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First published online March 16, 2007; 10.1104/pp.106.094748 Plant Physiology 144:432-444 (2007) © 2007 American Society of Plant Biologists Flavonoid Biosynthesis in Barley Primary Leaves Requires the Presence of the Vacuole and Controls the Activity of Vacuolar Flavonoid Transport1Zurich Basel Plant Science Center, University of Zurich, Plant Biology, CH–8008 Zurich, Switzerland (K.M., M.K.); and University of Cologne, Botanical Institute, D–50931 Cologne, Germany (K.K., G.W.)
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.
One of the major challenges of plant physiology in the postgenome era is to understand the overall regulation of metabolite flow in different pathways in response to developmental, tissue-, or cell-specific programs or environmental factors such as light or stress (Sweetlove and Fernie, 2005  ). Metabolic pathways often span more than one compartment at the cellular level. Compartmentation and controlled transmembrane metabolite transport hold the potential to establish the optimal concentrations of substances necessary for further enzymatic transformation in individual compartments. Subcellular compartmentation of metabolites results in individual pools of different sizes and is important for the local maintenance of high metabolite concentrations or for the separation of competing pathways such as anabolic and catabolic processes (Winkel, 2004). Thus, the analysis of biochemical pathways must also take account of transport processes and the spatial/subcellular organization of enzymes that catalyze consecutive synthetic reactions (Jorgensen et al., 2005). Despite the importance of transport steps for biosynthetic processes or the detoxification of xenobiotics, the mechanisms that allow a metabolic pathway to directly or indirectly control transport activities are largely unknown. This is reflected by the notion that under normal physiological conditions, transport steps are not considered rate limiting.
Flavonoid biosynthesis gives rise to structurally related, but functionally diverse, compounds such as flavonols, flavones, anthocyanins, proanthocyanidins, or isoflavonoids (Dixon and Paiva, 1995
Upon synthesis, many conjugated products of the flavonoid pathway such as flavonol and flavone glycosides or anthocyanins are found predominantly in the vacuole (Graham, 1998
It is generally accepted that flavonoid biosynthesis largely takes place in the cytosol. The prevailing spatial model suggests that the early proteins of the pathway are organized in a multienzyme complex centered around CytP450-dependent monoxygenases on the cytosolic side of the endoplasmic reticulum (ER) forming a functional metabolon (Saslowsky and Winkel-Shirley, 2001
In contrast to our knowledge on flavonoid biosynthetic enzymes and the transcriptional regulation of the corresponding genes, the transport of phenolic compounds into subcellular compartments, facilitating the protection of cytosolic processes against the intrinsic toxicity of these compounds, is only marginally understood both at the mechanistic and regulation levels. While transport of flavonoids across the plasma membrane has not been investigated so far, biochemical analysis of vacuolar uptake mechanisms led to the proposition of different transport and accumulation mechanisms (for review, see Grotewold, 2004
In barley (Hordeum vulgare), the 2-fold glucosylated saponarin (apigenin 6-C-glucosyl-7-O-glucoside) accumulates as the major compound during primary leaf development (Fig. 1
; Reuber et al., 1996
A model system that allows the analysis of a link between a metabolic pathway and the transport processes involved in metabolite translocation into compartments or organelles should fulfill several preconditions. First, it must be possible to control and manipulate the flow through the pathway experimentally. In other words, it must be possible to turn the pathway on and off. This can be done by selecting a mutant plant line with a lesion in a gene coding an enzyme involved in an early step in the pathway. This would allow the pathway to be switched on by applying the chemical product of the affected enzymatic reaction exogenously to effectively complement the mutation. Second, if the transport activity investigated is controlled by the flow through the metabolic pathway, transport is expected to decrease or increase in response to the absence (mutant) or presence of metabolites (chemically complemented mutant). Third, organelle or membrane isolation must be facile and quick to study transport activity changes and storage efficiency in response to alterations in the biosynthetic pathway.
