Search

The flavonoid pathway is characteristic of land plants and a central biosynthetic component enabling life in a terrestrial environment. Flavonoids provide tolerance to both abiotic and biotic stresses and facilitate beneficial relationships, such as signalling to symbiont microorganisms, or attracting pollinators and seed dispersal agents. The biosynthetic pathway shows great diversity across species, resulting principally from repeated biosynthetic gene duplication and neofunctionalization events during evolution. Such events may reflect a selection for new flavonoid structures with novel functions that enable occupancy of varied ecological niches. However, the biochemical and genetic diversity of the pathway also likely resulted from evolution along parallel trends across land plant lineages, producing variant compounds with similar biological functions. Analyses of the wide range of whole-plant genome sequences now available, particularly for archegoniate plants, have enabled proposals on which genes were ancestral to land plants and which arose within the land plant lineages. In this review, we discuss the emerging proposals for how the flavonoid pathway may have evolved and diversified. This article is part of the theme issue 'The evolution of plant metabolism'. [ABSTRACT FROM AUTHOR]

SUMMARY: Plant adaptation from aquatic to terrestrial environments required modifications to cell wall structure and function to provide tolerance to new abiotic and biotic stressors. Here, we investigate the nature and function of red auronidin pigment accumulation in the cell wall of the liverwort Marchantia polymorpha. Transgenic plants with auronidin production either constitutive or absent were analysed for their cell wall properties, including fractionation of polysaccharide and phenolic components. While small amounts of auronidin and other flavonoids were loosely associated with the cell wall, the majority of the pigments were tightly associated, similar to what is observed in angiosperms for polyphenolics such as lignin. No evidence of covalent binding to a polysaccharide component was found: we propose auronidin is present in the wall as a physically entrapped large molecular weight polymer. The results suggested auronidin is a dual function molecule that can both screen excess light and increase wall strength, hydrophobicity and resistance to enzymatic degradation by pathogens. Thus, liverworts have expanded the core phenylpropanoid toolkit that was present in the ancestor of all land plants, to deliver a lineage‐specific solution to some of the environmental stresses faced from a terrestrial lifestyle. [ABSTRACT FROM AUTHOR]

Flavonoids are plant-specific secondary metabolites that arose early during land-plant colonisation, most likely evolving for protection from UV-B and other abiotic stresses. As plants increased in complexity, so too did the diversity of flavonoid compounds produced and their physiological roles. The most conspicuous are the pigments, including yellow aurones and chalcones, and the red/purple/blue anthocyanins, which provide colours to flowers, fruits and foliage. Anthocyanins have been particularly well studied, prompted by the ease of identifying mutants of genes involved in biosynthesis or regulation, providing an important model system to study fundamental aspects of genetics, gene regulation and biochemistry. This has included identifying the first plant transcription factor, and later resolving how multiple classes of transcription factor coordinate in regulating the production of various flavonoid classes – each with different activities and produced at differing developmental stages. In addition, dietary flavonoids from fruits/vegetables and forage confer human- and animal-health benefits, respectively. This has prompted strong interest in generating new plant varieties with increased flavonoid content through both traditional breeding and plant biotechnology. Gene-editing technologies provide new opportunities to study how flavonoids are regulated and produced and to improve the flavonoid content of flowers, fruits, vegetables and forages. [ABSTRACT FROM AUTHOR]

Paeonia delavayi is listed as an endangered species in the southwest of China. The flower colours of cultivated examples of this species are most commonly yellow and red, but a much wider range of flower colours can be found in plants growing in the natural habitat. To elucidate the basis of the colour range, pigments were extracted from flower petals of seven selected colours and profiled and quantified using High Performance Liquid Chromatography (HPLC) and Liquid Chromatography–Mass Spectrometry (LC-MS). All the petal colours were found to share the same base pigment types as the yellow flower – a composite colour based on a mix of flavonoids, carotenoids and chlorophyll. Anthocyanin profiling revealed the same anthocyanin compositions in all red coloured flowers, namely, cyanidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, peonidin-3,5-diglucoside, peonidin-3-glucoside and peonidin 3-arabinoside. Quantitative analysis suggested the yellow colour was principally formed by the flavonoid naringenin chalcone 2'-glucoside (2',4',6',4-tetrahydroxychalcone 2'-glucoside). The various red/orange flower colours were the result of superimposing different amounts of anthocyanins onto the yellow flower background, with carotenoids and chlorophylls having only a minor contribution to the final colour. The results provide useful information for breeding programmes for ornamental varieties of P. delavayi. [ABSTRACT FROM AUTHOR]

