Kouichi Mizuno

Identification, cDNA Cloning, and Gene Expression of Soluble Starch Synthase in Rice (Oryza sativa L.) Immature Seeds

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatography. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in [lambda]gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.

Sequence conservation of the catalytic regions of anylolytic enzymes in maize branching enzyme-I

We have identified cDNA clones encoding branching enzyme-I (BE-I) from a maize kernel cDNA library. The combined nucleotide sequence of the cDNAs indicates that maize BE-I is initially synthesized as a precursor protein with a putative 64-residue transit peptide at the amino terminus, and that the mature enzyme contains 759 amino acid residues with a calculated molecular mass of 86,236 Da. The four regions, which constitute the catalytic site of amylolytic enzymes, are conserved in the sequences of BE-I and bacterial branching enzymes. This result demonstrates that branching enzyme belongs to a family of the amylolytic enzymes. The BE-I gene is highly expressed in the early stages of kernel development, and the level of the message concentration decreases slowly as kernel maturation proceeds.

Plant biotechnology: Caffeine synthase gene from tea leaves

Caffeine synthase is an enzyme that catalyses the final two steps in the caffeine biosynthesis pathway. We have cloned the gene encoding caffeine synthase from young leaves of tea (Camellia sinensis), opening up the possibility of creating tea and coffee (Coffea arabica) plants that are naturally deficient in caffeine. Consumers concerned about the possible adverse effects of caffeine consumption will welcome this development towards caffeine-free drinks that retain their flavour.

Isolation of a new dual‐functional caffeine synthase gene encoding an enzyme for the conversion of 7‐methylxanthine to caffeine from coffee (Coffea arabica L.)1

In coffee and tea plants, caffeine is synthesized from xanthosine via a pathway that includes three methylation steps. We report the isolation of a bifunctional coffee caffeine synthase (CCS1) clone from coffee endosperm by reverse transcription‐polymerase chain reaction (RT‐PCR) and rapid amplification of cDNA ends (RACE) technique using previously reported sequence information for theobromine synthases (CTSs). The predicted amino acid sequences of CCS1 are more than 80% identical to CTSs and are about 40% similar to those of tea caffeine synthase (TCS1). Interestingly, CCS1 has dual methylation activity like tea TCS1.

Caffeine synthase gene from tea leaves.

Caffeine synthase is an enzyme that catalyses the final two steps in the caffeine biosynthesis pathway. We have cloned the gene encoding caffeine synthase from young leaves of tea (Camellia sinensis), opening up the possibility of creating tea and coffee (Coffea arabica) plants that are naturally deficient in caffeine. Consumers concerned about the possible adverse effects of caffeine consumption will welcome this development towards caffeine-free drinks that retain their flavour.

Molecular analysis of the gene encoding a rice starch branching enzyme

SummaryThe sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5′ flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5′ half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.

Coordinated Regulation of the Genes Participating in Starch Biosynthesis by the Rice Floury-2 Locus

The recessive floury-2 (flo-2) locus of rice (Oryza sativa L.), which is located on chromosome 4, causes a strong reduction in expression of the gene encoding an isoform of branching enzyme RBE1 in immature seeds 10 d after flowering. Mapping of the RBE1 gene demonstrated the localization on rice chromosome 6, suggesting that the wild-type Floury-2 (Flo-2) gene regulates RBE1 gene expression in trans. However, reduced expression of the genes encoding some other starch-synthesizing enzymes, including another isoform of branching enzyme RBE3 and granule-bound starch synthase, was also found in the flo-2 seeds. In spite of the low level of RBE1 gene expression in the immature seeds of the flo-2 mutants, the RBE1 gene was equally expressed in the leaves of the wild type and flo-2 mutants. Thus, these results imply that the Flo-2 gene may co-regulate expression of some of the genes participating in starch synthesis possibly in a developing seed-specific manner.

Cloning, Expression, and Characterization of an Antifungal Chitinase from Leucaena leucocephala de Wit

Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by PCR amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening. Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids. Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli produced high chitinase activity in the cell lysate. The recombinant thioredoxin fusion protein was purified and cleaved to yield a 32-kDa chitinase. The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity. It also inhibited growth of 13 of the 14 fungal strains tested.

Regulation of tuber formation and ADP-glucose pyrophosphorylase (AGPase) in sweet potato (Ipomoea batatas (L.) Lam.) by nitrate

The relationship between nitrate fertilization and tuber-bulking inroots from single-leaf explants was examined to clarify the mechanism of tuberformation. Cuttings of sweet potato leaves were grown on a medium containing 1,10, 30, and 50 mM nitrate and analyzed with regard tomorphologicaland biochemical traits. The cuttings grown on higher concentrations (≥10mM) showed inhibited root growth, with no apparent effect on leafgrowth or root length. The roots accumulated starch in a medium containing alower nitrate concentration (≤10 mM). Under this culturecondition, both the ADP-glucose pyrophosphorylase (AGPase) transcript level andthe starch content in the root decreased, while there was no decrease in thestarch content in the leaf. The greater AGPase transcript level in rootsgrowingwith a higher nitrate concentration (≥10 mM) might be caused bynitrogen-mediated signalling and/or changes in the levels of starch metabolism.Starch synthesis via AGPase and cell proliferation may work together for tuberformation in sweet potato root.

Regulated expression of ADPglucose pyrophosphorylase and chalcone synthase during root development in sweet potato

The phase transition in sweet potato root during tuber differentiationis a complex developmental process that involves changes in gene expression andmorphogenesis. Among the three kinds of root in sweet potato (white fibrousroot, thick-pigmented root and lateral root), ADP-glucose pyrophosphorylase(AGPase) and chalcone synthases (CHS) are expressed only in thick-pigmentedroots after 3 weeks, and this also depends on the developmental stage. Sinceexposing roots to the light or culturing under hydroponic conditions inhibitstuber formation in sweet potato, the expression of AGPase and CHS was studiedunder light and dark conditions. AGPase and CHS expression in sweet potatorootswas suppressed very sensitively by light or water stress, similar to rootdevelopmental patterns. Based on an analysis of AGPase and CHS expression indifferent kinds of root tissues and in different developmental stages, thesegenes were shown to be closely associated with the differentiation ofthickeningpigmented roots.