Increased Anthocyanin Accumulation Research Articles

Fine-mapping of the BjPur gene for purple leaf color in Brassica juncea.

Purple leaves are rich in health-protecting anthocyanins and food colorants in Brassica juncea. But the causal gene, which is related to leaf color formation, have not been reported in B. juncea. Anthocyanins mainly accumulated throughout the adaxial and abaxial epidermal leaf cells of purple leaves. A genetic analysis indicated that an incompletely dominant gene controls the purple leaf trait in B. juncea. Furthermore, the BjPur gene, which increased anthocyanin accumulation in purple-leafmustard, was cloned. Blast and phylogenetic analyses revealed that BjPur encodes a new R2R3-MYB transcription factor. Sequence analysis of two alleles revealed a DNA sequence insertion in the first intron of BjPur in green leaves parent line (LY) when compared with the BjPur gene in the purple-leaf parent line (ZY). And this insertion greatly reduced the transcription of BjPur in green leaves. In purple-leaf plants, the transcript level of BjPur was significantly higher in leaves than in roots, stems, siliques, and flower buds. Additionally, molecular markers linked to leaf color were developed to distinguish different genotypes of B. juncea. These results will be helpful for the genetic improvement of the purple leaf color in B. juncea.

Blue Light Acclimation Reduces the Photoinhibition of Phalaenopsis aphrodite (Moth Orchid).

The moth orchid is an important ornamental crop. It is very sensitive to high light irradiation due to photoinhibition. In this study, young orchid tissue culture seedlings and 2.5” potted plants pretreated under blue light (BL, λmax = 450 nm) at 100 µmol m−2 s−1 for 12 days (BL acclimation) were found to have an increased tolerance to high light irradiation. After BL acclimation, orchids had an increased anthocyanin accumulation, enhanced chloroplast avoidance, and increased chlorophyll fluorescence capacity whenever they were exposed to high light of 1000 μmol m−2 s−1 for two weeks (HL). They had higher Fv/Fm, electron transport rate (ETR), chlorophyll content, catalase activity and sucrose content when compared to the control without BL acclimation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that transcript levels of phototropins, D1, RbcS, PEPCK, Catalase and SUT2 were upregulated in the BL-acclimated orchids. Consequently, BL acclimation orchids had better growth when compared to the control under long-term high light stress. In summary, this study provides a solution, i.e., BL acclimation, to reduce moth orchid photoinhibition and enhance growth before transplantation of the young tissue culture seedlings and potted plants into greenhouses, where they usually suffer from a high light fluctuation problem.

Exploring the Use of Fruit Callus Culture as a Model System to Study Color Development and Cell Wall Remodeling during Strawberry Fruit Ripening

Cell cultures derived from strawberry fruit at different developmental stages have been obtained to evaluate their potential use to study different aspects of strawberry ripening. Callus from leaf and cortical tissue of unripe-green, white, and mature-red strawberry fruits were induced in a medium supplemented with 11.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) under darkness. The transfer of the established callus from darkness to light induced the production of anthocyanin. The replacement of 2,4-D by abscisic acid (ABA) noticeably increased anthocyanin accumulation in green-fruit callus. Cell walls were isolated from the different fruit cell lines and from fruit receptacles at equivalent developmental stages and sequentially fractionated to obtain fractions enriched in soluble pectins, ester bound pectins, xyloglucans (XG), and matrix glycans tightly associated with cellulose microfibrils. These fractions were analyzed by cell wall carbohydrate microarrays. In fruit receptacle samples, pectins were abundant in all fractions, including those enriched in matrix glycans. The amount of pectin increased from green to white stage, and later these carbohydrates were solubilized in red fruit. Apparently, XG content was similar in white and red fruit, but the proportion of galactosylated XG increased in red fruit. Cell wall fractions from callus cultures were enriched in extensin and displayed a minor amount of pectins. Stronger signals of extensin Abs were detected in sodium carbonate fraction, suggesting that these proteins could be linked to pectins. Overall, the results obtained suggest that fruit cell lines could be used to analyze hormonal regulation of color development in strawberry but that the cell wall remodeling process associated with fruit softening might be masked by the high presence of extensin in callus cultures.

