Hisashi Miyagawa

Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield

Increases in the yield of rice, a staple crop for more than half of the global population, are imperative to support rapid population growth. Grain weight is a major determining factor of yield. Here, we report the cloning and functional analysis of THOUSAND-GRAIN WEIGHT 6 (TGW6), a gene from the Indian landrace rice Kasalath. TGW6 encodes a novel protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. In sink organs, the Nipponbare tgw6 allele affects the timing of the transition from the syncytial to the cellular phase by controlling IAA supply and limiting cell number and grain length. Most notably, loss of function of the Kasalath allele enhances grain weight through pleiotropic effects on source organs and leads to significant yield increases. Our findings suggest that TGW6 may be useful for further improvements in yield characteristics in most cultivars.

The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production

The upregulation of the tryptophan (Trp) pathway in rice leaves infected by Bipolaris oryzae was indicated by: (i) enhanced enzyme activity of anthranilate synthase (AS), which regulates metabolic flux in the Trp pathway; (ii) elevated levels of the AS (OASA2, OASB1, and OASB2) transcripts; and (iii) increases in the contents of anthranilate, indole, and Trp. The measurement of the contents of Trp-derived metabolites by high-performance liquid chromatography coupled with tandem mass spectrometry revealed that serotonin and its hydroxycinnamic acid amides were accumulated in infected leaves. Serotonin accumulation was preceded by a transient increase in the tryptamine content and by marked activation of Trp decarboxylase, indicating that enhanced Trp production is linked to the formation of serotonin from Trp via tryptamine. Feeding of radiolabeled serotonin to inoculated leaves demonstrated that serotonin is incorporated into the cell walls of lesion tissue. The leaves of a propagating-type lesion mimic mutant (sl, Sekiguchi lesion) lacked both serotonin production and deposition of unextractable brown material at the infection sites, and showed increased susceptibility to B. oryzae infection. Treating the mutant with serotonin restored deposition of brown material at the lesion site. In addition, the serotonin treatment suppressed the growth of fungal hyphae in the leaf tissues of the sl mutant. These findings indicated that the activation of the Trp pathway is involved in the establishment of effective physical defenses by producing serotonin in rice leaves.

LPT1 encodes a membrane-bound O-acyltransferase involved in the acylation of lysophospholipids in the yeast Saccharomyces cerevisiae.

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156–22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Δlpt1 mutant strain. Although the Δlpt1 mutant strain did not show other detectable defects, the Δlpt1 Δslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata

cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetle Leptinotarsa decemlineata (LdEcR and LdUSP) was conducted. Amino‐acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor. Northern hybridization analysis showed a 12.4‐kb message for the LdEcR A‐isoform, a 10.5‐kb message for the LdEcR B1‐isoform and a 5.7‐kb message for the LdUSP, in fat body, gut, integument, testis and ovaries. In developmental profile studies, expression of both the LdEcR and LdUSP transcript in integument changed dramatically. In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response element, although LdUSP alone did not, and this binding was dramatically enhanced by the addition of LdUSP. LdEcR/LdUSP complex also showed significant binding to an ecdysone agonist, ponasterone A (KD = 2.8 nm), while LdEcR alone showed only weak binding (KD = 73.4 nm), and LdUSP alone did not show any binding. The receptor‐binding affinity of various ecdysone agonists to LdEcR/LdUSP was not correlated to their larvicidal activity to L. decemlineata. From these results, it was suggested that multiple factors including the receptor binding affinity are related to the determination of the larvicidal activity of nonsteroidal ecdysone agonists in L. decemlineata.

Quantification of indole-3-acetic acid and amino acid conjugates in rice by liquid chromatography-electrospray ionization-tandem mass spectrometry.

A method for quantifying indole-3-acetic acid (IAA) and its conjugates with the six amino acids, Ala, -Asp, -Ile, -Glu, -Phe and -Val, in rice (Oryza sativa) by using high-performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (HPLC–ESI–MS/MS) is described. Samples from the rice plant or callus were treated with 80% acetone in water containing 2.5 mM diethyl dithiocarbamate. Each extract was partially purified in C18 cartridge column for solid-phase extraction (SPE) and subjected to HPLC–ESI–MS/MS without converting the product. The detection limit was 3.8 fmol for IAA, and 0.4–2.9 fmol for the IAA amino acid conjugates. The method was applied to the analysis of IAA and its conjugates in rice seedlings, dehulled rice and calli, using 20–100 mg tissue samples.

Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan.

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.

Induction of hydroxyanthranilate hydroxycinnamoyl transferase activity by oligo-N-acetylchitooligosaccharides in oats

Abstract An assay method for hydroxycinnamoyl-CoA: hydroxyanthranilate N -hydroxycinnamoyl transferase (HHT) in oat leaves ( Avena sativa L.), which is thought to be one of the key enzymes for the biosynthesis of avenanthramides, phytoalexins in this plant, was established. HHT activitiy was induced by treating the leaves with oligo- N -acetylchitooligosaccharides. Among the chitooligosaccharides tested, penta- N -acetylchitopentaose ((GlcNAC) 5 ) was the most effective in inducing activity. The induction by (GlcNAc) 5 was dose-dependent, in which case HHT activity was initially detected after 6 hr and reached a maximum by 12 hr. All of the putative precursors of avenanthramides acted as substrates for HHT, with 5-hydroxyanthranilic acid and feruloyl-CoA being the best substrates for the anthranilic moiety and the cinnamoyl moiety of avenanthramides, respectively.

Involvement of Ca2+ ion in phytoalexin induction in oats

Abstract The involvement of cytosolic Ca2+ in the signal transduction mechanism of phytoalexin induction was examined in oats. The treatment with a calcium ionophore, A23187, strongly induced the accumulation of avenanthramide A, a major phytoalexin in oats, in a dose-dependent manner. The induction of avenanthramide A by A23187 was suppressed by Mn2+ and EGTA, suggesting that the elevation of cytosolic Ca2+ by the ionophore caused phytoalexin production. The effects of various inhibitors of Ca2+-dependent processes on the elicitor activity of penta-N-acetylchitopentaose ((GlcNAc)5) were also examined. Treatment of oat leaf segments with three calcium channel blockers, LaCl3, verapamil and nifedipine inhibited the phytoalexin induction by (GlcNAc)5 at sub-millimolar levels. TMB-8 and ruthenium red, inhibitors of intracellular calcium movement, also abolished the phytoalexin induction. These findings indicate that Ca2+ is involved in the induction of the plant responses by elicitors.

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.

Three-dimensional quantitative structure: activity relationship analysis of acyclic and cyclic chloronicotinyl insecticides

The binding activity of chloronicotinyl insecticides, including acetamiprid, nitenpyram and related compounds, to the nicotinic acetylcholine receptors (nAChR) of houseflies was measured. These compounds were defined as ‘acyclic’ compounds. Variations in the binding activity were analysed using comparative molecular field analysis (CoMFA) which is a technique for the analysis of three-dimensional quantitative structure–activity relationships. The CoMFA results showed that steric interactions were more significant for the acyclic compounds than for imidacloprid and its derivatives (cyclic compounds). It was also shown that the acyclic compounds could bind to housefly-nAChR in a similar manner to the cyclic compounds, and that the electrostatic natures of the acyclic amino- and cyclic imdazolidine-moieties affected their binding activity. © 2000 Society of Chemical Industry