Controls Anthocyanin Accumulation Research Articles

Genetic variations at BBX24 and MYB110a loci regulated anthocyanin accumulation in pear bud sports

Genetic variation contributes to the phenotypic diversity of plants. Most red pear cultivars that accumulate anthocyanins originated from somatic variation and are termed bud sports. ‘Hongzaosu’ is a bud sport derived from the green pear ‘Zaosu’. A new genetic map was constructed from a population derived from the cross of ‘Yuluxiang’ and ‘Hongzaosu’, from which PpBBX24 was identified as a crucial gene controlling anthocyanin accumulation. Genetic and phylogenetic evidence revealed that PpBBX24 is a repressor of anthocyanin biosynthesis in pear. A 14 bp deletion was detected in the third exon of PpBBX24 in ‘Hongzaosu’, resulting in premature termination of protein translation. The truncated PpBBX24 protein was a positive regulator of anthocyanin biosynthesis by activating the transcription of PpCHS and PpUFGT. Three independent variations in the BBX24 coding region that led to premature termination of translation were detected in other pear bud sports and the progeny of a bud sport that all accumulate anthocyanins. The genome of ‘Hongzaosu’ was assembled using both long-read and short-read sequences. By combining genomic and transcriptomic data, allele-specific expression of PpMYB110a was observed in the fruit skin of ‘Hongzaosu’, which was likely caused by large variation in the promoter. This work enhances understanding of coloration in red pears and provides a novel genomic resource for further studies on ‘Hongzaosu’ pear.

Functional characterization of DcMYB11, an R2R3 MYB associated with the purple pigmentation of carrot petiole.

DcMYB11, an R2R3 MYB gene associated with petiole anthocyanin pigmentation in carrot, was functionally characterized. A putative enhancer sequence is able to increase DcMYB11 activity. The accumulation of anthocyanin pigments can exhibit different patterns across plant tissues and crop varieties. This variability allowed the investigation of the molecular mechanisms behind the biosynthesis of these pigments in several plant species. Among crops, carrots have a well-defined anthocyanin pigmentation pattern depending on the genic background. In this work, we report on the discovery of DNA structural differences affecting the activity of an R2R3 MYB (encoded by DcMYB11) involved in anthocyanin regulation in carrot petiole. To this end, we first verified the function of DcMYB11 using heterologous systems and identified three different alleles which may explain differences in petiole pigmentation. Characterization of the DcMYB11 alleles at the 5' upstream sequence unveiled a sequence that functions as a putative enhancer. In conclusion, this study provides novel insight into the molecular mechanisms controlling anthocyanin accumulation in carrot. By these outcomes, we expanded our knowledge on the cis-regulatory sequences in plants.

Transcriptome analysis reveals the crucial function of hyperoside in inhibiting anthocyanin accumulation in grape (Vitis vinifera L.) fruits by inducing VvMYB62.

The formation of color in plants is significantly dependent on anthocyaninpigments. Grape species vary in color due to the differences in anthocyanin accumulation. It is widely recognized that both biotic and abiotic conditions may have an impact on anthocyanin synthesis in plants. The underlying molecular mechanisms by which external application of hyperoside impacts anthocyanin formation in grapes, however, have received little attention. In the current study,the transcriptome of Gemstone seedless grape was examined using high-throughput RNA sequencing at various developmental stages reply to both control and hyperoside treatments. The results of this study suggested that the major genes controlling anthocyanin accumulation in response to the externalinjection of hyperoside could be VvMYB62, VvPAL, VvCHS, and VvF3'5'H.Quantitative reverse transcription PCR (RT-qPCR) results were used to confirm the changes in the expression levels of the genes encoding the anthocyanin biosynthesis pathway under the control and hyperoside treatments. Using a transient transformation system, it was discovered that VvMYB62 was shown to regulate the anthocyanin accumulation at both the transcriptional and posttranslational levels and could be influenced by the external administration of hyperoside. In grape embryogenic calli, hyperoside could specifically suppress theexpression of VvMYB62 and anthocyanin accumulation. In this instance, the VvMYB62 characterisation brought attention to the significance of exogenous hyperoside-induced anthocyanin accumulation. Therefore, the results demonstrated that VvMYB62 could be hindered in the process of grape during anthocyanin accumulation caused by hyperoside. These findings offer excellent candidate genes in the future breeding of novel grape varieties in addition to serving as a crucial reference for understanding the underlying molecular processes of hyperoside suppression of anthocyanin formation in plants.

