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.

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.

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.

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.

Functional Characterization of SmbHLH13 in Anthocyanin Biosynthesis and Flowering in Eggplant

Anthocyanin is abundant in a few vegetables,including eggplant.It protects plants from abiotic stress and benefits human health,making the research of anthocyanin biosynthesis increasingly important.Flowering time is an important reference for judging reproduction and adaptability,which can guide plant production.In this study,SmbHLH13 from eggplant was identified.Yeast one-hybrid and dual-luciferase assays showed that SmbHLH13 binded and activated the expression of structural genes SmCHS and SmF3H in anthocyanin biosynthesis and it also was bound to the promoter of the key gene SmFT in flowering.Furthermore,genetic transformation of Arabidopsis revealed that overexpression of SmbHLH13 enhanced anthocyanin accumulation and delayed flowering.These results demonstrated that SmbHLH13 might promote anthocyanin accumulation through positive regulation of SmCHS and SmF3H.Moreover,SmbHLH13 might have a role in delaying eggplant flowering.

The R2R3-MYB transcription factor GaPC controls petal coloration in cotton

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene(named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene,GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis,also has potential breeding value by engineering GaPC to develop colored petals or fibers for multifunctional utilization of cotton.

MdbHLH162 connects the gibberellin and jasmonic acid signals to regulate anthocyanin biosynthesis in apple

Anthocyanins are secondary metabolites induced by environmental stimuli and developmental signals.The positive regulators of anthocyanin biosynthesis have been reported,whereas the anthocyanin repressors have been neglected.Although the signal transduction pathways of gibberellin(GA)and jasmonic acid(JA)and their regulation of anthocyanin biosynthesis have been investigated,the cross-talk between GA and JA and the antagonistic mechanism of regulating anthocyanin biosynthesis remain to be investigated.In this study,we identified the anthocyanin repressor MdbHLH162 in apple and revealed its molecular mechanism of regulating anthocyanin biosynthesis by integrating the GA and JA signals.MdbHLH162 exerted passive repression by interacting with MdbHLH3 and MdbHLH33,which are two recognized positive regulators of anthocyanin biosynthesis.MdbHLH162 negatively regulated anthocyanin biosynthesis by disrupting the formation of the anthocyanin-activated MdMYB1-MdbHLH3/33complexes and weakening transcriptional activation of the anthocyanin biosynthetic genes MdDFR and MdUF3GT by MdbHLH3 and MdbHLH33.The GA repressor MdRGL2a antagonized MdbHLH162-mediated inhibition of anthocyanins by sequestering MdbHLH162 from the MdbHLH162-MdbHLH3/33 complex.The JA repressors MdJAZ1 and MdJAZ2 interfered with the antagonistic regulation of MdbHLH162 by MdRGL2a by titrating the formation of the MdRGL2a-MdbHLH162 complex.Our findings reveal that MdbHLH162 integrates the GA and JA signals to negatively regulate anthocyanin biosynthesis.This study provides new information for discovering more anthocyanin biosynthesis repressors and explores the cross-talk between hormone signals.

RHA2b-mediated MYB30 degradation facilitates MYB75-regulated,sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings

Anthocyanins play diverse roles in plant physiology and stress adaptation.In Arabidopsis,the MYB–bHLH–WD40(MBW)complex has a crucial role in the regulation of anthocyanin synthesis.Here,we report that the R2R3-MYB transcription factor MYB30 and the ubiquitin E3 ligase RHA2b participate in anthocyanin biosynthesis through regulation of the MBW complex.MYB30 was found to negatively regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings.Expression of multiple genes involved inflavo-noid or anthocyanin biosynthesis was affected in the myb30 mutant,and MYB30 directly repressed the expression of MYB75,which encodes a core component of the MBW complex,by binding to its promoter.Moreover,MYB30 physically interacted with MYB75 to inhibit its activity by repressing MBW complex as-sembly.In addition,sucrose treatment signicantly promoted MYB30 degradation via the action of RHA2b.The ubiquitination and degradation of MYB30 were signicantly attenuated in the rha2b mutant un-der high-sucrose treatment,and further analysis showed that MYB75 directly promoted RHA2b expression in response to high sucrose.Our work thus reveals an anthocyanin biosynthetic regulatory module,RHA2b–MYB30,that controls the function of the MBW complex via MYB75.The repression of MYB75 by MYB30 is released by MYB75-induced RHA2b expression,thus ensuring the self-activation of MYB75 when anthocy-anin synthesis is needed.

