A gain-of-function mutation in Msl10 triggers cell death and wound-induced hyperaccumulation of jasmonic acid in Arabidopsis.

Jasmonates (JAs) are rapidly induced after wounding and act as key regulators for wound induced signaling pathway. However, what perceives the wound signal and how that triggers JA biosynthesis remains poorly understood. To identify components involved in Arabidopsis wound and JA signaling pathway, we screened for mutants with abnormal expression of a luciferase reporter, which is under the control of a wound-responsive promoter of an ethylene response factor (ERF) transcription factor gene, RAP2.6 (Related to APetala 2.6). The rea1 (RAP2.6 expresser in shoot apex) mutant constitutively expressed the RAP2.6-LUC reporter gene in young leaves. Along with the typical JA phenotypes including shorter petioles, loss of apical dominance, accumulation of anthocyanin pigments and constitutive expression of JA response gene, rea1 plants also displayed cell death and accumulated high levels of JA in response to wounding. The phenotype of rea1 mutant is caused by a gain-of-function mutation in the C-terminus of a mechanosensitive ion channel MscS-like 10 (MSL10). MSL10 is localized in the plasma membrane and is expressed predominantly in root tip, shoot apex and vascular tissues. These results suggest that MSL10 is involved in the wound-triggered early signal transduction pathway and possibly in regulating the positive feedback synthesis of JA.

Soybean oil biosynthesis: role of diacylglycerol acyltransferases.

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to form seed oil triacylglycerol (TAG). To understand the features of genes encoding soybean (Glycine max) DGATs and possible roles in soybean seed oil synthesis and accumulation, two full-length cDNAs encoding type 1 diacylglycerol acyltransferases (GmDGAT1A and GmDGAT1B) were cloned from developing soybean seeds. These coding sequences share identities of 94 % and 95 % in protein and DNA sequences. The genomic architectures of GmDGAT1A and GmDGAT1B both contain 15 introns and 16 exons. Differences in the lengths of the first exon and most of the introns were found between GmDGAT1A and GmDGAT1B genomic sequences. Furthermore, detailed in silico analysis revealed a third predicted DGAT1, GmDGAT1C. GmDGAT1A and GmDGAT1B were found to have similar activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-diacylglycerol were preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. Both transcripts are much more abundant in developing seeds than in other tissues including leaves, stem, roots, and flowers. Both soybean DGAT1A and DGAT1B are highly expressed at developing seed stages of maximal TAG accumulation with DGAT1B showing highest expression at somewhat later stages than DGAT1A. DGAT1A and DGAT1B show expression profiles consistent with important roles in soybean seed oil biosynthesis and accumulation.

A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources.

Six-carbon (C(6)) aldehydes and alcohols are important components of the aroma and flavor of fruits and vegetables. Soybean lipoxygenase (LOX) isozyme LOX 3 was reported not only to produce less 13-hydroperoxides, precursors of C(6) aldehydes, but also to convert them to ketodiene products. Here, we examined the effects of LOX 3 on hexenal formation from linolenic acid homogenized with watermelon 13-hydroperoxide lyase (HL)-overexpressing Nicotiana tabacum leaves and soybean acetone powder. Compared to the wild type, which contains LOXs 1, 2, and 3, the elimination of LOX 3 in LOX 1 + 2 facilitates greater production of hexenals. The use of LOX 2 alone yielded the highest hexenal production, while a two-step conversion was required for LOX 1 to produce hexenals at high levels due to different pH optima of the enzymes involved. These results clearly demonstrate that the soybeans lacking LOX 3 in combination with watermelon HL-overexpressing leaf tissues greatly enhance hexenal formation.

Watermelon (Citrullus lanatus) hydroperoxide lyase greatly increases C6 aldehyde formation in transgenic leaves.

Fatty acid hydroperoxide lyase (HL) is the key enzyme for the production of the "green note"compounds, leaf aldehyde [(2E)-hexenal] and leaf alcohol [(3Z)-hexenol], in plant tissues. A cDNA encoding HL was cloned from leaves of watermelon (Citrullus lanatus) and expressed in Nicotiana tabacum. The enzyme is 3 times more active with 13-hydroperoxylinolenic acid than with 13-hydroperoxylinoleic acid. The activity against 9-hydroperoxides of polyunsaturated fatty acids is minimal. Enzyme activity of the watermelon HL in the transgenic leaves was approximately 50 times higher than endogenous HL activity in the wild-type N. tabacum plants. When compared with Arabidopsis HL also expressed in N. tabacum, the highest HL activity is 10 times higher in watermelon HL overexpressing leaves than in Arabidopsis HL overexpressers.

