Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis

Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDalpha, PLDbeta, and PLDgamma differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDalpha, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDalpha attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDalpha-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

Volatile Products of the Lipoxygenase Pathway Evolved from Phaseolus vulgaris (L.) Leaves Inoculated with Pseudomonas syringae pv phaseolicola

Activation of the "lipoxygenase pathway" in plants gives rise to a series of products derived from fatty acids. Analysis by gas chromatography-mass spectroscopy of volatile products produced by Phaseolus vulgaris (L.) cv Red Mexican leaves during a hypersensitive resistance response (HR) to the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola showed evolution of several lipid-derived volatiles, including cis-3-hexenol and trans-2-hexenal, which arise from the 13-hydroperoxide of linolenic acid. These compounds were not produced in detectable amounts by buffer-inoculated leaves, nor did they evolve to such a high degree during comparable stages of the susceptible response. The absence of trans-2,cis-6-nonadienal, a product expected from 9-hydroperoxide of linolenic acid, suggests that lipid peroxidation during the HR proceeded primarily enzymically via bean lipoxygenase, which produces the 13-hydroperoxide, and not via autoxidative processes. The effects of trans-2-hexenal, cis-3-hexenol, and traumatic acid on P.s pv phaseolicola were investigaed. trans-2-Hexenal appeared to be highly bactericidal at low concentrations, whereas cis-3-hexenol was bactericidal only at much higher concentrations. Traumatic acid appeared to have no effect on P.s. pv. phaseolicola at the concentrations tested. These results demonstrate that during plant defense responses against microbial attack, several lipid-derived compounds are produced by the plant, some of which possess antimicrobial activity and conceivably are involved in plant disease resistance. The time of production of these substances, in amounts that would be expected to be antibacterial in vitro, correlated with a slowing down of the growth rate of bacteria in the leaves and was seen at a time before the accumulation of isoflavonoid phytoalexins in the host.     

A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate.

We investigated the relationship between the expression of lipoxygenase (LOX) genes and the systemin-dependent wound response in tomato (Lycopersicon esculentum) leaves. A polymerase chain reaction-based approach was used to isolate two tomato Lox cDNAs, called TomLoxC and TomLoxD. Both TomLOXC and TomLOXD amino acid sequences possess an N-terminal extension of about 60 residues that were shown by in vitro uptake to function as transit peptides, targeting these proteins into the chloroplast. Within 30 to 50 min following wounding or systemin or methyl jasmonate treatments, the TomLoxD mRNA level increased and reached a maximum between 1 and 2 h. TomLoxC mRNA was not detectable in leaves and was not found following wounding, but it was found in ripening fruits, indicating that the two tomato Lox genes are regulated in different tissues by different processes. The results suggest that the TomLoxD gene is up-regulated in leaves in response to wounding and encodes a chloroplast LOX that may play a role as a component of the octadecanoid defense-signaling pathway.     

西花蓟马取食对菜豆防御基因表达的诱导作用

为探讨菜豆对西花蓟马Frankliniella occidentalis取食的防御反应分子机制, 本实验利用荧光定量PCR（RT-qPCR）研究了西花蓟马取食、 机械损伤及外源水杨酸甲酯（MeSA）和茉莉酸（JA）处理对菜豆叶片防御相关基因（LOX, AOS, PAL及PR-2）相对表达量的影响。结果表明： 脂氧合酶基因（LOX）的相对表达量在外源JA和机械损伤诱导后分别在24 h和48 h时达到最大值, 但与对照相比差异不显著（P>0.05）, 外源MeSA处理后LOX几乎没有表达, 西花蓟马取食后LOX的相对表达量在24 h显著升高, 约为对照的41.9倍, 且明显高于其余3个处理（P<0.05）。不同诱导处理后丙二烯氧化物合成酶基因（AOS）的相对表达量较低, 其中外源MeSA处理后在整个诱导时间内几乎没有表达。苯丙氨酸解氨酶基因（PAL）在机械损伤和外源JA处理后在整个诱导时间内几乎没有表达, 外源MeSA处理后PAL的表达量在24 h时最高, 为对照的1.9倍左右。PAL的相对表达量在遭受蓟马取食诱导后迅速升高, 在24 h时约为CK的4.3倍, 显著高于其他3种处理诱导的（P<0.05）。β-1, 3-葡聚糖酶基因（PR-2）的相对表达量在蓟马取食和外源JA处理的整个诱导时间内受到抑制, 机械损伤诱导后PR-2的表达水平在24 h时有所升高, 之后诱导期间内受到抑制。外源MeSA诱导后菜豆叶片中PR-2的相对表达量在24 h就急剧升高, 达到对照的6.63倍, 且显著高于其余3种处理诱导的（P<0.05）, 但48 h后却几乎检测不到。研究结果提示, 西花蓟马取食不仅能够诱导SA和JA介导的信号传导途径, 且两通路间存在交互作用。     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid (JA) in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase (LOX)-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves.LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid (NDGA), an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13(S)-H(P)ODE) was confirmed to be the main product of LOX throughout the experimental time. In addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from Its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant Pathogenic Pseudomonads