The flavonoid pathway is one of the best characterized specialized metabolite pathways of plants. In angiosperms, the flavonoids have varied roles in assisting with tolerance to abiotic stress and are also key for signaling to pollinators and seed dispersal agents. The pathway is thought to be specific to land plants and to have arisen during the period of land colonization around 550–470 million years ago. In this review we consider current knowledge of the flavonoid pathway in the bryophytes, consisting of the liverworts, hornworts, and mosses. The pathway is less characterized for bryophytes than angiosperms, and the first genetic and molecular studies on bryophytes are finding both commonalities and significant differences in flavonoid biosynthesis and pathway regulation between angiosperms and bryophytes. This includes biosynthetic pathway branches specific to each plant group and the apparent complete absence of flavonoids from the hornworts. [ABSTRACT FROM AUTHOR]

Summary: Damaging UVB radiation is a major abiotic stress facing land plants. In angiosperms the UV RESISTANCE LOCUS8 (UVR8) photoreceptor coordinates UVB responses, including inducing biosynthesis of protective flavonoids. We characterised the UVB responses of Marchantia polymorpha (marchantia), the model species for the liverwort group of basal plants. Physiological, chemical and transcriptomic analyses were conducted on wild‐type marchantia exposed to three different UVB regimes. CRISPR/Cas9 was used to obtain plant lines with mutations for components of the UVB signal pathway or the flavonoid biosynthetic pathway, and transgenics overexpressing the marchantia UVR8 sequence were generated. The mutant and transgenic lines were analysed for changes in flavonoid content, their response to UVB exposure, and transcript abundance of a set of 48 genes that included components of the UVB response pathway characterised for angiosperms. The marchantia UVB response included many components in common with Arabidopsis, including production of UVB‐absorbing flavonoids, the central activator role of ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV‐B PHOTOMORPHOGENESIS1 (RUP1). Notable differences included the greater importance of CHALCONE ISOMERASE‐LIKE (CHIL). Mutants disrupted in the response pathway (hy5) or flavonoid production (chalcone isomerase, chil) were more easily damaged by UVB. Mutants (rup1) or transgenics (35S:MpMYB14) with increased flavonoid content had increased UVB tolerance. The results suggest that UVR8‐mediated flavonoid induction is a UVB tolerance character conserved across land plants and may have been an early adaptation to life on land. Significance statement: Inducible flavonoid biosynthesis regulated by the photoreceptor UVR8 is key for tolerance to UVB radiation in flowering plants, but whether this is the case for other plant groups is unclear. In this study, CRISPR/Cas9 mutagenesis is used in the model species Marchantia polymorpha to show that both the UVR8 signal transduction pathway and the importance of flavonoids for UVB tolerance are conserved in one of the most distantly related groups of plants to flowering plants, the liverworts. [ABSTRACT FROM AUTHOR]

Summary: The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light‐ and nutrient‐deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress‐induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization. [ABSTRACT FROM AUTHOR]

Key message: The Md - MYB10 gene from apple is capable of self-regulating in heterologous host species and enhancing anthocyanin pigmentation, but the activity of MYB10 is dependent on endogenous protein partners. Abstract: Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples ( Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md- MYB10, are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md- MYB10 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md- MYB10 transgene ( MYB10- R6 :MYB10:MYB10) activated anthocyanin synthesis when transiently expressed in Antirrhinum rosea petals and petunia leaf discs. Stable transgenic petunias containing Md- MYB10 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of 'Mitchell' petunia. The absence of foliar pigmentation was due to the failure of the Md- MYB10 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md- MYB10 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals. [ABSTRACT FROM AUTHOR]