Here, we present an experimental model to investigate the metabolic control mechanism of the flavonoid biosynthetic pathway through the vacuolar transport system. We use the barley anthocyanin-less310 (ant310) mutant, which accumulates less than 5% of the flavonoids of the wild-type Ca33787 (Reuber et al., 1996
Feeding of ant310 Leaf Segments with Naringenin Fully Reconstitutes Saponarin Biosynthesis When naringenin was fed externally to segments of 4-d-old ant310 primary leaves, efficient saponarin biosynthesis was observed within 7 h of incubation (Fig. 2 ). In the presence of 50 µM naringenin in the medium, full reconstitution of saponarin biosynthesis was observed, because saponarin levels reached the amount present in the wild-type Ca33787 leaf segments incubated in the absence of naringenin. Saponarin production was absent in ant310 leaves on control medium, indicating that presence of naringenin was necessary for saponarin production. Furthermore, feeding of ant310 leaves with 100 µM naringenin resulted in less efficient saponarin production, which suggested that 50 µM naringenin was sufficient for efficient saponarin synthesis, while higher concentrations could be toxic. Saponarin production from naringenin in ant310 leaves reached similar levels in 5- and 6-d-old primary leaf segments, while the biosynthesis was reduced in 7- or 8-d-old leaves. In the latter leaves, about 30% of saponarin was formed from naringenin when compared to the corresponding Ca33787 flavonoid content (data not shown). In contrast, secretion of saponarin from leaf segments into the incubation medium never exceeded 5% of the amount formed within the tissue, suggesting that the flavonoids produced in ant310 leaves in the presence of naringenin were efficiently stored within the cells. This result prompted us to investigate the cell autonomy of saponarin reconstitution and vacuolar compartmentation.
For all experiments with protoplast and fractions derived thereof, 7- to 8-d-old primary leaves were used. Older primary leaves were chosen because: (1) protoplast and vacuole experiments required the preparation of large amounts of primary leaf material; and (2) large-scale isolation of protoplasts and vacuoles from younger leaves is difficult. Starch production is marginal in young leaves, which results in the loss of mesophyll protoplasts during density centrifugation and fraction purification. In any case, saponarin production was still accurately measurable by HPLC in protoplasts derived from 7- to 8-d-old leaves.
Mesophyll protoplasts isolated from 7-d-old ant310 primary leaves were incubated in the absence or presence of 50 µM naringenin and the content of saponarin in protoplasts was analyzed by HPLC in a time-course study. As for the leaf segments, ant310 protoplasts were able to synthesize saponarin in the presence of naringenin, reaching maximal levels after 2 to 3 h of incubation, while ant310 protoplasts kept without naringenin did not synthesize saponarin (Fig. 3
). As observed for leaf segments, protoplasts isolated from 7-d-old ant310 leaves reached about 25% to 30% of the saponarin content of Ca33787 protoplasts (data not shown). We investigated whether the saponarin synthesized in ant310 protoplasts during naringenin feeding was stored within the vacuole by performing a compartmentation analysis. The saponarin content was measured by HPLC in protoplast and vacuolar fractions isolated after 6 h of feeding ant310 protoplasts with naringenin. To compare protoplasts and vacuoles, we determined acid phosphatase activity as a vacuolar marker enzyme in the same samples. Reconstituted saponarin synthesized during naringenin incubation of ant310 protoplasts was efficiently transferred into the vacuole, because 95% ± 3% of the saponarin formed during naringenin feeding was found in the vacuolar fraction. Thus, in contrast to our former saponarin transport experiments into ant310 vacuoles (Frangne et al., 2002
We used diphenylboric acid-2-aminoethyl ester to microscopically visualize saponarin accumulation in ant310 protoplasts during reconstitution from naringenin but found that the fluorescence signal obtained does not appropriately detect the vacuolar localization of the barley flavonoids in mesophyll protoplasts (data not shown).
