Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways

Two cDNAs encoding geranylgeranyl pyrophosphate (GGPP) synthases from tomato (Lycopersicon esculentum) have been cloned and functionally expressed in Escherichia coli. LeGGPS1 was predominantly expressed in leaf tissue and LeGGPS2 in ripening fruit and flower tissue. LeGGPS1 expression was induced in leaves by spider mite (Tetranychus urticae)-feeding and mechanical wounding in wild type tomato but not in the jasmonic acid (JA)-response mutant def-1 and the salicylic acid (SA)-deficient transgenic NahG line. Furthermore, LeGGPS1 expression could be induced in leaves of wild type tomato plants by JA- or methyl salicylate (MeSA)-treatment. In contrast, expression of LeGGPS2 was not induced in leaves by spider mite-feeding, wounding, JA- or MeSA-treatment. We show that emission of the GGPP-derived volatile terpenoid (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) correlates with expression of LeGGPS1. An exception was MeSA-treatment, which resulted in induction of LeGGPS1 but not in emission of TMTT. We show that there is an additional layer of regulation, because geranyllinalool synthase, catalyzing the first dedicated step in TMTT biosynthesis, was induced by JA but not by MeSA.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Aspirin prevents wound-induced gene expression in tomato leaves by blocking jasmonic acid biosynthesis

Jasmonic acid (JA) and its methyl ester, like mechanical wounding, strongly induce accumulation of proteinase inhibitor II (Pin2) in tomato and potato leaves. In plants, JA is synthesized from α-linolenic acid by a lipoxygenase (LOX)-mediated oxygenation leading to 13-hydroxyperoxylinolenic acid (13-HPLA) which is then subsequently transformed to JA by the action of hydroperoxide-dehydrase activity and additional modification steps. Both the chemical structure as well as the biosynthetic pathway of JA resemble those of the mammalian eicosanoids (prostaglandins and leukotrienes) which are derived from LOX-and cyclooxygenase (COX)-mediated reactions. To assess the role of endogenous JA in the wound response, detached tomato (Lycopersicon esculentum Mill.) leaves were supplied with different LOX and COX inhibitors and the expression of the wound-induced genes for Pin2 (Pin2), cathepsin D inhibitor (Cdi) and threonine deaminase (Td) was analyzed. Lipoxygenase inhibitors as well as some COX inhibitors blocked the wound-induced accumulation of Pin2, Cdi and Td mRNA. Quantitation of endogenous levels of JA showed that aspirin blocks the increase of this phytohormone normally observed as a result of wounding. Linolenic acid and 13-HPLA do not induce the expression of Pin2, Cdi and Td in the presence of aspirin. However, 12-oxo-phytodienoic acid and jasmonic acid are able to overcome the inhibitory effect of this substance. These results strongly indicate that aspirin prevents wound-induced gene activation by inhibiting the hydroxyperoxide-dehydrase activity that mediates the conversion of 13-HPLA to 12-oxo-phytodienoic acid.     

Nitric oxide is induced by wounding and influences jasmonic acid signaling in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Nitric oxide (NO) has been associated with plant defense responses during microbial attack, and with induction and/or regulation of programmed cell death. Here, we addressed whether NO participates in wound responses in Arabidopsis thaliana (L.) Heynh.. Real-time imaging by confocal laser-scanning microscopy in conjunction with the NO-selective fluorescence indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) uncovered a strong NO burst after wounding or after treatment with JA. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive responses. Furthermore, we were able to detect NO in plants (here induced by wounding) by means of electron paramagnetic resonance measurements using diethyldithiocarbamate as a spin trap. When plants were treated with NO, Northern analyses revealed that NO strongly induces key enzymes of jasmonic acid (JA) biosynthesis such as allene oxide synthase (AOS) and lipoxygenase (LOX2). On the other hand, wound-induced AOS gene expression was independent of NO. Furthermore, JA-responsive genes such as defensin (PDF1.2) were not induced, and NO induction of JA-biosynthesis enzymes did not result in elevated levels of JA. However, treatment with NO resulted in accumulation of salicylic acid (SA). In transgenic NahG plants (impaired in SA accumulation and/or signaling), NO did induce JA production and expression of JA-responsive genes. Altogether, the presented data demonstrate that wounding in Arabidopsis induces a fast accumulation of NO, and that NO may be involved in JA-associated defense responses and adjustments.Abbreviations AOS Allene oxide synthase - cPTIO Carboxy-2-phenyl-4,4,5,5-tetramethylimidazolinone-3-oxide-1-oxyl - DAF-2 DA 4,5-Diaminofluorescein diacetate - DETC Diethyldithiocarbamate - EPR Electron paramagnetic resonance - iNOS Inducible nitric oxide synthase - JA Jasmonic acid - JIP Jasmonic acid-induced protein - LOX2 Lipoxygenase 2 - NO Nitric oxide - OPR3 12-Oxophytodienoate reductase - PDF1.2 Plant defensin - ROS Reactive oxygen species - SA Salicylic acid - SNP Sodium nitroprusside     

Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

Simultaneous quantitative LC-ESI-MS/MS analyses of salicylic acid and jasmonic acid in crude extracts of Cucumis sativus under biotic stress

Salicylic acid (SA) and jasmonic acid (JA) are plant hormones involved in basal resistance against plant pathogens and also in induced resistance. The aim of this study is to develop a fast and sensitive method to determine simultaneously the levels of both these hormones. The present paper proposes a method that includes hormone extraction with MeOH-H(2)O-HOAc (90:9:1, v/v), evaporation of the extracts, and injection into the liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system in multiple reaction monitoring (MRM). Endogenous SA and JA levels in noninfested control cucumber cotyledons were 30.96 and 0.73ngg(-1) fresh weight, respectively. In roots, the levels were 8.31 and 15.82ngg(-1) FW, respectively. In plants treated with the biological control agent Trichoderma asperellum strain T-34, the levels of SA and JA did not differ from control plants. Rhizoctonia solani-diseased cucumber plants showed higher levels of SA and JA compared to noninfested controls (up to 2 and 13-fold higher, respectively). Detection limits for SA and JA were 0.45 and 0.47ngg(-1) fresh weight, respectively. The results of our research include the development of a method that is both fast and highly sensitive in the simultaneous quantitation of SA and JA from crude cucumber plant extracts, avoiding any purification and derivatization steps.     

Phospholipid and phospholipase changes by jasmonic acid during stolon to tuber transition of potato

Potato tuber formation starts with the stolon swelling and is regulated by jasmonates. The cascade of events leading to tuber formation is not completely understood. The aim of this study was to evaluate phospholipid composition and phospholipase activities during four stages of stolon-to-tuber transition of Solanum tuberosum L., cv. Spunta, and involvement of phosphatidic acid (PA) in stolon cell expansion during early stages. Effects of jasmonic acid (JA) treatment on phospholipid content and activation of phospholipase D (PLD) (EC 3.1.4.4) and phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP₂-PLC) (EC 3.1.4.3) were studied in the early stages (first stage, hooked apex stolon; second stage, initial swelling stolon) of tuberization. All the phospholipid species identified, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), PA, and cardiolipin (CL), decreased as tuber formation progressed. PLD and PLC were activated in control tissues at an early stage. JA treatment caused a decrease of PC and PS in first stage stolons, accumulation of PA in second stage stolons, and modification of PLD and PLC activities. PA increased stolon cell area in the first and second stages. These findings indicate that phospholipid catabolism is activated from the early stages of tuber formation, and that JA treatment modifies the pattern of phospholipid (PC, PS, and PA) composition and phospholipase (PLD and PLC) activity. These phospholipids therefore may play a role in activation of an intracellular mechanism that switches the developmental fate of stolon meristem cells, causing differentiation into a tuber.     

The promoter of the gene Itr1 from barley confers a different tissue specificity in transgenic tobacco

Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, theItr1 promoter drives expression of the β-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed. These authors have contributed equally to this work     

Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice

Enzyme 12-oxophytodienoate (OPDA) reductase (EC1.3.1.42), which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of 10, 11-double bonds of OPDA to yield 3-oxo-2-(2′-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). The rice OsOPR1 gene encodes OPDA reductase (OPR) converting (−)-cis-OPDA preferentially, rather than (+)-cis-OPDA, a natural precursor of JA. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cis-OPDA, similar to the OPR3 protein in Arabidopsis thaliana (L.) Heynh. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. The GFP-OsOPR7 fusion protein was detected exclusively in peroxisomes in onion epidermal cells. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing the reduction of natural (+)-cis-OPDA for the JA biosynthesis in rice. Tomoyuki Tani and Hiroyuki Sobajima have equally contributed to this work.     

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.     