In vitro Anthocyanin Induction and Metabolite Analysis in Malus spectabilis Leaves Under Low Nitrogen Conditions

Absract Anthocyanins are the most widely produced secondary metabolites in plants, and they play an important role in plant growth and reproduction. The nitrogen source is an important factor affecting anthocyanin production, but the nitrogen concentrations on metabolism and the underlying genetic basis remain unclear. In this study, in vitro anthocyanin induction was conducted on Malus spectabilis. The leaf explants were cultivated in media containing different nitrogen concentrations. The results suggested that when the nitrogen contents decreased in limit, the color of leaf explants turned from green to red, and increased anthocyanin accumulation led to a change in phenotype. Furthermore, the content of other flavonoids, such as dihydroquercetin, epicatechin, and catechin, increased under low nitrogen conditions. The transcription levels of the general flavonoid pathway genes, from phenylalanine ammonia lyase (PAL) to anthocyanidin synthase (ANS), were associated with the concentration of corresponding flavonoid compounds and phenotype changes. In particular, the expression level of ANS increased substantially under a low nitrogen treatment, which was significantly and positively correlated with the anthocyanin levels (R2 = 0.72, P

PpGST1, an anthocyanin-related glutathione S-transferase gene, is essential for fruit coloration in peach.

SummaryAnthocyanins have crucial biological functions and affect quality of horticultural produce. Anthocyanins accumulate in ripe peach fruit; differential accumulation is observed in deep coloured cultivar ‘Hujingmilu’ and lightly pigmented cultivar ‘Yulu’. The difference was not fully explained by accumulation of total flavonoids and expression of anthocyanin biosynthetic genes. Expression analysis was conducted on a glutathione S‐transferase gene (PpGST1), and it was found that the expression correlated well with anthocyanin accumulation in peach fruit tissues. Functional complementation of the Arabidopsis tt19 mutant indicated that PpGST1 was responsible for transport of anthocyanins but not proanthocyanidins. PpGST1 was localized in nuclei and the tonoplast, including the sites at which anthocyanin vacuolar sequestration occurred. Transient overexpression of PpGST1 together with PpMYB10.1 in tobacco leaves and peach fruit significantly increased anthocyanin accumulation as compared with PpMYB10.1 alone. Furthermore, virus‐induced gene silencing of PpGST1 in a blood‐fleshed peach not only resulted in a reduction in anthocyanin accumulation but also a decline in expression of anthocyanin biosynthetic and regulatory genes. Cis‐element analysis of the PpGST1 promoter revealed the presence of four MYB binding sites (MBSs). Dual‐luciferase assays indicated that PpMYB10.1 bound to the promoter and activated the transcription of PpGST1 by recognizing MBS1, the one closest to the ATG start codon, with this trans‐activation being stronger against the promoter of deep coloured ‘Hujingmilu’ compared with lightly coloured cultivar ‘Yulu’. Altogether, our data provided molecular evidence supporting coordinative regulatory roles of PpGST1 and PpMYB10.1 in anthocyanin accumulation in peach.

