The same anthocyanins served four different ways: Insights into anthocyanin structure-function relationships from the wintergreen orchid, Tipularia discolor.

Over 500 unique anthocyanins have been described to date, which vary in color, antioxidant, light-attenuating, and antimicrobial properties. Identification of anthocyanin chemical structure may therefore serve as an important clue to their in situ function in plants. We characterized the histological and chemical structures of anthocyanins associated with diverse leaf color patterns in the terrestrial orchid, Tipularia discolor, as a step towards understanding their ultimate function. Tipularia discolor produces a single wintergreen leaf in autumn, which is drab brown in color during expansion. Upper (adaxial) surfaces of fully-expanded leaves may be green, purple-spotted, or solid purple, while lower (abaxial) surfaces are bright magenta. Our results showed that the same three cyanidin 3,7,3'-triglucosides, in similar concentrations and proportions, accounted for coloration in each of these cases, and that different colors result from differences in histological location of anthocyanins (i.e. abaxial/adaxial epidermis, mesophyll). Anthocyanins with 3,7,3' linkage positions are rare in plants, occurring only within the orchid subfamily Epidendroideae, to which Tipularia belongs. These results are important to the discussion of anthocyanin structure-function because they serve as a reminder that 1) plants may employ the same anthocyanins in different anatomical locations to achieve a broad range of colors (and potentially adaptive functions), and 2) anthocyanin chemical structure and anatomical location are influenced by phylogenetic inertia, as well as natural selection.

Unveiling the shade nature of cyanic leaves: A view from the "blue absorbing side" of anthocyanins.

Anthocyanins have long been suggested as having great potential in offering photoprotection to plants facing high light irradiance. Nonetheless, their effective ability in protecting the photosynthetic apparatus from supernumerary photons has been questioned by some authors, based upon the inexact belief that anthocyanins almost exclusively absorb green photons, which are poorly absorbed by chlorophylls. Here we focus on the blue light absorbing features of anthocyanins, a neglected issue in anthocyanin research. Anthocyanins effectively absorb blue photons: the absorbance of blue relative to green photons increases from tri- to mono-hydroxy B-ring substituted structures, reaching up to 50% of green photons absorption. We offer a comprehensive picture of the molecular events activated by low blue-light availability, extending our previous analysis in purple and green basil, which we suggest to be responsible for the "shade syndrome" displayed by cyanic leaves. While purple leaves display overexpression of genes promoting chlorophyll biosynthesis and light harvesting, in green leaves it is the genes involved in the stability/repair of photosystems that are largely overexpressed. As a corollary, this adds further support to the view of an effective photoprotective role of anthocyanins. We discuss the profound morpho-anatomical adjustments imposed by the epidermal anthocyanin shield, which reflect adjustments in light harvesting capacity under imposed shade and make complex the analysis of the photosynthetic performance of cyanic versus acyanic leaves.

Maize SRO1e represses anthocyanin synthesis through regulating the MBW complex in response to abiotic stress.

Plants experiencing abiotic stress react by generating reactive oxygen species (ROS), compounds that, if allowed to accumulate to excess, repress plant growth and development. Anthocyanins induced by abiotic stress are strong antioxidants that neutralize ROS, whereas their over-accumulation retards plant growth. Although the mechanism of anthocyanin synthesis has been revealed, how plants balance anthocyanin synthesis under abiotic stress to maintain ROS homeostasis is unknown. Here, ROS-related proteins, SIMILAR TO RCD-ONEs (SROs), were analysed in Zea mays (maize), and all six SRO1 genes were inducible by a variety of abiotic stress agents. The constitutive expression of one of these genes, ZmSRO1e, in maize as well as in Arabidopsis thaliana increased the sensitivity of the plant to abiotic stress, but repressed anthocyanin biosynthesis and ROS scavenging activity. Loss-of-function mutation of ZmSRO1e enhanced ROS tolerance and anthocyanin accumulation. We showed that ZmSRO1e competed with ZmR1 (a core basic helix-loop-helix subunit of the MYB-bHLH-WD40 transcriptional activation complex) for binding with ZmPL1 (a core MYB subunit of the complex). Thus, during the constitutive expression of ZmSRO1e, the formation of the complex was compromised, leading to the repression of genes, such as ZmA4 (encoding dihydroflavonol reductase), associated with anthocyanin synthesis. Overall, the results have revealed a mechanism that allows the products of maize SRO1e to participate in the abiotic stress response.

