Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.     

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.     

Differential gene expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit skin coloration

‘Granny Smith’ apples growing under normal sunlight develop green skin, whereas the peel turns red due to anthocyanin accumulation after the removal of a bagging treatment. Two anthocyanins, Cyanidin 3-O-galactoside (cy3-gal) and Cyanidin 3-O-arabinoside (cy3-ara), were detected in the red ‘Granny Smith’ apple peels, and cy3-gal was determined to be chiefly responsible for the red color. The content of cy3-gal was more than 98% of the total anthocyanin in the red ‘Granny Smith’ peels. To better understand the molecular basis of anthocyanin biosynthesis in ‘Granny Smith’ apples, we performed a quantitative real-time PCR (qRT-PCR) analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB1). Our results indicate that the expression of these genes (except MdCHS) was associated with increased anthocyanin accumulation in the skin of ‘Granny Smith’ apples. Four selected genes obtained from the ‘Granny Smith’ skin cDNA library, phytoene synthase (PSY), WD40 repeat protein, polygalacturonase (PG), and galactosidase (GAL), were also confirmed by qRT-PCR. We found that these genes were differently expressed during ‘Granny Smith’ apple skin coloration, suggesting that they are directly or indirectly involved in pigment accumulation. In conclusion, anthocyanin biosynthesis in ‘Granny Smith’ apples is the result of interactions between multiple enzymes in the anthocyanin biosynthesis pathway, and the coloring mechanism of ‘Granny Smith’ apples may be similar to that of red-skinned cultivars.     

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.