LcCOP1 and LcHY5 control the suppression and induction of anthocyanin accumulation in bagging and debagging litchi fruit pericarp

Bagging can improve fruit qualities and provide a useful method for studying fruit pigmentation. Bagging treatment regulates anthocyanin accumulation in the pericarp of litchi (Litchi chinensis Sonn.). However, how bagging treatment controls anthocyanin accumulation remains unclear. In this study, we analyzed the effects of bagging and debagging on the anthocyanin accumulation and its biosynthesis-related genes in litchi. To clarify the mechanism of light regulates anthocyanin biosynthesis, LcUVR8-5, LcCOP1-2 and LcHY5-1 genes in light signal transduction were identified. Gene expression analysis showed that LcUVR8-5 and LcHY5-1 were up-regulated after bag removal, while LcCOP1-2 was down-regulated. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that LcCOP1-2 interacts with LcUVR8-5, LcHY5-1 and LcMYB1. Moreover, LcCOP1-2 interacts with LcUVR8-5 in a light-dependent manner. Functional complementation of Arabidopsis cop1-4 mutant and transiently expressing experiments demonstrated that LcCOP1-2 negatively regulates anthocyanin accumulation. In addition, LcHY5-1 directly bound to the promoters of LcMYB1 and anthocyanin biosynthesis structural genes (LcCHS, LcCHI, LcF3H, and LcANS) and activated their expression. Taken together, our results preliminarily revealed that the light regulated anthocyanin biosynthesis in litchi through a relatively conserved UVR8-COP1-HY5 signal pathway.

Carrot Anthocyanins Genetics and Genomics: Status and Perspectives to Improve Its Application for the Food Colorant Industry.

Purple or black carrots (Daucus carota ssp. sativus var. atrorubens Alef) are characterized by their dark purple- to black-colored roots, owing their appearance to high anthocyanin concentrations. In recent years, there has been increasing interest in the use of black carrot anthocyanins as natural food dyes. Black carrot roots contain large quantities of mono-acylated anthocyanins, which impart a measure of heat-, light- and pH-stability, enhancing the color-stability of food products over their shelf-life. The genetic pathway controlling anthocyanin biosynthesis appears well conserved among land plants; however, different variants of anthocyanin-related genes between cultivars results in tissue-specific accumulations of purple pigments. Thus, broad genetic variations of anthocyanin profile, and tissue-specific distributions in carrot tissues and organs, can be observed, and the ratio of acylated to non-acylated anthocyanins varies significantly in the purple carrot germplasm. Additionally, anthocyanins synthesis can also be influenced by a wide range of external factors, such as abiotic stressors and/or chemical elicitors, directly affecting the anthocyanin yield and stability potential in food and beverage applications. In this study, we critically review and discuss the current knowledge on anthocyanin diversity, genetics and the molecular mechanisms controlling anthocyanin accumulation in carrots. We also provide a view of the current knowledge gaps and advancement needs as regards developing and applying innovative molecular tools to improve the yield, product performance and stability of carrot anthocyanin for use as a natural food colorant.

Transcriptomic profiling of purple broccoli reveals light-induced anthocyanin biosynthetic signaling and structural genes.

Purple Broccoli (Brassica oleracea L. var italica) attracts growing attention as a functional food. Its purple coloration is due to high anthocyanin amounts. Light represents a critical parameter affecting anthocyanins biosynthesis. In this study, ‘Purple Broccoli’, a light-responding pigmentation cultivar, was assessed for exploring the mechanism underlying light-induced anthocyanin biosynthesis by RNA-Seq. Cyanidin, delphinidin and malvidin derivatives were detected in broccoli head samples. Shading assays and RNA-seq analysis identified the flower head as more critical organ compared with leaves. Anthocyanin levels were assessed at 0, 7 and 11 days, respectively, with further valuation by RNA-seq under head-shading and light conditions. RNA sequences were de novo assembled into 50,329 unigenes, of which 38,701 were annotated against four public protein databases. Cluster analysis demonstrated that anthocyanin/phenylpropanoid biosynthesis, photosynthesis, and flavonoid biosynthesis in cluster 8 were the main metabolic pathways regulated by light and had showed associations with flower head growth. A total of 2,400 unigenes showed differential expression between the light and head-shading groups in cluster 8, including 650 co-expressed, 373 specifically expressed under shading conditions and 1,377 specifically expressed under normal light. Digital gene expression (DGE) analysis demonstrated that light perception and the signal transducers CRY3 and HY5 may control anthocyanin accumulation. Following shading, 15 structural genes involved in anthocyanin biosynthesis were downregulated, including PAL, C4H, 4CL, CHS, CHI, F3H and DFR. Moreover, six BoMYB genes (BoMYB6-1, BoMYB6-2, BoMYB6-3, BoMYB6-4, BoMYBL2-1 and BoMYBL2-2) and three BobHLH genes (BoTT8_5-1, BoTT8_5-2 and BoEGL5-3) were critical transcription factors controlling anthocyanin accumulation under light conditions. Based on these data, a light-associated anthocyanin biosynthesis pathway in Broccoli was proposed. This information could help improve broccoli properties, providing novel insights into the molecular mechanisms underpinning light-associated anthocyanin production in purple vegetables.

