Biomedical subjects

Takaya Moriguchi

Publications and source records attributed to Takaya Moriguchi.

10 recordsLinked to original sources

PubMed2006

Polyamine biosynthesis of apple callus under salt stress: importance of the arginine decarboxylase pathway in stress response.

To clarify the involvement of the arginine decarboxylase (ADC) pathway in the salt stress response, the polyamine titre, putrescine biosynthetic gene expression, and enzyme activities were investigated in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] in vitro callus under salt stress, during recovery after stress, and when ADC was inhibited by D-arginine, an inhibitor of ADC. Salt stress (200 mM NaCl) caused an increase in thiobarbituric acid-reactive substances (TBARS) and electrolyte leakage (EL) of the callus, which was accompanied by an increase in free putrescine content, during 7 d of treatment. Conjugated putrescine was also increased, but this increase was limited to the early stage of salt stress. Accumulation of putrescine was in accordance with induction of ADC activity and expression of the apple ADC gene (MdADC). When callus that had been treated with 200 mM NaCl was transferred to fresh medium with (successive stress) or without (recovery) NaCl, TBARS and EL were significantly reduced in the recovery treatment, indicating promotion of formation of new callus cells, compared with the successive stress treatment. Meanwhile, MdADC expression and ADC activity were also decreased in the callus undergoing recovery, whereas those of the callus under successive stress were increased. Ornithine decarboxylase (ODC) activity showed a pattern opposite to that of ADC in these conditions. D-Arginine treatment led to more serious growth impairment than no treatment under salt stress. In addition, accumulation of putrescine, induction of MdADC, and activation of ADC in D-arginine-treated callus were not comparable with those of the untreated callus. Exogenous addition of putrescine could alleviate salt stress in terms of fresh weight increase and EL. All of these findings indicated that the ADC pathway was tightly involved in the salt stress response. Accumulation of putrescine under salt stress, the possible physiological role of putrescine in alleviating stress damage, and involvement of MdADC and ADC in response to salt stress are discussed.

Arginine↗

PubMed2006

Role of polyamines in peach fruit development and storage.

To investigate the role of polyamines in pre- and post-harvest fruit development of 'Akatsuki' peach (Prunus persica (L.) Batsch.) we measured polyamine concentrations, activities of polyamine biosynthetic enzymes and expression of genes encoding these enzymes. Concentrations of the free polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) in pre-harvest fruit peaked 16 days after full bloom (DAF) and then progressively decreased until harvest with the exception of Put, which showed a second peak at 94 DAF, just before the onset of ethylene production. In post-harvest fruit, minor changes in concentrations of Spd and Spm were observed, whereas Put concentration peaked on the harvest day, followed by an abrupt decrease and a subsequent 2-fold increase, which was opposite to the fluctuating pattern of ethylene production. Activities of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) peaked during the first stage of fruit development and then decreased until 80 DAF, after which the activities were below detection limits, suggesting that Put is synthesized during the early stage of fruit development. Activity of S-adenosylmethionine decarboxylase (SAMDC) decreased progressively until the end of S2. Expression levels of five putative polyamine biosynthetic genes, ADC, ODC, SAMDC, spermidine synthase (SPDS) and spermine synthase (SPMS), in pre-harvest and post-harvest fruit did not coincide precisely with the observed changes in enzymatic activities and polyamine concentrations. The possible role of polyamines during peach fruit development and the relationship between polyamines and ethylene biosynthesis are discussed.

Adenosylmethionine Decarboxylase↗

PubMed2005

Two types of spermine synthase gene: MdACL5 and MdSPMS are differentially involved in apple fruit development and cell growth.

Three cDNAs with high homology to spermine (Spm) synthases in Arabidopsis were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. MdACL5-1 and MdACL5-2 have high homology with ACL5 and MdSPMS has high homology with AtSPMS. The similarity of MdSPMS to spermidine synthases (SPDSs) was higher than that of MdACL5s, despite the fact that both are putative Spm synthases. However, MdSPMS could be discriminated from SPDSs by the presence of several characteristic amino acids, i.e., Val-149, Ser-161, Ala-205, and Val-235, in the decarboxylated S-adenosylmethionine (dcSAM)-binding motif of MdSPMS. Both MdACL5-1 and MdSPMS complemented Spm biosynthesis in a yeast mutant deficient in Spm synthase, and ectopic expression of MdACL5-1 in the Arabidopsis dwarf mutant acl5 allowed recovery of the normal phenotype. RNA gel blot analysis showed that MdACL5 and MdSPMS are differentially expressed in tissues and suspension cells. These results suggest that functional MdACL5 and MdSPMS are independently involved in apple fruit development and cell growth.

Amino Acid Sequence↗

PubMed2005

Combinatorial biosynthesis of flavones and flavonols in Escherichia coli.

(2S)-Flavanones (naringenin and pinocembrin) are key intermediates in the flavonoid biosynthetic pathway in plants. Recombinant Escherichia coli cells containing four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:CoA ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-CoA carboxylase, have been established for efficient production of (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. Further introduction of the flavone synthase I gene from Petroselinum crispum under the control of the T7 promoter and the synthetic ribosome-binding sequence in pACYCDuet-1 caused the E. coli cells to produce flavones: apigenin (13 mg/l) from tyrosine and chrysin (9.4 mg/l) from phenylalanine. Introduction into the E. coli cells of the flavanone 3beta-hydroxylase and flavonol synthase genes from the plant Citrus species led to production of flavonols: kaempferol (15.1 mg/l) from tyrosine and galangin (1.1 mg/l) from phenylalanine. The combinatorial biosynthesis of the flavones and flavonols in E. coli is promising for the construction of a library of various flavonoid compounds and un-natural flavonoids in bacteria.