Identification of PAL genes related to anthocyanin synthesis in tea plants and its correlation with anthocyanin content

The phenylalanine ammonia-lyase(PAL)gene family in tea plants(Camellia sinensis L.)encodes the enzyme that catalyzes the first reaction of the phenylpropane metabolic pathway.The present study aimed to characterize the PAL genes in tea plants,and get better insights on the CsPALs in anthocyanins accumulation.Seven CsPAL genes were identified and characterized in tea plants by bioinformatics analysis.Systematic analysis of CsPALs was conducted for its phylogenetic relationship,gene structure,chromosomal location,and protein conserved motifs based on tea plant genome.The cis-elements of CsPALs were responsive to light,abiotic stress,hormone,and MYB-binding site.Furthermore,tissuespecific expression analysis showed that CsPAL4 was expressed preferentially in young leaves and buds.Correlation analysis was performed in purple-leaf tea with anthocyanin components,and it was suggested that CsPAL4 was closely related with different anthocyanin accumulated,especially with cyanidin 3-O-galactoside,cyanidin 3-O-glucoside,and delphinidin 3-O-glucoside.Additionally,the putative upstream regulation factors CsMYBs(CsMYB59,CsARR1,CsSRM1,CsMYB101,and CsMYB52)and CsbHLHs(CsbHLH104,CsbHLH3,CsbIM1,CsTCP14,and CsPIF4)could bind to the promoter of CsPALs,thereby activating its transcription.This study provides a theoretical basis for further research to elucidate the functions of the CsPAL genes.

High temperature enhances anthocyanin coloration in Asiatic hybrid lily flowers via upregulation of the MYB12 positive regulator

Although high atmospheric temperatures suppress anthocyanin accumulation in most plant species,we show that high temperatures accelerate anthocyanin pigmentation in flower tepals of Asiatic hybrid lilies(Lilium spp.).After incubation at high temperatures(35℃)for two days,anthocyanin color became deeper in the most of tepal parts of two Asiatic hybrid lily cultivars,although the basal parts of’Montreux’tepals and top parts of’Toronto’outer tepals were whitened.Environmental stimuli often affect the expression of R2R3-MYB positive regulators that control anthocyanin biosynthesis.Although their expression is often suppressed by hot temperatures in other species,the expression of lily MYB12 in tepals was upregulated by high temperatures.MicroRNA828(miR828),which suppresses the action of MYB12 post-transcriptionally,exhibited reduced accumulation levels under high temperature,indicating that miR828 regulation is involved in MYB12 upregulation.In addition,transcription levels of MYB12,estimated by unspliced MYB12 transcript accumulation,were also activated by high temperatures.Thus,both suppressed miR828 accumulation and increased MYB12 transcription are likely to be involved in MYB12 activation at high temperatures.In the whitened basal parts of’Montreux’tepals at 35℃,expression of bHLH2 was severely suppressed while that of MYB12 was not affected.The present results demonstrate that plants display diverse responses to hot climatic conditions and shed new light on anthocyanin regulation under various environmental conditions.

Genome-wide identification and expression profiling of MYB transcription factor genes in radish(Raphanus sativus L.)

Radish(Raphanus sativus L.), an important root vegetable crop of the Brassicaceae family, has a high level of anthocyanin accumulation in its pigment root tissues. It was reported that MYB transcription factors(TFs) play vital roles in plant development and anthocyanin metabolism, and the PAP1/2 could promote expression of anthocyanin biosynthesis genes. In this study, a total of 187 radish MYB genes(Rs MYBs) were identified in the radish genome and clustered into 32 subfamilies. Among them, 159 Rs MYBs were localized on nine radish chromosomes. Interestingly, 14 Rs MYBs exhibited differential expression profiles in different taproot developmental stages among four differently colored radish lines. A number of Rs MYBs were highly expressed in the pigmented root tissues at the maturity stage, several genes including Rs MYB41, Rs MYB117, and Rs MYB132 being homologous to PAP1/2, showed high expression levels in the red skin of NAU-YH(red skin-white flesh) taproot, while Rs MYB65 and Rs MYB159 were highly expressed in the purple root skin of NAU-YZH(purple skin-red flesh), indicating that these Rs MYBs might positively regulate the process of anthocyanin accumulation in radish taproot. These results would provide valuable information for further functional characterization of Rs MYBs, and facilitate clarifying the molecular mechanism underlying anthocyanin biosynthesis in radish.

Transcription factor GLK1 promotes anthocyanin biosynthesis via an MBW complex-dependent pathway in Arabidopsis thaliana

Anthocyanins are important natural plant pigments and play diverse roles in plant growth and adaptation.Anthocyanins function as screens to protect photosynthetic tissues from photoinhibition.However,the regulatory mechanisms underlying the biosynthesis and spatial accumulation pattern of anthocyanins remain some unresolved issues.Here,we demonstrate that the GARP-type transcription factor GOLDEN2-LIKE 1(GLK1)functions as a positive factor in anthocyanin accumulation.GLK1 enhances the transcriptional activation activities of MYB75,MYB90,and MYB113 via direct proteinprotein interactions to increase the expression of anthocyanin-specific biosynthetic genes.Anthocyanins accumulate in an acropetal manner in Arabidopsis.We also found that the expression pattern of GLK1 overall mimicked the accumulation pattern of anthocyanin from the base of the main stem to the shoot apex.Based on these findings,we established a working model for the role of GLK1 in anthocyanin accumulation and propose that GLK1mediates the spatial distribution pattern of anthocyanins by affecting the transcriptional activation activities of MYB75,MYB90,and MYB113.

