Flavonoid biosynthesis in barley primary leaves requires the presence of the vacuole and controls the activity of vacuolar flavonoid transport

Abstract

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.

Figure 1.

Scheme of the biosynthesis and compartmentation of saponarin in barley and the effect of the ant310 mutation. Naringenin is formed by the condensation of three molecules of malonyl-CoA and one molecule of 4-coumaroyl-CoA via the CHS followed by ring closure catalyzed by CHI. The following steps of flavone formation toward isovitexin (apigenin 6-C-glucoside) have not been demonstrated in barley up to now, to our knowledge, and are adapted from buckwheat (Kerscher and Franz, 1987, 1988) and marked by a gray background. Isovitexin is detected in barley leaf extracts in low amounts and is processed to saponarin via an UDP-Glc:isovitexin OGT, which is soluble and cytosolic. OGT activity does not appear to be rate limiting (Blume et al., 1979). Finally, saponarin and isovitexin are transported into the vacuole via a flavone glucoside/H⁺-antiporter that is energized by the pH gradient across the tonoplast generated by the activity of the two vacuolar proton pumps, the H⁺-pumping ATPase and pyrophosphatase. The ant310 mutation (italics) causes absence of CHI leading to the formation of a novel substance, isosalipurposide (boxed). CGT, UDP-Glc-dependent C-glucosyltransferase; DIOX, dioxygenase; FNS, flavone synthase (hypothetical).

Figure 2.

Naringenin incubation of ant310 leaf segments fully reconstitutes saponarin biosynthesis. The 4-d-old primary leaf segments of ant310 were incubated in the absence (triangles) or presence of 50 (black circles) or 100 μm (white circles) naringenin for the indicated times. Saponarin content in Ca33787 leaf segments without naringenin (gray squares) are depicted as a control. Saponarin content was measured by HPLC.

Figure 3.

Naringenin incubation of ant310 mesophyll protoplasts leads to cell-autonomous synthesis of saponarin. ant310 mesophyll protoplasts isolated from 7-d-old primary leaves lacking flavonoids were incubated with or without 50 μm naringenin (black and white circles, respectively) in the light for the times indicated. Saponarin content in triplicate samples was determined by HPLC and is expressed based on the chlorophyll content measured in the same sample.

Figure 4.

The presence of the vacuole as a destination compartment is necessary for saponarin reconstitution. ant310 mesophyll protoplasts from 7-d-old primary leaves (white circles) and evacuolated miniprotoplasts (black circles) were incubated with 50 μm naringenin. Depicted is the reconstituted saponarin content based on chlorophyll after different times of naringenin incubation.

Figure 5.

Inhibitor sensitivity of saponarin biosynthesis reconstitution by naringenin in ant310 protoplasts. Isolated ant310 protoplasts were incubated in the absence (control set to 100%) or presence of the inhibitors indicated together with 50 μm naringenin for 5 h at room temperature before protoplast-associated saponarin content was measured by HPLC. As a further control, naringenin incubation of protoplasts on ice was analyzed (cold). NH₄Cl was added at a final concentration of 5 mm, while CCCP and valinomycin concentrations were 10 μm.

Figure 6.

Naringenin incubation of ant310 leaf segments or protoplasts reactivates the vacuolar flavone glucoside transport activity. Vacuoles isolated from 7-d-old ant310 primary leaves incubated in the absence (A) or presence (B) of 50 μm naringenin for 3 h were subjected to transport experiments in the presence of 5 mm MgATP and 100 μm isovitexin for 16 min. Depicted are representative HPLC traces where comparable amounts of vacuole extracts were injected. Isovitexin (peak 1) was only taken up into vacuoles after naringenin preincubation of leaf segments (B). Peak 2 designates the ant310-specific compound isosalipurposide. C and D, Quantification of vacuolar transport experiments with 100 μm saponarin (C, time dependency) and isovitexin (D, uptake after 16 min) as substrates. White circles/bar, Vacuoles isolated from untreated ant310 leaves. Black circles/bar, Vacuoles from ant310 leaves incubated with naringenin. Efficient flavonoid transport reoccurs after treatment of leaf segments or protoplasts with naringenin. vv, Vacuolar volume.

Figure 7.

The reactivation of the vacuolar saponarin transport activity is not immediate. Protoplasts isolated from 7-d-old ant310 leaves were in parallel incubated in the absence (control) or presence of 50 μm naringenin for 10 or 120 min, as indicated, followed by immediate vacuole isolation and saponarin uptake experiment.