Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

MAIN CONCLUSION: A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Identification and characterization of the gene BraANS.A03 associated with purple leaf color in pak choi (Brassica rapa L. ssp. chinensis).

MAIN CONCLUSION: BraANS.A3 was the key gene controlling purple leaf color in pak choi, and two short fragments of promoter region in green pak choi might be interfering its normal expression. Pak choi (B. rapa L. ssp. chinensis) is an influential and important vegetable with green, yellow, or purple leaves that is cultivated worldwide. The purple leaves are rich in anthocyanins, but the underlying genetics and evolution have yet to be extensively studied. Free-hand sections of the purple leaves indicated that anthocyanins mainly accumulate throughout the adaxial and abaxial epidermal leaf cells. Segregation analyses of an F2 population of a B. rapa ssp. chinensis L. purple leaf mutant ZBC indicated that the purple trait is controlled by an incompletely dominant nuclear gene. Bulked segregant analysis (BSA) showed that the key genes controlling the trait were between 24.25 and 38.10 Mb on chromosome A03 of B. rapa. From the annotated genes, only BraA03g050560.3C, homologous to Arabidopsis AtANS, was related to the anthocyanin synthesis pathway. Genome annotation results and transcriptional sequencing analyses revealed that the BraANS.A3 gene was involved in the purple leaf trait. qRT-PCR analyses showed that BraANS.A3 was highly upregulated in ZBC but hardly expressed in the leaves of an inbred homozygous line of B. campestris ssp. chinensis L. green leaf mutant WTC, indicating that BraANS.A3 played a key role catalyzing anthocyanin synthesis in ZBC. Full-length sequence alignment of BraANS.A3 in WTC and ZBC showed that it was highly conserved in the gene region, with significant variation in the promoter region. In particular, the insertion of two short fragments of the promoter region in WTC may interfere with its normal expression. The promoter regions of ANS in six Brassica species all had multiple cis-elements involved in responses to abscisic acid, light, and stress, suggesting that ANS may be involved in multiple metabolic pathways or biological processes. Protein-protein interactions predicted that BraANS.A3 interacts with virtually all catalytic proteins in the anthocyanin synthesis pathway and has a strong relationship with Transparent Testa 8 (TT8). These results suggest that BraANS.A3 promotes anthocyanin accumulation in purple pak choi and provide new insights into the functional analysis of anthocyanin-related genes in Chinese cabbage and transcriptional regulatory networks.

Two genes, ANS and UFGT2, from Vaccinium spp. are key steps for modulating anthocyanin production.

Anthocyanins are a major group of red to blue spectrum plant pigments with many consumer health benefits. Anthocyanins are derived from the flavonoid pathway and diversified by glycosylation and methylation, involving the concerted action of specific enzymes. Blueberry and bilberry (Vaccinium spp.) are regarded as 'superfruits' owing to their high content of flavonoids, especially anthocyanins. While ripening-related anthocyanin production in bilberry (V. myrtillus) and blueberry (V. corymbosum) is regulated by the transcriptional activator MYBA1, the role of specific structural genes in determining the concentration and composition of anthocyanins has not been functionally elucidated. We isolated three candidate genes, CHALCONE SYNTHASE (VmCHS1), ANTHOCYANIDIN SYNTHASE (VmANS) and UDP-GLUCOSE : FLAVONOID-3-O-GLYCOSYLTRANSFERASE (VcUFGT2), from Vaccinium, which were predominantly expressed in pigmented fruit skin tissue and showed high homology between bilberry and blueberry. Agrobacterium-mediated transient expression of Nicotiana benthamiana showed that overexpression of VcMYBA1 in combination with VmANS significantly increased anthocyanin concentration (3-fold). Overexpression of VmCHS1 showed no effect above that induced by VcMYBA1, while VcUFGT2 modulated anthocyanin composition to produce delphinidin-3-galactosylrhamnoside, not naturally produced in tobacco. In strawberry (Fragaria × ananassa), combined transient overexpression of VcUFGT2 with a FLAVONOID 3´,5´-HYDROXYLASE from kiwifruit (Actinidia melanandra) modulated the anthocyanin profile to include galactosides and arabinosides of delphinidin and cyanidin, major anthocyanins in blueberry and bilberry. These findings provide insight into the role of the final steps of biosynthesis in modulating anthocyanin production in Vaccinium and may contribute to the targeted breeding of new cultivars with improved nutritional properties.