Viburnum tinus Fruits Use Lipids to Produce Metallic Blue Structural Color.

Viburnum tinus is an evergreen shrub that is native to the Mediterranean region but cultivated widely in Europe and around the world. It produces ripe metallic blue fruits throughout winter [1]. Despite its limited fleshy pulp [2], its high lipid content [3] makes it a valuable resource to the small birds [4] that act as its seed-dispersers [5]. Here, we find that the metallic blue appearance of the fruits is produced by globular lipid inclusions arranged in a disordered multilayer structure. This structure is embedded in the cell walls of the epicarp and underlaid with a dark layer of anthocyanin pigments. The presence of such large, organized lipid aggregates in plant cell walls represents a new mechanism for structural coloration and may serve as an honest signal of nutritional content.

Isolation and identification of MYB transcription factors (MYB19Long and MYB19Short) involved in raised spot anthocyanin pigmentation in lilies (Lilium spp.).

Although anthocyanin color patterns on flowers are among the most attractive characteristics, the genetic mechanisms through which color patterns are developed are not well understood, especially for color patterns associated with altered petal structure. Lilium species and cultivars often develop raised spots, where the interior surfaces of tepals increase to develop bumps with accompanying anthocyanin accumulation. The aim of this study was to identify transcription factors regulating pigmentation of the bumps. We identified two R2R3-MYB genes, MYB19Long and MYB19Short, in Lilium leichtlinii, L. lancifolium, and Asiatic hybrid lily cultivars. Their amino acid sequences were similar; however, part of the C-terminal region was triplicated in MYB19Long. Spatial and temporal expression profiles in lilies were strongly associated with anthocyanin biosynthesis gene expression in the bumps, and some defects were found in these genes in L. lancifolium 'Pure Gold' that developed colorless bumps. Thus, both MYB19Long and MYB19Short were likely to be involved in the bump pigmentation. MYB19Long had a stronger ability to stimulate target gene expression than MYB19Short, and expression levels of MYB19Long were greater than those of MYB19Short in lily tepals; thus, the ability to biosynthesize anthocyanin pigments was greater for MYB19Long than for MYB19Short. Among the F1 population, MYB19Short expression was found only in the tepals of F1 plants that developed bumps, although all of the F1 plants possessed the MYB19Short gene, indicating that MYB19 expression followed bump development. These findings helped to elucidate the genetic mechanisms underlying raised spot development.

An anthocyanin marker for direct visualization of plant transformation and its use to study nitrogen-fixing nodule development.

The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis.

Seasonal changes in amino acids and phenolic compounds in fruits from hybrid cross populations of American grapes differing in disease resistance.

