Gottfried Weissenböck

Differential flavonoid response to enhanced uv-b radiation in brassica napus

Abstract We have examined the qualitative and quantitative differences in methanol-soluble flavonoids of leaves of two cultivars of Brassica napus , which were grown with or without (control) supplemental UV-B radiation. The flavonoids were identified using HPLC-diode array spectroscopy (-DAS), -electrospray ionization-mass spectroscopy (-ESI-MS) and 1 H and 13 C NMR, and quantitatively analysed by HPLC-DAS. After exposure to supplementary UV-B radiation, the overall amount of soluble flavonoids, kaempferol and quercetin glycosides, increased by ca 150% in cv. Paroll, compared to control plants. Cultivar Stallion showed a 70% increase, and also a lower overall content of soluble flavonoids compared to Paroll. The supplementary UV-B radiation resulted in a marked, specific increase in the amount of quercetin glycosides relative to the kaempferol glycosides with a 36- and 23-fold increase in cvs Paroll and Stallion, respectively. Four of the flavonol glycosides appearing after supplemental UV-B exposure were identified as quercetin- and kaempferol 3-sophoroside-7-glucoside and 3-(2″′- E -sinapoylsophoroside)-7-glucoside.

Contribution of phenolic compounds to the UV-B screening capacity of developing barley primary leaves in relation to DNA damage and repair under elevated UV-B levels

Epidermally located UV-absorbing hydroxycinnamic acid conjugates and flavonoid glycosides are known to be efficient UV-B protectants in higher plants, although important biological molecules are not always fully protected. However, repair mechanisms also exist, such as repair of damaged DNA by photolyases. To distinguish between the relative importance of the phenolic compounds and of DNA repair, developing primary leaves of two barley lines, mutant ant 30-310, deficient in flavonoids, and its parent line Ca 33787, were grown under relatively high visible light (650-700 micromol m(-2) s(-1) max for 6 h in a 13 h photoperiod) and supplemented with (+ UV-B) or without (-UV-B) 12 kJ m(-2) UV-B(BE) for 6 h daily. UV-B screening capacity of the leaf phenolics was determined at 315 nm during leaf development and compared with thymine dimers (TD) accumulation, as an indicator of UV-B-induced DNA damage and potential subsequent repair. The degree of damage was related to the phenolic contents of the leaves. UV-B screening capacity was increased ca. 4-fold in the parent line (+ UV-B), mainly due to UV-induced flavonoid (saponarin, lutonarin) accumulation in epidermal and subepidermal mesophyll tissue, relative to the flavonoid-deficient mutant. Nevertheless, in the parent line an 8-fold increase in TD levels occurred over the growth period of 18 days, whereas the mutant accumulated additional DNA damage, with 6- to 9-fold higher TD amounts. Surprisingly, under the high UV-B irradiation, growth and development of the primary leaves in both lines were only slightly reduced.

Different Energization Mechanisms Drive the Vacuolar Uptake of a Flavonoid Glucoside and a Herbicide Glucoside

Glycosylation of endogenous secondary plant products and abiotic substances such as herbicides increases their water solubility and enables vacuolar deposition of these potentially toxic substances. We characterized and compared the transport mechanisms of two glucosides, isovitexin, a native barley flavonoid C-glucoside and hydroxyprimisulfuron-glucoside, a herbicide glucoside, into barley vacuoles. Uptake of isovitexin is saturable (Km = 82 μM) and stimulated by MgATP 1.3-1.5-fold. ATP-dependent uptake was inhibited by bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, but not by vanadate. Transport of isovitexin is strongly inhibited after dissipation of the ΔpH or the ΔΨ across the vacuolar membrane. Uptake experiments with the heterologue flavonoid orientin and competition experiments with other phenolic compounds suggest that transport of flavonoid glucosides into barley vacuoles is specific for apigenin derivatives. In contrast, transport of hydroxyprimisulfuron-glucoside is strongly stimulated by MgATP (2.5-3 fold), not sensitive toward bafilomycin, and much less sensitive to dissipation of the ΔpH, but strongly inhibited by vanadate. Uptake of hydroxyprimisulfuron-glucoside is also stimulated by MgGTP or MgUTP by about 2-fold. Transport of both substrates is not stimulated by ATP or Mg2+ alone, ADP, or the nonhydrolyzable ATP analogue 5′-adenylyl-β,γ-imidodiphosphate. Our results suggest that different uptake mechanisms exist in the vacuolar membrane, a ΔpH-dependent uptake mechanism for specific endogenous flavonoid-glucosides, and a directly energized mechanism for abiotic glucosides, which appears to be the main transport system for these substrates. The herbicide glucoside may therefore be transported by an additional member of the ABC transporters.

Flavone Glucoside Uptake into Barley Mesophyll and Arabidopsis Cell Culture Vacuoles. Energization Occurs by H+-Antiport and ATP-Binding Cassette-Type Mechanisms

In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a K m of 50 to 100 μm. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H+antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta.

Tissue-distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L.

Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.

Secondary phenolic products in isolated guard cell, epidermal cell and mesophyll cell protoplasts from pea (Pisum sativum L.) leaves: Distribution and determination

SummaryGuard cells and epidermal cells of the abaxial (lower) and adaxial (upper) epidermis ofPisum sativum L., mutant Argenteum, are the predominant sites of flavonoid accumulation within the leaf. This was demonstrated by the use of a new method of simultaneous isolation and separation of intact, highly-purified guard cell and epidermal cell protoplasts from both epidermal layers and of protoplasts from the mesophyll. Isolated guard and epidermal protoplasts retained flavonoid patterns of the parent epidermal tissue; quercetin 3-triglucoside and its p-coumaric acid ester as major constituents, kaempferol 3-triglucoside and its p-coumaric acid ester as minor compounds. Total flavonoid content in the lower epidermis was estimated to be ca. 80 fmol per guard cell protoplast and 500 fmol per epidermal cell protoplast. Protoplasts isolated from the upper epidermis had about 20–30% as much of these flavonoids. Mesophyll protoplasts retained only about 25 fmol total flavonoid per protoplast.By fluorescence microscopy, using the alkaline-induced yellow-green fluorescence characteristics of flavonols, we suggest that these flavonol glycosides are present in cell vacuoles. There was no indication for the presence of flavine-like compounds.

Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll.

Vacuoles were isolated by osmotic rupture of mesophyll protoplasts from the primary leaves of 4-d- and 7-d-old plants of rye (Secale cereale L.). Their content of two flavones, luteolin 7-O-[β-d-glucuronosyl-(1→2)β-d-glucuronide] (R2) and luteolin 7-O-[β-d-glucuronosy 1 (1→2) β-d-glucuronide]-4′-O-β-d-glucuronide (R1), as well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific β-glucuronidase was determined in comparison to the parent protoplasts. The two flavonoids were found to be entirely located in the vacuolar fraction, together with 70% of the activity of UDP-glucuronate: luteolin 7-O-diglucuronide-4′-O-glucuronosyl-transferase (LDT; EC 2.4.1.), the third enzyme of the sequence of three transferases in the anabolic pathway. The activities of the first and second anabolic enzymes, UDP-glucuronate: luteolin 7-O-glucuronosyltransferase (LGT; EC 2.4.1.) and UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase (LMT; EC 2.4.1.) could not be found in the vacuolar fraction in appreciable amounts. The specific β-glucuronidase (EC 3.2.1.), catalyzing the deglucuronidation of luteolin triglucuronide to luteolin diglucuronide, was present with 90% of its activity in the digestion medium after isolation of mesophyll protoplasts, indicating an apoplastic localization of this enzyme. The data presented indicate a directed anabolic and subsequent catabolic pathway for the luteolin glucuronides in the mesophyll cells of rye primary leaves. This includes two cytosolic and a last vacuolar step of glucuronidation of luteolin, and the vacuolar storage of the luteolin triglucuronide. We propose the transport of the latter into the cell wall, after which the triglucuronide is deglucuronidated, this being the first step for further turnover.

Flavonoid Biosynthesis in Barley Primary Leaves Requires the Presence of the Vacuole and Controls the Activity of Vacuolar Flavonoid Transport

Barley (Hordeum vulgare) primary leaves synthesize saponarin, a 2-fold glucosylated flavone (apigenin 6-C-glucosyl-7-O-glucoside), which is efficiently accumulated in vacuoles via a transport mechanism driven by the proton gradient. Vacuoles isolated from mesophyll protoplasts of the plant line anthocyanin-less310 (ant310), which contains a mutation in the chalcone isomerase (CHI) gene that largely inhibits flavonoid biosynthesis, exhibit strongly reduced transport activity for saponarin and its precursor isovitexin (apigenin 6-C-glucoside). Incubation of ant310 primary leaf segments or isolated mesophyll protoplasts with naringenin, the product of the CHI reaction, restores saponarin biosynthesis almost completely, up to levels of the wild-type Ca33787. During reconstitution, saponarin accumulates to more than 90% in the vacuole. The capacity to synthesize saponarin from naringenin is strongly reduced in ant310 miniprotoplasts containing no central vacuole. Leaf segments and protoplasts from ant310 treated with naringenin showed strong reactivation of saponarin or isovitexin uptake by vacuoles, while the activity of the UDP-glucose:isovitexin 7-O-glucosyltransferase was not changed by this treatment. Our results demonstrate that efficient vacuolar flavonoid transport is linked to intact flavonoid biosynthesis in barley. Intact flavonoid biosynthesis exerts control over the activity of the vacuolar flavonoid/H+-antiporter. Thus, the barley ant310 mutant represents a novel model system to study the interplay between flavonoid biosynthesis and the vacuolar storage mechanism.

C-Glycosylflavones from Avena sativa

Abstract Avena sativa leaves, stems and inflorescences contain a range of new C -glycosylflavone 2″- O -glycosides, including vitexin and isoswertisin 2″-rhamnosides, isovitexin and isoorientin 2″-arabinosides. The structure of ‘vitexin 4′-rhamnoside’ from Crataegus oxyacantha is revised in vitexin 2″-rhamnoside.

Partial purification and characterization of a luteolin-triglucuronide-specific β-glucuronidase from rye primary leaves (Secale cereale)

Abstract Primary leaves of rye contain a β-glucuronidase with high specificity for luteolin 7- O -[β- D -glucuronosyl (1 → 2)β- D -glucuronide]-4′- O -β- D -glucuronide, the major flavonoid of the leaf. The enzyme hydrolyses the glucuronic acid moiety in position 4′ (K m = 7 μM; V max = 1093 μkat/kg protein). A 330-fold purification was obtained by protein fractionation with ammonium sulphate, Sephadex G 150, hydroxylapatite and CM-Sepharose CL-6B. The enzyme has a pH optimum of 4.3 in 0.01 M citrate buffer. The molecular weight was determined to be 280 kD with active subunits of 67 kD. Isoelectric focusing indicates subunits of different isoelectric points at pH 5.5 and 6.3. The β-glucuronidase shows a temperature optimum at 55° and is inhibited by heavy metal ions such as Cu (0.85 mM) and Ag (0.25 mM), but is activated by ethyleneglycol monomethylether up to 15%. The enzyme is stable in 50% glycerol at −20° for at least 2 months. The results suggest that this β-glucuronidase is involved in the turnover of luteolin triglucuronide in vivo .