Kosaku Takahashi

Identification of the bacterial alarmone guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in plants

Stringent control mediated by the bacterial alarmone guanosine 5′-diphosphate 3′-diphosphate (ppGpp) is a key regulatory process governing bacterial gene expression. By devising a system to measure ppGpp in plants, we have been able to identify ppGpp in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to such biotic and abiotic stresses as wounding, heat shock, high salinity, acidity, heavy metal, drought, and UV irradiation. Abrupt changes from light to dark also caused a substantial elevation in ppGpp levels. In vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp, demonstrating the existence of a bacteria-type stringent response in plants. Elevation of ppGpp levels was elicited also by treatment with plant hormones jasmonic acid, abscisic acid, and ethylene, but these effects were blocked completely by another plant hormone, indole-3-acetic acid. On the basis of these findings, we propose that ppGpp plays a critical role in systemic plant signaling in response to environmental stresses, contributing to the adaptation of plants to environmental changes.

Arabidopsis CYP94B3 encodes jasmonyl-l-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate

The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants.

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium, Thermus thermophilus

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product

Natural products display impressive activities against a wide range of targets, including viruses, microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A, and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.

Biosynthesis of jasmonic acid in a plant pathogenic fungus, Lasiodiplodia theobromae

Jasmonic acid (JA) is a plant hormone that plays an important role in a wide variety of plant physiological processes. The plant pathogenic fungus, Lasiodiplodia theobromae also produces JA; however, its biosynthesis in this fungus has yet to be explored. Administration of [1-(13)C] and [2-(13)C] NaOAc into L. theobromae established that JA in this fungus originates from a fatty acid synthetic pathway. The methyl ester of 12-oxo-phytodienoic acid (OPDA) was detected in the culture extracts of L. theobromae by GC-MS analysis. This finding indicates the presence of OPDA (a known intermediate of JA biosynthesis in plants) in L. theobromae. (2)H NMR spectroscopic data of JA produced by L. theobromae with the incorporation of [9,10,12,13,15,16-(2)H(6)] linolenic acid showed that five deuterium atoms remained intact. In plants, this is speculated to arise from JA being produced by the octadecanoid pathway. However, the observed stereoselectivity of the cyclopentenone olefin reduction in L. theobromae was opposite to that observed in plants. These data suggest that JA biosynthesis in L. theobromae is similar to that in plants, but differing in the facial selectivity of the enone reduction.

Bacterial Alarmone, Guanosine 5′-Diphosphate 3′-Diphosphate (ppGpp), Predominantly Binds the β′ Subunit of Plastid-Encoded Plastid RNA Polymerase in Chloroplasts

Its alarming: Bacterial alarmone guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp), which is a key regulatory molecule that controls the stringent response, also exists in chloroplasts of plant cells. Cross‐linking experiments with 6‐thioguanosine 5′‐diphosphate 3′‐diphosphate (6‐thioppGpp) and chloroplast RNA polymerase indicate that ppGpp binds the β′ subunit of plastid‐encoded plastid RNA polymerase that corresponds to the Escherichia coli β′ subunit.

Novel cyclic peptide, epichlicin, from the endophytic fungus, Epichloe typhina.

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC50 value of 22 nM.

Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha

12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JA-dependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha.

Cloning and Functional Analysis of an Allene Oxide Synthase in Physcomitrella patens

Jasmonic acid (JA) is a plant hormone that plays important roles in a large number of processes in stress adaptation and development in flowering plants. A search of genome database indicated the existence of allene oxide synthase (AOS), an enzyme of JA biosynthesis, in Physcomitrella patens, a model plant among mosses. In this study, the presence of JA was detected in P. patens. The recombinant AOS of P. patens, which was overexpressed in Escherichia coli, showed AOS activity. These data suggest that the octadecanoid pathway also exists in P. patens.

Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid.

Tuberonic acid (12-hydroxy epi-jasmonic acid, TA) and its glucoside (TAG) were isolated from potato leaflets (Solanumtuberosum L.) and shown to have tuber-inducing properties. The metabolism of jasmonic acid (JA) to TAG in plant leaflets, and translocation of the resulting TAG to the distal parts, was demonstrated in a previous study. It is thought that TAG generated from JA transmits a signal from the damaged parts to the undamaged parts by this mechanism. In this report, the metabolism of TA in higher plants was demonstrated using [12-(3)H]TA, and a glucosyltransferase active toward TA was purified from the rice cell cultures. The purified protein was shown to be a putative salicylic acid (SA) glucosyltransferase (OsSGT) by MALDI-TOF-MS analysis. Recombinant OsSGT obtained by overexpression in Escherichia coli was active not only toward TA but also toward SA. The OsSGT characterized in this research was not specific, but this is the first report of a glucosyltransferase active toward TA. mRNA expressional analysis of OsSGT and quantification of TA, TAG, SA and SAG after mechanical wounding indicated that OsSGT is involved in the wounding response. These results demonstrated a crucial role for TAG not only in potato tuber formation, but also in the stress response in plants and that the SA glucosyltransferase can work for TA glucosylation.