Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3′4′5′-hydroxylated forms through the differential regulation of F3′H and F3′5′H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.     

Flower colour and cytochromes P450

Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3′-hydroxylase (F3′H, CYP75B) and flavonoid 3′,5′-hydroxylase (F3′5′H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue flower colour due to deficiency of F3′5′H. Successful expression of heterologous F3′5′H genes in roses and carnations results in delphinidin production, causing a novel blue/violet flower colour. Down-regulation of F3′H and F3′5′H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of flavones that contribute to the bluing of flower colour, and modulation of FNSII gene expression in petunia and tobacco changes their flower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of flower colours.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

Co-expression of<Emphasis Type="Italic">flavonoid 3′ 5′-hydroxylase</Emphasis> and<Emphasis Type="Italic">flavonoid 3′-hydroxylase</Emphasis> Accelerates Decolorization in Transgenic Chrysanthemum Petals

Theflavonoid 3′,5′-hydroxylase (F3′,5′H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation. Cotyledon expiants were co-cultured withA. tumefaciens LBA 4404 harboring the vector pMBP that carriesF3′,5′H under the control of the CaMV 35S promoter andnptll as a selectable marker gene. After 72 h of co-cultivation, the expiants were placed on an MS medium supplemented with 4 mg L^-1 BA, 0.1 mg L^-1 NAA, 400 mg L^-1 carbenicillin, and 100 mg L^-1; kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were then rooted and acclimatized in potting soil. Integration ofF3′,5′H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed thatF3′,5′H andflavonoid 3′hydroxylase (F3′H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color results from 1) competition between levels of transgenicF3′,5′H and endogenousF3′H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of chrysanthemumDFR (dihydroflavonol 4-reductase).     

Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas)

Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3′H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3’′H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3′H was highly homologous with F3′Hs from other plant species. Conserved domain search revealed that IbF3′H was a cytochrome P450 dependent enzyme. Three F3′H-specific motifs (V₇₅VVAAS₈₀, G₄₂₇GEK₄₃₀ and V₄₃₃DVKG₄₃₇) were conserved in IbF3′H. Phylogenetic analysis revealed that IbF3′H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3′H gene in the genome of I. batatas. IbF3′H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3′H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3′H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3′H could encode a functional protein and had a special catalytic activity of flavonoid 3′-hydroxylase.     

Functional characterization of key structural genes in rice flavonoid biosynthesis

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl ^w locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T⁶⁴ M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3′H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3′H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering of red cells of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and comparative genome-wide gene expression analysis of red cells versus wild-type cells

We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC–MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1–GL3/TT8–PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.     

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3′5′ hydroxylase (F3′5′H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3′ hydroxylation (F3′H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3′5′H to F3′H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3′5′H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.