Metabolomic and transcriptomic analyses reveal the effects of grafting on anthocyanin synthesis in grapevine

The grafting has been commonly used in viticulture, which joints the scion from a cultivar with the stem of a rootstock. Grafting has crucial impacts on various phenotypes of the cultivar including berry metabolome and berry coloring, however, the genetics and regulation mechanisms are largely unexplored. In this study, we analyzed the phenotypic, metabolomic and transcriptomic profiles at three stages (45, 75 and105 days after flowering) of the Crimson Seedless (Vitis vinifera, CS) cultivar grafted to four rootstocks (three heterografting: CS/101-14MG, CS/SO4, CS/110R and one self-grafting CS/CS) with an own-rooted grafting-free Crimson Seedless (CS) as a control. All the heterografting plants had a significant influence on berry reddening as early as ~45 days after flowering. The grafting of rootstocks promoted anthocyanin synthesis and accumulation in grape berries. The metabolomic features showed that Cyanidin 3-O-glucoside, Delphinidin 3-O-glucosid, Malvidin 3-O-glucoside, Peonidin 3-O-glucoside and Petunidin 3-O-glucoside were the pigments responsible for the purplish-red color peels. Transcriptomic analyses revealed that the anthocyanins biosynthetic related genes from the upstream (phenylalanine ammonia-lyase) to the downstream (anthocyanidin 3-O-glucosyltransferase and anthocyanidin synthase) were upregulated with the accumulations of anthocyanins in CS/101-14MG, CS/SO4 and CS/110R. At the same time, all these genes were also highly expressed and more anthocyanin was accumulated in CS/CS samples compared to CS samples, suggesting that self-grafting rootstocks might also have promoted berry reddening in grapevine. Our results provide global transcriptomic and metabolomic features in berry coloring regulation under different grafting conditions for improving the berry quality in grapevine production.

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.

Functional validation of Winged bean (Psophocarpus tetragonolobus (L.) DC.) anthocyanidin synthase (Wb ANS) gene through virus-induced gene silencing

Background Histochemical and microscopic observations of various tissues of the underutilized legume winged bean (Psophocarpus tetragonolobus (L.) DC.) indicated that the plant and its various parts are highly infested with condensed tannin (CT). Characterization of CT was carried out through the quantification of its structural-monomeric units catechin and epicatechin. The responsible candidate gene for anthocyanidin synthase (ANS) biosynthesis was identified, phylogenetically mapped and manipulated for lowering the CT-content. Results Virus-induced gene silencing (VIGS) was employed for silencing of WbANS transcript. WbANS-VIGS induction resulted in four-fold decrease in condensed tannin biosynthesis in P. tetragonolobus. Conclusion As condensed tannin adversely affects digestion and considered as an anti-nutrient, so this study might be helpful in future for altering the biosynthesis of condensed tannin by manipulating the ANS-encoding molecular factors.

Functional characterisation of banana (Musa spp.) 2-oxoglutarate-dependent dioxygenases involved in flavonoid biosynthesis

Bananas (Musa) are 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.

Understanding Alstroemeria pallida Flower Colour: Links between Phenotype, Anthocyanins and Gene Expression

Flower colour is mainly due to the accumulation of flavonoids, carotenoids and betalains in the petals. Of these pigments, flavonoids are responsible for a wide variety of colours ranging from pale yellow (flavones, flavonols and flavanodiols) to blue-violet (anthocyanins). This character plays a crucial ecological role by attracting and guiding pollinators. Moreover, in the ornamental plants market, colour has been consistently identified as the main feature chosen by consumers when buying flowers. Considering the importance of this character, the aim of this study was to evaluate flower colour in the native Chilean geophyte Alstroemeria pallida, by using three different approaches. Firstly, the phenotype was assessed using both a colour chart and a colourimeter, obtaining CIELab parameters. Secondly, the anthocyanin content of the pigmented tepals was evaluated by high-performance liquid chromatography (HPLC), and finally, the expression of two key flavonoid genes, chalcone synthase (CHS) and anthocyanidin synthase (ANS) was analysed using real-time polymerase chain reaction (PCR). Visual evaluation of A. pallida flower colour identified 5 accessions, ranging from white (Royal Horticultural Society (RHS) N999D) to pink (RHS 68C). Moreover, this visual evaluation of the accessions correlated highly with the CIELab parameters obtained by colourimetry. An anthocyanidin corresponding to a putative 6-hydroxycyanidin was identified, which was least abundant in the white accession (RHS N999D). Although CHS was not expressed differentially between the accessions, the expression of ANS was significantly higher in the accession with pink flowers (RHS 68C). These results suggest a correlation between phenotype, anthocyanin content and ANS expression for determining flower colour of A. pallida, which could be of interest for further studies, especially those related to the breeding of this species with ornamental value.