Reducing the biosynthesis of condensed tannin in winged bean (Psophocarpus tetragonolobus (L.) DC.) by virus-induced gene silencing of anthocyanidin synthase (ANS) gene.

The Underutilized legume-winged bean (Psophocarpus tetragonolobus (L.) DC.) and its various parts are infested with condensed tannin (CT) or proanthocyanidin (PA). CT has anti-nutritional effect as it adversely affects the digestion of proteins, minerals and vitamin among ruminants and humans. It is also responsible for low protein digestibility and decreased amino acid availability. One of the probable reasons of underutilization of P. tetragonolobus is due to its infestation with CT. Histochemical staining of various tissues of P. tetragonolobus with dimethylcinnmaldehyde (DMACA) developed a deep-blue colour indicating the presence of polyphenolic condensed tannin. Structural monomeric unit catechin and epi-catechin were reported to be responsible for biosynthesis of CT in P. tetragonolobus. The enzyme anthocyanidin synthase (ANS) and its corresponding transcripts were identified and phylogenetically mapped. The transcript was subjected to virus-induced gene silencing (VIGS) through agro-infiltration in P. tetragonolobus for reducing the CT-content. The WbANS-VIGS induced P. tetragonolobus resulted in four-fold decrease of CT as compared to the control P. tetragonolobus. A decrease of 73% of CT level was reported in VIGS silenced Wb-ANS line of P. tetragonolobus. This study resulted and confirmed that, the silencing of (ANS) gene in P. tetragonolobus has a regulatory effect on the condensed tannin biosynthesis. This study will pave way for further manipulation of ANS enzyme for reducing the biosynthesis of the anti-nutrient CT. Reducing the CT content will make this underutilized legume more acceptable. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03435-5.

Exogenous γ-aminobutyric acid strengthens phenylpropanoid and nitrogen metabolism to enhance the contents of flavonoids, amino acids, and the derivatives in edamame.

γ-aminobutyric acid (GABA) has been reported to improve stress resistance in plants. Nonetheless, little is known about the effects of GABA on the nutritional quality and regulatory mechanisms of edamame. Therefore, we analyzed the flavonoid and amino acid (AA) metabolism and the effects of GABA on the nutrient content of edamame seeds through physiological and metabolomic analyses. Exogenous GABA increased endogenous GABA metabolism and GABA transaminase activity and enhanced the oxoglutarate content, which entered into nitrogen metabolism and increased the activity and expression of nitrogen metabolism-related enzymes, to accumulate AAs and bioactive peptides. Meanwhile, exogenous GABA induced the metabolism of flavonoids, including total flavonoids, anthocyanins, 6''-o-malonyglycitin, glycitin, ononin, cyanin, and ginkgetin, by increasing the activity and expression of flavonoid biosynthetic enzymes. This is the first study to reveal that GABA effectively improves the nutritional quality of edamame through the accumulation of AAs, bioactive peptides, isoflavones, anthocyanins, sugars, and organic acids.

Oxidative Transformation of Dihydroflavonols and Flavan-3-ols by Anthocyanidin Synthase from Vitis vinifera.

Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate-cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C3-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed.

Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis.

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

The Regulation of Floral Colour Change in Pleroma raddianum (DC.) Gardner.

