Production and function of jasmonates in nodulated roots of soybean plants inoculated with Bradyrhizobium japonicum

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation."     

Changes in jasmonates and 12-oxophytodienoic acid contents of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during somatic embryogenesis

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.     

Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis

Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of beta-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis.     

Octadecanoid signaling component "burst" in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor chitosan

Octadecanoid pathway components, 12-oxo-phytodieonic acid (OPDA) and jasmonic acid (JA), are key biologically active regulators of plant self-defense response(s). However, to date these compounds have been studied mostly in dicots, and used large (1-10 g fresh weight, FW) samples for quantification, even when examined in mature rice plants, which is a drawback considering their rapid responsiveness to stress. Focusing on rice--a monocot cereal crop research model--this work describes an efficient and simultaneous quantification of both OPDA and JA using a minimum amount of 200mg FW seedling leaf tissue upon wounding (by cut) and treatment with fungal elicitor, chitosan (CT) by high-pressure liquid chromatography-turboionspray tandem mass spectrometry. Transient OPDA/JA "burst" was consistently and reproducibly detected within 3 min in wounded and CT treated leaves. OPDA peaked dramatically around 5 min and returned to its basal level within 15 min, whereas JA induction upon wounding and CT treatment were in parallel to OPDA production, peaking at 30 and 60 min, respectively. Present results mark a major advance in our understanding of key inducible octadecanoid pathway components in rice, and strongly suggest a role for the octadecanoid pathway downstream of perception of at least these two fundamentally different extracellular stimuli.     

Do stress-related phytohormones,abscisic acid and jasmonic acid play a role in the regulation of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. somatic embryogenesis?

This study examined the role of endogenous abscisic acid (ABA) and jasmonic acid (JA) in indirect somatic embryogenesis of Medicago sativa L. A multiplex GC-MS/MS technique allowed quantitative single-run analyses of ABA, JA, 12-oxophytodienoic acid (OPDA) and indole-3-acetic acid (IAA). The preparation of initial explants led to a strong accumulation of ABA, JA and OPDA but not of IAA. Substantially higher levels of ABA, JA and OPDA were detected in developing somatic embryos than in callus or embryogenic suspension. Fluridone (FLD) decreased ABA, JA and OPDA levels. Indoprofen (INP) appeared to be a specific inhibitor of octadecanoid biosynthesis. Somatic embryo production and development were negatively affected by FLD or INP. Only INP (0.5 μM) applied during proliferation phase increased the number of cotyledonary embryos. The results strongly indicate the involvement of ABA and JA in somatic embryogenesis of M. sativa. Surprisingly, low IAA contents in comparison to stress-related compounds (ABA, JA and OPDA) were detected in explants, embryogenic tissues and somatic embryos.     

Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-phytodienoic acid-containing galactolipid, arabidopside E

Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (>90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7-8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.     

Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis(+)12-oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato. In stems, young leaves and young flowers, AOC mRNA accumulates to a low level, contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g-1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue-specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue-specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink-source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin-mediated wound response of tomato.     

Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways.

Jasmonates mediate various physiological events in plant cells such as defense responses, flowering, and senescence through intracellular and intercellular signaling pathways, and the expression of a large number of genes appears to be regulated by jasmonates. In order to obtain information on the regulatory network of jasmonate-responsive genes (JRGs) in Arabidopsis thaliana (Arabidopsis), we screened 2880 cDNA clones for jasmonate responsiveness by a cDNA macroarray procedure. Since many of the JRGs reported so far have been identified in leaf tissues, the cDNA clones used were chosen from a non-redundant EST library that was prepared from above-ground organs. Hybridization to the filters was achieved using alpha-33P-labeled single-strand DNAs synthesized from mRNAs obtained from methyl jasmonate (MeJA)-treated and untreated Arabidopsis seedlings. Data analysis identified 41 JRGs whose mRNA levels were changed by more than three fold in response to MeJA. This was confirmed by Northern blot analysis by using eight representatives. Among the 41 JRGs identified, 5 genes were JA biosynthesis genes and 3 genes were involved in other signaling pathways (ethylene, auxin, and salicylic acid). These results suggest the existence of a positive feedback regulatory system for JA biosynthesis and the possibility of crosstalk between JA signaling and other signaling pathways.     

Oxo-phytodienoic acid-containing galactolipids in Arabidopsis: jasmonate signaling dependence

The jasmonate family of phytohormones, as represented by 12-oxo-phytodienoic acid (OPDA), dinor-phytodienoic acid (dn-OPDA), and jasmonic acid in Arabidopsis (Arabidopsis thaliana), has been implicated in a vast array of different developmental processes and stress responses. Recent reports indicate that OPDA and dn-OPDA occur not only as free acids in Arabidopsis, but also as esters with complex lipids, so-called arabidopsides. Recently, we showed that recognition of the two bacterial effector proteins AvrRpm1 and AvrRpt2 induced high levels of a molecule consisting of two OPDAs and one dn-OPDA esterified to a monogalactosyl diacylglycerol moiety, named arabidopside E. In this study, we demonstrate that the synthesis of arabidopsides is mainly independent of the prokaryotic lipid biosynthesis pathway in the chloroplast, and, in addition to what previously has been reported, arabidopside E as well as an all-OPDA analog, arabidopside G, described here accumulated during the hypersensitive response and in response to wounding. We also show that different signaling pathways lead to the formation of arabidopsides during the hypersensitive response and the wounding response, respectively. However, the formation of arabidopsides during both responses is dependent on an intact jasmonate signaling pathway. Additionally, we report inhibition of growth of the fungal necrotrophic pathogen Botrytis cinerea and in planta release of free jasmonates in a time frame that overlaps with the observed reduction of arabidopside levels. Thus, arabidopsides may have a dual function: as antipathogenic substances and as storage compounds that allow the slow release of free jasmonates.     

