Functional characterization and vacuolar localization of fructan exohydrolase derived from onion (Allium cepa).

Fructans such as inulin and levan accumulate in certain taxonomic groups of plants and are a reserve carbohydrate alternative to starch. Onion (Allium cepa L.) is a typical plant species that accumulates fructans, and it synthesizes inulin-type and inulin neoseries-type fructans in the bulb. Although genes for fructan biosynthesis in onion have been identified so far, no genes for fructan degradation had been found. In this study, phylogenetic analysis predicted that we isolated a putative vacuolar invertase gene (AcpVI1), but our functional analyses demonstrated that it encoded a fructan 1-exohydrolase (1-FEH) instead. Assessments of recombinant proteins and purified native protein showed that the protein had 1-FEH activity, hydrolyzing the ß-(2,1)-fructosyl linkage in inulin-type fructans. Interestingly, AcpVI1 had an amino acid sequence close to those of vacuolar invertases and fructosyltransferases, unlike all other FEHs previously found in plants. We showed that AcpVI1 was localized in the vacuole, as are onion fructosyltransferases Ac1-SST and Ac6G-FFT. These results indicate that fructan-synthesizing and -degrading enzymes are both localized in the vacuole. In contrast to previously reported FEHs, our data suggest that onion 1-FEH evolved from a vacuolar invertase and not from a cell wall invertase. This demonstrates that classic phylogenetic analysis on its own is insufficient to discriminate between invertases and FEHs, highlighting the importance of functional markers in the nearby active site residues.

Confirmation of Fructans biosynthesized in vitro from [1-13C]glucose in asparagus tissues using MALDI-TOF MS and ESI-MS.

Accumulation of Fructans was confirmed in asparagus tissues that had been cultured for 2 days on media supplemented with glucose. It is very common that Fructans are biosynthesized from sucrose. We hypothesized however that Fructans could also be biosynthesized from glucose. Stem tissues of in vitro-cultured asparagus were subcultured for 72 h on a medium containing 0.5M of [1-(13)C]glucose. A medium containing 0.5M of normal ((12)C) glucose was used as control. Carbohydrates were extracted from the tissues and analyzed using HPLC, MALDI-TOF MS and ESI-MS. HPLC results indicated that the accumulation of short-chain Fructans was similar in both (13)C-labelled and control samples. Short-chain Fructans of DP=3-7 were detected using MALDI-TOF MS. The molecular mass of each oligomer in the (13)C-labelled sample was higher than the mass of the natural sample by 1 m/z unit per sugar moiety. The results of ESI-MS on the HPLC fractions of neokestose and 1-kestose showed that these oligomers (DP=3) were biosynthesized from exogenous glucose added to the medium. We conclude that not only exogenous sucrose but glucose can induce Fructan biosynthesis; fructans of both inulin type and inulin neoseries are also biosynthesized from glucose accumulated in asparagus tissues; the glucose molecules (or its metabolic products) were incorporated into Fructans as structural monomers.

Effects of various asparagus production methods on rutin and protodioscin contents in spears and cladophylls.

The purpose of this study was to clarify the relationship between various cultivation conditions and the amounts of the rutin (RT) and protodioscin (PD) in asparagus spears. Green and white spears were grown in open culture and under two different blanching conditions. Although RT was detected only in the green spears, PD was detected mainly in white spears produced by covering with soil. The RT and PD contents of cladophylls grown in an open field and in a closed cultivation system were also investigated, and the closed system resulted in cladophylls with low RT and high PD, unlike the open field.

Identification of anthocyanins in the sprouts of buckwheat.

The anthocyanin profiles and varieties/breeding line differences of anthocyanin concentrations in common/tartary buckwheat sprouts have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-galactoside, and cyanidin 3-O-galactopyranosyl-rhamnoside, were isolated from the sprouts of common buckwheat, were separated using high-performance liquid chromatography (HPLC), and were identified using reversed-phase liquid chromatography (LC)/electrospray ionization-mass spectrometry (ESI-MS)/MS techniques. In tartary buckwheat sprouts, two anthocyanins (cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside) were identified. Among 19 common/tartary buckwheat varieties/breeding lines, Hokkai T10 contained the highest amounts of anthocyanins. Cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside concentrations in 6-10 days after seeding sprouts of Hokkai T10 ranged from 0.16 to 0.20 mg/g dry wt and from 5.55 to 6.57 mg/g dry wt, respectively. In addition, dark-grown sprouts of Hokkai T10 accumulated 0.091 and 2.77 mg/g dry wt of cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside whereas other varieties/breeding lines accumulated trace amounts of anthocyanins. Given its anthocyanin-rich red cotyledons, Hokkai T10 is a promising line for use as "Moyashi" type sprouts and is strongly recommended as a new functional food, rich in dietary anthocyanins.