The production of wine grapes in upstate New York (USA) is limited by diseases that are promoted by the cool and sometimes rainy climate. A breeding program has been introducing disease resistance from related species into the cultivated stock. Previous work has indicated that such resistance may be based on biochemical reactions rather than on a hypersensitive reaction. We therefore undertook metabolic profiling of amino acids and phenolic compounds in berries from collections of susceptible and resistant hybrids over the course of berry development to determine whether any of these compounds could be causal in disease resistance. The most abundant amino acids were GLN, ARG, PRO and THR. The amount of amino acids in ripe berries was from 3 to 4.7-fold higher compared to earlier stages. The concentrations of total phenolics were variable through the season with no consistent trend between susceptible and resistant fruits. Notable changes in phenolic compounds, especially anthocyanins, were recorded, especially during the ripening phase, when phenolics and anthocyanins increased following veraison. The most abundant phenolic compounds were catechin and epi-catechin; the most abundant anthocyanin was delphinidin-3-glucoside, which had a slightly greater concentration in resistant fruit at harvest, followed by malvidin-3-glucoside and petunidin-3-glucoside. The content of both amino acids and phenolic compounds in white-fruited parent cv. Horizon was equal to several-fold lower than the progeny plants, whether susceptible or resistant, depending on the harvest time. While no major differences between susceptible and resistant lines were found, multivariate analyses showed that it is possible to discriminate the susceptibility or resistance of grapes by analyzing their combined concentrations of amino acids, polyphenols and anthocyanins. Therefore, these compounds are influenced by the resistance capacity of grapes and could be used as a chemical fingerprint of this ability. However, it is likely that these are associations with disease resistance rather than their cause as no major consistent differences were noted.

Enhanced anthocyanin accumulation confers increased growth performance in plants under low nitrate and high salt stress conditions owing to active modulation of nitrate metabolism.

Plants require nitrogen (N) for growth and development. However, they are frequently exposed to conditions of nitrogen deficiency. In addition, anthocyanin accumulation is induced under salt stress and nitrate deficiency. To date, most studies have revealed that nitrate deficiency under high sucrose levels induce high levels of anthocyanin accumulation in plants. However, the underlying mechanisms remain unclear. Under nitrate-starved conditions, plant growth rapidly worsens and cells eventually die. In addition, plants are severely affected by salt exposure. Therefore, in this study, we determined whether increased levels of anthocyanin could improve plant growth under salt stress and nitrate-starved conditions. We used PAP1-D/fls1ko and ttg1 plants which have a perturbed anthocyanin biosynthesis pathway to explore the role of anthocyanin in plant adaptation to nitrate-deficient conditions and salt stress. Our results demonstrate that high anthocyanin accumulation in PAP1-D/fls1ko plants confers enhanced tolerance to nitrate-deficient conditions combined with high salinity. PAP1-D/fls1ko plants appeared to use absorbed nitrate efficiently during the nitrate reduction process. In addition, nitrate-related genes such as NRT1.1, NiA1 and NiA2 were upregulated in the PAP1-D/fls1ko plants. On the basis of these findings, it can be concluded that high anthocyanin accumulation helps plants to cope with salt stress under nitrate-deficient conditions via the effective utilization of nitrate metabolism.

Characterisation of flower colouration in 30 Rhododendron species via anthocyanin and flavonol identification and quantitative traits.

Floral colour is a key reproductive character, often associated with environmental adaptation, and subject to human intervention. A large number of Rhododendron species differ widely in flower colour, providing a good model for flower colouration. The chromatic features and anthocyanin compositions of 30 species from seven subgenera were systematically analysed. The Royal Horticultural Society Colour Chart and CIE L*a*b* system were employed to describe and investigate flower colours. The UPLC-PDA/ESI-MSn system was used to identify and quantify anthocyanins in petal extracts. The flower colours of 30 Rhododendron species were categorised into four groups - red, purplish pink, purple and white. Seven anthocyanins were identified and quantified in petals: delphinidin, cyanidin and malvidin 3-O-arabinoside-5-O-glucosides, cyanidin 3,5-di-O-glucoside, 3-O-galactoside and 3-O-arabinoside, and delphinidin 3-O-glucoside. The red-flowered species mainly contained cyanidin monoglycosides and had much higher total anthocyanin content than purplish pink- and purple-flowered species. Purplish pink- and purple-flowered species had similar anthocyanin types and content. The chromatic differences were significant among groups, except the purplish pink and purple groups. Statistical analysis showed that Cy3Gal and Cy3Arb are characteristic for red-flowered species, and Mv3Arb5G and Dp3Arb5G play important roles in purple colouration; their contents were major components that greatly affected the chromatic parameters. In total, 21 flavonol derivates were identified. However, total flavonol content and co-pigmentation index showed no significant difference or correlation among/with colour groups, suggesting that flavonols might not play a major role in colouration. These results enhance our knowledge of the biochemical basis of flower colouration in Rhododendron species, and provide a foundation for genetic variation studies and aid in breeding cultivars with novel flower colours.