Petunia line Mitchell [MP, Petunia axillaris × (P. axillaris × P. hybrida)] and Eustoma grandiflorum (lisianthus) plants were produced containing a transgene for over-expression of the R2R3-MYB transcription factor [TF; ROSEA1 (ROS1)] that up-regulates flavonoid biosynthesis in Antirrhinum majus. The petunia lines were also crossed with previously produced MP lines containing a Zea mays flavonoid-related basic helix-loop-helix TF transgene (LEAF COLOR, LC), which induces strong vegetative pigmentation when these 35S:LC plants are exposed to high-light levels. 35S:ROS1 lisianthus transgenics had limited changes in anthocyanin pigmentation, specifically, precocious pigmentation of flower petals and increased pigmentation of sepals. RNA transcript levels for two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were increased in the 35S:ROS1 lisianthus petals compared to those of control lines. With MP, the 35S:ROS1 calli showed novel red pigmentation in culture, but this was generally not seen in tissue culture plantlets regenerated from the calli or young plants transferred to soil in the greenhouse. Anthocyanin pigmentation was enhanced in the stems of mature 35S:ROS1 MP plants, but the MP white-flower phenotype was not complemented. Progeny from a 35S:ROS1 × 35S:LC cross had novel pigmentation phenotypes that were not present in either parental line or MP. In particular, there was increased pigment in the petal throat region, and the anthers changed from yellow to purple pigmentation. An outdoor field trial was conducted with the 35S:ROS1, 35S:LC, 35S:ROS1 × 35S:LC and control MP lines. Field conditions rapidly induced intense foliage pigmentation in 35S:LC plants, a phenotype not observed in control MP or equivalent 35S:LC plants maintained in a greenhouse. No difference in plant stature, seed germination, or plant survival was observed between transgenic and control plants. [ABSTRACT FROM AUTHOR]

Background: With the explosive numbers of sequences generated by next generation sequencing, the demand for high throughput screening to understand gene function has grown. Plant viral vectors have been widely used as tools in down-regulating plant gene expression. However, plant viral vectors can also express proteins in a very efficient manner and, therefore, can also serve as a valuable tool for characterizing proteins and their functions in metabolic pathways in planta. Results: In this study, we have developed a Gateway®-based high throughput viral vector cloning system from Narcissus Mosaic Virus (NMV). Using the reporter genes of GFP and GUS, and the plant genes PAP1 (an R2R3 MYB which activates the anthocyanin pathway) and selenium-binding protein 1 (SeBP), we show that NMV vectors and the model plant Nicotiana benthamiana can be used for efficient protein expression, protein subcellular localization and secondary metabolite production. Conclusions: Our results suggest that not only can the plant viral vector system be employed for protein work but also can potentially be amenable to producing valuable secondary metabolites on a large scale, as the system does not require plant regeneration from seed or calli, which are stages where certain secondary metabolites can interfere with development. [ABSTRACT FROM AUTHOR]

Aurones are bright yellow flavonoids produced in petals of a limited range of plant species, including Antirrhinum majus. The biosynthesis of aurones is thought to occur by the action of aureusidin synthase (AUS), and possibly aureusidin 7- O-glucosyltransferase (A7GT). The temporal and spatial occurrence of AUS and A7GT transcript was examined in wild-type A. majus and two mutant lines; sulfurea, which has increased aurone production in petals, and violacea, which has reduced aurone production. AUS and A7GT transcript abundance was similar in all three lines, increasing during flower development coincident with yellow coloration. The spatial pattern of AUS occurrence was also similar in all three lines, being spatially restricted to the inner epidermis of the face and throat of the lower petal. A new recessive line ( CFR1011) with greatly reduced aurone production in all parts of the petal was identified by ethylmethanesulfonate mutagenesis of the homozygous recessive sulfurea line. Transcript abundance for AUS was not changed in the CFR1011 line compared with the wild-type line, and neither were any point mutations detected in the coding sequences for AUS or A7GT. Thus, the sulfurea, violacea and CFR1011 mutations do not seem to control aurone production through a change in transcript abundance of the predicted biosynthetic genes AUS or A7GT. To examine AUS gene regulation further, the putative AUS gene promoter region was isolated and compared with other A. majus flavonoid gene promoters. A number of conserved potential regulatory regions were identified, in particular a consensus site for the MYB-type transcription factors. [ABSTRACT FROM AUTHOR]