The E3 ubiquitin ligase SINA1 and the protein kinase BIN2 cooperatively regulate PHR1 in apple anthocyanin biosynthesis

PHR1(PHOSPHATE STARVATION RESPONSE1)plays key roles in the inorganic phosphate(Pi)starvation response and in Pi deficiency-induced anthocyanin biosynthesis in plants. However, the post-translational regulation of PHR1 is unclear,and the molecular basis of PHR1-mediated anthocyanin biosynthesis remains elusive. In this study, we determined that MdPHR1 was essential for Pi deficiency-induced anthocyanin accumulation in apple(Malus × domestica). MdPHR1 interacted with MdWRKY75, a positive regulator of anthocyanin biosynthesis, to enhance the MdWRKY75-activated transcription of MdMYB1,leading to anthocyanin accumulation. In addition,the E3 ubiquitin ligase SEVEN IN ABSENTIA1(MdSINA1) negatively regulated MdPHR1-promoted anthocyanin biosynthesis via the ubiquitination-mediated degradation of MdPHR1.Moreover, the protein kinase apple BRASSINOSTEROID INSENSITIVE2(MdBIN2) phosphorylated MdPHR1 and positively regulated MdPHR1-mediated anthocyanin accumulation by attenuating the MdSINA1-mediated ubiquitination degradation of MdPHR1. Taken together,these findings not only demonstrate the regulatory role of MdPHR1 in Pi starvation induced anthocyanin accumulation, but also provide an insight into the post-translational regulation of PHR1.

Regulation of Anthocyanin during Storage Root Development Stage in Sweet Potato(Ipomoea batatas(L.) Lam.)

Anthocyanin accumulation during storage root development in purple-fleshed sweet potato was analyzed by detection of anthocyanin concentration, accumulation rate and the expression pattern of anthocyanin biosynthetic genes by semi-quantitative RT-PCR. Anthocyanin concentration in sweet potato cvs Jishu 18 and Ayamurasaki increased steadily during storage root development stage. The accumulation rate in two genotypes peaked at 50 to 65 d after transplanting, and then declined rapidly. During storage root development of Ayamurasaki, the anthocyanin biosynthesis gene, IbCHS, was constitutively expressed, the genes IbF3H, IbDFR, IbANS were induced steadily, reaching a maximum at the later stage of root thickening, and IbPAL steadily decreased. Therefore, the mechanism of anthocyanin accumulation differed between the two cultivars, and anthocyanin biosynthesis was regulated through regulation of its synthetic enzymes.

DELLA Proteins Promote Anthocyanin Biosynthesis via Sequestering MYBL2 and JAZ Suppressors of the MYB/bHLH/WD40 Complex in Arabidopsis thaliana

Anthocyanin accumulation is recognized as a visible biomarker of plants that have suffered from environmental stresses. However, the molecular mechanisms underlying stress-induced anthocyanin biosynthesis remain unclear. Expression of anthocyanin-specific genes is regulated by the conserved MBW complex, which is composed of the MYB, bHLH, and WD40 subunRs in higher plants. MBW activity is repressed by MYBL2 and the JAZ family proteins, which bind competitively to bHLH and MYB/bHLH, respectively. Here, we found that MYBL2 and JAZs mediate gibberellic acid-inhibRed anthocyanin biosynthesis in Arabidopsis. Competitive pull-down and dual-lucifarase assays showed that DELLA proteins directly sequester MYBL2 and JAZ repressors, leading to the release of bHLH/MYB subunits and subsequently to the formation of active MBW complex, which then activates the anthocyanin biosynthetic pathway. The JAZ-DELLA-MYBL2 module also plays an Important role in abiotic stress-induced anthocy- anin biosynthesis. Furthermore, we found that the DELLA protein RGA accumulates upon plant exposure to abiotic stresses. Altogether, our data reveal that DELLA-promoted anthocyanin biosynthesis is mediated at least in part by MYBL2 and JAZ regulatory proteins, providing new insights into the coordinated regulation of plant growth and defense through metabolic pathway regulation.

