SummaryAureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrosedensity gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H þ -translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing ␤-glucosidase. Here we report on the purification and cDNA cloning of this important ␤-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing ␤-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6؆-O-malonyl-␤-D-glucoside) to produce free genistein (k cat , 98 s ؊1 ; K m , 25 M at 30°C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous ␤-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.

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Two-dimensional nanoarrays of Ge quantum dots (QDs) with the ability to self-repair were epitaxially grown by self-organization on Si substrates using an ultrathin SiO(2) film technique. Nanometer-sized voids were patterned on ultrathin SiO(2) films by transcription of the pattern of block copolymer films using a selective etching method and worked as nucleation sites for QD growth. The epitaxial QDs were elastically strain-relaxed without misfit dislocations and of uniform size. The epitaxial structures of Si-capped QD nanoarrays exhibited strong photoluminescence near 1.5 microm.

The formation mechanism for the potent antioxidative o-dihydroxyisoflavones, 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), was studied by incubating whole soybeans in a solid culture and a soybean extract in a liquid culture with Aspergillus saitoi. Analyses of changes in the isoflavone analogue content, β-glucosidase activity, and isoflavone hydroxylation ability indicated that 8-OHD and 8-OHG were formed from daidzein and genistein, respectively, by microbial hydroxylation, being respectively liberated from daidzin and genistin by β-glucosidase from A. saitoi during incubation. No selective hydroxylation reaction at the 8-position of daidzein and genistein were apparent during the vegetative stage, but were induced at the stage of sporulation.