JAZ4 is involved in plant defense, growth, and development in Arabidopsis

Jasmonate zim-domain (JAZ) proteins comprise a family of transcriptional repressors that modulate jasmonate (JA) responses. JAZ proteins form a co-receptor complex with the F-box protein coronatine insensitive1 (COI1) that recognizes both jasmonoyl-l-isoleucine (JA-Ile) and the bacterial-produced phytotoxin coronatine (COR). Although several JAZ family members have been placed in this pathway, the role of JAZ4 in this model remains elusive. In this study, we observed that the jaz4-1 mutant of Arabidopsis is hyper-susceptible to Pseudomonas syringae pv. tomato (Pst) DC3000, while Arabidopsis lines overexpressing a JAZ4 protein lacking the Jas domain (JAZ4∆Jas) have enhanced resistance to this bacterium. Our results show that the Jas domain of JAZ4 is required for its physical interaction with COI1, MYC2 or MYC3, but not with the repressor complex adaptor protein NINJA. Furthermore, JAZ4 degradation is induced by COR in a proteasome- and Jas domain-dependent manner. Phenotypic evaluations revealed that expression of JAZ4∆Jas results in early flowering and increased length of root, hypocotyl, and petiole when compared with Col-0 and jaz4-1 plants, although JAZ4∆Jas lines remain sensitive to MeJA- and COR-induced root and hypocotyl growth inhibition. Additionally, jaz4-1 mutant plants have increased anthocyanin accumulation and late flowering compared with Col-0, while JAZ4∆Jas lines showed no alteration in anthocyanin production. These findings suggest that JAZ4 participates in the canonical JA signaling pathway leading to plant defense response in addition to COI1/MYC-independent functions in plant growth and development, supporting the notion that JAZ4-mediated signaling may have distinct branches.

Effects of salicylic acid and sucrose on pigment content in Pistacia chinensis leaves

Pistacia chinensis leaves turn red or yellow in autumn due to changes in pigments. We investigated the effect of salicylic acid and sucrose on pigment in P. chinensis leaves. Different concentrations of salicylic acid (0.07 g/L, 0.14 g/L, and 0.21 g/L) and sucrose (6 g/L, 15 g/L, and 30 g/L) were sprayed on leaves at the hardening period, and the contents of total chlorophyll, carotenoids, anthocyanins, soluble sugar and soluble protein were determined. After spraying for 7 and 17 days, the total chlorophyll content following salicylic acid treatment (0.07 g/L) was higher than that following water treatment. After spraying for 24 days, the carotenoid content following most treatments reached the maximum, and the carotenoid content following the 0.14 g/L salicylic acid and 6 g/L sucrose treatments was significantly higher than that following water treatment. After spraying for 47 days, the anthocyanin content following salicylic acid and water treatments reached the crest value, and the anthocyanin content following the 0.07 g/L salicylic acid treatment was significantly higher than that following water treatment. After spraying for 55 days, the anthocyanin content of sucrose treatments reached the peak value, and the anthocyanin content following the 15 g/L and 30 g/L sucrose treatments was significantly higher than that follwing water treatment. In addition, the soluble sugar and soluble protein contents showed a significantly positive correlation with the anthocyanin content and a significantly negative correlation with total chlorophyll content. In summary, salicylic acid applied at 0.07 g/L at the onset of hardening period increased chlorophyll content of P. chinensis seedlings. Meanwhile, salicylic acid and sucrose applied at 0.14 g/L and 6 g/L respectively promoted the synthesis of carotenoids. Salicylic acid applied at 0.07 g/L and sucrose applied at 15 g/L or 30 g/L at the later hardening period increased anthocyanin accumulation of P. chinensis seedlings.

Methyl Jasmonate and Salinity Increase Anthocyanin Accumulation in Radish Sprouts

Plant secondary metabolites with antioxidant properties, such as anthocyanins, are considered to have an important commercial value for some crops. Although anthocyanin concentration increases in response to various stimuli in plants, the mechanism of anthocyanin accumulation under multiple stimuli is not yet well understood. Here, we examined the effects of methyl jasmonate (MJ) and salinity on anthocyanin accumulation in radish (Raphanus sativus) sprouts. MJ treatments induced anthocyanin accumulation, which was enhanced by simultaneous treatment with salinity (200 mM NaCl), accompanied by growth restrictions. Sprouts treated with salinity alone did not induce anthocyanin accumulation, although sprout growth was restricted. Co-treatment with MJ and salinity increased hydrogen peroxide, total phenol content, and radical scavenging capacity more strongly than was achieved when each treatment was applied singly. Accumulation of anthocyanin was dependent on NaCl concentration and light intensity. Changing MJ and salinity treatment periods had different effects on anthocyanin accumulation and growth restriction, indicating that these phenomena might be differentially regulated. These results may provide an effective anthocyanin accumulation method without reducing plant biomass.