Saponarin was formed during naringenin feeding of ant310 protoplasts and was efficiently stored in the vacuole. We therefore examined whether presence of the vacuole was a prerequisite for saponarin biosynthesis from naringenin. We prepared evacuolated miniprotoplasts from ant310 mesophyll protoplasts lacking the central, acidic vacuole as seen by the absence of a neutral red-stained compartment in miniprotoplasts (data not shown; Hörtensteiner et al., 1992
Because saponarin biosynthesis from naringenin in ant310 was a rather quick process, already observed within up to 30 min (Figs. 3 and 4), we reasoned that the absence of CHI in the mutant did not affect the amount or in situ activity of the biosynthetic enzymes downstream of the isomerase. Indeed, addition of cycloheximid to ant310 protoplasts did not affect saponarin production from externally fed naringenin, suggesting that de novo protein biosynthesis was not required for saponarin biosynthesis and vacuolar storage (Fig. 5 ). In contrast, when carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or NH4Cl, which dissipate pH gradients across membranes, were added to ant310 protoplasts in the presence of naringenin, saponarin production was reduced by about 50% to values observed with protoplasts incubated on ice during naringenin treatment. Changes in the electrical membrane potential, caused by the addition of the K+ ionophore, valinomycin, did not affect saponarin biosynthesis in naringenin-fed ant310 protoplasts (Fig. 5). Thus, the overall inhibitor sensitivity of saponarin reconstitution in ant310 protoplasts resembled the pharmacological profile of the vacuolar saponarin/H+-antiporter (Frangne et al., 2002 ). Based on the absence of saponarin production in miniprotoplasts together with our previous observation of strongly decreased saponarin transport activity in isolated ant310 vacuoles, we hypothesized that the absence of the vacuole or a decrease in the activity of vacuolar transport reduces the capacity to synthesize saponarin.
Naringenin Feeding of ant310 Reactivates the Vacuolar Flavone Glucoside Transporter
Saponarin was efficiently stored in the vacuole during incubation of ant310 protoplasts with naringenin. Thus, the vacuolar transport system should not have limited the overall biosynthetic capacity. In view of our previous data demonstrating strongly reduced saponarin transport in ant310 vacuoles (Frangne et al., 2002
We incubated ant310 leaf segments and protoplasts without or with 50 µM naringenin for 3 h and isolated vacuoles in parallel followed by transport experiments. From our earlier experiments, it was known that the precursor isovitexin as well as saponarin itself were transported into vacuoles of the barley wild type by an H+-antiport with Km values of about 100 µM. Furthermore, saponarin and isovitexin competitively inhibited each other's transport, suggesting that both substances were taken up in barley vacuoles by the same transporter (Klein et al., 1996
In accordance with our former observation (Frangne et al., 2002
The latter notion prompted us to investigate whether naringenin feeding-induced vacuolar transport reactivation in ant310 was a quick or slow process. For this experiment, ant310 protoplasts were isolated and incubated in parallel for 10 min and 2 h with naringenin, before vacuoles were isolated and saponarin transport activities were determined. As a control, vacuoles were prepared from ant310 protoplasts, omitting naringenin treatment (2 h). As can be seen from Figure 7 , 10 min of naringenin treatment of protoplasts was not sufficient for an activation of the vacuolar saponarin transport activity when compared to vacuoles isolated after 2 h of protoplast feeding with naringenin. This result indicated that a metabolite derived from or induced by naringenin reactivated the vacuolar transport activity and not naringenin itself. We concluded that resumed flavone glucoside biosynthesis in ant310 leaves by naringenin feeding restored the vacuolar transporter for these flavone glucosides to a high degree.
Naringenin Feeding Does Not Affect the Activity of the UDP-Glu:Isovitexin 7-O-Glucosyltransferase
Finally, we examined whether a metabolic regulation of a step in flavone glucoside biosynthesis was specific for the vacuolar transport system or also extended to other enzymes. As the only unequivocally defined step in late flavone glucoside biosynthesis in barley, we measured the activity of the UDP-Glc:isovitexin 7-O-glucosyltransferase (OGT) converting isovitexin to saponarin (Fig. 1; Blume et al., 1979
We have previously shown that the vacuolar transport activity for the major barley flavone glucoside saponarin was strongly reduced when vacuoles isolated from ant310 leaves were compared to the corresponding wild type (Frangne et al., 2002 ). The discovery that the mutation affected the CHI gene (Reuber et al., 1997; Druka et al., 2003) suggested that the absence of CHI activity in ant310 in some way inhibited the vacuolar flavonoid transport activity. To examine whether intermediate(s) or products of flavonoid biosynthesis stimulate vacuolar flavonoid transport, we first analyzed whether it is possible to chemically complement the chi mutation in ant310 by external application of naringenin. We demonstrate that full chemical reconstitution of the flavonoid pathway by naringenin is possible in the ant310 mutant, that naringenin conversion to saponarin needs the presence of an intact vacuole, and that reconstitution of the flavonoid pathway by naringenin in the ant310 mutant reactivates the vacuolar flavonoid transporter to a high degree. Thus, we present an experimental model system that allows the detailed analysis of a metabolic linkage between a biosynthetic pathway whose activity can be externally manipulated and a transport step that appears to be necessary for the full activity of the pathway.
