The ABC-like vacuolar transporter for rye mesophyll flavone glucuronides is not species-specific.

In many cases, the vacuolar uptake of secondary metabolites has been demonstrated to be strictly specific for a given compound and plant species. While most plants contain glycosylated secondary substances, few cases are known where flavonoids may also carry negative charges, e.g. as glucuronide conjugates. Vacuolar transport of glucosylated phenylpropanoid derivatives has been shown to occur by proton substrate antiport mechanisms (Klein, M., Weissenböck. G., Dufaud, A., Gaillard, C., Kreuz, K., Martinoia, E., 1996. Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside. J. Biol. Chem. 271, 29,666-29,671). In contrast, flavone glucuronides appearing specifically in rye mesophyll vacuoles are taken up by direct energisation utilising MgATP, strongly arguing for the presence of an ATP-binding cassette (ABC) transporter belonging to the subfamily of multidrug resistance-associated proteins (MRP) on the rye vacuolar membrane (Klein, M., Martinoia, E., Hoffmann-Thoma, G., Weissenböck, G., 2000. A membrane-potential dependent, ubiquitous ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides regulation of glucturonide uptake by glutathione and its conjugates. Plant Journal 21, 289-304). MRPs are known to transport negatively charged organic anions. Results presented here suggest that the vacuolar directly energised MRP-like glucuronate pump for plant-specific flavone glucuronides is ubiquitously present in diverse plant species since rye flavone glucuronides are taken up into vacuoles isolated from the barley mesophyll or from the broccoli stalk parenchyma representing two species which do not synthesise glucuronidated secondary compounds. According to the transport characteristics and inhibition profile observed we propose the existence of a high-capacity, uncoupler-insensitive vacuolar ABC transporter for flavone glucuronides and possibly other negatively charged organic compounds -- plant-born or xenobiotic -- irrespective of the plant's capability to endogenously produce glucuronidated compounds.

A membrane-potential dependent ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides: regulation of glucuronide uptake by glutathione and its conjugates.

In this paper we present results on the vacuolar uptake mechanism for two flavone glucuronides present in rye mesophyll vacuoles. In contrast to barley flavone glucosides (Klein et al. (1996) J. Biol. Chem. 271, 29666-29671), the flavones luteolin 7-O-diglucuronyl-4'-O-glucuronide (R1) and luteolin 7-O-diglucuronide (R2) were taken up into vacuoles isolated from rye via a directly energized mechanism. Kinetic studies suggested that the vacuolar glucuronide transport system is constitutively expressed throughout rye primary leaf development. Competition experiments argued for the existence of a plant MRP-like transporter for plant-specific and non-plant glucuronides such as beta-estradiol 17-(beta-D-glucuronide) (E217G). The interaction of ATP-dependent vacuolar glucuronide uptake with glutathione and its conjugates turned out to be complex: R1 transport was stimulated by dinitrobenzene-GS and reduced glutathione but was inhibited by oxidized glutathione in a concentration-dependent manner. In contrast, R2 uptake was not increased in the presence of reduced glutathione. Thus, the transport system for plant-derived glucuronides differed from the characteristic stimulation of vacuolar E217G uptake by glutathione conjugates but not by reduced glutathione (Klein et al. (1998) J. Biol. Chem. 273, 262-270). Using tonoplast vesicles isolated with an artificial K+ gradient, we demonstrate for the first time for plant MRPs that the ATP-dependent uptake of R1 is membrane-potential dependent. We discuss the kinetic capacity of the ABC-type glucuronide transporter to explain net vacuolar flavone glucuronide accumulation in planta during rye primary leaf development and the possibility of an interaction of potential substrates at both the substrate binding and allosteric sites of the MRP transporter regulating the activity towards a certain substrate.

Directly energized uptake of beta-estradiol 17-(beta-D-glucuronide) in plant vacuoles is strongly stimulated by glutathione conjugates.