Development of ＂Purple Endosperm Rice＂ by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice （Oryza sativa L.）, could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes （two regulatory genes from maize and six structural genes from Coleus） driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm ＂Purple Endosperm Rice＂ （called ＂Zijingmi＂ in Chinese）, which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation （or enhancement） of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis

Single-repeat R3 MYB transcription factors like CPC （CAPRICE） are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S：：CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs （e.g. root hairs, trichomes）, but can influence anthocyanin pigment synthesis.

Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation

Anthocyanins are a class of products of plant secondary metabolism and are responsible for tubers color in potato. The biosynthesis of anthocyanins is a complex biological process, in which multiple genes are involved including structural genes and regulatory genes. In this study, StAN11, a WD4o-repeat gene, was cloned from potato cultivar Chieftain （Solanum tuberosum L.）. StAN11 （HQ5995o6） contained no intron and its open reading frame （ORF） was 1,o29 bp long, encoding a putative protein of 342 amino acids. In order to verify its role in anthocyanin biosynthesis, StAN11 was inserted behind the CaMV-35S promoter of pCMBIA1304 and the recombination vector was introduced into the potato cultivar Desiree plants by Agrobacterium-mediated transfor- mation. The color of transgenic tuber skin was significantly deepened, compared to the wild-type control, which was highly consistent with the accumulation of anthocyanin and expression of StAN11 in transgenic lines tuber skin. Further analysis on the expression of Flavonone-3-hydroxylase （F3H）,Dihydroflavonol reductase （DFR）, Anthocyanidin synthase （ANS）, and F1avonoid 3-O-glucosyl transferase （3GT） in transgenic plants revealed that only DFR was upregulated. This result suggested that StAN11 regulated anthocyanin biosynthesis in potato by controlling DFR expression and accumulation of anthocyanin could be increased through overexpression of .StAN11 in the tubers with the genetic background of anthocyanin biosynthesis.

An ubiquitin-like protein SDE2 negatively affects sucrose-induced anthocyanin biosynthesis in Arabidopsis

Anthocyanin biosynthesis is regulated by a conserved transcriptional MBW complex composed of MYB,b HLH and WD40 subunits. However, molecular mechanisms underlying transcriptional regulation of these MBW subunits remain largely elusive. In this study, we isolated an Arabidopsis mutant that displays a constitutive red color in aboveground tissues with retarded growth phenotypes. In the presence of sucrose, the mutant accumulates more than 3-fold anthocyanins of the wild type(WT), but cannot produce anthocyanins as WT in the absence of sucrose. Map-based cloning results demonstrated that the mutation occurs in the locus At4 G01000, which encodes a conserved nuclear-localized ubiquitin-like(UBL) superfamily protein, silencing defective 2(SDE2), in eukaryotes. SDE2 is ubiquitously expressed in various tissues. In the sucrose-induced anthocyanin biosynthesis, SDE2 expression was not responded to sucrose treatment at the early stage but was enhanced at the late stage. SDE2 mutations result in upregulation of anthocyanin biosynthetic and regulatory genes. Yeast-two hybrid analysis indicated that SDE2 has no direct interaction with the MYB transcription factor PAP1 and b HLH factor TT8, indicating that SDE2 is a indirect factor to affect anthocyanin accumulation. Taking together, our data suggest that SDE2 may play a role in finely coordinating anthocyanin biosynthesis with other biological processes.

The Upregulation of NtAN2 Expression at Low Temperature is Required for Anthocyanin Accumulation in Juvenile Leaves of Lc-transgenic Tobacco(Nicotiana tabacum L.)

Anthocyanins often accumulate in plants subjected to environmental stress, including low temperature. However, the molecular regulatory mechanism of anthocyanin biosynthesis at low temperature is largely unknown. Here, tobacco was transformed with a maize anthocyanin regulatory gene Lc driven by AtSPX3 promoter to investigate the effect of Lc upon the anthocyanin-biosynthesis pathway. We found that the anthocyanin-biosynthesis pathway could not be activated in wild type, while Lc-transgenic tobacco lines exhibited purple pigmentation in juvenile leaves at low temperature. Accordingly, the total anthocyanin contents increased specifically in juvenile leaves in Lc-transgenic lines. Transcriptional analysis showed that NtCHS and NtCHI were induced by low temperature in leaves of wild type and transgenic lines. NtDFR was uniquely expressed in Lc-transgenic lines, but its transcript was not detected in wild type, implying that NtDFR expression in tobacco leaves was dependent on Lc. Furthermore, the expression of NtAN2 （regulatory gene） and NtANS （anthocyanidin synthase gene） was coordinately upregulated in Lc-transgenic lines under low temperature, suggesting that both Lc and NtAN2 might activate the expression of NtANS. Based on our findings and previous reports, we postulated that Lc interacted with NtAN2 induced by low-temperature stress and consequently stimulated anthocyanin biosynthesis in juvenile leaves of Lc-transgenic tobacco lines.