The flavonoid biosynthetic pathway represents an important branch of the general phenylpropanoid pathway that also gives rise to other structurally and functionally diverse phenolic compounds, including soluble and cell wall-bound hydroxycinnamic acids, monolignols/lignin, sinapate esters, coumarins, and simple phenolics such as benzoic acid and salicylic acid (Dixon and Paiva, 1995 Here, we demonstrate that exogenous addition of naringenin resulted in efficient synthesis of saponarin, the major flavone glucoside in barley ant310 primary leaves lacking functional CHI (Figs. 2 and 3). In the case of 4-d-old primary leaves, 50 µM naringenin in the medium led to the production of levels of saponarin within 7 h of incubation that are present in the wild type. This result suggests that naringenin is very efficiently taken up by barley leaf cells, is readily converted by all catalytic activities acting downstream of CHI, and is not, therefore, limiting the rate of metabolite flow in the ant310 mutant. Furthermore, flavonoid production occurred in a cell-autonomous manner, because saponarin was synthesized from naringenin in isolated ant310 mesophyll protoplasts (Figs. 3 and 4). Finally, the major end product is efficiently transferred into the vacuole, while in evacuolated miniprotoplasts, lacking the vacuole, saponarin production from naringenin it was completely blocked (Fig. 4).
Studies where exogenous feeding of naringenin is used to analyze the functionality of the flavonoid pathway in intact plants are rare. Using illuminated buckwheat (Fagopyrum esculentum) hypocotyls, Amrhein (1979)
A clear advantage of the barley ant310 primary leaf experimental system to study linkage between flavonoid biosynthesis and vacuolar transport of its products is the relative ease and speed of organelle isolation following the supply of the metabolic precursor. Although Arabidopsis or petunia (Petunia hybrida), as genetically well-defined systems with regard to flavonoid biosynthesis, offer more mutants in important biosynthetic genes, exogenous feeding of precursors followed by the isolation of lytic vacuoles in sufficient amounts for transport experiments is presently only well established in barley. Furthermore, the primary leaf is a well-defined system with respect to flavonoid biosynthesis (Klein et al., 1996
Here, we provide experimental evidence that the vacuolar flavonoid transport system in barley is linked to functional flavonoid biosynthesis. In vacuoles isolated from ant310 leaves, uptake of saponarin and its precursor isovitexin was strongly reduced (Frangne et al., 2002 If the flavonoid pathway generates a metabolite regulating the transport step, it cannot be naringenin itself, because reactivation required a naringenin incubation period of several hours while short (10 min) exposure of ant310 protoplasts to naringenin was not sufficient for efficient transport reactivation (Fig. 7). Furthermore, this metabolite must be absent in ant310 plants and should be specific for the flavonoid pathway. Transport reactivation occurred after naringenin feeding of ant310 leaves and therefore in the presence of the mutant-specific compound isosalipurposide accumulating as a consequence of the absence of CHI. Consequently, isosalipurposide does not likely act as an inhibitor of vacuolar saponarin transport in ant310 leaves (Figs. 1 and 6).
