been reported regarding the non-photosensitive chrysanthemum in anthocya-nins light-independent synthesis pathways.Through the phenotype analysis of 44 F1 generations,we found that light-independence is a dominant trait which can be stable inherited by progeny.The transcriptome of the rayflorets of‘2021135’and‘2001402’under light and bagging treatment were sequenced and analyzed.Based on weighted gene co-expression network analysis(WGCNA),K-means analysis,and Real-Time Quantitative Poly-merase Chain Reaction(RT-qPCR)analysis,16 genes were highly correlated with the anthocyanin content.The anthocyanin content of rayflorets treated with different light-quality conditions indicated that blue light signifi-cantly affected anthocyanin accumulations.Through Yeast one-hybrid analysis,CmBIC1.1 and CmBIC1.2 can directly regulate the anthocyanin structural gene CmCHS2.In our study,we revealed the important characteristics of light-independent anthocyanin synthesis in chrysanthemums and screened regulatory factors in light-depen-dent and light-independent anthocyanin synthesis pathways.The results laid the groundwork for subsequent ana-lysis of the molecular mechanism involved in the light-independent synthesis of anthocyanins in chrysanthemums.

Metabolomic Analysis of the Anthocyanins Associated with Different Colors of Cymbidium goeringii in Guizhou, China

Cymbidium goeringii is an economically important ornamental plant,and flower color is one of the main features of C.goeringii that contributes to its high economic value.To clarify the molecular mechanisms underlying the role of anthocyanins in mediating differences in color among varieties,liquid chromatography–tandem mass spectrometry was used to perform anthocyanin-targeted metabolomics of seven C.goeringii varieties,including‘Jin Qian Yuan’(JQY),‘Jin Xiu Qian Yuan’(JXQY),‘Miao Jiang Su Die’(MJSD),‘Qian Ming Su’(QMS),‘Shi Chan’(SC),and‘Yang Ming Su’(YMS),as well as the C.goeringii.We detected 64 anthocyanins,including cyanidins,delphinidins,malvidins,pelargonidins,peonidins,petunidins,procyanidins,and flavonoids.We identified six shared differentially accumulated metabolites(DAMs),including cyanidin-3-O-rutinoside,delphinidin-3-Osophoroside,pelargonidin-3-O-rutinoside,peonidin-3-O-(6-O-malonyl-beta-D-glucoside),peonidin-3-Osophoroside,and chalcone.Most DAMs were enriched in the anthocyanin biosynthesis pathway.Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed metabolites were significantly enriched in the anthocyanin biosynthesis pathway.Analysis of the content of differentially expressed metabolites indicated that peonidin-3-O-(6-O-malonyl-beta-D-glucoside)was the key metabolite underlying color differences among C.goeringii varieties.Procyanidin B2,pelargonidin-3-O-galactoside,and naringenin might also affect the color formation of JQY and QMS,SC,and MJSD,respectively.The results of this study shed light on the metabolic mechanism underlying flower color differences in C.goeringii at the molecular level.Our findings will aid future studies of the mechanism of flower color regulation in C.goeringii and have implications for the breeding of new varieties.

Quantification of Total Phenols, Total Flavonoids, Total Anthocyanins and Evaluation of Antioxidant and Antiradical Activities of Detarium Senegalense Extracts from Chad

The aim of the present work is to assess the value of Detarium Senegalense by determining the content of total phenols, total flavonoids and total anthocyanins, and by evaluating the free radical scavenging activity of Detarium Senegalense extracts. For this purpose, sequential extraction using solvents of increasing polarity was essential. The various extracts obtained underwent phytochemical and biochemical analyses. Phytochemical screening revealed the presence of flavonoids, alkaloids, tannins, polyphenols, anthocyanins and steroids/terpenes. Quantitative analysis of total polyphenols, total flavonoids and total anthocyanins yielded the following results: total flavonoids (0.803 ± 0029 mg EQ/100g P for acetone extract of roots and 0.871 ± 0.401 mg EQ/100g P for methanol extract of leaves);total polyphenols (23.298 ± 12.68 mg EAG/100g P for acetone extract of roots and 24.69 ± 0.49 401 mg EAG/100g P for methanol extract of leaves);total monomeric anthocyanins (44.697 ± 0.939 mg EC3G/100g P and 16.699 ± 0.193 mg EC3G/100g P respectively for acetone and methanol extracts of stem bark). DPPH free radical scavenging activity was 1.674 ± 0.023 mg/mL for the acetone extract and 0.934 ± 0.24 mg/mL for the methanol extract of roots. .