12-Hydroxy-Jasmonoyl-l-Isoleucine Is an Active Jasmonate That Signals through CORONATINE INSENSITIVE 1 and Contributes to the Wound Response in Arabidopsis.

12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile) is a metabolite in the catabolic pathway of the plant hormone jasmonate, and is synthesized by the cytochrome P450 subclade 94 enzymes. Contrary to the well-established function of jasmonoyl-isoleucine (JA-Ile) as the endogenous bioactive form of jasmonate, the function of 12OH-JA-Ile is unclear. Here, the potential role of 12OH-JA-Ile in jasmonate signaling and wound response was investigated. Exogenous application of 12OH-JA-Ile mimicked several JA-Ile effects including marker gene expression, anthocyanin accumulation and trichome induction in Arabidopsis thaliana. Genome-wide transcriptomics and untargeted metabolite analyses showed large overlaps between those affected by 12OH-JA-Ile and JA-Ile. 12OH-JA-Ile signaling was blocked by mutation in CORONATINE INSENSITIVE 1. Increased anthocyanin accumulation by 12OH-JA-Ile was additionally observed in tomato and sorghum, and was disrupted by the COI1 defect in tomato jai1 mutant. In silico ligand docking predicted that 12OH-JA-Ile can maintain many of the key interactions with COI1-JAZ1 residues identified earlier by crystal structure studies using JA-Ile as ligand. Genetic alternation of jasmonate metabolic pathways in Arabidopsis to deplete both JA-Ile and 12OH-JA-Ile displayed enhanced jasmonate deficient wound phenotypes and was more susceptible to insect herbivory than that depleted in only JA-Ile. Conversely, mutants overaccumulating 12OH-JA-Ile showed intensified wound responses compared with wild type with similar JA-Ile content. These data are indicative of 12OH-JA-Ile functioning as an active jasmonate signal and contributing to wound and defense response in higher plants.

BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear.

SummaryThe red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.

Regulation of anthocyanin accumulation via MYB75/HAT1/TPL-mediated transcriptional repression

Anthocyanin is part of secondary metabolites, which is induced by environmental stimuli and developmental signals, such as high light and sucrose. Anthocyanin accumulation is activated by the MYB-bHLH-WD40 (MBW) protein complex in plants. But the evidence of how plants maintain anthocyanin in response to signals is lacking. Here we perform molecular and genetic evidence to display that HAT1 plays a new breaker of anthocyanin accumulation via post-translational regulations of MBW protein complex. Loss of function of HAT1 in the Arabidopsis seedlings exhibits increased anthocyanin accumulation, whereas overexpression of HAT1 significantly repressed anthocyanin accumulation. We found that HAT1 interacted with MYB75 and thereby interfered with MBW protein complex. Overexpression of HAT1 suppresses abundant anthocyanin phenotype of pap1-D plant. HAT1 is characterized as a transcriptional repressor possessing an N-terminal EAR motif, which determines to interact with TOPLESS corepressor. Repression activity of HAT1 in regulation of gene expression and anthocyanin accumulation can be abolished by deletion or mutation of the EAR motif 1. Chromatin immunoprecipitation assays revealed that MYB75 formed a transcriptional repressor complex with HAT1-TPL by histone H3 deacetylation in target genes. We proposed that HAT1 restrained anthocyanin accumulation by inhibiting the activities of MBW protein complex through blocking the formation of MBW protein complex and recruiting the TPL corepressor to epigenetically modulate the anthocyanin late biosynthetic genes (LBGs).

Over-expression of the red plant gene R1 enhances anthocyanin production and resistance to bollworm and spider mite in cotton.