For an alternative proposition, a physical integration of the transport step into the hypothesized metabolon, an intriguing problem has to be addressed: How can an ER-associated biosynthetic complex interact with a vacuolar transporter? In this respect it is interesting to note that cellular inclusions containing flavonoids that may move between different compartments have been repeatedly described in different plant species, e.g. in pathogen-challenged Sorghum bicolor (Snyder and Nicholson, 1990 We did not observe any changes in vacuolar morphology in ant310 leaves or protoplasts, and also, isolation of vacuoles was possible from flavonoid-free protoplasts. It must be assumed, therefore, that the morphology of the central vacuole responsible for saponarin storage is not altered in ant310. Nevertheless, it is possible that the molecularly unidentified flavonoid transporter could travel between two compartments: the ER where it could be activated by a component of the metabolon and where it also could accept the flavonoid substrate as a cargo and the vacuole where transport would occur. Alternatively, flavonoids produced at the ER could be accepted by soluble glutathione S-transferases and handed over to the vacuolar transporter after traveling through the cytosol. Clearly, experiments testing these hypotheses will need to address the cellular localization of the transporter in response to naringenin treatment and the availability of substrates.
The absence and reduction of naringenin-induced saponarin biosynthesis in evacuolated ant310 miniprotoplasts (Fig. 4) and NH4Cl- or CCCP-treated ant310 protoplasts (Fig. 5) suggest that an intact and acidic vacuole is required for saponarin biosynthesis. These experiments do not help determine which hypothesis, described above, is true; however, they do suggest that a destination compartment is necessary for efficient flavonoid production. The efficiency of the elimination of products of the flavonoid pathway from the cytosol to the vacuole appears to affect overall biosynthesis, which presumably involves feedback inhibition. Feedback inhibition of flavonoid biosynthesis resulting from the failure to store flavonoid end products in the vacuole agrees with observations made in antisense experiments of the anthocyanin transporter ZmMRP3 or using the Arabidopsis tt12 mutation, which causes a lesion in the gene encoding a presumptive transporter for proanthocyanidin precursors (Debeaujon et al., 2001
Chemicals
Naringenin was obtained from Sigma (Buchs) and dissolved in 40% (v/v) ethanol/water (stock 1 mg mL–1). The glucosylated apigenins, saponarin and isovitexin, were obtained from Extrasynthese, and flavonoid as well as inhibitor stocks were dissolved in dimethyl sulfoxide. Stock concentrations for saponarin and isovitexin were calculated after measuring the A340 using the molar extinction coefficient for apigenin (
The barley (Hordeum vulgare) lines Ca33787 (wild type) and the ant310 mutant (Jende-Strid, 1993
To achieve maximal turgescence, plants were well watered approximately 30 min before primary leaf segments were cut once above the coleoptile and 1 cm from the tip. This resulted in leaf segments of 3 to 4 and 5 to 6 cm for 4- or 5-d-old primary leaves, respectively. The abaxial epidermis was removed mechanically and leaf segments (4–8 segments per experiment depending on leaf age) were transferred with their abaxial side on 5 or 10 mL 0.1 M KPi buffer, pH 7, in 5- or 9-cm petri dishes, respectively. Reconstitution started by the addition of naringenin solution or solvent to the medium followed by incubation at room temperature on a shaker (50 rpm) close to a window with daylight for the times indicated. Following incubation, the segments were quickly dried with filter paper and the sample fresh weight (FW) was determined. For flavonoid extraction, segment samples were pulverized in the presence of liquid N2 and 5 mL/g FW of 50% (v/v) MeOH/water were added followed by 15-min extraction at room temperature with occasional vortexing. Prior to HPLC analysis, extracts were centrifuged in 1.5-mL reaction tubes at 18,000g for 10 min. Investigation of flavonoid exudates during leaf segment incubation was performed by directly subjecting incubation media aliquots to HPLC analysis.