Purification and identification of linoleic acid hydroperoxides generated by soybean seed lipoxygenases 2 and 3.

It has been known that lipoxygenase (LOX) isozymes exhibit differences in product formation, but most product information to date is for LOX 1 among soybean (Glycine max) LOX isozymes. In this study, LOXs 2 and 3 were purified and used to generate hydroperoxide (HPOD) products in an in vitro system using linoleic acid as a substrate in the presence of either air or O2. The products were analyzed to determine their stereoisomeric characteristics. The control (no enzyme) showed only low levels of hydroperoxide production and no stereoisomeric specificity. LOX 2 shows high specificity in product formation, producing roughly 4 times more 13-HPOD than 9-HPOD, nearly all of which was 13-S(Z,E)-HPOD. LOX 3 produced more 9-HPOD than 13-HPOD at approximately a 2:1 ratio. No single stereoisomer was predominant among LOX 3 products. These results demonstrate that different isozymes of LOX have characteristic product profiles in in vitro reactions.

Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant.

Salicylic acid (SA), ethylene, and jasmonic acid (JA) are important signaling molecules in plant defense to biotic stress. An intricate signaling network involving SA, ethylene, and JA fine tunes plant defense responses. SA-dependent defense responses in Arabidopsis thaliana are mediated through NPR1-dependent and -independent mechanisms. We have previously shown that activation of an NPR1-independent defense mechanism confers enhanced disease resistance and constitutive expression of the pathogenesis-related (PR) genes in the Arabidopsis ssi1 mutant. In addition, the ssi1 mutant constitutively expresses the defensin gene PDF1.2. Moreover, SA is required for the ssi1-conferred constitutive expression of PDF1.2 in addition to PR genes. Hence, the ssi1 mutant appears to target a step common to SA- and ethylene- or JA-regulated defense pathways. In the present study, we show that, in addition to SA, ethylene and JA signaling also are required for the ssi1-conferred constitutive expression of PDF1.2 and the NPR1-independent expression of PR-1. Furthermore, the ethylene-insensitive ein2 and JA-insensitive jar1 mutants enhance susceptibility of ssi1 plants to the necrotrophic fungus Botrytis cinerea. However, defects in either the ethylene- or JA-signaling pathways do not compromise ssi1-conferred resistance to the bacterial pathogen Pseudomonas synringae pv. maculicola and the oomycete pathogen Peronospora parasitica. Interestingly, ssi1 exhibits a marginal increase in the levels of ethylene and JA, suggesting that low endogenous levels of these phytohormones are sufficient to activate expression of defense genes. Taken together, our results indicate that although cross talk in ssi1 renders expression of ethylene- or JA-responsive defense genes sensitive to SA and vice versa, it does not affect downstream signaling leading to resistance.

Labeling of major plant lipids and jasmonic acid using [1-(14C)] lauric acid.

A medium chain length fatty acid, [1-(14C)] lauric acid (12:0) was administered to the detached leaves of Artemisia and was incorporated into major lipids, including phospholipids and galactolipids. [1-(14C)]12:0 was elongated and desaturated into linolenic acid (18:3). In detached leaves of both Artemisia and Arabidopsis thaliana ecotype Columbia, radioactivity from [14C]18:3 was incorporated into jasmonic acid (JA) and methyl jasmonate (MJ). Higher amounts of [14C]JA were measured in Artemisia than Arabidopsis leaves. In Artemisia, [14C]JA was actively metabolized into [14C]MJ. Extracts prepared from the leaves of Artemisia, exhibited higher in vitro JA methyltransferase activity than those from Arabidopsis.

Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence.

In this work, the role of jasmonic acid (JA) in leaf senescence is examined. Exogenous application of JA caused premature senescence in attached and detached leaves in wild-type Arabidopsis but failed to induce precocious senescence of JA-insensitive mutant coi1 plants, suggesting that the JA-signaling pathway is required for JA to promote leaf senescence. JA levels in senescing leaves are 4-fold higher than in non-senescing ones. Concurrent with the increase in JA level in senescing leaves, genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway are differentially activated during leaf senescence in Arabidopsis, except for allene oxide synthase, which is constitutively and highly expressed throughout leaf development. Arabidopsis lipoxygenase 1 (cytoplasmic) expression is greatly increased but lipoxygenase 2 (plastidial) expression is sharply reduced during leaf senescence. Similarly, AOC1 (allene oxide cyclase 1), AOC2, and AOC3 are all up-regulated, whereas AOC4 is down-regulated with the progression of leaf senescence. The transcript levels of 12-oxo-PDA reductase 1 and 12-oxo-PDA reductase 3 also increase in senescing leaves, as does PED1 (encoding a 3-keto-acyl-thiolase for beta-oxidation). This represents the first report, to our knowledge, of an increase in JA levels and expression of oxylipin genes during leaf senescence, and indicates that JA may play a role in the senescence program.

