Katja Karppinen

Light-controlled flavonoid biosynthesis in fruits

Light is one of the most important environmental factors affecting flavonoid biosynthesis in plants. The absolute dependency of light to the plant development has driven evolvement of sophisticated mechanisms to sense and transduce multiple aspects of the light signal. Light effects can be categorized in photoperiod (duration), intensity (quantity), direction and quality (wavelength) including UV-light. Recently, new information has been achieved on the regulation of light-controlled flavonoid biosynthesis in fruits, in which flavonoids have a major contribution on quality. This review focuses on the effects of the different light conditions on the control of flavonoid biosynthesis in fruit producing plants. An overview of the currently known mechanisms of the light-controlled flavonoid accumulation is provided. R2R3 MYB transcription factors are known to regulate by differential expression the biosynthesis of distinct flavonoids in response to specific light wavelengths. Despite recent advances, many gaps remain to be understood in the mechanisms of the transduction pathway of light-controlled flavonoid biosynthesis. A better knowledge on these regulatory mechanisms is likely to be useful for breeding programs aiming to modify fruit flavonoid pattern.

Octaketide‐producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins

Hypericins are biologically active constituents of Hypericum perforatum (St John’s wort). It is likely that emodin anthrone, an anthraquinone precursor of hypericins, is biosynthesized via the polyketide pathway by type III polyketide synthase (PKS). A PKS from H. perforatum, HpPKS2, was investigated for its possible involvement in the biosynthesis of hypericins. Phylogenetic tree analysis revealed that HpPKS2 groups with functionally divergent non‐chalcone‐producing plant‐specific type III PKSs, but it is not particularly closely related to any of the currently known type III PKSs. A recombinant HpPKS2 expressed in Escherichia coli resulted in an enzyme of ∼ 43 kDa. The purified enzyme catalysed the condensation of acetyl‐CoA with two to seven malonyl‐CoA to yield tri‐ to octaketide products, including octaketides SEK4 and SEK4b, as well as heptaketide aloesone. Although HpPKS2 was found to have octaketide synthase activity, production of emodin anthrone, a supposed octaketide precursor of hypericins, was not detected. The enzyme also accepted isobutyryl‐CoA, benzoyl‐CoA and hexanoyl‐CoA as starter substrates producing a variety of tri‐ to heptaketide products. In situ RNA hybridization localized the HpPKS2 transcripts in H. perforatum leaf margins, flower petals and stamens, specifically in multicellular dark glands accumulating hypericins. Based on our results, HpPKS2 may have a role in the biosynthesis of hypericins in H. perforatum but some additional factors are possibly required for the production of emodin anthrone in vivo.

Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit development

Bilberry (Vaccinium myrtillus L.) possesses a high antioxidant capacity in berries due to the presence of anthocyanins and ascorbic acid (AsA). Accumulation of AsA and the expression of the genes encoding the enzymes of the main AsA biosynthetic route and of the ascorbate-glutathione cycle, as well as the activities of the enzymes involved in AsA oxidation and recycling were investigated for the first time during the development and ripening of bilberry fruit. The results showed that the AsA level remained relatively stable during fruit maturation. The expression of the genes encoding the key enzymes in the AsA main biosynthetic route showed consistent trends with each other as well as with AsA levels, especially during the first stages of fruit ripening. The expression of genes and activities of the enzyme involved in the AsA oxidation and recycling route showed more prominent developmental stage-dependent changes during the ripening process. Different patterns of activity were found among the studied enzymes and the results were, for some enzymes, in accordance with AsA levels. In fully ripe berries, both AsA content and gene expression were significantly higher in skin than in pulp.

Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry (Vaccinium myrtillus L.)