Floral colour change is a widespread phenomenon in angiosperms, but poorly understood from the genetic and chemical point of view. This article investigates this phenomenon in Pleroma raddianum, a Brazilian endemic species whose flowers change from white to purple. To this end, flavonoid compounds and their biosynthetic gene expression were profiled. By using accurate techniques (Ultra Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UPLC-HRMS)), thirty phenolic compounds were quantified. Five key genes of the flavonoid biosynthetic pathway were partially cloned, sequenced, and the mRNA levels were analysed (RT-qPCR) during flower development. Primary metabolism was also investigated by gas chromatography coupled to mass spectrometry (GC-EIMS), where carbohydrates and organic acids were identified. Collectively, the obtained results suggest that the flower colour change in P. raddianum is determined by petunidin and malvidin whose accumulation coincides with the transcriptional upregulation of early and late biosynthetic genes of the flavonoid pathway, mainly CHS and ANS, respectively. An alteration in sugars, organic acids and phenolic co-pigments is observed together with the colour change. Additionally, an increment in the content of Fe3+ ions in the petals, from the pink to purple stage, seemed to influence the saturation of the colour.

Engineering Lactococcus lactis for the production of unusual anthocyanins using tea as substrate.

Plant material rich in anthocyanins has been historically used in traditional medicines, but only recently have the specific pharmacological properties of these compounds been the target of extensive studies. In addition to their potential to modulate the development of various diseases, coloured anthocyanins are valuable natural alternatives commonly used to replace synthetic colourants in food industry. Exploitation of microbial hosts as cell factories is an attractive alternative to extraction of anthocyanins and other flavonoids from plant sources or chemical synthesis. In this study, we present the lactic acid bacterium Lactococcus lactis as an ideal host for the production of high-value plant-derived bioactive anthocyanins using green tea as substrate. Besides the anticipated red-purple compounds cyanidin and delphinidin, orange and yellow pyranoanthocyanidins with unexpected methylation patterns were produced from green tea by engineered L. lactis strains. The pyranoanthocyanins are currently attracting significant interest as one of the most important classes of anthocyanin derivatives and are mainly formed during the aging of wine, contributing to both colour and sensory experience.

Oxidative Transformation of Leucocyanidin by Anthocyanidin Synthase from Vitis vinifera Leads Only to Quercetin.

Anthocyanidin synthase from Vitis vinifera ( VvANS) catalyzes the in vitro transformation of the natural isomer of leucocyanidin, 2 R,3 S,4 S- cis-leucocyanidin, into 2 R,4 S-flavan-3,3,4-triol ([M + H]+, m/ z 323) and quercetin. The C3-hydroxylation product 2 R,4 S-flavan-3,3,4-triol is first produced and its C3,C4-dehydration product is in tautomeric equilibrium with (+)-dihydroquercetin. The latter undergoes a second VvANS-catalyzed C3-hydroxylation leading to a 4-keto-2 R-flavan-3,3-gem-diol which upon dehydration gives quercetin. The unnatural isomer of leucocyanidin, 2 R,3 S,4 R- trans-leucocyanidin, is similarly transformed into quercetin upon C3,C4-dehydration, but unlike 3,4- cis-leucocyanidin, it also undergoes some C2,C3-dehydration followed by an acid-catalyzed hydroxyl group extrusion at C4 to give traces of cyanidin. Overall, the C3,C4- trans isomer of leucocyanidin is transformed into 2 R,4 R-flavan-3,3,4-triol (M + 1, m/ z 323), (+)-DHQ, (-)-epiDHQ, quercetin, and traces of cyanidin. Our data bring the first direct observation of 3,4- cis-leucocyanidin- and 3,4- trans-leucocyanidin-derived 3,3-gem-diols, supporting the idea that the generic function of ANS is to catalyze the C3-hydroxylation of its substrates. No cyanidin is produced with the natural cis isomer of leucocyanidin, and only traces with the unnatural trans isomer, which suggests that anthocyanidin synthase requires other substrate(s) for the in vivo formation of anthocyanidins.

