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Two cultivars of Perilla frutescens, red and green formas are known to differ in anthocyanin accumulation in leaves and stems. cDNA clones encoding the enzymes involved in anthocyanin biosynthesis, chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and UDP glucose: flavonoid 3-O-glucosyltransferase (3GT), were isolated from cDNA libraries derived from the leaves of a red forma of P. frutescens by screening with partial fragments amplified by means of polymerase chain reaction (PCR) and heterologous cDNAs as probes. The deduced amino acid sequences of these four genes exhibited 40-90% identity with those reported for the corresponding gene from other unrelated species. Southern blot analysis for these genes and two other structural genes, the leucoanthocyanidin dioxygenase (LDOX, anthocyanidin synthase) and anthocyanin acyltransferase (AAT) genes, indicated that each gene comprises a small multi-gene family. More than three copies of the CHS gene are present, two copies of the other genes being present. The expression of five genes, the exception being the CHS gene, was detected only in red leaves of the red forma of P. frutescens, i.e. not in green leaves of the green forma plant. The CHS gene was expressed in both red and green leaves, but 10-fold more in red leaves than in green leaves. These results suggest that the expression of all structural genes examined is coordinately regulated in a forma-specific manner. Under weak-light conditions, the accumulation of both anthocyanin and mRNAs of biosynthetic enzymes was lower in leaves of the red forma. High-intensity white light coordinately induced the accumulation of transcripts of all six genes examined in the mature leaves of red P. frutescens.

Alliinase [S-alk(en)yl-L-cysteine sulfoxide lyase], a pyridoxal-phosphate-(Pxy-P)-dependent enzyme, is responsible for the degradative conversion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfurcontaining metabolites in Allium plants. We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis. A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A. tuberosum by hybridization screening with a synthetic 50-residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic. The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54 083 Da. The deduced amino acid sequence exhibited 66Ϫ69% identities with those of reported alliinases from onion, garlic and shallot. The partial amino acid sequence, which was determined for a V8 protease-digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the cDNA. An expression vector of recombinant alliinase cDNA was constructed in yeast. The catalytically active protein was in the soluble fraction of transformed yeast. Site-directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy-P-binding residue. The mRNA expression of the alliinase gene comprising a multigene family in the shoots of green plants was twofold higher than that in the roots of green plants ; however, the expression in the shoots of etiolated plants was only 13% that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants. Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A. tuberosum.Keywords : Allium; alliinase ; cDNA cloning; pyridoxal 5′-phosphate; sulfur metabolism.The plants belonging to genus Allium generally produce characteristic odorous sulfur-containing compounds. These volatile sulfur compounds are formed from non-volatile S-alk(en)yl-L-cysteine sulfoxide (ACS), as a precursor, through enzymatic degradation, which is catalyzed by so-called alliinase [S-alk-(en)yl-L-cysteine sulfoxide lyase] [1, 2]. In onion (Allium cepa) cells, alliinase is assumed to be localized in vacuoles, thereby being separated from ACS present in the cytosol [3]. Upon rupture of the cells, the enzyme can react with the substrate to yield sulfenic acid as the first sulfur-containing volatile compound (Scheme 1). Numerous sulfur-containing metabolites, such as thiosulfinates, thiols, disulfides, etc. are secondarily produced from sulfenic acid through non-enzymatic reactions [4]. These

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