Identification of Candidate HY5-Dependent and -Independent Regulators of Anthocyanin Biosynthesis in Tomato.

High quantities of anthocyanins in plants confer potential protective benefits against biotic and abiotic stressors. Studies have shown that the bZIP transcription factor HY5 plays a key role in controlling anthocyanin accumulation in response to light. However, in hy5 mutants, residual anthocyanins have been detected, indicating that other regulators exist to regulate anthocyanin biosynthesis in an HY5-independent manner. Here, we employed the CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9) system specifically to induce targeted mutagenesis of SlHY5 in the purple tomato cultivar 'Indigo Rose'. The T2 generation of tomato plants homozygous for the null allele of the SlHY5 frameshift mutated by a 1 bp insertion contained a lower anthocyanin content. Transcriptional analysis showed that most of the anthocyanin biosynthesis structural genes and several regulatory genes were down-regulated in the hy5 mutant lines. With transcriptome analyses of the various tissues from hy5 mutant lines, eight candidate transcription factors were identified that may regulate anthocyanin biosynthesis in an HY5-independent manner. These findings deepen our understanding of how light controls anthocyanin accumulation and facilitate the identification of the regulators of anthocyanin biosynthesis in an HY5-independent manner.

AgMYB2 transcription factor is involved in the regulation of anthocyanin biosynthesis in purple celery (Apium graveolens L.).

This study showed that an R2R3-MYB transcription factor, AgMYB2, functions in anthocyanin biosynthesis and accumulation in purple celery. Anthocyanins are involved in tissue coloration and stress response in plants. Foods containing high anthocyanin content are also beneficial to human health. Purple celery accumulated amounts of anthocyanins in the petioles. The biosynthesis of anthocyanin in plants is mainly regulated by the R2R3-MYB transcription factor (TF). However, the R2R3-MYB TF that controls anthocyanin accumulation in purple celery remains unclear. In this study, an R2R3-MYB TF gene, AgMYB2, was cloned from purple celery and characterized as anthocyanin biosynthetic regulator. Sequence analysis indicated that AgMYB2 contained highly conserved R2R3 domain and two anthocyanin characteristic motifs, ANDV motif and KPRPR[S/T]F motif. The relative expression level of AgMYB2 in purple celery was significantly higher than that in non-purple celery at three developmental stages. Heterologous expression of AgMYB2 in Arabidopsis generated more anthocyanins and resulted in dark-purple leaves and flowers. The expression levels of anthocyanin biosynthetic genes and the antioxidant activity of transgenic Arabidopsis carrying AgMYB2 were up-regulated. The determination of anthocyanin glycosylation activity of Arabidopsis crude enzyme verified the anthocyanin biosynthesis regulatory function of AgMYB2 at the protein level. The interaction between AgMYB2 and bHLH proteins was shown by yeast two-hybrid assay. The results will help to elucidate the molecular mechanism of anthocyanin biosynthesis in purple celery and provide an approach for cultivating plants with high anthocyanin content.