Background: Although the biosynthetic pathways for anthocyanins and their regulation have been well studied, the mechanism of anthocyanin accumulation in the cell is still poorly understood. Different models have been proposed to explain the transport of anthocyanins from biosynthetic sites to the central vacuole, but cellular and subcellular information is still lacking for reconciliation of different lines of evidence in various anthocyanin sequestration studies. Here, we used light and electron microscopy to investigate the structures and the formation of anthocyanic vacuolar inclusions (AVIs) in lisianthus (Eustoma grandiflorum) petals. Results: AVIs in the epidermal cells of different regions of the petal were investigated. Three different forms of AVIs were observed: vesicle-like, rod-like and irregular shaped. In all cases, EM examinations showed no membrane encompassing the AVI. Instead, the AVI itself consisted of membranous and thread structures throughout. Light and EM microscopy analyses demonstrated that anthocyanins accumulated as vesicle-like bodies in the cytoplasm, which themselves were contained in prevacuolar compartments (PVCs). The vesicle-like bodies seemed to be transported into the central vacuole through the merging of the PVCs and the central vacuole in the epidermal cells. These anthocyanin-containing vesicle-like bodies were subsequently ruptured to form threads in the vacuole. The ultimate irregular AVIs in the cells possessed a very condensed inner and relatively loose outer structure. Conclusion: Our results strongly suggest the existence of mass transport for anthocyanins from biosynthetic sites in the cytoplasm to the central vacuole. Anthocyanin-containing PVCs are important intracellular vesicles during the anthocyanin sequestration to the central vacuole and these specific PVCs are likely derived directly from endoplasmic reticulum (ER) in a similar manner to the transport vesicles of vacuolar storage proteins. The membrane-like and thread structures of AVIs point to the involvement of intravacuolar membranes and/or anthocyanin intermolecular association in the central vacuole. [ABSTRACT FROM AUTHOR]

The expression of anthocyanin biosynthesis genes during flower colour development inAnthurium andraeanum(anthurium) was studied. A cDNA library was constructed from mRNA from the anthurium spathe, and full-length cDNA clones identified for the flavonoid biosynthetic enzymes chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). These were used to measure transcript levels in the spathe during flower development, in cultivars with different flower colours, over the diurnal cycle, and in the spadix.CHS,F3HandANSwere expressed at all stages of spathe and spadix development. However,DFRtranscript levels varied significantly between stages, and DFR may represent a key point of regulation. A diurnal rhythm ofDFRtranscript abundance in the spathe was also observed, with transcript levels high at dawn and dusk and low at noon. Control of anthocyanin biosynthesis in anthurium spathe differs from that described for flowers of other species, withDFRa key regulatory point and a complex mix of developmental and environmental control signals. [ABSTRACT FROM AUTHOR]

Flavonoids, in particular the anthocyanins, are responsible for flower colour in many species. The dihydroflavonols represent a branch point in flavonoid biosynthesis, being the intermediates for production of both the coloured anthocyanins, through the action of the enzyme dihydroflavonol 4-reductase (DFR), and the colourless flavonols, produced by flavonol synthase (FLS). In this study the white-flowered, flavonol accumulating Mitchell line of petunia was used as a model to examine the interaction between DFR and FLS enzyme activities and possibilities for redirecting flavonoid biosynthesis away from production of flavonols and towards anthocyanins. Introduction of a 35S CaMV-DFR sense transgene construct caused the production of anthocyanins, resulting in a pink-flowered phenotype. Furthermore, inhibition of FLS production through introduction of an FLS antisense RNA construct also led to anthocyanin production and a pink-flowered phenotype. A combination of both transgenes gave the highest level of anthocyanin formation. Anthocyanins were produced in the DFR-sense and FLS-antisense transgenic lines in spite of the greatly reduced levels of gene expression in the Mitchell line for three enzymes late in anthocyanin biosynthesis, anthocyanindin synthase, UDP-glucose: flavonoid 3-O-glucosyltransferase and UDP-rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase. Thus, the level of gene activity required for visible anthocyanin formation is much lower than the high levels normally induced during petal development. Altering the balance between the DFR and FLS enzyme activities, using genetic modification, may be a useful strategy for introducing or increasing anthocyanin production in target ornamental species. [ABSTRACT FROM AUTHOR]