Specific patterns of changes in wheat gene expression after treatment with three antifungal compounds

The two fungicides azoxystrobin and fenpropimorph are used against powdery mildew and rust diseases in wheat (Triticum aestivumL). Azoxystrobin, a strobilurin, inhibits fungal mitochondrial respiration and fenpropimorph, a morpholin, represses biosynthesis of ergosterol, the major sterol of fungal membranes. Although the fungitoxic activity of these compounds is well understood, their effects on plant metabolism remain unclear. In contrast to the fungicides which directly affect pathogen metabolism, benzo(1,2,3) thiadiazole-7-carbothioic acid S-methylester (BTH) induces resistance against wheat pathogens by the activation of systemic acquired resistance in the host plant. In this study, we monitored gene expression in spring wheat after treatment with each of these agrochemicals in a greenhouse trial using a microarray containing 600 barley cDNA clones. Defence-related genes were strongly induced after treatment with BTH, confirming the activation of a similar set of genes as in dicot plants following salicylic acid treatment. A similar gene expression pattern was observed after treatment with fenpropimorph and some defence-related genes were induced by azoxystrobin, demonstrating that these fungicides also activate a defence reaction. However, less intense responses were triggered than with BTH. The same experiments performed under field conditions gave dramatically different results. No gene showed differential expression after treatment and defence genes were already expressed at a high level before application of the agrochemicals. These differences in the expression patterns between the two environments demonstrate the importance of plant growth conditions for testing the impact of agrochemicals on plant metabolism.     

Systemic induction of a Phytolacca insularis antiviral protein gene by mechanical wounding, jasmonic acid, and abscisic acid

We have isolated a gene encoding a ribosome-inactivating protein (RIP) from Phytolacca insularis, designated as P. insularis antiviral protein 2 (PIP2). The PIP2 gene contained an open reading frame encoding a polypeptide of 315 amino acids. The deduced amino acid sequence of PIP2 was similar to those of other RIPs from Phytolacca plants. Recombinant PIP2 was expressed in Escherichia coli and was used to investigate its biological activities. Recombinant PIP2 inhibited protein synthesis in rabbit reticulocyte lysate by inactivating ribosomes through N-glycosidase activity. It also exhibited antiviral activity against tobacco mosaic virus (TMV). Expression of the PIP2 gene was developmentally regulated in leaves and roots of P. insularis. Furthermore, expression of the PIP2 gene was induced in leaves by mechanical wounding. The wound induction of the PIP2 gene was systemic. Expression of the PIP2 gene also increased in leaves in a systemic manner after treatment with jasmonic acid (JA) and abscisic acid (ABA), but not with salicylic acid (SA). These results imply that plants have employed the systemic synthesis of the defensive proteins to protect themselves more efficiently from infecting viruses.     

Is there crosstalk between extracellular and intracellular calcium mobilization in jasmonic acid signaling

The changes in cytosolic Ca²⁺ levels play an important role in the jasmonic acid (JA) signal transduction pathway. We demonstrate that an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis leaf cells was affected by pretreatment with heparin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). With pretreatment of heparin, an antagonist of inositol 1,4,5-trisphosphate (IP₃) sensitive channels, the basal and JA induced fluorescence of [Ca²⁺]_cyt were both decreased. Furthermore, heparin and TMB-8, another antagonist of IP₃ sensitive channels, enhanced the JA-induced gene expression of JR1. These data suggest that there may be a fine tune control system between extracellular and intracellular Ca²⁺ mobilization in JA signaling pathway.     

Induction of tuberisation in vitro with jasmonic acid and sucrose in an Australian terrestrial orchid, Pterostylis sanguinea

Effectsof jasmonic acid (JA) and sucrose on tuber formation were studied in a commontuberous terrestrial orchid Pterostylis sanguinea D.L.Jones& M. Clements (dark banded greenhood orchid) from southwestern Australia.Seeds were germinated symbiotically in vitro on an oatmealagar (OMA) in the presence of a mycorrhizal fungus isolated from the orchidhost. Ten week-old seedlings were transferred into culture vessels containingOMA supplemented with JA (at concentrations 0, 0.1, 1, 5 or 10M), sucrose (at concentrations 0, 5, 10 or 20 gl^–1) or combinations of each. Tuber development in alltreatments occurred approximately twenty-six weeks after seed sowing. Theaddition of 5 or 10 g l^–1 sucrose with JA to themedia resulted in higher frequencies of tuber formation in comparison to thecontrol. Significantly higher proportion of seedlings produced tuber when themedium was supplemented with 5 g l^–1 sucrose incombination with 5 M JA, compared to the control.In vitro tuber formation of Pterostylissanguinea can be improved by combined addition of appropriate levelsof JA and sucrose to OMA, which will aid in rapid in vitrotuberisation of terrestrial orchids.