Plastidial fatty acid signaling modulates salicylic acid- and jasmonic acid-mediated defense pathways in the Arabidopsis ssi2 mutant.

A mutation in the Arabidopsis gene ssi2/fab2, which encodes stearoyl-acyl carrier protein desaturase (S-ACP-DES), results in the reduction of oleic acid (18:1) levels in the mutant plants and also leads to the constitutive activation of NPR1-dependent and -independent defense responses. By contrast, ssi2 plants are compromised in the induction of the jasmonic acid (JA)-responsive gene PDF1.2 and in resistance to the necrotrophic pathogen Botrytis cinerea. Although S-ACP-DES catalyzes the initial desaturation step required for JA biosynthesis, a mutation in ssi2 does not alter the levels of the JA precursor linolenic acid (18:3), the perception of JA or ethylene, or the induced endogenous levels of JA. This finding led us to postulate that the S-ACP-DES-derived fatty acid (FA) 18:1 or its derivative is required for the activation of certain JA-mediated responses and the repression of the salicylic acid (SA) signaling pathway. Here, we report that alteration of the prokaryotic FA signaling pathway in plastids, leading to increased levels of 18:1, is required for the rescue of ssi2-triggered phenotypes. 18:1 levels in ssi2 plants were increased by performing epistatic analyses between ssi2 and several mutants in FA pathways that cause an increase in the levels of 18:1 in specific compartments of the cell. A loss-of-function mutation in the soluble chloroplastic enzyme glycerol-3-phosphate acyltransferase (ACT1) completely reverses SA- and JA-mediated phenotypes in ssi2. In contrast to the act1 mutation, a loss-of-function mutation in the endoplasmic reticulum-localized omega6 oleate desaturase (FAD2) does not alter SA- or JA-related phenotypes of ssi2. However, a mutation in the plastidial membrane-localized omega6 desaturase (FAD6) mediates a partial rescue of ssi2-mediated phenotypes. Although ssi2 fad6 plants are rescued in their morphological phenotypes, including larger size, absence of visible lesions, and straight leaves, these plants continue to exhibit microscopic cell death and express the PR-1 gene constitutively. In addition, these plants are unable to induce the expression of PDF1.2 in response to the exogenous application of JA. Because the act1 mutation rescues all of these phenotypes in ssi2 fad6 act1 triple-mutant plants, act1-mediated reversion may be mediated largely by an increase in the free 18:1 content within the chloroplasts. The reversion of JA responsiveness in ssi2 act1 plants is abolished in the ssi2 act1 coi1 triple-mutant background, suggesting that both JA- and act1-generated signals are required for the expression of the JA-inducible PDF1.2 gene. Our conclusion that FA signaling in plastids plays an essential role in the regulation of SSI2-mediated defense signaling is further substantiated by the fact that overexpression of the N-terminal-deleted SSI2, which lacks the putative plastid-localizing transit peptide, is unable to rescue ssi2-triggered phenotypes, as opposed to overexpression of the full-length protein.

Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants.

Plants under stress from both biotic and abiotic sources produce increased levels of ethylene, which is perceived by ethylene receptors and triggers cellular responses further downstream. Protein phosphorylation and dephosphorylation were implicated in the regulation of ethylene induction by stresses based on studies using protein kinase and phosphatase inhibitors. However, the kinase(s) involved remains to be determined. Using a conditional gain-of-function transgenic system, we demonstrate that the activation of SIPK, a tobacco mitogen-activated protein kinase (MAPK), by NtMEK2DD, an active mutant of the upstream kinase of SIPK, resulted in a dramatic increase in ethylene production. The increase in ethylene after the activation of SIPK coincided with a dramatic increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, which was followed by the activation of a subgroup of ACS and ACC oxidase (ACO) genes, suggesting that either the activation of unidentified ACS(s) or post-transcriptional regulation is involved. Infection with Tobacco mosaic virus (TMV), which is known to activate the SIPK cascade and induce ethylene biosynthesis, also induced the same ACSs and ACOs. After ethylene production in NtMEK2DD plants, strong activation of ETHYLENE-RESPONSE FACTOR (ERF) genes was observed, similar to the effect in NN tobacco plants infected with TMV. In contrast to previous reports, no major increase in jasmonic acid (JA) and methyl jasmonate (MJ) was detected after the activation of SIPK/WIPK in NtMEK2DD transgenic plants. These results suggest that the induction of ethylene but not JA/MJ is involved in plant defense responses mediated by the NtMEK2-SIPK/WIPK pathway.