Role of cis-12-oxo-phytodienoic acid in tomato embryo development

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.     

Effects of methyl jasmonate (MeJA) on the dark-induced senescence in oat (Avena sativa L.) leaf segments

To investigate the relationship between methyl jasmonate (MeJA) and ethylene in leaf senescence, we studied the effects of MeJA on ethylene production and ethylene biosynthetic enzyme activities in oat(Avena sativa L.) leaf segments incubated in darkness. MeJA promoted dark-induced senescence judged from the contents of chlorophyll and protein, and increased ethylene production 6 times of the control. MeJA also increased the activities of ethylene biosynthetic enzymes, 1-aminocyclopropane carboxylic acid (ACC) synthase and ACC oxidase as compared to control. In MeJA-treated leaf segments, ACC synthase activity reached its maximum level at 24 h of incubation and ACC oxidase activity peaked at 6 h of incubation. Aminoethoxyvinylglycine (AVG) and Co²⁺, inhibitors of ACC synthase and ACC oxidase respectively, reduced MeJA-induced ethylene production. They also delayed leaf senescence that was promoted by the treatment of MeJA. From these results, we can suggest that MeJA increased the activities of ACC synthase and ACC oxidase, these increased activities lead to increase in ethylene production and this increased ethylene production might promote dark-induced leaf senescence.     

Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.     

Methyl jasmonate and cis-jasmone do not dispose of the herbivore-induced jasmonate burst in Nicotiana attenuata

The oxylipin pathway mediates wound- and herbivore-induced defense reactions in Nicotiana attenuata as evidenced by a transient jasmonic acid (JA)-burst that precedes these defense responses. The fate of this induced JA-burst remains unknown. Two derivatives of JA, its methylester, methyl jasmonate (MeJA) and cis -jasmone ( cis J), are thought to be a means of disposing of JA through volatilization at the plant surface. In N. attenuata, the headspace quantities of these compounds did not change over 3 days, although levels of MeJA and cis J increased 100- and 70-fold, respectively, in surface extracts of attacked leaves after feeding of Manduca sexta larvae or application of larval regurgitant to mechanical wounds. Inhibition of the wound-induced increase in JA with indole-3-acetic acid (IAA) revealed an association between the JA accumulation and subsequent increases in MeJA and cis J. Induced systemic increases of MeJA were not of local origin and therefore do not contribute to the inactivation of the JA-burst in the wounded leaf. The total amount of MeJA and cis J produced could only account for 9% of the JA-burst elicited by herbivore attack and therefore their production do not represent major disposal pathways of JA in N. attenuata .     

Apple BT2 protein negatively regulates jasmonic acid‐triggered leaf senescence by modulating the stability of MYC2 and JAZ2

Jasmonic acid (JA) is shown to induce leaf senescence. However, the underlying molecular mechanism is not well understood, especially in woody plants such as fruit trees. In this study, we are interested in exploring the biological role of MdBT2 in JA‐mediated leaf senescence. We found that MdBT2 played an antagonistic role in MdMYC2‐promoted leaf senescence. Our results revealed that MdBT2 interacted with MdMYC2 and accelerated its ubiquitination degradation, thus negatively regulated MdMYC2‐promoted leaf senescence. In addition, MdBT2 acted as a stabilizing factor to improve the stability of MdJAZ2 through direct interaction, thereby inhibited JA‐mediated leaf senescence. Furthermore, our results also showed that MdBT2 interacted with a subset of JAZ proteins in apple, including MdJAZ1, MdJAZ3, MdJAZ4 and MdJAZ8. Our investigations provide new insight into molecular mechanisms of JA‐modulated leaf senescence. The dynamic JA‐MdBT2‐MdJAZ2‐MdMYC2 regulatory module plays an important role in JA‐modulated leaf senescence.     

Three positive regulators of leaf senescence in Arabidopsis, ORE1, ORE3 and ORE9, play roles in crosstalk among multiple hormone-mediated senescence pathways

Leaf senescence is a developmentally programmed event, but the initiation and progression of leaf senescence are affected by a range of plant hormones including abscisic acid (ABA), ethylene and methyl jasmonate (MeJA). To investigate plant hormone crosstalk during leaf senescence, hormone-induced senescence phenotypes were analyzed in three leaf senescence mutants [ore1 (oresara1), ore3 and ore9] showing delayed senescence phenotypes in age-dependent and dark-induced senescence. The ore mutants exhibited delayed leaf senescence phenotypes following treatment with ABA, ACC (aminocyclo-propane-1-carboxylic acid) or MeJA. After each hormone treatment, the photochemical efficiency of photosystem II and chlorophyll content were significantly higher in the ore mutant leaves than in the wild-type leaves. The expression of CAB2 and SEN4 in the wild-type was rapidly altered following each hormone treatment. However, the decrease in CAB2 expression and the induction of SEN4 expression in the mutants were less affected by ABA, ACC or MeJA treatment. It is suggested that ORE1, ORE3 and ORE9 are required for the proper progression of leaf senescence mediated by ABA, ethylene and MeJA. This implies that ORE1, ORE3 and ORE9 may be linked to the crosstalk among senescence pathways induced by ABA, ethylene and MeJA, as well as age and darkness.