Overexpression of RelA/SpoT homologs, PpRSH2a and PpRSH2b, induces the growth suppression of the moss Physcomitrella patens.

Two genes encoding RelA/SpoT homologs, PpRSH2a and PpRSH2b, which are involved in the synthesis of bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) for the stringent response, were isolated from the moss, Physcomitrella patens. A complementary analysis of PpRSH2a and PpRSH2b in Escherichia coli showed that these genes had ppGpp biosynthetic activity. The recombinant PpRSH2a and PpRSH2b were also shown to synthesize ppGpp in vitro. Both proteins were localized to the chloroplasts of P. patens. Expression of the PpRSH genes was induced upon treatment with abscisic acid or abiotic stresses, such as dehydration and UV irradiation. Overexpression of PpRSH2a and PpRSH2b caused suppression of the growth in response to 1% (w/v) of glucose. The present study suggests the existence of a mechanism to regulate the growth of P. patens, which is governed by plant RSH in chloroplasts.

CYP94B3 activity against jasmonic acid amino acid conjugates and the elucidation of 12-O-ß-glucopyranosyl-jasmonoyl-L-isoleucine as an additional metabolite.

The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and inactivation. Oxidation of jasmonoyl-L-isoleucine at the C-12 position, which is catalyzed by cytochrome P450s CYP94B3 and CYP94C1, is thought to be one of the main inactivation pathways. In this study, an additional function of CYP94B3 was elucidated, as well additional jasmonoyl-L-isoleucine metabolites being investigated. It was found that CYP94B3 also catalyzes the hydroxylation of jasmonoyl-L-valine and jasmonoyl-L-phenylalanine, and that these hydroxyl compounds accumulated after wounding and possessed lower activity than non-hydroxylated compounds. Additionally, 12-O-ß-glucopyranosyl-jasmonoyl-L-isoleucine accumulated after wounding, suggesting that it is a metabolite of jasmonoyl-L-isoleucine.

Identification of rice ß-glucosidase with high hydrolytic activity towards salicylic acid ß-D-glucoside.

ß-Glucosidases (EC 3.2.1.21) split ß-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release ß-D-glucose. One of the important functions of plant ß-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonate-related compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid ß-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1 L of culture medium, 2.1 mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 °C. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 °C. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid ß-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid ß-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.

Transportation of de novo synthesized jasmonoyl isoleucine in tomato.

In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[¹³C6]Ile was recovered in distal untreated leaves after wounded leaves were treated with [¹³C6]Ile. However, as [¹³C6]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [¹³C6]Ile and [²H6]JA were applied separately into the wounds on two different leaves, JA-[¹³C6]Ile and [²H6]JA-Ile were detected in the stem exudates but [²H6]JA-[¹³C6]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [²H3]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10 min. Additionally, applying [²H3]JA-Ile to the wounded leaves at concentrations of 10 and 60 nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal.

Biological activity and tissue specific accumulation of fluorescently labeled methyl jasmonate.

The C-7 position of jasmonate is practical for synthesis of a probe to use for chemical biological studies. To confirm the utility, we synthesized fluorescent-labeled methyl jasmonate. The synthesized compound exhibited Arabidopsis thaliana root growth inhibitory and meandering activity, and potent fluorescence was observed inside the root and root hairs.

Antitrypanosomal activities of acetylated bruceines A and C; a structure-activity relationship study.

The crude extract of Brucea javanica showed strong in vitro inhibitory activity against Trypanosoma evansi. Among the isolated quassinoids, bruceines A, C, and bruceantinol were found to be the most potent compounds against T. evansi. To gain a deeper understanding of the relationship between the free hydroxyl groups and the activity, several O-acetylated derivatives of bruceines A and C were synthesized and their in vitro antitrypanosomal activities against trypomastigotes of T. evansi were examined and compared with those of the original compounds. The following structure-activity relationships were observed: (1) the free hydroxyl groups at positions C-3, C-11, and C-12 are essential for antitrypanosomal activity; (2) the C-11 and C-12 hydroxyl groups are more important for the activity than the enolic hydroxyl group at C-3, and; (3) the free hydroxyl group at C-4' of bruceine C does not have any significant effect on the activity.

Tobacco salicylic acid glucosyltransferase is active toward tuberonic acid (12-hydroxyjasmonic acid) and is induced by mechanical wounding stress.

Recently we reported that rice salicylic acid (SA) glucosyltransferase (OsSGT) is active toward 12-hydroxyjasmonic acid (tuberonic acid, TA) and that OsSGT gene expression is induced by wounding stress. Here we report that tobacco SA glucosyltransferase (NtSGT), which is thought to be an ortholog of OsSGT, is also active toward TA. Although NtSGT expression is known to be induced by biotrophic stress, it was also induced by wounding stress in the same manner as OsSGT. These results indicate that this glucosyltransferase is important not only in biotrophic stress but also for wounding stress. It was found that this enzyme is dually functional, with activity both toward TA and SA.

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.

OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice.