Function analysis of anthocyanidin synthase from Morus alba L. by expression in bacteria and tobacco

Background: Flavonoids are a kind of important secondary metabolite and are commonly considered to provide protection to plants against stress and UV-B for a long time. Anthocyanidin synthase (ANS), which encodes a dioxygenase in the flavonoid pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanidins, but there is no direct evidence indicating that it provides tolerance to stress in plants. Results: To investigate whether ANS can increase tolerance to abiotic stress, MaANS was isolated from mulberry fruits and transformed into tobacco. Our results suggested that the bacterially expressed MaANS protein can convert dihydroquercetin to quercetin. Overexpression of MaANS remarkably increased the accumulation of total flavonoids in transgenic lines and anthocyanins in corollas of flowers. Transgenic lines showed higher tolerance to NaCl and mannitol stress. Conclusions: These results indicated that MaANS participates in various dioxygenase activities, and it can protect plants against abiotic stress by improving the ROS-scavenging ability. Thus, this alternative approach in crop breeding can be considered in the improvement of stress tolerance by enriching flavonoid production in plants

Differential Accumulation of Anthocyanins in Dendrobium officinale Stems with Red and Green Peels.

Dendrobium officinale stems, including red and green stems, are widely used as a dietary supplement to develop nutraceutical beverages and food products. However, there is no detailed information on pigment composition of red and green stems. Here, we investigated the content and composition of pigments in red and green stems by Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and assessed the differential accumulation of anthocyanins at the molecular level. The color of peels in red stems was caused by the presence of anthocyanins in epidermal cells unlike the peels of green stems. The glucoside derivatives delphinidin and cyanidin are responsible for the red color. Within the D. officinale anthocyanidin biosynthetic pathway, DoANS and DoUFGT, coding for anthocyanidin synthase and UDP-glucose flavonoid-3-O-glucosyltransferase, respectively, are critical regulatory genes related to the differential accumulation of anthocyanidin. These findings provide a more complete profile of pigments, especially anthocyanin, in D. officinale stems, and lay a foundation for producing functional foods.

Fine Mapping Identifies SmFAS Encoding an Anthocyanidin Synthase as a Putative Candidate Gene for Flower Purple Color in Solanum melongena L.

Anthocyanins are the main pigments in flowers and fruits. These pigments are responsible for the red, red-purple, violet, and purple color in plants, and act as insect and animal attractants. In this study, phenotypic analysis of the purple flower color in eggplant indicated that the flower color is controlled by a single dominant gene, FAS. Using an F2 mapping population derived from a cross between purple-flowered 'Blacknite' and white-flowered 'Small Round', FlowerAnthocyanidin Synthase (FAS) was fine mapped to an approximately 165.6-kb region between InDel marker Indel8-11 and Cleaved Amplified Polymorphic Sequences (CAPS) marker Efc8-32 on Chromosome 8. On the basis of bioinformatic analysis, 29 genes were subsequently located in the FAS target region, among which were two potential Anthocyanidin Synthase (ANS) gene candidates. Allelic sequence comparison results showed that one ANS gene (Sme2.5_01638.1_g00003.1) was conserved in promoter and coding sequences without any nucleotide change between parents, whereas four single-nucleotide polymorphisms were detected in another ANS gene (Sme2.5_01638.1_g00005.1). Crucially, a single base pair deletion at site 438 resulted in premature termination of FAS, leading to the loss of anthocyanin accumulation. In addition, FAS displayed strong expression in purple flowers compared with white flowers and other tissues. Collectively, our results indicate that Sme2.5_01638.1_g00005.1 is a good candidate gene for FAS, which controls anthocyanidin synthase in eggplant flowers. The present study provides information for further potential facilitate genetic engineering for improvement of anthocyanin levels in plants.

Overexpression of the Anthocyanidin Synthase Gene in Strawberry Enhances Antioxidant Capacity and Cytotoxic Effects on Human Hepatic Cancer Cells.

Food fortification through the increase and/or modulation of bioactive compounds has become a major goal for preventing several diseases, including cancer. Here, strawberry lines of cv. Calypso transformed with a construct containing an anthocyanidin synthase (ANS) gene were produced to study the effects on anthocyanin biosynthesis, metabolism, and transcriptome. Three strawberry ANS transgenic lines (ANS L5, ANS L15, and ANS L18) were analyzed for phytochemical composition and total antioxidant capacity (TAC), and their fruit extracts were assessed for cytotoxic effects on hepatocellular carcinoma. ANS L18 fruits had the highest levels of total phenolics and flavonoids, while those of ANS L15 had the highest anthocyanin concentration; TAC positively correlated with total polyphenol content. Fruit transcriptome was also specifically affected in the polyphenol biosynthesis and in other related metabolic pathways. Fruit extracts of all lines exerted cytotoxic effects in a dose/time-dependent manner, increasing cellular apoptosis and free radical levels and impairing mitochondrial functionality.

