| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Two cDNAs encoding 4-hydroxybeazoic acid (4HB) : geranyltransferase involved in shikonin biosynthesis, which accept 4HB as the prenyl acceptor and geranylpyrophosphate as the prenyl donor, were isolated by nested RT-PCR from cultured cells of Lithospermum erythrorhizon. The identity of the putative amino acid sequences of them, designated LePGT-1 and LePGT-2, was 90 %, whereas the identities with the 4HB : hexaprenyltranseferase, a biosynthetic enzyme of ubiquinone in yeast, was only 38 %. Contrary to the yeast prenyltransferase, the mitochondrial targeting sequence is lacking in the Lithospermum proteins. Northern analyses showed that the expression pattern of both cDNA is in conformity with that of geranyltransferase activity which are regulated by addition of ammonium ion, auxin, and methyl jasmonate, as well as light irradiation, a strong inhibitor of geranyltransferase expression. The enzyme activity of both clones were measured by HPLC with the recombinant ptotein expressed in ye
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ast whose 4HB : hexaprenyltranseferase was disrupted, and a strong 4HB : geranyltransferase activity was detected in the microsomal fractions of the transformants for both cDNAs. They showed strict substrate specificity to geranyl diphosphate, while the mitochondrial 4HB : prenyltransferase showed higher preference to geranylgeranyl diphosphate as the substrate. This is the first geranyltransferase which takes an aromatic acceptor in plant secondary metabolism.
In the intact plant of L. erythrorhizon, shikonin derivatives are exclusively accumulated in the root epidermis, whereas the other secondary metabolites as rosmarinic acid are also localized in cortex. This tissue specificity for shikonin accumulation was further examined by in situ hybridization to assess the contribution of LePGTs' expression using Lithospermum hairy root as a model material. The results showed that LePGT-1-specific signal was solely detected in the epidermis and root hairs. This data indicates that the expression pattern of LePGT-1 largely contribute to the tissue specificity of shikonin accumulation as a key biosynthetic enzyme. Less
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