The MYB transcription factor RcMYB1 plays a central role in rose anthocyanin biosynthesis

Rose(Rosa hybrida)is one of most famous ornamental plants in the world,and its commodity value largely depends on its flower color.However,the regulatory mechanism underlying rose flower color is still unclear.In this study,we found that a key R2R3-MYB transcription factor,RcMYB1,plays a central role in rose anthocyanin biosynthesis.Overexpression of RcMYB1 significantly promoted anthocyanin accumulation in both white rose petals and tobacco leaves.In 35S:RcMYB1 transgenic lines,a significant accumulation of anthocyanins occurred in leaves and petioles.We further identified two MBW complexes(RcMYB1-RcBHLH42-RcTTG1;RcMYB1-RcEGL1-RcTTG1)associated with anthocyanin accumulation.Yeast one-hybrid and luciferase assays showed that RcMYB1 could active its own gene promoter and those of other EBGs(early anthocyanin biosynthesis genes)and LBGs(late anthocyanin biosynthesis genes).In addition,both of the MBW complexes enhanced the transcriptional activity of RcMYB1 and LBGs.Interestingly,our results also indicate that RcMYB1 is involved in the metabolic regulation of carotenoids and volatile aroma.In summary,we found that RcMYB1 widely participates in the transcriptional regulation of ABGs(anthocyanin biosynthesis genes),indicative of its central role in the regulation of anthocyanin accumulation in rose.Our results provide a theoretical basis for the further improvement of the flower color trait in rose by breeding or genetic modification.

Dietary supplementation of bilberry anthocyanin on growth performance,intestinal mucosal barrier and cecal microbes of chickens challenged with Salmonella Typhimurium

Background Anthocyanins(AC)showed positive effects on improving the intestinal health and alleviating intestinal pathogen infections,therefore,an experiment was conducted to explore the protective effects of supplemented AC on Salmonella-infected chickens.Methods A total of 240 hatchling chickens were randomly allocated to 4 treatments,each with 6 replicates.Birds were fed a basal diet supplemented with 0(CON,and ST),100(ACL)and 400(ACH)mg/kg of AC for d 60,and orally challenged with PBS(CON)or 10^(9) CFU/bird(ST,ACL,ACH)Salmonella Typhimurium at d 14 and 16.Results(1)Compared with birds in ST,AC supplementation increased the body weight(BW)at d 18 and the average daily gain(ADG)from d 1 to 18 of the Salmonella-infected chickens(P<0.05);(2)AC decreased the number of Salmonella cells in the liver and spleen,the contents of NO in plasma and inflammatory cytokines in ileal mucosa of Salmonella-infected chickens(P<0.05);(3)Salmonella infection decreased the ileal villi height,villi height to crypt depth(V/C),and the expression of zonulaoccludins-1(ZO-1),claudin-1,occludin,and mucin 2(MUC2)in ileal mucosa.AC supplementation relieved these adverse effects,and decreased ileal crypt depth(P<0.05);(4)In cecal microbiota of Salmonella-infected chickens,AC increased(P<0.05)the alpha-diversity(Chao1,Pd,Shannon and Sobs indexes)and the relative abundance of Firmicutes,and decreased(P<0.05)the relative abundance of Proteobacteria and Bacteroidota and the enrichment of drug antimicrobial resistance,infectious bacterial disease,and immune disease pathways.Conclusions Dietary AC protected chicken against Salmonella infection via inhibiting the Salmonella colonization in liver and spleen,suppressing secretion of inflammatory cytokines,up-regulating the expression of ileal barrier-related genes,and ameliorating the composition and function of cecal microbes.Under conditions here used,100 mg/kg bilberry anthocyanin was recommended.