Effect of phosphate deficiency-induced anthocyanin accumulation on the expression of Solanum lycopersicum GLABRA3 (SlGL3) in tomato

ABSTRACTIn Arabidopsis thaliana, the bHLH transcription factor, GLABRA3 (AtGL3), is an important regulator of epidermal cell differentiation and positively controls anthocyanin accumulation. In contrast, we previously showed that Solanum lycopersicum GLABRA3 (SlGL3), the AtGL3 homolog, suppressed anthocyanin accumulation in Arabidopsis. To clarify this functional discrepancy in anthocyanin accumulation, we analyzed the SlGL3 expression pattern in anthocyanin-induced tomato. The SlGL3 expression was significantly reduced in tomato seedlings rich in anthocyanin as a result of inorganic phosphate (Pi) starvation. This was consistent with the previous result obtained in Arabidopsis, wherein the overexpression of SlGL3 was shown to inhibit anthocyanin accumulation. Our study suggests that the function of SlGL3 is different from that of AtGL3, and it might inhibit anthocyanin accumulation in tomato.

Overexpression of snapdragon Delila (Del) gene in tobacco enhances anthocyanin accumulation and abiotic stress tolerance

BackgroundRosea1 (Ros1) and Delila (Del) co-expression controls anthocyanin accumulation in snapdragon flowers, while their overexpression in tomato strongly induces anthocyanin accumulation. However, little data exist on how Del expression alone influences anthocyanin accumulation.ResultsIn tobacco (Nicotiana tabacum ‘Xanthi’), Del expression enhanced leaf and flower anthocyanin production through regulating NtCHS, NtCHI, NtF3H, NtDFR, and NtANS transcript levels. Transgenic lines displayed different anthocyanin colors (e.g., pale red: T0-P, red: T0-R, and strong red: T0-S), resulting from varying levels of biosynthetic gene transcripts. Under salt stress, the T2 generation had higher total polyphenol content, radical (DPPH, ABTS) scavenging activities, antioxidant-related gene expression, as well as overall greater salt and drought tolerance than wild type (WT).ConclusionWe propose that Del overexpression elevates transcript levels of anthocyanin biosynthetic and antioxidant-related genes, leading to enhanced anthocyanin production and antioxidant activity. The resultant increase of anthocyanin and antioxidant activity improves abiotic stress tolerance.

Mapping and expression profiling reveal an inserted fragment from purple mustard involved anthocyanin accumulation in Chinese cabbage

Anthocyanins are the major pigments responsible for purple coloration in flowers, fruits and leaves, and the genes involved in their biosynthetic pathway have been identified in many plants. A purple-leaf Chinese cabbage (Brassica rapa L. ssp. pekinensis) was bred by interspecies crossing between Chinese cabbage and purple-leaf mustard [Brassica juncea (L.) Coss. var. foliosa L. H. Bailey]. In this study, high-performance liquid chromatographic analysis indicated purple coloration in Chinese cabbage is due to the accumulation of the same kind of cyaninin as in purple mustard. To elucidate the genetic factors controlling anthocyanin accumulation in this purple-leaf Chinese cabbage, we mapped the anthocyanin gene from the mustard (Anm) locus in an F2 population and performed expression profiling of anthocyanin-related genes. A genetic analysis revealed that the purple-leaf phenotype is a qualitative trait and that its inheritance is unstable in purple-leaf Chinese cabbage. Mapping insertion/deletion markers from 288 individuals of the F2 population located the Anm locus within a 2.5-cM interval on B. rapa chromosome A02. The sequencing and alignment of the amplified fragments demonstrated that purple Chinese cabbage contains fragments of purple mustard on chromosome A02. We evaluated the expression profiles of 12 anthocyanin-related genes on A02 by reverse-transcription and quantitative real-time PCR methods, which revealed that the expression levels of five genes were higher in purple Chinese cabbage than in the non-purple variety. These results offer insights into the molecular mechanism of anthocyanin biosynthesis and improve the knowledge on molecular breeding of purple-type Chinese cabbage.

Novel insight into the mechanism underlying light-controlled anthocyanin accumulation in eggplant (Solanum melongena L.)

