A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Allocation of carbon to growth and secondary metabolites in birch seedlings under UV-B radiation and CO2 exposure

In plants, the allocation of carbon to secondary metabolites has been shown to be determined by both the availability of resources (e.g., CO₂ concentration) and by specific stress factors (e.g., ultraviolet [UV]‐radiation). It has been suggested that, in combination, CO₂ and UV‐B radiation may differentially affect plant growth and morphogenic parameters, and elevated CO₂ may ameliorate the effects of UV‐B radiation. In the present work, the effects of increased atmospheric CO₂ concentration and UV‐B radiation on growth and the accumulation of different types of secondary metabolites were studied in silver birch (Betula pendula Roth). Seedlings were exposed to 350 and 700 μmol mol^?1 of CO₂ in a greenhouse. At both CO₂ levels, additional UV‐B was either present (8.16 kJ m^?2 day^?1 of biologically effective UV‐B irradiance) or absent. The time course of accumulation of individual secondary compounds and the shifts in allocation of carbon between biomass and the secondary metabolites (phenolic acids, flavonoids, condensed tannins) were studied during a 1‐month‐long exposure. Additionally, the activities of enzymes ( l ‐phenylalanine ammonia‐lyase [PAL], EC 4.3.1.5; peroxidase, EC 1.11.1.7; polyphenol oxidase, EC 1.10.3.1) were determined for leaves. UV‐B radiation significantly increased biomass, PAL activity, and the accumulation of phenolic acids and flavonoids in seedlings. Elevated CO₂ concentration increased the activities of all the enzymes studied and the accumulation of condensed tannins in leaves, especially with UV‐B radiation. Because the observed UV‐B induction of flavonoids was smaller under a high CO₂ concentration, it was suggested that the excess of carbon in the atmosphere may moderate the effect of UV‐B by increasing the metabolic activity of leaves (high enzyme activities) and by changing the allocation of internal carbon between different primary and secondary metabolites in the plant. Our results demonstrate the significant increase in the allocation of carbon to secondary metabolites without any large change in growth due to the elevation of CO₂ concentration and UV‐B radiation. There also was a stronger impact of CO₂ than UV‐B on the phenolic metabolism of birch seedlings.     

Responses of the flavonoid pathway to UV-B radiation stress and the correlation with the lipid antioxidant characteristics in the desert plant Caryopteris mongolica

Caryopteris mongolica is a dwarf shrub mainly found in grassland and desert areas of north-west China, and which can survive severe environmental stress. This study aimed to assess the responses of the flavonoid pathway to UV-B radiation treatments and its correlation to the lipid peroxide and antioxidant systems in C. mongolica. In UV-B radiation experiments, plants were exposed to UV-B radiation treatments with a intensity of 30 J/s for 1, 4 and 24 h, respectively. A control group without UV-B radiation treatment was also used. The chlorophyll fluorescence parameters, contents of chlorophyll and carotenoid, levels of lipid peroxidation, activities of antioxidant system enzymes, accumulations of total flavonoids and anthocyanins, and activities of phenylalanine ammonialyase (PAL) and chalcone isomerase (CHI) under different UV-B radiation treatments were investigated. The correlations between products and key enzymes in the flavonoid pathway and the lipid peroxide and antioxidant systems were also analyzed. The results showed that chlorophyll fluorescence parameters decreased within 24 h of treatment. The chlorophyll contents decreased within 4 h and remained stable after 24 h. Carotenoid content significantly increased. The level of MDA, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) and the contents of total flavonoids and anthocyanidins increased, while catalase (CAT) activity decreased under UV-B stress. The activities of PAL and CHI also increased with the increased content of total flavonoids. The flavonoid products anthocyanidins had a significant positive correlation with MDA level, as well as the activities of antioxidant enzyme SOD. In conclusion, UV-B radiation induced the degradation of photosynthetic pigments and decreased photochemical efficiency of Photosystem II; increased the contents of MDA, total flavonoids and anthocyanidins; and also enhanced activities of antioxidant enzymes (SOD, APX and POD) and key enzymes (PAL and CHI) in the flavonoid pathway in C. mongolica. Thus, we speculate that the flavonoid pathway were involved in the regulation of stress resistance in C. mongolica.     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

Chloroplastic ATP synthase builds up a proton motive force preventing production of reactive oxygen species in photosystem I

Over‐reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H⁺ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over‐reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)‐treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over‐reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ‐subunit of chloroplastic CF₀CF₁‐ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H⁺ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF₀CF₁‐ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild‐type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF₀CF₁‐ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H⁺ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF₀CF₁‐ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.     

Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet‐B (UV‐B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV‐B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV‐B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV‐B radiation exposure activated antioxidants and stress‐responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV‐B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV‐B irradiation.     