UV-vis spectroscopy and colorimetric models for detecting anthocyanin-metal complexes in plants: An overview of in vitro and in vivo techniques.

Although anthocyanin (ACN) biosynthesis is one of the best studied pathways of secondary metabolism in plants, the possible physiological and ecological role(s) of these pigments continue to intrigue scientists. Like other dihydroxy B-ring substituted flavonoids, ACNs have an ability to bind metal and metalloid ions, a property that has been exploited for a variety of purposes. For example, the metal binding ability may be used to stabilize ACNs from plant food sources, or to modify their colors for using them as food colorants. The complexation of metals with cyanidin derivatives can also be used as a simple, sensitive, cheap, and rapid method for determination concentrations of several metals in biological and environmental samples using UV-vis spectroscopy. Far less information is available on the ecological significance of ACN-metal complexes in plant-environment interactions. Metalloanthocyanins (protocyanin, nemophilin, commelinin, protodelphin, cyanosalvianin) are involved in the copigmentation phenomenon that leads to blue-pigmented petals, which may facilitate specific plant-pollinator interactions. ACN-metal formation and compartmentation into the vacuole has also been proposed to be part of an orchestrated detoxification mechanism in plants which experience metal/metalloid excess. However, investigations into ACN-metal interactions in plant biology may be limited because of the complexity of the analytical techniques required. To address this concern, here we describe simple methods for the detection of ACN-metal both in vitro and in vivo using UV-vis spectroscopy and colorimetric models. In particular, the use of UV-vis spectra, difference absorption spectra, and colorimetry techniques will be described for in vitro determination of ACN-metal features, whereas reflectance spectroscopy and colorimetric parameters related to CIE L*a*b* and CIE XYZ systems will be detailed for in vivo analyses. In this way, we hope to make this high-informative tool more accessible to plant physiologists and ecologists.

A magic red coat on the surface of young leaves: anthocyanins distributed in trichome layer protect Castanopsis fissa leaves from photoinhibition.

The presence of anthocyanins in young leaves plays an important role in mitigation against photodamage and allows leaves to grow and develop normally. Many studies have reported that foliar anthocyanins are distributed within the vacuoles of mesophyll cells, so we explored the novel defence style of anthocyanin-coated young leaves of Castanopsis fissa, a dominant subtropical forest tree species, via removable trichomes. Anthocyanins were distributed in C. fissa leaf trichomes, which produced a red coating for the young leaves. As young leaves developed and then matured, the thickness and density of the anthocyanin trichomes progressively decreased, the coating finally disappearing, allowing greater utilization of light by mature leaves. In addition to anthocyanins, the trichomes contained a remarkably high amount of phenolics, which enable the red coating to be more efficient in screening ultraviolet light. Compared with mature leaves, the young leaves exhibited lower photosynthetic ability, which was attributable to the reduced chlorophyll and Rubisco contents. Removal of the red coating had little effect on the photosynthetic capacity of young leaves. However, the young leaves without the coating suffered greater light-induced photoinhibition due to greater excess light entering the chloroplast and the production of H2O2 Our results suggest that the anthocyanin coating is photoprotective and this anthocyanin defence style may be a metabolically cost-effective way of adjusting the anthocyanin content in response to demand.

Examining the photoprotection hypothesis for adaxial foliar anthocyanin accumulation by revisiting comparisons of green- and red-leafed varieties of coleus (Solenostemon scutellarioides).