Anthocyanins are a class of pigments ubiquitously distributed in plants and play roles in adoption to several stresses. The red plant gene (R1) promotes light-induced anthocyanin accumulation and red/purple pigmentation in cotton. Using 11 markers developed via genome resequencing, the R1 gene was located in an interval of approximately 136kb containing three annotated genes. Among them, a PAP1 homolog, GhPAP1D (Gohir.D07G082100) displayed differential transcript level in the red- and green-plant leaves. GhPAP1D encoded a R2R3-MYB transcription factor and its over-expression resulted in increased anthocyanin accumulation in transgenic tobaccos and cottons. Dual luciferase assay indicated that GhPAP1D activated the promoters of several cotton anthocyanin structural genes in tobacco leaves. Importantly, we found that the GhPAP1D-overexpressing cotton leaves had increased resistance to both bollworm and spite mite. Our data demonstrated that GhPAP1D was the controlling gene of the red plant phenotype in cotton, and as the major anthocyanin regulator, this gene was potential to create transgenic cottons with resistance to a broad spectrum of herbivores.

Accretion of Anthocyanin in Rice Cultivars under Salinity Stress

High soil salinity is a major environmental stress in the world’s cultivated land that limits plant growth, metabolism and productivity of crop plants. Anthocyanins are water soluble pigments found in all plant tissues throughout the plant kingdom, derived from flavonoids via the shikimic acid pathway and are known to be inducible under stress. Anthocyanin can act as antioxidant by donating hydrogen to highly reactive radicals, thereby preventing further radical formation. The objective of this study was to determine effect of salinity on total anthocyanin content of five native rice cultivars collected from a traditional saline rice tract and two collected from a traditional non-saline rice tract of Kerala State of India. All the cultivars showed proportionate and significant increase in anthocyanin accumulation in relation to increase in salt stress. The variation was cultivar specific showing their differential response to stress.

Postharvest light irradiation and appropriate temperature treatment increase anthocyanin accumulation in grape berry skin

Grape skin color is important because consumers generally prefer well pigmented grapes, and the high marketability of these fruit is important for farmers. Poor coloration, caused mainly by high temperatures during maturation, is a common problem. We investigated the effect of 7- to 9-day light irradiation and temperature treatment at 10, 15, 20, and 25 °C on anthocyanin accumulation in harvested berries of grape accessions. Anthocyanin accumulation of berries, mainly in red- to purple-skinned accessions, could be increased by combining postharvest light irradiation (white light + UV light, or blue LED light) with 15–25 °C treatments. Our data suggest that the optimal temperature (15–20 °C) improves anthocyanin accumulation without a decline in titratable acidity and berry weight. The coordinated induction of anthocyanin biosynthesis–related genes under these conditions might explain the accumulation of anthocyanins. These findings will help us to develop techniques for stable production of well colored grapes.

A Rice B-Box Protein, OsBBX14, Finely Regulates Anthocyanin Biosynthesis in Rice.

Anthocyanins are responsible pigments for giving attractive colors of plant organs and nutraceutical benefits of grains. Anthocyanin biosynthesis is known to be regulated by transcription factors and other regulatory proteins. In rice (Oryza sativa), the R2R3 MYB transcription factor (TF) OsC1 and a bHLH TF, OsB2, were previously reported to control anthocyanin biosynthesis in vegetative tissues and seeds, respectively; however, the regulatory mechanisms of the anthocyanin biosynthesis by TFs remain largely unknown. In this study, we identified OsBBX14, a homolog of Arabidopsis thaliana B-box domain protein 22 (AtBBX22), and investigated its function. The transcript level of OsBBX14 was high in pigmented rice seeds and gradually increased as the seeds matured. The ectopic expression of OsBBX14 in Arabidopsis resulted in a dramatic increase in anthocyanin accumulation in its seedlings. Using a steroid receptor-based inducible activation system, OsBBX14 and OsHY5 were found to directly activate OsC1 or OsB2 in an independent or collaborative manner. Yeast two hybrid revealed that the second B-box domain of OsBBX14 physically interacts with the bZIP domain of OsHY5. These results suggest that the anthocyanin biosynthesis in rice is induced and finely tuned by OsBBX14 in collaboration with OsHY5.