Cellulase applied to the leaves of sweet pepper (Capsicum annuum L. var. grossum) upregulates the production of salicylic and azelaic acids.

Treating the leaves of sweet pepper (Capsicum annuum L. var. grossum) with an aqueous solution of cellulase resulted in a four-fold increase in the salicylic acid level compared to a control plant. The level of endogenous azelaic acid was also elevated by the cellulase treatment. Azelaic acid has recently been reported to act as a mobile "priming" agent to arm plants against pathogenic attack. Our results are consistent with this and that the cellulase treatment enhanced the ability of sweet pepper to withstand viral attack.

Distal transport of exogenously applied jasmonoyl-isoleucine with wounding stress.

Determining the mobile signal used by plants to defend against biotic and abiotic stresses has proved elusive, but jasmonic acid (JA) and its derivatives appear to be involved. Using deuterium-labeled analogs, we investigated the distal transport of JA and jasmonoyl-isoleucine (JA-Ile) in response to leaf wounding in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) plants. We recovered [(2)H(2)-2]JA ([(2)H(2)]JA) and [(2)H(3)-12]JA-Ile ([(2)H(3)]JA-Ile) in distal leaves of N. tabacum and S. lycopersicum after treating wounded leaves with [(2)H(2)]JA or [(2)H(3)]JA-Ile. We found that JA-Ile had a greater mobility than JA, despite its lower polarity, and that application of exogenous JA-Ile to wounded leaves of N. tabacum led to a higher accumulation of JA and JA-Ile in distal leaves compared with wounded control plants. We also found that exudates from the stem of S. lycopersicum plants with damaged leaflets contained JA and JA-Ile at higher levels than in an undamaged plant, and a significant difference in the levels of JA-Ile was observed 30 min after wounding. Based on these results, it was found that JA-Ile is a transportable compound, which suggests that JA-Ile is a signaling cue involved in the resistance to biotic and abiotic stresses in plants.

Bioconversion of proposed precursors into theobroxide and related compounds.

We have previously reported a tetraketide origin for theobroxide and its related compound. In the present study, bioconversion of natural and deuterium-labeled precursors of this proposed biosynthetic pathway by Lasiodipoldia theobromae was investigated. Theobroxide was quantified after bioconversion from each proposed precursor. The transformation of the isotopically labeled precursor to products was tracked by 2H NMR measurement.

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.

Chemical inhibitors of viviparous germination in the fruit of watermelon.

It is well known that the seeds of watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai] have a high potential to germinate when the fruit has ripened. When removed from the mature fruit, the seeds can germinate under appropriate conditions. However, it is unclear why they cannot germinate in the flesh of the fruit. Here, we show that cis-ABA and its ß-D-glucopyranosyl ester (ABA-ß-GE) accumulate in the flesh of the fruit at levels high enough to inhibit seed germination. This result indicates the existence of chemical factors that inhibit viviparous seed germination of watermelon.

Identification of a beta-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.).

Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing beta-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an approximately 4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS-PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K(m) and V(max) values of TAG were 31.7 microM and 0.25 microkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific beta-glucosidase hydrolyzing TAG, which releases the physiologically active TA.

Biosynthesis of resorcylic acid lactone (5S)-5-hydroxylasiodiplodin in Lasiodiplodia theobromae.

An administration study of (2)H-labeled precursors showed that the 9-hydroxydecanoyl unit, the acyl intermediate of lasiodiplodin (1), was also the intermediate of (5S)-5-hydroxylasiodiplodin (2) in Lasiodiplodia theobromae. The incorporation of [O-methyl-(2)H(3)]-lasiodiplodin (6) into 2 indicated that hydroxylation at C-5 occurred after cyclization.

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.

Kinetics of the accumulation of jasmonic acid and its derivatives in systemic leaves of tobacco (Nicotiana tabacum cv. Xanthi nc) and translocation of deuterium-labeled jasmonic acid from the wounding site to the systemic site.

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.

Potato tuber cell expansion-inducing activity of stereochemically restricted JA analogs.

Stereochemically restricted analogues of C-7 substituted 7-epi-jasmonate, together with 12-hydroxy jasmonic acid, 12-hydroxy jasmonic acid glucoside, and jasmonic acid conjugated with L-isoleucine (JA-Ile), were synthesized and then tested for potato tuber cell expansion-inducing activity. JA-Ile showed almost the same activity as JA, while the C-7 substituted 7-epi-jasmonates exhibited weaker activity than JA and showed an antagonist effect against JA.

Isolation and structural elucidation of a new cyclohexenone compound from Lasiodiplodia theobromae.

A new cyclohexenone compound was isolated as a mixture of enantiomers from a culture filtrate of Lasiodiplodia theobromae. The relative structure was determined to be 4,5-dihydroxy-3-methyl-cyclohex-2-enone on the basis of MS, (1)H-NMR, and (13)C-NMR spectroscopic analyses, including 2D-NMR experiments. Resolution of the enantiomers was conducted by a coupling reaction with (S)-MTPA-Cl followed by HPLC separation.