A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species. Since glucuronate conjugates also occur in plants, we addressed the question whether plant vacuoles take up the abiotic glucuronate conjugate estradiol 17-(beta-glucuronide) (E217G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrates, or via a distinct glucuronate transporter. Uptake studies into vacuoles from rye and barley were performed with E217G and metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to compare glucuronate conjugate transport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively. Our results indicate that E217G and MOC-GS are taken up into vacuoles of both plants via a directly energized mechanism since transport was (i) strictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar DeltapH or DeltaPsi; (iii) E217G uptake into rye vacuoles was partially driven by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent with CTP, whereas the non-hydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate, ADP, or PPi did not energize uptake. E217G transport into rye vacuoles was saturable (Km approximately 0.2 mM). The rye-specific luteolin glucuronides decreased uptake rates of E217G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono- and diglucuronidated derivatives (40-60% inhibition) being more effective than the triglucuronide. Inhibition of E217G uptake by luteolin 7-O-diglucuronide was competitive (Ki = 120 microM). Taurocholate had no effect on E217G transport, and uptake of MOC-GS was not inhibited by E217G. Although GS conjugates and oxidized GS decreased MOC-GS transport, E217G uptake into rye and barley vacuoles was stimulated up to 7-fold in a concentration-dependent manner by these substances, with dinitrobenzene-GS being most effective. The stimulation of the GS conjugates was not due to detergent or redox effects and was specific for the E217G pump. GS conjugate stimulation of glucuronate uptake was unique for plants as E217G uptake into yeast microsomal vesicles was not affected. By comparison with a DeltaYCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediated by YCF1, it was shown that E217G was taken up into yeast vesicles via a YCF1-independent directly energized pump. These results indicate that E217G as a glucuronate conjugate is transported across the vacuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase.

Transport of lucifer yellow CH into plant vacuoles--evidence for direct energization of a sulphonated substance and implications for the design of new molecular probes.

Contrasting observations exist which indicate that in plants the fluorescent dye lucifer yellow CH (LYCH) either can be used as a tracer for endocytosis or as a substrate for an anion transporter located at the vacuolar membrane. In addition, LYCH as a disulphonated substance may represent an analogue of sulphonated or sulfated natural compounds like some flavonoids. We performed uptake experiments with LYCH into isolated rye vacuoles and observed saturable (Km = 0.3-0.6 mM) vacuolar transport and accumulation of the dye against the concentration gradient only when MgATP was present. GTP and, to a low extent, UTP could substitute for ATP, while the non-hydrolysable ATP analogue AMP-PNP did not drive LYCH uptake. Vanadate and probenecid, the latter substance is known to inhibit organic anion transport at the liver canalicular membrane, both strongly decreased the vacuolar uptake of LYCH, while bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, had no effect. Together with the fact that abolishment of the delta pH via CCCP had only a weak influence on LYCH accumulation, our results indicate that this compound is taken up into rye vacuoles by a directly energized process. Uptake of LYCH was strongly inhibited by other sulfated compounds including sulfobromophthalein and the flavones apigenin 7,4'-disulfate and luteolin 7,4'-disulfate arguing for the presence of a vacuolar transporter for structurally different sulphonated or sulfated compounds. Glucuronates like the rye-specific flavone luteolin 7-O-diglucuronide also strongly decreased uptake of the dye, whereas only a weak effect was observed in the presence of glutathione and a glutathione conjugate, suggesting that LYCH uptake is not mediated via the vacuolar glutathione conjugate pump.

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 microM) 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 DeltapH or the DeltaPsi 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 DeltapH, 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-beta,gamma-imidodiphosphate. Our results suggest that different uptake mechanisms exist in the vacuolar membrane, a DeltapH-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.

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-[ß-D-glucuronosyl-(1→2)ß-D-glucuronide] (R2) and luteolin 7-O-[ß-D-glucuronosy 1 (1→2) ß-D-glucuronide]-4'-O-ß-D-glucuronide (R1), as well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific ß-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 ß-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.

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves.

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (λmax 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (λmax 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.

The role of chalcone synthase in the regulation of flavonoid biosynthesis in developing oat primary leaves.

The role of chalcone synthase in the regulation of flavonoid biosynthesis during organogenesis of oat primary leaves has been investigated at the level of enzyme activity and mRNA translation in vitro. Chalcone synthase was purified about 500-fold. The apparent Km values were 1.5 and 6.3 microM for 4-coumaroyl-CoA and malonyl-CoA, respectively. The end products of oat flavonoid biosynthesis, three C-glucosylflavones, did not inhibit the reaction at concentrations as measured up to 60 microM each. Apigenin (4',5,7-trihydroxyflavone), a stable structural analog of the reaction product, 2',4,4',6'-tetrahydroxychalcone, was found to be a strong competitive inhibitor of 4-coumaroyl-CoA binding and a strong noncompetitive inhibitor of malonyl-CoA binding. Although apigenin is not supposed to be an intermediate of C-glucosylflavone biosynthesis, this compound might be a valuable tool for future kinetic studies. To date, there is no indication of chalcone synthase regulation by feedback or similar mechanisms which modulate enzyme activity. Mathematical correlation of chalcone synthase activity and flavonoid accumulation during leaf development, however, indicates that chalcone synthase is the rate-limiting enzyme of the pathway. By in vitro translation studies using preparations of total RNA from different leaf stages, we could demonstrate for the first time that the translational activity of chalcone synthase mRNA undergoes marked daily changes. The high values found at the end of the dark phase suggest that light does not exert direct influence on flavonoid biosynthesis but probably functions by controlling the basic diurnal rhythm.