Systematic analysis of MYB transcription factors and the role of LuMYB216 in regulating anthocyanin biosynthesis in the flowers of flax(Linum usitatissimum L.)

Anthocyanin is an important pigment that affects plant color and nutritional quality.MYBs play an important role in plant anthocyanin synthesis and accumulation.However,the regulatory function of MYB transcription factors in anthocyanin synthesis in flax flowers is still unclear.In this study,402 MYB transcription factors were identified in the flax genome.These MYB members are unevenly distributed on 15 chromosomes.The R2R3-LuMYB members were divided into 32phylogenetic subfamilies.qRT-PCR analysis showed that seven R2R3-LuMYB genes in the adjacent subfamily of the evolutionary tree had similar expression patterns,among which Lu MYB216 was highly expressed in the petals of different colors.Moreover,gene editing of LuMYB216 in flax showed that the petal color,anther color and seed coat color of mutant plants were significantly lighter than those of wild-type plants,and the anthocyanin content of lumyb216 mutant plants was significantly reduced.Correlation analysis indicated that LuMYB216 was significantly positively correlated with the upstream regulator bHLH30.This study systematically analyzed the MYB gene family in flax,laying a foundation for studying the regulation of LuMYB216 in flax flower anthocyanin synthesis.

Interaction of AcMADS68 with transcription factors regulates anthocyanin biosynthesis in red-fleshed kiwifruit

In red-fleshed kiwifruit,anthocyanin pigmentation is a crucial commercial trait.The MYB-bHLH-WD40(MBW)complex and other transcription factors regulate its accumulation.Herein,a new SEP gene,AcMADS68,was identified as a regulatory candidate for anthocyanin biosynthesis in the kiwifruit by transcriptome data and bioinformatic analyses.AcMADS68 alone could not induce the accumulation of anthocyanin both in Actinidia arguta fruit and tobacco leaves.However,in combination with AcMYBF110,AcMYB123,and AcbHLH1,AcMADS68 co-overexpression increased anthocyanin biosynthesis,whereas its silencing reduced anthocyanin accumulation.The results of the dual-luciferase reporter,firefly luciferase complementation,yeast two-hybrid and co-immunoprecipitation assays showed that AcMADS68 could interact with both AcMYBF110 and AcMYB123 but not with AcbHLH1,thereby co-regulating anthocyanin biosynthesis by promoting the activation of the target genes,including AcANS,AcF3GT1,and AcGST1.Moreover,AcMADS68 also could activate the promoter of AcbHLH1 surported by dual-luciferase reporter and yeast one-hybrid assays,thereby further amplifying the regulation signals from the MBW complex,thus resulting in enhanced anthocyanin accumulation in the kiwifruit.These findings may facilitate better elucidation of various regulatory mechanisms underlying anthocyanin accumulation and contribute to the quality enhancement of red-fleshed kiwifruit.

The PcERF5 promotes anthocyanin biosynthesis in red-fleshed pear(Pyrus communis)through both activating and interacting with PcMYB transcription factors

As there is a strong interest in red-skinned pears,the molecular mechanism of anthocyanin regulation in red-skinned pears has been widely investigated;however,little is known about the molecular mechanism of anthocyanin regulation in red-fleshed pears due to limited availability of such germplasm,primarily found in European pears(Pyrus communis).In this study,based on transcriptomic analysis in red-fleshed and white-fleshed pears,we identified an ethylene response factor(ERF)from P.communis,PcERF5,of which expression level in fruit flesh was significantly correlated with anthocyanin content.We then verified the function of PcERF5 in regulating anthocyanin accumulation by genetic transformation in both pear skin and apple calli.PcERF5 regulated anthocyanin biosynthesis by different regulatory pathways.On the one hand,PcERF5 can activate the transcription of flavonoid biosynthetic genes(PcDFR,PcANS and PcUFGT)and two key transcription factors encoding genes PcMYB10 and PcMYB114.On the other hand,PcERF5 interacted with PcMYB10 to form the ERF5-MYB10 protein complex that enhanced the transcriptional activation of PcERF5 on its target genes.Our results suggested that PcERF5 functioned as a transcriptional activator in regulating anthocyanin biosynthesis,which provides new insights into the regulatory mechanism of anthocyanin biosynthesis.This new knowledge will provide guidance for molecular breeding of red-fleshed pear.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed,and anthocyanin and proanthocyanidins(PAs)are major seed-coat pigments.Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed.In this study,we identified a transcription factor gene BnbHLH92a(BnaA06T0441000ZS)in rapeseed.Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs.Correspondingly,the expression profiles of anthocyanin and PA biosynthesis genes(TT3,BAN,TT8,TT18,and TTG1)were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds,indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis.BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed.BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression.These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B.napus.