The blue light signal transduction pathway is involved in anthocyanin accumulation in 'Red Zaosu' pear.

A conserved blue light sensing and transduction pathway contributes to blue light-induced anthocyanin accumulation in the peel of red pear. Peel color is an economically important characteristic that influences the appearance quality of red pear, whose red color is due to anthocyanin accumulation. The process of coloration in the fruit peel is strongly influenced by light. However, how light quality influences color development remains unclear. In this study, we analyzed the effects of different light qualities on color development in the red pear 'Red Zaosu', a mutant of the hybrid cultivar 'Zaosu' of Pyrus pyrifolia and P. communis. The results showed that blue light increased anthocyanin accumulation after 72h of light treatment, while red light had almost no effect. The expression of anthocyanin biosynthesis-related genes showed a similar trend to the anthocyanin accumulation. To clarify the mechanism of blue-light induced coloration, PpCRYs, PpCOP1 and PpHY5 genes were cloned. Gene expression analysis showed that their transcript abundance did not correlate with the expression of anthocyanin-related genes or anthocyanin content, but the yeast two-hybrid system revealed conserved physical interactions among these proteins. In addition, PpHY5 directly bound to the promoters of the anthocyanin biosynthesis genes PpCHS, PpDFR, PpANS and PpMYB10, and activated the transcription of PpCHS in a Nicotiana benthamiana-based dual-luciferase assay. In summary, our results preliminarily revealed that the conserved blue light signal transduction module CRY-COP1-HY5 contributed to the anthocyanin biosynthesis induced by blue light in red pear. However, our results did not provide evidence for why red light had no effect on anthocyanin accumulation, which needs further study.

Medium composition potentially regulates the anthocyanin production from suspension culture of Daucus carota.

In the present study, an effort has been made to optimize various culture conditions for enhanced production of anthocyanin. Nutrient content of MS medium (ammonium to potassium nitrate ratio and phosphate concentration) had a profound influence on the cell biomass and anthocyanin accumulation in cell suspension cultures of Daucus carota. Suspension cultures were carried out in shake flasks for 18days and examined for cell growth, anthocyanin synthesis, anthocyanin yield and development of pigmented cells in relation to the uptake of total sugar, extracellular phosphate, nitrate and ammonia. The addition of NH4NO3 to KNO3 ratio (20.0mM: 37.6mM) in the suspension culture media resulted in a 2.85-fold increase in anthocyanin content at day 3. Similarly, a lower concentration of KH2PO4 (0.45mM) in the MS medium resulted in 1.63-fold increase in anthocyanin content at day 9. The total sugar uptake was closely associated with a significant increase in anthocyanin accumulation. Total sugar and nitrate were consumed until 9-12days, while ammonia and phosphate were completely consumed within 3days after inoculation. After 9days, cell lysis was observed and resulted in the leakage of intracellular substances. These observations suggest that anthocyanin was synthesized only by viable pigmented cells and degraded rapidly after cell death and lysis. This study signifies the utility of D. carota suspension culture for further up-scaling studies of anthocyanin.

Research Article Expression analysis of anthocyanin gene induced under phosphorus starvation in maize genotypes with contrasting phosphorus use efficiency