Geometric morphometrics reveals shifts in flower shape symmetry and size following gene knockdown of CYCLOIDEA and ANTHOCYANIDIN SYNTHASE.

BACKGROUND: While floral symmetry has traditionally been assessed qualitatively, recent advances in geometric morphometrics have opened up new avenues to specifically quantify flower shape and size using robust multivariate statistical methods. In this study, we examine, for the first time, the ability of geometric morphometrics to detect morphological differences in floral dorsoventral asymmetry following virus-induced gene silencing (VIGS). Using Fedia graciliflora Fisch. & Meyer (Valerianaceae) as a model, corolla shape of untreated flowers was compared using canonical variate analysis to knockdown phenotypes of CYCLOIDEA2A (FgCYC2A), ANTHOCYANIDIN SYNTHASE (FgANS), and empty vector controls. RESULTS: Untreated flowers and all VIGS treatments were morphologically distinct from each other, suggesting that VIGS may cause subtle shifts in floral shape. Knockdowns of FgCYC2A were the most dramatic, affecting the position of dorsal petals in relation to lateral petals, thereby resulting in more actinomorphic-like flowers. Additionally, FgANS knockdowns developed larger flowers with wider corolla tube openings. CONCLUSIONS: These results provide a method to quantify the role that specific genes play in the developmental pathway affecting the dorsoventral axis of symmetry in zygomorphic flowers. Additionally, they suggest that ANS may have an unintended effect on floral size and shape.

Expression of Key Structural Genes of the Phenylpropanoid Pathway Associated with Catechin Epimerization in Tea Cultivars.

Catechin epimerization is an important factor affecting tea catechin compositions and thereby tea quality. However, a lack of tea germplasms with high non-epicatechins limits relative research. Here, a tea cultivar Y510 with high non-epicatechins was firstly reported and used for catechin and RNA sequencing (RNA-Seq) analysis. Results showed that the (-)-gallocatechin gallate and (+)-catechin (C) contents in Y510 were at least 136 and 6 times higher than those in Fudingdabaicha and 0306I, but the epicatechins (-)-epigallocatechin and (-)-epicatechin (EC) were significantly lower. Eleven unigenes potentially involved in catechin epimerization were identified by RNA-Seq analysis. Based on a combination of catechin and gene expression analysis, it was hypothesized that two anthocyanidin reductase genes (CsANR1, CsANR2) and an anthocyanidin synthase gene (CsANS) are the key genes affecting catechin epimerization in tea. Non-epicatechin formations were hypothesized to be mainly influenced by the expression ratio of CsANR2 to CsANR1 and the expression of CsANS. Overexpression of CsANS in an Arabidopsis mutant tds4-2 led to a significant increase of EC accumulation in seeds, revealing CsANS is important for catechin epimerization. These results shed new light on breeding tea cultivars with special catechin compositions.

Production of anthocyanins in metabolically engineered microorganisms: Current status and perspectives.

Microbial production of plant-derived natural products by engineered microorganisms has achieved great success thanks to large extend to metabolic engineering and synthetic biology. Anthocyanins, the water-soluble colored pigments found in terrestrial plants that are responsible for the red, blue and purple coloration of many flowers and fruits, are extensively used in food and cosmetics industry; however, their current supply heavily relies on complex extraction from plant-based materials. A promising alternative is their sustainable production in metabolically engineered microbes. Here, we review the recent progress on anthocyanin biosynthesis in engineered bacteria, with a special focus on the systematic engineering modifications such as selection and engineering of biosynthetic enzymes, engineering of transportation, regulation of UDP-glucose supply, as well as process optimization. These promising engineering strategies will facilitate successful microbial production of anthocyanins in industry in the near future.

Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages.