Summary: Chalcones are intermediates in the biosynthesis of all flavonoids. In addition, in some species they constitute the major yellow flower pigments. There are two types of chalcones, distinguished by the presence (6′-hydroxychalcones) or absence (6′-deoxychalcones) of a hydroxyl group at the 6′ position of the A-ring. The 6′-deoxychalcones are formed when the enzyme chalcone reductase (CHR) is active in conjunction with chalcone synthase (CHS). In Petunia, only 6′-hydroxychalcones are synthesized, and except in the pollen of some genotypes, they are ephemeral intermediates in flavonoid metabolism. By introducing a CHR cDNA from Medicago sativa under the control of the 35S CaMV promoter into acyanic- or cyanic-flowered lines of Petunia, flower colour was changed from either white to pale yellow or deep purple to pale purple, respectively. Lines were generated that accumulated up to 60% of their petal flavonoids as 6′-deoxychalcones. Several different 6′-deoxychalcones accumulated in the petals of the CHR transgenics. The structures of three of these were determined: one, butein 4-O-glucoside, is a novel plant chalcone. Another chalcone compound was identified in the pollen of the transgenics. The results show that the Petunia chalcone isomerase is unable to use 6′-deoxychalcones as substrates so that 6′-deoxychalcones are stable in Petunia petals, leaves and pollen, but some Petunia flavonoid enzymes can use 6′-deoxychalcones as substrates to modify their structures. The introduction of CHR provides a method to redirect the flavonoid pathway into chalcone production, in order to modify flower colour or to reduce the biosynthesis of other flavonoid types. [ABSTRACT FROM AUTHOR]

The emergence of land plants around 450 million years ago transformed the terrestrial environment. The transition of plants to land required adaptations to a host of abiotic environmental challenges, such as drought, UV-B light, temperature fluctuations, and nutrient deficiency. Key for coping with these stresses was the evolution of specialist metabolite pathways. Of particular note for stress tolerance functions are the flavonoids, which are thought to have arisen with land plants. Recently, a new group of red flavonoid compounds named auronidins were identified in the liverwort model species Marchantia polymorpha (hereafter, Marchantia). The functions of auronidin in abiotic stress resistance are uncharacterised. To address this, we generated near-isogenic lines of Marchantia with either greatly increased or no auronidin production, by overexpression (35 S :Mp MYB14) or CRISPR/Cas9 knockout (Mp myb14), respectively, of Mp MYB14 , the key R2R3MYB transcription factor regulating auronidin biosynthesis. Among a range of abiotic stresses tested, auronidin production was specifically induced by carbon/nitrogen (C/N) imbalance and by light stress. The presence of auronidin was associated with delayed plant senescence, enhanced photo-protection, and improved plant survival rates under C/N imbalance stress. Generation and analysis of loss-of-function and over-expression Marchantia lines for the homologue to the angiosperm BT2 gene, which induces flavonoid production in response to C/N balance in Arabidopsis, showed that the Mp BT gene is not involved in C/N signalling. Thus, the function of BT2 in the regulation of anthocyanin biosynthesis may have arisen through neofunctionalisation during angiosperm evolution. Our study furthers understanding of the function of auronidins in enhancing plant abiotic stress tolerance, revealing that they improve Marchantia tolerance to nutrient imbalance, potentially by serving as a carbon sink. • Nutrient imbalance induces auronidin pigmentation in Marchantia polymorpha. • Auronidin production delays senescence and enhances plant survival under stress. • Auronidins may function as carbon sinks and light screens during stress. • The Mp BT protein in liverworts lacks angiosperm BTB/TAZ function in C/N signalling. [ABSTRACT FROM AUTHOR]

Highlights: [•] New identifications of anthocyanins in the reproductive and vegetative organs of Rhabdothamnus solandri. [•] The differential accumulation of pelargonidin-based and cyanidin-based anthocyanins underlies the colour patterns observed in the bird-pollinated Gesneriaceae species. [•] Anthocyanin patterning in R. solandri is likely regulated through the differential activity of the flavonoid-3′-hydroxylase. [•] Anthocyanic vacuolar inclusions (AVIs) in the flowers suggest that basic anthocyanin structures without aromatic acylation can also form AVIs. [•] A valuable system for studying regulatory mechanisms of anthocyanins, providing new insights into ornamental breeding. [Copyright &y& Elsevier]