Lipoxygenase (LOX) mRNA, enzyme protein, and enzyme activity were found to be induced in leaves of tomato (Lycopersicon esculentum Mill. cv Moneymaker) on inoculation with plant pathogenic bacteria. The rate of enzyme activity with linoleic or linolenic acid as substrate was approximately 10 times greater than that with arachidonic acid. Optimum activity was at pH 7.0. In the incompatible interaction, which was associated with a hypersensitive reaction (HR), a single band with relative molecular weight approximately 100,000 was revealed by probing western blots of enzyme extracts with antiserum raised against a pea lipoxygenase. Changes in the intensity of this band reflected the changes observed in LOX enzyme activity after bacterial inoculations. In the hypersensitive reaction, i.e. after inoculation with Pseudomonas syringae pv syringae, LOX mRNA was induced by 3 hours and enzyme activity began to increase between 6 and 12 hours and had reached maximum levels by 24 to 48 hours. In tomato leaves inoculated with P. syringae pv tomato (compatible interaction), LOX mRNA was induced later and enzyme activity changed only marginally in the first 24 hours, then increased steadily up to 72 hours, reaching the levels seen in the HR.     

Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity.

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His(6)-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K(m) value of 4.02+/-0.64 microM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His(6)-HbAOS enzyme activity. Although JA had no effect on His(6)-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His(6)-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.     

A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.     

Volatile C6-aldehydes and Allo-ocimene activate defense genes and induce resistance against Botrytis cinerea in Arabidopsis thaliana

Green leafy volatiles or isoprenoids are produced after mechanical wounding or pathogen/herbivore attacks in higher plants. We monitored expression profiles of the genes involved in defense responses upon exposing Arabidopsis thaliana to the volatiles. Among the genes investigated, those known to be induced by mechanical wounding and/or jasmonate application, such as chalcone synthase (CHS), caffeic acid-O-methyltransferase (COMT), diacylglycerol kinase1 (DGK1), glutathione-S-transferase1 (GST1) and lipoxygenase2 (LOX2), were shown to be induced with (E)-2-hexenal, (Z)-3-hexenal, (Z)-3-hexenol or allo-ocimene (2,6-dimethyl-2,4,6-octatriene). A salicylic acid-responsive gene, pathogenesis-related protein2 (PR2), was not induced by the volatiles. Detailed analyses of the expression profiles showed that the manner of induction varied depending on either the gene monitored or the volatile used. A chemically inert compound, (Z)-3-hexenol, was also potent, which suggested that chemical reactivity was not the sole requisite for the inducing activity. With a jasmonate-insensitive mutant (jar1), the induction by the volatiles was mostly suppressed, however, that of LOX2 was unaltered. An ethylene-insensitive mutant (etr1) showed responses almost identical to the wild type, with minor exceptions. From these observations, it was suggested that both the jasmonate-dependent and -independent pathways were operative upon perception of the volatiles, while the ETR1-dependent pathway was not directly involved. When Botrytis cinerea was inoculated after the volatile treatment, retardation of disease development could be seen. It appears that volatile treatment could make the plants more resistant against the fungal disease.     

The biochemical origin of pentenol emissions from wounded leaves

Large releases of 1-penten-3-ol (pentenol) and 1-penten-3-one (pentenone) were recently observed from a variety of leaves subjected to freeze-thaw damage in the presence of oxygen. In order to understand the biochemical origins of these volatiles, soybean leaf extracts were used to determine if the formation of pentenol and pentenone can be explained by known O(2)-dependent lipoxygenase (LOX) reactions. Enzymatic formation of these C5 volatiles was found to be dependent on alpha-linolenic acid or the 13(S)-hydroperoxide of alpha-linolenic acid [13(S)-HPOT] and blocked by LOX inhibitors. Five soybean leaf LOX isozyme genes (VLXA, VLXB, VLXC, VLXD, and VLXE) were then expressed in Escherichia coli and used in in vitro incubations with 13(S)-HPOT to test for volatile formation. Each of the LOX isozymes catalyzed the formation of low levels of pentenol, but not pentenone. It therefore seems likely that the C5,13-cleavage activity of LOX is the direct source of abundant pentenol and the indirect source of pentenone observed upon leaf wounding.     

Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid*

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.