In comparison to naringenin feeding of 4- or 5-d-old leaf segments, 7-d-old primary leaves were used for all protoplast experiments. Isolation of barley protoplasts and vacuoles followed published procedures (Rentsch and Martinoia, 1991
When protoplasts were used for naringenin feeding without subsequent vacuole isolation, protoplasts were purified using the Percoll step gradient described by Rentsch and Martinoia (1991)
Naringenin feeding experiments with Ca33787 and ant310 protoplasts and miniprotoplasts were performed by adding naringenin to a final concentration of 50 µM to the illuminated cells kept in medium A. At times indicated, triplicate 0.25-mL aliquots were removed and added to 1.5 mL 0.5 M Gly betaine 1 mM CaCl2, 10 mM MES KOH, pH 5.8 (medium B), followed by gentle centrifugation (50g, 5 min). The pellet was resuspended in 0.3 mL medium B, presence of protoplasts or miniprotoplasts was checked by microscopy, and flavonoids were extracted by the addition of 0.2 mL resuspended cells to 0.2 mL MeOH. After 2 h at –20°C, samples were centrifuged for 10 min at 18,000g, and the supernatant was subjected to HPLC analysis. To compare different isolations, 10-µL aliquots were taken from the remaining cell suspension in medium B, added to 1 mL of 80% (v/v) acetone, and total chlorophyll content was determined by spectrophotometric measurement of absorbances at 470, 647, and 663 nm using the equations of Lichtenthaler (1987) Two different naringenin-feeding conditions were used for the demonstration of vacuolar flavonoid transport reactivation following naringenin treatment of ant310 without significant differences in their effectivity. For standard long-term naringenin incubation (2–3 h), 50 µM naringenin (control, solvent only) was present in all media during the preparation of leaf segments, cell wall digestion, and protoplast isolation up to the moment of vacuole lysis. Alternatively, only purified protoplasts were incubated in the presence or absence of naringenin. To avoid cell wall regeneration that would subsequently complicate vacuole isolation, medium A was supplied with 0.5% (w/v) cellulase Y-C and 0.05% (w/v) pectolyase Y-23. In all cases, flavonoid transport experiments were performed immediately after vacuole isolation. For vacuole uptake studies, naringenin-feeding experiments and corresponding controls were in all cases performed in parallel, resulting in a minimum of two independent protoplast and vacuole isolations on the same day.
Vacuolar compartmentation of saponarin reconstituted during naringenin incubation of ant310 protoplasts (3 h) was analyzed by comparing the amount of saponarin in protoplasts and vacuoles isolated from the same naringenin-treated protoplast preparation. To compare both fractions, the activity of the acid phosphatase was determined as a vacuolar marker according to Hörtensteiner et al. (1992)
Flavonoid uptake experiments into vacuoles isolated from ant310 leaves or protoplasts that were incubated either in the presence or absence of naringenin were performed in parallel using the silicone oil centrifugation technique as described previously (Klein et al., 1996
The flavonoid composition of methanolic extracts of leaf segments, protoplasts, miniprotoplasts, and vacuoles was analyzed by HPLC performed identically either on a Shimadzu (experiments performed in Cologne) or on a Gynkothek HPLC system using the following reverse-phase conditions: Nucleosil 100 to 5 C18 column (125 x 4.6 mm; Macherey-Nagel); constant flow rate of 1 mL min–1; solvent A, water/1% (v/v) H3PO4; solvent B, acetonitrile; gradient (B over A in % [v/v]; all changes linear) 0 to 1 min 10% to 10%, 1 to 16 min 10% to 14%, 16 to 26 min 14% to 14%, 26 to 41 min 14% to 22%, 41 to 42 min 22% to 100%, 42 to 43 min 100% to 10%, 43 to 48 min 10% to 10%; detection at 315 nm. The Gynkothek HPLC system was connected to a Dionex diode array detector. Peaks were therefore identified by coelution with authentic standards and due to identity of absorption spectra (220–370 nm).
The activity of the uridine-diphosphate-Glc:isovitexin OGT was measured in crude extracts as follows: segments of 4- to 5-d-old Ca33787 and ant310 primary leaves were incubated in the absence or presence of 50 µM naringenin for 4 h as described above. Liquid N2-frozen samples were pulverized in a mortar and extracted with 6.25 mL/g FW of extraction buffer (0.1 M KPi, pH 7.5, 0.5 mM 1,4-dithioerythritol supplied with 0.2 g Polyclar AT, and 0.2 g Dowex Cl–/g FW) for 15 min on ice. The homogenate was filtered through Miracloth and centrifuged (4°C, 20 min, 15,000g). The supernatant was purified on a PD-10 column that was previously equilibrated with extraction buffer and used immediately for the activity assay. A total of 10 to 50 µL of extract was incubated at 30°C in 0.1 M KPi, pH 7.5, 0.5 mM 1,4-dithioerythritol, 0.4 mg bovine serum albumin, 3 mM UDP-Glc, and 0.2 mM isovitexin in a total volume of 0.1 mL. Assays were stopped by the addition of 1 vol MeOH/1% (v/v) HCl, centrifuged, and the amount of saponarin formed from isovitexin was analyzed by HPLC. The glucosyltransferase activity was linear over time for at least 60 min, and for the protein concentrations, up to 45 µg per assay was used.
