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-Oglucoside), 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 1 -antiporter. Thus, the barley ant310 mutant represents a novel model system to study the interplay between flavonoid biosynthesis and the vacuolar storage mechanism.

MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides and Glycoside Malonates inMedicago truncatula

“…Several known flavonoid MATE transporters, including AM1, AM3, MATE1, and TT12, are localized to the tonoplast membrane, as shown by imaging of green fluorescent protein (GFP)-tagged transporters or membrane proteomics studies (Marinova et al, 2007a;Gomez et al, 2009;Zhao and Dixon, 2009). Topological prediction suggests that MATE2 has 12 putative transmembrane domains in the same pattern as other known MATE transporters (see Supplemental Figure 12 online).…”

“…Dysfunction of the vacuole or loss of function of vacuolar flavonoid transporters can reduce flavonoid accumulation (Marinova et al, 2007a(Marinova et al, , 2007b. The ABC transporter MRP3 and a few MATE transporters (AM1 and AM3) have been suggested to transport anthocyanins into the vacuole (Goodman et al, 2004;Gomez et al, 2009).…”

“…MATE2 is phylogenetically related to the known flavonoid transporters MATE1, TT12, AM1, and AM3, all of which appear to be localized to the vacuolar membrane on the basis of GFP fusion imaging (Marinova et al, 2007a;Gomez et al, 2009;Zhao and Dixon, 2009). Using the same approach, MATE2-GFP also appears to be targeted to the vacuolar membrane in plant cells (tobacco, Arabidopsis, and onion epidermal cells) as well as in yeast cells.…”

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

“…These metabolites may otherwise display adverse activities for the producing plant cell. Intracellular storage of such biologically active metabolites in the vacuole is well established for water-soluble compounds and for compounds that become water soluble through conjugation (Marinova et al, 2007;Ferreres et al, 2011;Zhao et al, 2011;Li et al, 2012b). Similarly, for the large class of often-lipophilic terpenoids, it has been suggested that their intracellular accumulation may be limited by nonspecific interference with cellular Figure 6.…”

ABCC1, an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin 3-O-Glucosides

“…Once the biosynthesis is completed, including the additional chemical modifications (glycosylation, methylation, and acylation;Winkel-Shirley, 2008), anthocyanins are transported into the vacuole. A report has shown that vacuolar accumulation of flavonoids is a prerequisite for their biosynthesis and thus fulfils a regulatory function during synthesis (Marinova et al, 2007a). The mechanisms by which anthocyanins are transported into the vacuole have long been debated.…”