BACKGROUND: Catechins are the main polyphenol compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, the levels of catechins and relative expressions of key genes in tea leaves of different developmental stages were analyzed. RESULTS: The amounts of catechins differed significantly in leaves of different stages, except for gallocatechin gallate. Close correlations between the expression of synthesis genes and the accumulation of catechins were identified. Correlation analysis showed that the expressions of chalcone synthase 1, chalcone synthase 3, anthocyanidin reductase 1, anthocyanidin reductase 2 and leucoanthocyanidin reductase genes were significantly and positively correlated with total catechin contents, suggesting their expression may largely affect total catechin accumulation. Anthocyanidin synthase was significantly correlated with catechin. While both ANRs and LAR were significantly and positively correlated with the contents of (-)-epigallocatechin gallate and (-)-epicatechin gallate. CONCLUSION: Our results suggest synergistic changes between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanidin synthase may regulate earlier steps in the conversion of catechin, while the anthocyanidin reductase and leucoanthocyanidin reductase genes may both play important roles in the biosynthesis of galloylated catechins.

Nonsense Mutation Inside Anthocyanidin Synthase Gene Controls Pigmentation in Yellow Raspberry (Rubus idaeus L.).

Yellow raspberry fruits have reduced anthocyanin contents and offer unique possibility to study the genetics of pigment biosynthesis in this important soft fruit. Anthocyanidin synthase (Ans) catalyzes the conversion of leucoanthocyanidin to anthocyanidin, a key committed step in biosynthesis of anthocyanins. Molecular analysis of the Ans gene enabled to identify an inactive ans allele in a yellow fruit raspberry ("Anne"). A 5 bp insertion in the coding region was identified and designated as ans+5. The insertion creates a premature stop codon resulting in a truncated protein of 264 amino acids, compared to 414 amino acids wild-type ANS protein. This mutation leads to loss of function of the encoded protein that might also result in transcriptional downregulation of Ans gene as a secondary effect, i.e., nonsense-mediated mRNA decay. Further, this mutation results in loss of visible and detectable anthocyanin pigments. Functional characterization of raspberry Ans/ans alleles via complementation experiments in the Arabidopsis thaliana ldox mutant supports the inactivity of encoded protein through ans+5 and explains the proposed block in the anthocyanin biosynthetic pathway in raspberry. Taken together, our data shows that the mutation inside Ans gene in raspberry is responsible for yellow fruit phenotypes.

A cis-element responsible for cGMP in the promoter of the soybean chalcone synthase gene.

The cyclic nucleotides cGMP and cAMP have been reported to play key roles in the regulation of plant processes and responses. We have previously reported that several genes encoding flavonoid biosynthetic enzymes, including chalcone synthase (CHS) in soybean (Glycine max L.), were induced by cGMP but not cAMP. The soybean genome contains nine CHS gene copies (GmCHS1-9). We investigated the responsiveness of several GmCHS genes to cGMP, cAMP, NO, and white light. Quantitative RT-PCR analysis showed that the transcript levels of GmCHS7 and GmCHS8 were increased by 3.6- and 3.8-fold, respectively, with cGMP whereas the transcript levels of GmCHS2 remained constant. Although cAMP had no effect on the transcript levels of the three genes, NO had an activation effect on all three. White light activated the three genes in a transient manner, with GmCHS2, GmCHS7, and GmCHS8 transcript levels increasing 3-fold after 3 h and decreasing to basal levels after 9 h. The GmCHS8 promoter contains several important cis-elements, including the G-box and H-box forming the Unit-I-like sequence and the MYB binding sequence, a target of the GmMYB176 transcription factor regulating the expression of GmCHS8. A transient gene expression assay revealed the activation of the Unit-I-like sequence, but not of the MYB binding sequence, by cGMP. The combination of G-box and H-box was necessary for cGMP responsiveness. Taken together, these results suggest that the Unit-I-like sequence in the promoters of GmCHS7 and GmCHS8 is a cGMP responsive cis-element in these genes and that NO exerts its effect via cis-elements other than the Unit-I-like sequence.