Identification of the Genes Involved in Anthocyanin Biosynthesis and Accumulation in Taxus chinensis

Taxus chinensis is a precious woody species with significant economic value. Anthocyanin as flavonoid derivatives plays a crucial role in plant biology and human health. However, the genes involved in anthocyanin biosynthesis have not been identified in T. chinensis. In this study, twenty-five genes involved in anthocyanin biosynthesis were identified, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, anthocyanidin synthase, flavonoid 3’-hydroxylase, flavonoid 3’,5’-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin reductase, and leucoanthocyanidin reductase. The conserved domains and phylogenetic relationships of these genes were characterized. The expression levels of these genes in different tissues and different ages of xylem were investigated. Additionally, the anthocyanin accumulation in xylem of different ages of T. chinensis was measured. The results showed the anthocyanin accumulation was correlated with the expression levels of dihydroflavonol 4-reductase, anthocyanidin synthase, flavonoid 3’-hydroxylase, and flavonoid 3’,5’-hydroxylase. Our results provide a basis for studying the regulation of the biosynthetic pathway for anthocyanins and wood color formation in T. chinensis.

Differential Regulation of Anthocyanins in Green and Purple Turnips Revealed by Combined De Novo Transcriptome and Metabolome Analysis

Purple turnip Brassica rapa ssp. rapa is highly appreciated by consumers but the metabolites and molecular mechanisms underlying the root skin pigmentation remain open to study. Herein, we analyzed the anthocyanin composition in purple turnip (PT) and green turnip (GT) at five developmental stages. A total of 21 anthocyanins were detected and classified into the six major anthocynanin aglycones. Distinctly, PT contains 20 times higher levels of anthocyanins than GT, which explain the difference in the root skin pigmentation. We further sequenced the transcriptomes and analyzed the differentially expressed genes between the two turnips. We found that PT essentially diverts dihydroflavonols to the biosynthesis of anthocyanins over flavonols biosynthesis by strongly down-regulating one flavonol synthase gene, while strikingly up-regulating dihydroflavonol 4-reductase (DFR), anthocyanidin synthase and UDP-glucose: flavonoid-3-O-glucosyltransferase genes as compared to GT. Moreover, a nonsense mutation identified in the coding sequence of the DFR gene may lead to a nonfunctional protein, adding another hurdle to the accumulation of anthocyanin in GT. We also uncovered several key members of MYB, bHLH and WRKY families as the putative main drivers of transcriptional changes between the two turnips. Overall, this study provides new tools for modifying anthocyanin content and improving turnip nutritional quality.

Overexpression of SmANS Enhances Anthocyanin Accumulation and Alters Phenolic Acids Content in Salvia miltiorrhiza and Salvia miltiorrhiza Bge f. alba Plantlets

Flavonoids play multiple roles in plant coloration and stress resistance and are closely associated with human health. Flavonoids and non-flavonoids (such as phenolic acids) are produced via the phenylpropanoid-derived pathway. Anthocyanidin synthase (ANS) catalyzes the synthesis of anthocyanins from leucoanthocyanidin in the flavonoids branched pathway. In this study, SmANS from Salvia miltiorrhiza was cloned and mainly localized in the endoplasmic reticulum (ER), plastids, Golgi, plasma membrane, and nucleus of tobacco epidermal cells, and was most highly expressed in purple petals in S. miltiorrhiza, whereas it showed almost no expression in white petals, green calyxes, and pistils in S. miltiorrhiza Bge f. alba. Overexpressed SmANS enhanced anthocyanin accumulation but reduced salvianolic acid B (SAB) and rosmarinic acid (RA) biosynthesis in S. miltiorrhiza and S. miltiorrhiza Bge f. alba plantlets, meanwhile, it restored the purple-red phenotype in S. miltiorrhiza Bge f. alba. These changes were due to reallocation of the metabolic flow, which was influenced by the SmANS gene. These findings indicate that SmANS not only plays a key role in anthocyanin accumulation in S. miltiorrhiza, but also acts as a “switch” for the coloration of S. miltiorrhiza Bge f. alba. This study provides baseline information for further research on flavonoids metabolism and improvement of anthocyanin or phenolic acid production by genetic engineering.