SUSCEPTIBILITY AND PROTECTIVE MECHANISMS OF MOTILE AND NON MOTILE CELLS OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) TO PHOTOOXIDATIVE STRESS1

The life cycle of the unicellular green alga Haematococcus pluvialis consists of motile and nonmotile stages under typical growing conditions. In this study, we observed that motile cells were more susceptible than nonmotile cells to high light, resulting in a decrease in population density and photo‐bleaching. Using two Haematococcus strains, CCAP 34/12 (a motile cell dominated strain) and SAG 34/1b (a nonmotile cell dominated strain), as model systems we investigated the cause of cell death and the protective mechanisms of the cells that survived high light. The death of motile cells under high light was attributed to the generation of excess reactive oxygen species (ROS), which caused severe damage to the photosynthetic components and the membrane system. Motile cells were able to dissipate excess light energy by nonphotochemical quenching and to relax ROS production by a partially up‐regulated scavenging enzyme system. However, these strategies were not sufficient to protect the motile cells from high light stress. In contrast, nonmotile cells were able to cope with and survive under high light by (i) relaxing the over‐reduced photosynthetic electron transport chain (PETC), thereby effectively utilizing PETC‐generated NADPH to produce storage starch, neutral lipid, and astaxanthin, and thus preventing formation of excess ROS; (ii) down‐regulating the linear electron transport by decreasing the level of cytochrome f; and (iii) consuming excess electrons produced by PSII via a significantly enhanced plastid terminal oxidase pathway.     

Do UV‐A radiation and blue light during growth prime leaves to cope with acute high light in photoreceptor mutants of Arabidopsis thaliana?

We studied how plants acclimated to growing conditions that included combinations of blue light (BL) and ultraviolet (UV)‐A radiation, and whether their growing environment affected their photosynthetic capacity during and after a brief period of acute high light (as might happen during an under‐canopy sunfleck). Arabidopsis thaliana Landsberg erecta wild‐type were compared with mutants lacking functional blue light and UV photoreceptors: phototropin 1, cryptochromes (CRY1 and CRY2) and UV RESISTANT LOCUS 8 (uvr8). This was achieved using light‐emitting‐diode (LED) lamps in a controlled environment to create treatments with or without BL, in a split‐plot design with or without UV‐A radiation. We compared the accumulation of phenolic compounds under growth conditions and after exposure to 30 min of high light at the end of the experiment (46 days), and likewise measured the operational efficiency of photosystem II (?PSII, a proxy for photosynthetic performance) and dark‐adapted maximum quantum yield (F_v/F_m to assess PSII damage). Our results indicate that cryptochromes are the main photoreceptors regulating phenolic compound accumulation in response to BL and UV‐A radiation, and a lack of functional cryptochromes impairs photosynthetic performance under high light. Our findings also reveal a role for UVR8 in accumulating flavonoids in response to a low UV‐A dose. Interestingly, phototropin 1 partially mediated constitutive accumulation of phenolic compounds in the absence of BL. Low‐irradiance BL and UV‐A did not improve ?PSII and F_v/F_m upon our acute high‐light treatment; however, CRYs played an important role in ameliorating high‐light stress.     

Dynamic changes in plant secondary metabolites during UV acclimation in Arabidopsis thaliana

Plants respond to environmental stress by synthesizing a range of secondary metabolites for defense purposes. Here we report on the effect of chronic ultraviolet (UV) radiation on the accumulation of plant secondary metabolites in Arabidopsis thaliana leaves. In the natural environment, UV is a highly dynamic environmental parameter and therefore we hypothesized that plants are continuously readjusting levels of secondary metabolites. Our data show distinct kinetic profiles for accumulation of tocopherols, polyamines and flavonoids upon UV acclimation. The lipid‐soluble antioxidant α‐tocopherol accumulated fast and remained elevated. Polyamines accumulated fast and transiently. This fast response implies a role for α‐tocopherol and polyamines in short‐term UV response. In contrast, an additional sustained accumulation of flavonols took place. The distinct accumulation patterns of these secondary metabolites confirm that the UV acclimation process is a dynamic process, and indicates that commonly used single time‐point analyses do not reveal the full extent of UV acclimation. We demonstrate that UV stimulates the accumulation of specific flavonol glycosides, i.e. kaempferol and (to a lesser extent) quercetin di‐ and triglycosides, all specifically rhamnosylated at position seven. All metabolites were identified by Ultra Performance Liquid Chromatography (UPLC)‐coupled tandem mass spectrometry. Some of these flavonol glycosides reached steady‐state levels in 3–4 days, while concentrations of others are still increasing after 12 days of UV exposure. A biochemical pathway for these glycosides is postulated involving 7‐O‐rhamnosylation for the synthesis of all eight metabolites identified. We postulate that this 7‐O‐rhamnosylation has an important function in UV acclimation.     