Genome-wide identification of the MATE gene family and functional characterization of PbrMATE9 related to anthocyanin in pear

Plant multidrug and toxic compound extrusion(MATE) genes play an important role in the process of detoxification, plant morphogenesis, and anthocyanin accumulation. However, whether the MATE gene family functions in pear peel coloration is still unknown. To evaluate and identify the MATE gene family members which are involving in anthocyanin accumulation and coloration in pear. In this study, 85 MATE genes were identified in the reference pear genome of ‘Dangshansuli’ through genome-wide identification. Based on gene structure and phylogenetic tree analysis, the MATE family was divided into five subfamilies. RNA sequencing and quantitative real-time polymerase chain reaction(qRTPCR) indicated that the expression patterns of PbrMATEs were tissue-specific. 28.24%(24) of PbrMATE genes were expressed in the fruits, and44.71%(38) of PbrMATE genes were expressed in the leaves. Additionally, we found that the expression levels of PbrMATE9, PbrMATE26,PbrMATE50, and PbrMATE69 in debagged fruits with red peel were significantly higher than those in bagged fruits without red peel, according to our bagging/debagging treatment of ‘Mantianhong’. The expression pattern of PbrMATE9 was consistent with the variation trend in anthocyanin content, suggesting that it might play an important role in anthocyanin accumulation in response to light exposure. Subcellular localization showed that PbrMATE9 was a membrane protein. More strikingly, the transient overexpression of PbrMATE9 promoted anthocyanin accumulation in the peel of pear, and the expression of structural genes(PbrCHI, PbrANS, PbrDFR, and PbrUFGT) in the anthocyanin biosynthesis pathway also increased significantly. Through co-expression network analysis, the transcription factors were identified, such as WRKY, COL,GATA, and BBX, which might be involved in the regulation of PbrMATE9. The study has enriched the genetic resources and improved the understanding of the regulation network of anthocyanin accumulation in pear.

The PcHY5 methylation is associated with anthocyanin biosynthesis and transport in ‘Max Red Bartlett' and ‘Bartlett' pears

The red coloring of pear fruits is mainly caused by anthocyanin accumulation. Red sport, represented by the green pear cultivar ‘Bartlett’(BL) and the red-skinned derivative ‘Max Red Bartlett’(MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear. Genetic analysis has previously revealed a quantitative trait locus(QTL) associated with red skin color in MRB. However, the key gene in the QTL and the associated regulatory mechanism remain unknown. In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB. These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars;MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL. These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB. We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport. Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5. The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color. Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.

Down-regulation of MeMYB2 leads to anthocyanin accumulation and increases chilling tolerance in cassava(Manihot esculenta Crantz)

Chilling-induced accumulation of reactive oxygen species(ROS) is harmful to plants,which usually produce anthocyanins to scavenge ROS as protection from chilling stress.As a tropical crop,cassava is hypersensitive to chilling,but the biochemical basis of this hypersensitivity remains unclear.We previously generated Me MYB2-RNAi transgenic cassava with increased chilling tolerance.Here we report that Me MYB2-RNAi transgenic cassava accumulated less ROS but more cyanidin-3-O-glucoside than the wild type under early chilling stress.Under this stress,the anthocyanin biosynthesis pathway was more active in Me MYB2-RNAi lines than in the wild type,and several genes involved in the pathway,including Me TT8,were up-regulated by Me MYB2-RNAi in the transgenic cassava.Me MYB2 bound to the Me TT8 promoter and blocked its expression under both normal and chilling conditions,thereby inhibiting anthocyanin accumulation.Me TT8 was shown to bind to the promoter of Dihydroflavonol 4-reductase(Me DFR-2)and increased Me DFR-2 expression.Me MYB2 appears to act as an inhibitor of chilling-induced anthocyanin accumulation during the rapid response of cassava to chilling stress.