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.

Acifluorfen-induced isoflavonoids and enzymes of their biosynthesis in mature soybean leaves : whole leaf and mesophyll responses.

Mature soybean (Glycine max L. cv Harosoy 63) leaves normally contain kaempferol-3-glycosides but they accumulate no other flavonoids. Whole leaves sprayed with the diphenyl ether herbicide Acifluorfen and maintained in the light developed small necrotic lesions and accumulated isoflavone aglycones, isoflavone glucosides, and pterocarpans. Isoflavonoid accumulation was preceded by induced activity for chalcone synthase (CHS) and by increased activity for phenylalanine ammonia-lyase (PAL) and UDP-glucose:isoflavone 7-O-glucosyl transferase (IGT). PAL and CHS activity was highest between 24 and 30 hours after treatment, isoflavone aglycones and pterocarpans at 48 hours, IGT at 72 hours, and isoflavone glucosides at 96 hours.Mesophyll cells isolated from control leaves contained no activity for PAL, CHS, or IGT and no flavonoids of any class. Cells isolated from treated leaves at the stage of maximal enzyme activity or isoflavonoid content contained PAL (12% of the whole leaf activity), CHS (24%), IGT (20%), and 25% of the whole leaf isoflavone glucosides, but only traces, presumably as contaminants, of the other flavonoids. We suggest that the isoflavone glucosides were synthesized and accumulated in intact mesophyll cells as soluble detoxification products, while the isoflavone aglycones and pterocarpans accumulated in the epidermis or extracellularly within the mesophyll. To our knowledge this is the first report of tissue-specific induction of isoflavonoid glucosides and key enzymes of their biosynthesis in any plant.

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine: vitexin 2"-O-rhamnoside 7-O-methyltransferase of Avena sativa L.

An O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteine-sepharose. Km values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 microM and 15 microM, respectively. The lack of methyltransfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-O-methylvitexin 2"-O-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.

Application of liquid chromatography to a study on 4-coumarate: coenzyme A ligase activity.

This report describes the separation of components from a 4-coumarate:CoA ligase assay by means of liquid chromatography. With the aid of polyamide column chromatography it is possible to enrich and isolate chromatographically and UV spectroscopically pure p-coumaroyl-CoA using as a solvent 0.01% NH4OH in methanol subsequent to water and methanol alone. High performance liquid chromatography on octadecylsilane-bonded silica stationary phase allows a discontinuous determination of ligase activity. All components - ATP, Coenzyme A, p-coumaric acid, and the products AMP and p-coumaroyl-CoA - can be separated and accurately quantified within 20 min using a water-acetonitrile gradient, containing 1% phosphoric acid. The presented HPLC method may be used to affirm the accuracy of optical tests.

On the localization of enzymes related to flavonoid metabolism in sections and tissues of oat primary leaves.

During growth of the primary leaves of Avena sativa L., the distribution of extractable L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone-flavanone isomerase (CFI, EC 5.5.1.6) activities in distinct leaf sections (top section, medium section and meristematic basal section) and in the epidermal and mesophyll tissues were investigated in relation to C-glycosylflavone accumulation. Characteristic changes have been observed in the levels of PAL and CFI activities within the three leaf sections, depending upon their stage of development. An increase in both enzyme activities accompanies a strong flavone accumulation in the section of the leaf that derives from the basal meristem. Highest specific PAL activity is localized in the meristem itself, which is poor in both flavones and CFI activity. Total flavone accumulation was found to be nearly the same in all three leaf tissues, lower and upper epidermis and mesophyll. Similarly, PAL activity is distributed about equally in these tissues in young leaves; in older ones, activity is relatively higher in the lower leaf epidermis. In contrast, CFI is found to be localized almost entirely in the mesophyll and not in the epiderms. Therefore the question arises whether CFI is involved at all in flavone metabolism and whether it may represent, as a marker enzyme, the localization of other specific C15-enzymes of the flavonoid biosynthetic pathway in oat primary leaves.