Although plants rely on light to drive energy production via photosynthesis, excess light can be harmful. Plants have evolved photoprotective mechanisms to mitigate this threat, including thermal energy dissipation, the most common form of which involves de-epoxidized constituents of the xanthophyll cycle facilitating the conversion of excess excitation energy to heat. A role in photoprotection has also been proposed for red anthocyanins when they accumulate near the adaxial leaf surface. Here, we compared the response to experimental light stress of a red-leafed (anthocyanin rich) and a green-leafed variety of coleus [Solenostemon scutellarioides (L.) Codd], examining chlorophyll fluorescence emission and pigment composition. After experimentally imposed intense white light, red- and green-leafed coleus exhibited manifestations of light stress (decreased photosystem II quantum efficiency) of a similar magnitude. This, considered alone, could be interpreted as evidence that anthocyanins do not serve a photoprotective role. However, during excess light exposure, the green-leafed variety employed a greater level of thermal energy dissipation and possessed correspondingly higher xanthophyll cycle pool sizes and de-epoxidation states. During exposure to red light, which anthocyanins absorb very poorly, levels of thermal energy dissipation did not differ between coleus varieties. Taken together, our findings suggest that adaxial anthocyanins minimize stress associated with excess light absorption and that the green-leafed variety of coleus compensated for its much lower levels of adaxial anthocyanins by invoking higher levels of energy dissipation. Thus, anthocyanin accumulation should be considered alongside the suite of photoprotective mechanisms employed by photosynthetic tissues.

Demographic consequences of chromatic leaf defence in tropical tree communities: do red young leaves increase growth and survival?

BACKGROUND: Many tropical forest tree species delay greening their leaves until full expansion. This strategy is thought to provide newly flushing leaves with protection against damage by herbivores by keeping young leaves devoid of nutritive value. Because young leaves suffer the greatest predation from invertebrate herbivores, delayed greening could prevent costly tissue loss. Many species that delay greening also produce anthocyanin pigments in their new leaves, giving them a reddish tint. These anthocyanins may be fungicidal, protect leaves against UV damage or make leaves cryptic to herbivores blind to the red part of the spectrum. METHODS: A comprehensive survey was undertaken of seedlings, saplings and mature trees in two diverse tropical forests: a rain forest in western Amazonia (Yasuní National Park, Ecuador) and a deciduous forest in Central America (Barro Colorado Island, Panamá). A test was made of whether individuals and species with delayed greening or red-coloured young leaves showed lower mortality or higher relative growth rates than species that did not. KEY RESULTS: At both Yasuní and Barro Colorado Island, species with delayed greening or red young leaves comprised significant proportions of the seedling and tree communities. At both sites, significantly lower mortality was found in seedlings and trees with delayed greening and red-coloured young leaves. While there was little effect of leaf colour on the production of new leaves of seedlings, diameter relative growth rates of small trees were lower in species with delayed greening and red-coloured young leaves than in species with regular green leaves, and this effect remained when the trade-off between mortality and growth was accounted for. CONCLUSIONS: Herbivores exert strong selection pressure on seedlings for the expression of defence traits. A delayed greening or red-coloured young leaf strategy in seedlings appears to be associated with higher survival for a given growth rate, and may thus influence the species composition of later life stages.

The role of anthocyanin in photoprotection and its relationship with the xanthophyll cycle and the antioxidant system in apple peel depends on the light conditions.

The synthesis of anthocyanin, the xanthophyll cycle, the antioxidant system and the production of active oxygen species (AOS) were compared between red and non-red apple cultivars, in response to either long-term sunlight exposure (high light intensity) during fruit development, or to exposure of bagged fruits to lower light intensity late in fruit development. During fruit development of red and non-red apples, the xanthophyll cycle pool size decreased much more in red apple peel late in development. With accumulation of AOS induced by long-term sunlight exposure, enhancement of the antioxidant system was found. However, this change became significantly lower in red apple than non-red apple as fruit developed, which might serve to accelerate the anthocyanin synthesis in red apple peel. When, late in fruit development, bagged fruits were exposed to sunlight, the accumulation of AOS was lower in red apple peel than in non-red peel. This could be due to the higher anthocyanin concentration in the red peels. Meanwhile, compared with that in non-red cultivar, the xanthophyll cycle and the antioxidant system in red apple peel were protected first but then down-regulated by its higher anthocyanin concentration during sunlight exposure. In conclusions, red and non-red apples peel possess different photoprotective mechanisms under high light conditions. The relationship between anthocyanin synthesis and the xanthophyll cycle, and the antioxidant system, depends on the light conditions that fruit undergoes.