Phosphate (Pi) unavailability is a growth-limiting factor for plants. Under Pi-limited conditions, plants activate molecular mechanisms for better acquisition and utilization of this nutrient. In maize, changes in the expression pattern of several Pi starvation-induced genes, including the A1 coding for dihydroflavonol 4-reductase (DFR) involved in anthocyanin biosynthesis, were identified through microarray analysis. In order to elucidate the molecular determinants with a potential role in P use efficiency, we carried out a study on gene expression analysis of the A1 phosphate responsive gene by northern blot analysis of total RNA from maize genotypes contrasting for Pi efficiency. Two Pi-efficient (L-03 and L-161-1) and five inefficient (L-11, L-16, L-22, L-53, and L-5046) genotypes of maize were grown for 15 days in hydroponic culture in the presence (250 ,uM Pi) or absence (0 ,uM Pi) of phosphate. All genotypes showed an increase in anthocyanin accumulation in roots in the absence of Pi (0 ,uM Pi). The Pi-efficient genotype L-36 and the Pi-inefficient genotypes L-16, L22, and L-5046 showed the highest levels of anthocyanin accumulation. The A1 gene exhibits temporal and spatial expression patterns associated with Pi deficiency. Although there were differences in the expression profile of Pi starvation induced genes, no consistent expression patterns could be associated with either Pi-efficient or Pi-inefficient genotypes. It appears that Pi efficiency in tropical maize is a complex trait mediated by a coordinated action of genes that are either induced or suppressed in response to Pi-deficiency.

Drought Stress Enhances Up-Regulation of Anthocyanin Biosynthesis in Grapevine leafroll-associated virus 3-Infected in vitro Grapevine (Vitis vinifera) Leaves.

Reddish-purple coloration on the leaf blades and downward rolling of leaf margins are typical symptoms of grapevine leafroll disease (GLD) in red-fruited grapevine cultivars. These typical symptoms are attributed to the expression of genes encoding enzymes for anthocyanins synthesis, and the accumulation of flavonoids in diseased leaves. Drought has been proven to accelerate development of GLD symptoms in virus-infected leaves of grapevine. However, it is not known how drought affects GLD expression nor how anthocyanin biosynthesis in virus-infected leaves is altered. The present study used HPLC to determine the types and levels of anthocyanins, and applied reverse transcription quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of genes encoding enzymes for anthocyanin synthesis. Plantlets of Grapevine leafroll-associated virus 3 (GLRaV-3)-infected Vitis vinifera 'Cabernet Sauvignon' were grown in vitro under PEG-induced drought stress. HPLC found no anthocyanin-related peaks in the healthy plantlets with or without PEG-induced stress, while 11 peaks were detected in the infected plantlets with or without PEG-induced drought stress, but the peaks were significantly higher in infected drought-stressed plantlets. Increased accumulation of total anthocyanin compounds was related to the development of GLD symptoms in the infected plantlets under PEG stress. The highest level of up-regulated gene expression was found in GLRaV-3-infected leaves with PEG-induced drought stress. Analyses of variance and correlation of anthocyanin accumulation with related gene expression levels found that GLRaV-3-infection was the key factor in increased anthocyanin accumulation. This accumulation involved the up-regulation of two key genes, MYBA1 and UFGT, and their expression levels were further enhanced by drought stress.

The Arabidopsis ANGUSTIFOLIA3-YODA Gene Cascade Induces Anthocyanin Accumulation by Regulating Sucrose Levels.

Anthocyanin accumulation specifically depends on sucrose (Suc) signaling/levels. However, the gene cascades specifically involved in the Suc signaling/level-mediated anthocyanin biosynthetic pathway are still unknown. Arabidopsis ANGUSTIFOLIA3 (AN3), a transcription coactivator, is involved in the regulation of leaf shape and drought tolerance. Recently, an AN3-CONSTITUTIVE PHOTOMORPHOGENIC 1 gene cascade has been reported to regulate the light signaling-mediated anthocyanin accumulation. Target gene analysis indicates that AN3 is associated with the YODA (YDA) promoter, a mitogen-activated protein kinase kinase kinase, in vivo for inducing anthocyanin accumulation. Indeed, loss-of-function mutants of YDA showed significantly increased anthocyanin accumulation. YDA mutation can also suppress the decrease in an3-4 anthocyanin accumulation. Further analysis indicates that the mutations of AN3 and YDA disrupt the normal Suc levels because of the changes of invertase activity in mutants of an3 or yda, which in turn induces the alterations of anthocyanin accumulation in mutants of an3 or yda via unknown regulatory mechanisms.