MdWRKY40is directly promotes anthocyanin accumulation and blocks MdMYB15L, the repressor of MdCBF2, which improves cold tolerance in apple

Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337' and ‘60-160', which are resistant and sensitive to cold stress, respectively. The enriched pathways of differentially expressed genes(DEGs) and physiological changes in ‘M9T337' and ‘60-160' plantlets were clearly different after cold stress(1°C) treatment for 48 h, suggesting that they have differential responses to cold stress. The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance. When we overexpressed MdWRKY40is and MdWRKY48in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2. Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter. Yeast twohybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2inhibitor MdMYB15L through the leucine zipper(LZ). When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L. In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

VvBBX44 and VvMYBA1 form a regulatory feedback loop to balance anthocyanin biosynthesis in grape

Anthocyanins are essential for the quality of perennial horticultural crops,such as grapes.In grapes,ELONGATED HYPOCOTYL 5(HY5)and MYBA1 are two critical transcription factors that regulate anthocyanin biosynthesis.Our previous work has shown that Vitis vinifera B-box protein 44(VvBBX44)inhibits anthocyanin synthesis and represses VvHY5 expression in grape calli.However,the regulatory mechanism underlying this regulation was unclear.In this study,we found that loss of VvBBX44 function resulted in increased anthocyanin accumulation in grapevine callus.VvBBX44 directly represses VvMYBA1,which activates VvBBX44.VvMYBA1,but not VvBBX44,directly modulates the expression of grape UDP flavonoid 3-O-glucosyltransferase(VvUFGT).We demonstrated that VvBBX44 represses the transcriptional activation of VvUFGT and VvBBX44 induced by VvMYBA1.However,VvBBX44 and VvMYBA1 did not physically interact in yeast.The application of exogenous anthocyanin stimulated VvBBX44 expression in grapevine suspension cells and tobacco leaves.These findings suggest that VvBBX44 and VvMYBA1 form a transcriptional feedback loop to prevent overaccumulation of anthocyanin and reduce metabolic costs.Our work sheds light on the complex regulatory network that controls anthocyanin biosynthesis in grapevine.

Multifaceted roles of LhWRKY44 in promoting anthocyanin accumulation in Asiatic hybrid lilies (Lilium spp.)

The Asiatic hybrid lily(Lilium spp.)is a horticultural crop with high commercial value and diverse anthocyanin pigmentation patterns.However,the regulatory mechanism underlying lily flower color has been largely unexplored.Here,we identified a WRKY transcription factor from lily tepals,LhWRKY44,whose expression was closely associated with anthocyanin accumulation.Functional verification indicated that LhWRKY44 positively regulated anthocyanin accumulation.LhWRKY44 physically interacted with LhMYBSPLATTER and directly bound to the LhMYBSPLATTER promoter,which enhanced the effect of the LhMYBSPLATTER-LhbHLH2 MBW complex activator on anthocyanin accumulation.Moreover,EMSA and dual-luciferase assays revealed that LhWRKY44 activated and bound to the promoters of gene LhF3H and the intracellular anthocyanin-related glutathione S-transferase gene LhGST.Interestingly,our further results showed that LhWRKY44 participated in light and drought-induced anthocyanin accumulation,and improved the drought tolerance in lily via activating stress-related genes.These results generated a multifaceted regulatory mechanism for the LhWRKY44-meditaed enhancement by the environmental signal pathway of anthocyanin accumulation and expanded our understanding of the WRKY-mediated transcriptional regulatory hierarchy modulating anthocyanin accumulation in Asiatic hybrid lilies.