[Photoprotective mechanisms of leaf anthocyanins: research progress].

Anthocyanin is widely distributed in plant organs such as root, stem, leaf, flower and fruit, being a kind of secondary metabolites generated in plant morphogenesis or for stress response. Leaf anthocyanin has special chemical structure and spectral properties, playing important roles in plant photoprotection, and becomes a hotspot in plant photosynthetic physiological ecology. This paper summarized the recent research progress in the effects of leaf anthocyanin on plant photosynthesis, including the distribution of leaf anthocyanin, its spectral properties, and its relationships with photosynthetic pigments, with the focus on the potential mechanisms of anthocyanins photoprotection, including light absorption, antioxidation, and osmotic regulation. The further research directions on the effects of leaf anthocyanin on photoprotection were proposed.

Blue flower color development by anthocyanins: from chemical structure to cell physiology.

Blue flower colors are primarily due to anthocyanin, a flavonoid pigment. Anthocyanin itself is purple in neutral aqueous solutions, ans its color is very unstable and quickly fades. Therefore, the mechanism of blue color development in living flower petals is one of the most intriguing problems in natural product chemistry. Much progress has been made in understanding blue flower coloration since the comprehensive review by Goto and Kondo in 1991. This review focuses on the advances in the last 15 years, and cites 149 references.

Shift in polyphenol profile and sublethal phenotype caused by silencing of anthocyanidin synthase in apple (Malus sp.).

We have investigated the consequences of blocking anthocyanin biosynthesis by silencing a key enzyme, anthocyanidin synthase, in transgenic plants of a red-leaved apple cultivar. This is complementary to a previous study of induction of anthocyanin biosynthesis by overexpressing a heterologous transcription factor. Analysis of these opposite phenotypes allows one to study anthocyanin functions in apple and to test the influence of the genetic manipulation on other, related metabolites. As expected, anthocyanin biosynthesis was almost completely blocked and this was accompanied by a shift in the profile of flavonoids and related polyphenols. Most interestingly, a rise in epicatechin was found. A severe reduction of viability by necrotic leaf lesions was also observed, suggesting an essential function of anthocyanins in apple.

Optical effects of abaxial anthocyanin on absorption of red wavelengths by understorey species: revisiting the back-scatter hypothesis.

A red/purple coloration of lower (abaxial) leaf surfaces is commonly observed in deeply-shaded understorey plants, especially in the tropics. However, the functional significance of red abaxial coloration, including its role in photosynthetic adaptation, remains unclear. The objective of this study was to test the back-scatter hypothesis for abaxial leaf coloration, which posits that red pigments internally reflect/scatter red light transmitted by the upper leaf surface back into the mesophyll, thereby enhancing photon capture in light-limited environments. Abaxially red/non-red variegated leaves of Begonia heracleifolia (Cham. & Schltdl.) were used to compare reflectance spectra and chlorophyll fluorescence profiles of abaxially anthocyanic (red) and acyanic (non-red) tissues under red light. Photosynthetic gas exchange in response to red light was also compared for abaxially red/non-red leaf sections. The results did not support a back-scattering function, as anthocyanic leaf surfaces were not more reflective of red light than acyanic surfaces. Anthocyanic tissues also did not exhibit any increases in the mesophyll absorbance of red light, or increased photosynthetic gas exchange under red light at any intensity, relative to acyanic tissues. These results suggest that abaxial anthocyanins do not significantly enhance the absorption of red light in the species tested, and alternative functions are discussed.