UV‐radiation and elevated temperatures induce formation of reactive oxygen species in gametophytes of cold‐temperate/Arctic kelps (Laminariales,Phaeophyceae)

Enhanced UV‐radiation (UVR) through stratospheric ozone depletion and global warming are crucial stressors to marine macroalgae. Damages may arise through formation of reactive oxygen species (ROS) in gametophytes of ecologically important kelps, brown algae of the order Laminariales, Such stress‐induced damages may have a negative impact on their fitness and further impact their following life stages. In our study, gametophytes of three kelp species Alaria esculenta (L.) Grev., Laminaria digitata (Huds.) Lamour., Saccharina latissima (L.) Lane, Mayes, Druehl, Saunders from the Arctic, and of L. hyperborea (Gunnerus) Foslie from the North Sea were exposed to photosynthetically active radiation, UV‐A, and UV‐B radiation and four temperatures (2–18°C). ROS are formed predominantly in the peripheral cytoplasm and in chloroplasts especially after exposure to UVR. Superoxide (O₂*^‐) is additionally formed in small, globular cytoplasmic structures, possibly mitochondria. In the surrounding medium O₂*^‐‐concentration increased markedly at elevated temperatures and under UV stress in some cases. Ultrastructural damage was negligible pointing to a high stress tolerance of this developmental stage. Our data indicate that stress tolerant gametophytes of three Arctic kelp species should sustain their crucial function as seed bank for kelp populations even under prospective rising environmental perturbations.     

Production of yellow colour in flowers: redirection of flavonoid biosynthesis in Petunia

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.     

On the role of flavonoids in the integrated mechanisms of response of Ligustrum vulgare and Phillyrea latifolia to high solar radiation

The role of flavonoids in mechanisms of acclimation to high solar radiation was analysed in Ligustrum vulgare and Phillyrea latifolia, two Mediterranean shrubs that have the same flavonoid composition but differ strikingly in their leaf morpho-anatomical traits. In plants exposed to 12 or 100% solar radiation, measurements were made for surface morphology and leaf anatomy; optical properties, photosynthetic pigments, and photosystem II efficiency; antioxidant enzymes, lipid peroxidation and phenylalanine ammonia lyase; synthesis of hydroxycinnamates and flavonoids; and the tissue-specific distribution of flavonoid aglycones and ortho-dihydroxylated B-ring flavonoid glycosides. A denser indumentum of glandular trichomes, coupled with both a thicker cuticle and a larger amount of cuticular flavonoids, allowed P. latifolia to prevent highly damaging solar wavelengths from reaching sensitive targets to a greater degree than L. vulgare. Antioxidant enzymes in P. latifolia were also more effective in countering light-induced oxidative load than those in L. vulgare. Consistently, light-induced accumulation of flavonoids in L. vulgare, particularly ortho-dihydroxylated flavonoids in the leaf mesophyll, greatly exceeded that in P. latifolia. We conclude that the accumulation of flavonoid glycosides associated with high solar radiation-induced oxidative stress and, hence, biosynthesis of flavonoids appear to be unrelated to 'tolerance' to high solar radiation in the species examined.     

Similar stress responses are elicited by copper and ultraviolet radiation in the aquatic plant Lemna gibba: implication of reactive oxygen species as common signals

Metals and ultraviolet (UV) radiation are two environmental stressors that can cause damage to plants. These two types of stressors often impact simultaneously on plants and both are known to promote reactive oxygen species (ROS) production. However, little information is available on the potential parallel stress responses elicited by metals and UV radiation. Using the aquatic plant Lemna gibba, we found that copper and simulated solar radiation (SSR, a light source containing photosynthetically active radiation (PAR) and UV radiation) induced similar responses in the plants. Both copper and SSR caused ROS formation. The ROS levels were higher when copper was combined with SSR than when applied with PAR. Higher concentrations of copper plus PAR caused toxicity as monitored by diminished growth and chlorophyll content. This toxicity was more pronounced when copper was combined with SSR. Because the generation of ROS was also higher when copper was combined with SSR, we attributed this enhanced toxicity to elevated levels of ROS. In comparison to PAR-grown plants, SSR treated plants exhibited elevated levels of superoxide dismutase (SOD) and glutathione reductase (GR). These enzyme levels were further elevated under both PAR and SSR when copper was added at concentrations that generated ROS. Interestingly, copper treatment in the absence of SSR (i.e. copper plus PAR) induced synthesis of the same flavonoids as those observed in SSR without copper. Finally, addition of either dimethyl thiourea or GSH (two common ROS scavengers) lowered in vivo ROS production, alleviated toxicity and diminished induction of GR as well as accumulation of UV absorbing compounds. Thus, the potential of ROS being a common signal for acclimation to stress by both copper and UV can be considered.     

Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth

In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.