Abscisic acid (ABA) is a natural plant hormone playing an important role in many physiological processes including fruit ripening and is also recently found to be potential for biomedical applications. This study was aimed to measure ABA levels and its biosynthesis in bilberry (Vaccinium myrtillus L.), which is one of the best sources of anthocyanins. Five ABA biosynthetic genes were isolated from bilberry and their expression profiles were studied in bilberry tissues, particularly during berry development. The level of ABA highly increased at the onset of bilberry fruit ripening, at the stage when expression of anthocyanin biosynthetic genes, chalcone synthase (VmCHS) and anthocyanidin synthase (VmANS), also increased. In fully ripe berries and leaves, ABA levels were lower but none was detected in bilberry stem or rhizome. The expression of 9-cis-epoxycarotenoid dioxygenase (VmNCED1) and putative neoxanthin synthase (VmNSY) was high in berry tissues and their expression increased markedly at the onset of berry ripening along with the accumulation of ABA. In contrast, the expression of zeaxanthin epoxidase (VmZEP), short-chain dehydrogenase/reductase (VmSDR/ABA2) and aldehyde oxidase (VmAO) were most highly associated with leaf tissues with no obvious relation to ABA content during berry development. The obtained results indicate that the ABA biosynthesis may play an important role in the regulation of ripening of non-climacteric bilberry fruits through transcriptional regulation of key ABA biosynthetic genes.

Monochromatic light increases anthocyanin content during fruit development in bilberry.

BackgroundLight is one of the most significant environmental factors affecting to the accumulation of flavonoids in fruits. The composition of the light spectrum has been shown to affect the production of phenolic compounds during fruit ripening. However, specific information on the biosynthesis of flavonoids in fruits in response to different wavelengths of light is still scarce. In the present study bilberry (Vaccinium myrtillus L.) fruits, which are known to be rich with anthocyanin compounds, were illuminated with blue, red, far-red or white light during the berry ripening process. Following the illumination, the composition of anthocyanins and other phenolic compounds was analysed at the mature ripening stage of fruits.ResultsAll the three monochromatic light treatments had significant positive effect on the accumulation of total anthocyanins in ripe fruits compared to treatment with white light or plants kept in darkness. The elevated levels of anthocyanins were mainly due to a significant increase in the accumulation of delphinidin glycosides. A total of 33 anthocyanin compounds were detected in ripe bilberry fruits, of which six are novel in bilberry (cyanidin acetyl-3-O-galactose, malvidin acetyl-3-O-galactose, malvidin coumaroyl-3-O-galactose, malvidin coumaroyl-3-O-glucose, delphinidin coumaroyl-3-O-galactose, delphinidin coumaroyl-3-O-glucose).ConclusionsOur results indicate that the spectral composition of light during berry development has significant effect on the flavonoid composition of ripe bilberry fruits.

The hyp-1 gene is not a limiting factor for hypericin biosynthesis in the genus Hypericum

Biosynthesis of the hypericins that accumulate in the dark glands of some members of the genus Hypericum is poorly understood. The gene named hyp-1, isolated from Hypericum perforatum L. has been proposed as playing an important role in the final steps of hypericin biosynthesis. To study the role of this candidate gene in relation to the production of hypericins, the expression of this gene was studied in 15 Hypericum species with varying ability to synthesise hypericin. While the accumulation of hypericins and emodin, an intermediate in the respective pathway, was associated with the dark glands in the hypericin-producing species, the hyp-1 gene was expressed in all studied species regardless of whether hypericins and emodin were detected in the plants. The coding sequences of hyp-1 cDNA were isolated from all species and showed more than 86% similarity to each other. Although, in general, an increased level of the hyp-1 gene transcript was detected in hypericin-producing species, several of the hypericin-lacking species expressed comparable levels as well. Our results question the role of the hyp-1 gene product as a key enzyme responsible for biosynthesis of hypericins in the genus Hypericum. The function of the hyp-1 gene may not be restricted to hypericin biosynthesis only, or some additional factors are necessary for completion of hypericin biosynthesis.

Carotenoid metabolism during bilberry (Vaccinium myrtillus L.) fruit development under different light conditions is regulated by biosynthesis and degradation