The authors thank Christian Frey and Karl Huwiler for taking care of barley plants and Enrico Martinoia and Mark Curtis for discussion and help on the manuscript (all University of Zurich). Received December 13, 2006; accepted March 9, 2007; published March 16, 2007.
1 This work was supported by the Forschungskredit of the University of Zurich (M.K.) and the Deutsche Forschungsgemeinschaft (G.W.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Markus Klein (markus.klein{at}botinst.unizh.ch). www.plantphysiol.org/cgi/doi/10.1104/pp.106.094748 * Corresponding author; e-mail markus.klein{at}botinst.unizh.ch; fax 41–1–634–8204.
Abrahams S, Lee E, Walker AR, Tanner GJ, Larkin PJ, Ashton AR (2003) The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development. Plant J 35: 624–636[CrossRef][ISI][Medline] Achnine L, Blancaflor EB, Rasmussen S, Dixon RA (2004) Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16: 3098–3109 Alfenito MR, Souer E, Goodman CD, Buell R, Mol J, Koes R, Walbot V (1998) Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases. Plant Cell 10: 1135–1149 Amrhein N (1979) Biosynthesis of cyanidin in buckwheat hypocotyls. Phytochemistry 18: 585–589[CrossRef][ISI] Baxter IR, Young JC, Armstrong G, Foster N, Bogenschutz N, Cordova T, Peer WA, Hazen SP, Murphy AS, Harper JF (2005) A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana. Proc Natl Acad Sci USA 102: 2649–2654 Block A, Dangl JL, Hahlbrock K, Schulze-Lefert P (1990) Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter. Proc Natl Acad Sci USA 87: 5387–5391 Blume DE, Jaworski JG, McClure JW (1979) Uridinediphosphate-glucose: isovitexin 7-O-glucosyltransferase from barley protoplasts: subcellular localization. Planta 146: 199–202[CrossRef][ISI] Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA, Taiz L, Muday GK (2001) Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis. Plant Physiol 126: 524–535 Buer CS, Muday GK (2004) The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. Plant Cell 16: 1191–1205 Burbulis IE, Winkel-Shirley B (1999) Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci USA 96: 12929–12934 Chappell J, Hahlbrock K (1984) Transcription of plant defence genes in response to UV light or fungal elicitor. Nature 311: 76–78[CrossRef] Christie JM, Jenkins GI (1996) Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. Plant Cell 8: 1555–1567[Abstract] Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245: 35–47[CrossRef][ISI] Dean JV, Mohammed LA, Fitzpatrick T (2005) The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures. Planta 221: 287–296[CrossRef][ISI][Medline] Debeaujon I, Peeters AJ, Leon-Kloosterziel KM, Koornneef M (2001) The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell 13: 853–871 Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085–1097[CrossRef][ISI][Medline] Dixon RA, Xie D-Y, Sharma SB (2005) Proanthocyanidins: a final frontier in flavonoid research? New Phytol 165: 9–28[CrossRef][ISI][Medline] Druka A, Kudrna D, Rostoks N, Brueggeman R, von Wettstein D, Kleinhofs A (2003) Chalcone isomerase gene from rice (Oryza sativa) and barley (Hordeum vulgare): physical, genetic and mutation mapping. Gene 302: 171–178 |
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ABSTRACT
RESULTS
-N-acetylglucosaminidase, and 
-mannosidase, respectively. In contrast to some dicotyledonous plants, barley was not able to regenerate a new vacuole. When evacuolated miniprotoplasts isolated from ant310 leaves were incubated with naringenin, only very low amounts of saponarin were detected within 6 h, while the corresponding protoplasts reached maximal saponarin levels again after 2 h (
apigenin, 340 nm = 20,800 M–1; Weast, 1982
exc = 380 ± 20 nm;