Citrus↗

PubMed2005

Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses.

Two full-length S-adenosylmethionine decarboxylase (SAMDC) cDNAs, MdSAMDC1 and MdSAMDC2, were isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Both cDNAs encoded tiny and small ORFs in addition to the SAMDC ORFs, and genomic sequences of MdSAMDC1 and MdSAMDC2 contained two or three introns in the 5' upstream regions, respectively. Yeast complementation experiment indicated that two MdSAMDCs encoded functional proteins, and that the tiny and small ORFs possibly repressed their translation efficiency. RNA gel blot analysis showed that MdSAMDC1 were differentially regulated in fruits depending on the developmental stage and in cell suspension during the culture period, but MdSAMDC2 did not. In contrast, MdSAMDC2 was positively induced by cold and salt stresses, but MdSAMDC1 was not. These results suggest that MdSAMDC1 is mainly involved in fruit development and cell growth while MdSAMDC2 in stress responses, compared with their respective counterpart.

Adenosylmethionine Decarboxylase↗

PubMed2005

Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots.

Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are two important enzymes responsible for putrescine biosynthesis. In this study, a full-length ADC cDNA (MdADC) was isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Meanwhile, a partial ODC (pMdODC) could be amplified only by a second RCR from the RT-PCR products, whereas a full-length ODC could not be obtained by either cDNA library screening or 5'- and 3'-RACEs, suggesting quite low expression. Moreover, D-arginine, an ADC inhibitor, caused a decrease in ADC activity and severely inhibited the growth of apple callus, which could be partially resumed by exogenous addition of putrescine, whereas alpha-difluoromethylornithine (DFMO), an inhibitor for ODC, caused the incomplete repression of callus growth without changing ODC activity. RNA gel blot showed that the expression level of MdADC was high in young tissues/organs with rapid cell division and was positively induced by chilling, salt, and dehydration, implying its involvement in both cell growth and these stress responses. By contrast, the transcript of ODC could not be detected by RNA gel blot analysis. Based on the present study, it is possible to conclude that (i) the ODC pathway is active in apple, although the expression level of the pMdODC gene homologous with its counterparts found in other plant species is quite low; and (ii) MdADC expression correlates with cell growth and stress responses to chilling, salt, and dehydration, suggesting that ADC is a primary biosynthetic pathway for putrescine biosynthesis in apple.

Amino Acid Sequence↗

PubMed2003

Putative PIP1 genes isolated from apple: expression analyses during fruit development and under osmotic stress.

To gain insight into the function of plasma membrane intrinsic protein (PIP) genes in apple, two genes, MdPIP1a and MdPIP1b, were isolated. MdPIP1 expression was in accordance with the volume increase during fruit development, which is a loading process of water and solutes. In addition, the expression of MdPIP1 was up-regulated in the stems by osmotic stress. These results indicate that MdPIP1 may play important roles not only in fruit expansion, but also in maintaining water homeostasis under stress conditions.

Carrier Proteins↗

PubMed2002

Flavonol synthase gene expression during citrus fruit development.

We isolated a cDNA clone (CitFLS) encoding flavonol synthase (FLS) from the satsuma mandarin (Citrus unshiu Marc.) fruit and investigated the steady state of CitFLS RNA expression during the fruit development. The CitFLS was 1274 bp long, encoded 335 amino acid residues, and belonged to a family of 2-oxoglutarate-dependent dioxygenases. The level of CitFLS transcript was higher in the young leaves than in the old leaves, and it was high at the early developmental stage and low at the mature stage in the juice sacs/segment epidermis (edible part). On the other hand, the CitFLS transcript increased in the peel during fruit maturation. These results indicated that the satsuma mandarin CitFLS was differentially regulated in the developmental stage and in a tissue-specific manner. Additionally, satsuma mandarin peel tissues produced rutin (a flavonol glycoside) from an exogenous dihydroquercetin (taxifolin), indicating the ability of these tissues to produce flavonols.

Journal Article↗

PubMed2002

Functional expression and mutational analysis of flavonol synthase from Citrus unshiu.

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C. Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.

Amino Acid Sequence↗

PubMed2002

Analysis of sucrose synthase genes in citrus suggests different roles and phylogenetic relationships.

The purpose of this work was 2-fold; first, a molecular/evolutionary characterization of three sucrose synthase genes from citrus, and second, an analysis of their differential expression related to potential physiological function. Three non-allelic genes (CitSUS1, CitSUSA and CitSUS2) encoding sucrose synthase were isolated from citrus fruit (Citrus unshiu Marc.). Phylogenetic analysis from the deduced amino acid sequences showed that CitSUS1 and CitSUS2 could be classified into a dicot group. However, CitSUSA, together with Arabidopsis SSA, sugar beet SS and pea SusA defined another dicot group designated SUSA. Unlike other dicot sucrose synthases, these show a distinctive, monocot-like arrangement of introns and exons. The CitSUS1 and CitSUSA were also differentially expressed in leaf, flower and fruit tissues. Contrasting expression patterns were observed for CitSUS1 and CitSUSA in edible tissue (juice sacs/segment epidermis) and peel tissue (albedo/flavedo) of fruit: CitSUS1 mRNA levels decreased throughout fruit development, whereas those of CitSUSA increased. Various sugars also influenced the transcript levels of the CitSUS1 and CITSUSA: These results indicate that the CitSUS1 and CitSUSA genes for sucrose synthase in citrus differ markedly in their molecular structure and potential physiological roles. Sucrose synthase activity in edible tissue was high in the early stages and decreased until mid-develoment, then rapidly increased during maturation. The increase in activity during maturation paralleled that of sucrose accumulation. This result suggests that sucrose synthase has important roles on sugar metabolism when sucrose is accumulated in fruit.

Citrus↗