BackgroundCarotenoids are important pigments and precursors for central signaling molecules associated in fruit development and ripening. Carotenoid metabolism has been studied especially in the climacteric tomato fruit but the content of carotenoids and the regulation of their metabolism have been shown to be highly variable between fruit species. Non-climacteric berries of the genus Vaccinium are among the best natural sources of health-beneficial flavonoids but not studied previously for carotenoid biosynthesis.ResultsIn this study, carotenoid biosynthetic genes, PSY, PDS, ZDS, CRTISO, LCYB, LCYE, BCH and CYP450-BCH, as well as a carotenoid cleavage dioxygenase CCD1 were identified from bilberry (V. myrtillus L.) fruit and their expression was studied along with carotenoid composition during fruit development under different photoperiod and light quality conditions. Bilberry was found to be a good source of carotenoids among fruits and berries. The most abundant carotenoids throughout the berry development were lutein and β-carotene, which were accompanied by lower amounts of 9Z-β-carotene, violaxanthin, neoxanthin, zeaxanthin, antheraxanthin and β-cryptoxanthin. The expression patterns of the biosynthetic genes in ripening fruits indicated a metabolic flux towards β-branch of the carotenoid pathway. However, the carotenoid levels decreased in both the β-branch and ε,β-branch towards bilberry fruit ripening along with increased VmCCD1 expression, similarly to VmNCED1, indicating enzymatic carotenoid cleavage and degradation. Intense white light conditions increased the expression of the carotenoid biosynthetic genes but also the expression of the cleavage genes VmCCD1 and VmNCED1, especially in unripe fruits. Instead, mature bilberry fruits responded specifically to red/far-red light wavelengths by inducing the expression of both the carotenoid biosynthetic and the cleavage genes indicating tissue and developmental stage specific regulation of apocarotenoid formation by light quality.ConclusionsThis is the first report of carotenoid biosynthesis in Vaccinium berries. Our results indicate that both transcriptional regulation of the key biosynthetic genes and the enzymatic degradation of the produced carotenoids to apocarotenoids have significant roles in the determination of the carotenoid content and have overall effect on the metabolism during the bilberry fruit ripening.

Metabolic and molecular analyses of white mutant Vaccinium berries show down-regulation of MYBPA1-type R2R3 MYB regulatory factor

AbstractMain conclusionMYBPA1-type R2R3 MYBtranscription factor shows down-regulation in white mutant berries ofVaccinium uliginosumdeficient in anthocyanins but not proanthocyanidins suggesting a role in the regulation of anthocyanin biosynthesis. Berries of the genus Vaccinium are among the best natural sources of flavonoids. In this study, the expression of structural and regulatory flavonoid biosynthetic genes and the accumulation of flavonoids in white mutant and blue-colored wild-type bog bilberry (V. uliginosum) fruits were measured at different stages of berry development. In contrast to high contents of anthocyanins in ripe blue-colored berries, only traces were detected by HPLC–ESI–MS in ripe white mutant berries. However, similar profile and high levels of flavonol glycosides and proanthocyanidins were quantified in both ripe white and ripe wild-type berries. Analysis with qRT-PCR showed strong down-regulation of structural genes chalcone synthase (VuCHS), dihydroflavonol 4-reductase (VuDFR) and anthocyanidin synthase (VuANS) as well as MYBPA1-type transcription factor VuMYBPA1 in white berries during ripening compared to wild-type berries. The profiles of transcript accumulation of chalcone isomerase (VuCHI), anthocyanidin reductase (VuANR), leucoanthocyanidin reductase (VuLAR) and flavonoid 3′5′ hydroxylase (VuF3′5′H) were more similar between the white and the wild-type berries during fruit development, while expression of UDP-glucose: flavonoid 3-O-glucosyltransferase (VuUFGT) showed similar trend but fourfold lower level in white mutant. VuMYBPA1, the R2R3 MYB family member, is a homologue of VmMYB2 of V. myrtillus and VcMYBPA1 of V. corymbosum and belongs to MYBPA1-type MYB family which members are shown in some species to be related with proanthocyanidin biosynthesis in fruits. Our results combined with earlier data of the role of VmMYB2 in white mutant berries of V. myrtillus suggest that the regulation of anthocyanin biosynthesis in Vaccinium species could differ from other species studied.

Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis – phylogeny, site‐directed mutagenesis, and expression in nonanther tissues

Anther‐specific chalcone synthase‐like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala‐clade and the Val‐clade, and HpPKS1 belongs to the Ala‐clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium‐chain (C6) to very long‐chain (C24) fatty acyl‐CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl‐CoA esters over the analogous unsubstituted fatty acyl‐CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl‐binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl‐CoA, a poor substrate for the wild‐type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium‐chain to long‐chain fatty acyl‐CoA substrates. The R202L/D211V double mutant was selective for acyl‐CoA with chain lengths of C16–C18, and showed a diminished preference for the hydroxylated acyl‐CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala‐clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues.

Optimization of Protein Extraction from Hypericum perforatum Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.