Eggplant is rich in anthocyanins, which are the major secondary metabolites and beneficial to human health. We discovered that the anthocyanin biosynthesis of eggplant cultivar ‘Lanshan Hexian’ was regulated by light. In this study, we isolated two blue light receptor genes, SmCRY1 and SmCRY2, and negative/positive anthocyanin regulatory factors SmCOP1 and SmHY5 from eggplant. In terms of transcript levels, SmCRY1, SmCRY2 and SmHY5 were up-regulated by light, while SmCOP1 was down-regulated. Subsequently, the four genes were functionally complemented in phenotype of corresponding mutants, indicating that they act as counterparts of Arabidopsis genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that SmCRY1 and SmCRY2 interact with SmCOP1 in a blue-light-dependent manner. It also obtained the result that SmCOP1 interacts with SmHY5 and SmMYB1. Furthermore, using yeast one-hybrid assay, we found that SmHY5 and SmMYB1 both bind the promoters of anthocyanin biosynthesis structural genes (SmCHS and SmDFR). Taken together, blue-light-triggered CRY1/CRY2-COP1 interaction creates the condition that HY5 and MYB1 combine with the downstream anthocyanin synthesis genes (CHS and DFR) in eggplant. Our finding provides a new working model by which light controls anthocyanin accumulation in eggplant.

Fine mapping and candidate gene analysis of an anthocyanin-rich gene, BnaA.PL1, conferring purple leaves in Brassica napus L.

Because of the advantages of anthocyanins, the genetics and breeding of crops rich in anthocyanins has become a hot research topic. However, due to the lack of anthocyanin-related mutants, no regulatory genes have been mapped in Brassica napus. In this study, we first report the characterization of a B. napus line with purple leaves and the fine mapping and candidate screening of the BnaA.PL1 gene. The amount of anthocyanins in the purple leaf line was six times higher than that in a green leaf line. A genetic analysis indicated that the purple character was controlled by an incomplete dominant gene. Through map-based cloning, we localized the BnaA.PL1 gene to a 99-kb region at the end of B. napus chromosome A03. Transcriptional analysis of 11 genes located in the target region revealed that the expression level of only the BnAPR2 gene in seedling leaves decreased from purple to reddish green to green individuals, a finding that was consistent with the measured anthocyanin accumulation levels. Molecular cloning and sequence analysis of BnAPR2 showed that the purple individual-derived allele contained 17 variants. Markers co-segregating with BnaA.PL1 were developed from the sequence of BnAPR2 and were validated in the BC4P2 population. These results suggested that BnAPR2, which encodes adenosine 5'-phosphosulfate reductase, is likely to be a valuable candidate gene. This work may lay the foundation for the marker-assisted selection of B. napus vegetables that are rich in anthocyanins and for an improved understanding of the molecular mechanisms controlling anthocyanin accumulation in Brassica.

Anthocyanin profile characterization and quantitative trait locus mapping in zicaitai (Brassica rapa L. ssp. chinensis var. purpurea)

Anthocyanins have several biological functions in plants and are beneficial to human health. To elucidate the metabolic profile of anthocyanins and determine the genetic basis controlling anthocyanin accumulation in zicaitai (Brassica rapa L. ssp. chinensis var. purpurea), we conducted anthocyanin profile characterization and quantitative trait locus (QTL) analysis. Seventeen anthocyanin compounds were identified as cyanidin glycosides in zicaitai. A genetic linkage map based on 200 F2 lines was constructed using 161 insertion/deletion markers. Total anthocyanin content (TAC) was determined by pH differential spectrophotometry for the F2 lines. Using the map and phenotypic data, a major QTL which explained 56.7 % of phenotypic variation was identified for TAC on chromosome A09. Two genes, BrEGL3.1 and BrEGL3.2, as syntenic orthologs of AtEGL3 encoding basic helix–loop–helix transcription factors in this QTL region, are candidate genes for a key role in the control of anthocyanin accumulation in zicaitai.

MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple.

MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species.

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.

Anp, a genetic locus controlling organ-specific accumulation of anthocyanins in Phaseolus vulgaris L.

A genetic locus, Anp, controls anthocyanin accumulation in the epidermal cell layers of bean (Phaseo-lus vulgaris L.) pods and pulvini, and in the standard petals of bean flowers. The expression of the Anp-dependent phenotype requires normal purple pigmentation of the hypocotyl and of the cotyledons. In the Anp-deficient bean genotype, La Victoire, the mRNA transcripts of chalcone synthase and chalcone isomerase accumulated to levels comparable to those of the Anp-carrying genotype AFN. The two genotypes had similar amounts of chalcone synthase enzyme in the epidermal cells where anthocyanins are accumulated in AFN, as shown by immunocytolocalization. The genotype La Victoire accumulated low levels of flavones, flavonols and proanthocyanins but absolutely no anthocyanins in the organs in which pigmentation is controlled by Anp. The data indicate that Anp represents a locus involved in the organ-specific expression of the late steps of the anthocyanin pathway.