Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.     

The jasmonic acid-amino acid conjugates JA-Val and JA-Leu are involved in rice resistance to herbivores

The phytohormone jasmonic acid (JA) plays a core role in plant defence against herbivores. When attacked by herbivores, JA and its bioactive derivatives are accumulated at the damage site, and subsequently perceived by the jasmonate co-receptors COI1 and JAZ proteins. The (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) is known to be the main active JA derivative controlling vascular plant responses to herbivores as well as other JA-regulated processes. However, whether other endogenous JA-amino acid conjugates (JA-AAs) are involved in herbivore-induced defence responses remain unknown. Here, we investigated the role of herbivore-elicited JA-AAs in the crop plant rice. The levels of five JA-AAs were significantly increased under the armyworm, leaf folder and brown planthopper attack. Of the elicited JA derivatives, JA-Ile, JA-Val and JA-Leu could serve as ligands to promote the interaction between rice COI1 and JAZs, inducing OsJAZ4 degradation in vivo. JA-Val or JA-Leu treatment increased the expression of JA- and defence-related pathway genes but not JA-Ile levels, suggesting that these JA-AAs may directly function in JA signalling. Furthermore, the application of JA-Val or JA-Leu resulted in JA-mediated plant growth inhibition, while enhancing plant resistance to herbivore attack. This study uncovers that JA-Val and JA-Leu also play a role in rice defence against herbivores.     

Light induces jasmonate‐isoleucine conjugation via OsJAR1‐dependent and ‐independent pathways in rice

The bioactive form of jasmonate is the conjugate of the amino acid isoleucine (Ile) with jasmonic acid (JA), which is biosynthesized in a reaction catalysed by the GH3 enzyme JASMONATE RESISTANT 1 (JAR1). We examined the biochemical properties of OsJAR1 and its involvement in photomorphogenesis of rice (Oryza sativa). OsJAR1 has a similar substrate specificities as its orthologue in Arabidopsis. However, osjar1 loss‐of‐function mutants did not show as severe coleoptile phenotypes as the JA‐deficient mutants coleoptile photomorphogenesis 2 (cpm2) and hebiba, which develop long coleoptiles in all light qualities we examined. Analysis of hormonal contents in the young seedling stage revealed that osjar1 mutants are still able to synthesize JA‐Ile conjugate in response to blue light, suggesting that a redundantly active enzyme can conjugate JA and Ile in rice seedlings. A good candidate for this enzyme is OsJAR2, which was found to be able to catalyse the conjugation of JA with Ile as well as with some additional amino acids. In contrast, if plants in the vegetative stage were mechanically wounded, the content of JA‐Ile was severely reduced in osjar1, demonstrating that OsJAR1 is the most important JA‐Ile conjugating enzyme in the wounding response during the vegetative stage.     

Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid

Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood. One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M. grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves. Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm). Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA. These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis. JA acts as an enhancer of both types of INA-induced reactions in rice.     

Uptake of jasmonic acid and related compounds by mesophyll protoplasts of the barley leaf

Jasmonic acid (JA) permeates the plasma membrane of mesophyll cells by diffusion as the lipophilic undissociated JAH molecule probably without the participation of a saturable uptake component. The mesophyll plasma membrane is nearly impermeable to the JA anion. The permeability coefficients of JA and several JA derivatives (its methyl ester (JAMe), 7-iso-cucurbic acid (7-iso-CA), 6-epi-7-iso-cucurbic acid (6-epi-7-iso-CA), and both stereoisomers of the JA leucine conjugate ((+)-JA-Leu and (-)-JA-Leu)) were determined and used in a simplified mathematical model to predict stressdependent JA redistribution between cytosol and apoplast in comparison with ABA. The redistribution of JA takes place similar to ABA; however, its velocity is much higher because of the high JA membrane permeability. When the permeability coefficients for the mesophyll plasma membrane are plotted double-logarithmically against the ratio of the distribution coefficient and the molecular ratio to the power of 1.5 (K_DM_r ^–1.5), two straight lines result for two different classes of compounds. The permeability coefficients of JA conjugates are lower than that of the free acid by approximately one order of magnitude, but they are still significantly higher than that of ABA.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Light-dependent regulation of the jasmonate pathway

Jasmonates (JAs) are plant hormones which are crucial for the response of plants to several biotic and abiotic stresses. Beside this important function, they are involved in several developmental processes throughout plant life. In this short review, we would like to summarize the recent findings about the function of JAs in photomorphogenesis with a main focus on the model plant rice. Early plant development is determined to a large extent by light. Depending on whether seedlings are raised in darkness or in light, they show a completely different appearance which led to the terms skoto- and photomorphogenesis, respectively. The different appearance depending on the light conditions has been used to screen for mutants in photoperception and signalling. By this approach, mutants for several photoreceptors and in the downstream signalling pathways could be isolated. In rice, we and others isolated mutants with a very intriguing phenotype. The mutated genes have been cloned by map-based cloning, and all of them encode for JA biosynthesis genes. The most bioactive form of JAs identified so far is the amino acid conjugate jasmonoyl-isoleucin (JA-Ile). In order to conjugate JA to Ile, an enzyme of the GH3 family, JASMONATE RESISTANT 1, is required. We characterized mutants of OsJAR1 on a physiological and biochemical level and found evidence for redundantly active enzymes in rice.     

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.     

Light-dependent regulation of the jasmonate pathway

Jasmonates (JAs) are plant hormones which are crucial for the response of plants to several biotic and abiotic stresses. Beside this important function, they are involved in several developmental processes throughout plant life. In this short review, we would like to summarize the recent findings about the function of JAs in photomorphogenesis with a main focus on the model plant rice. Early plant development is determined to a large extent by light. Depending on whether seedlings are raised in darkness or in light, they show a completely different appearance which led to the terms skoto- and photomorphogenesis, respectively. The different appearance depending on the light conditions has been used to screen for mutants in photoperception and signalling. By this approach, mutants for several photoreceptors and in the downstream signalling pathways could be isolated. In rice, we and others isolated mutants with a very intriguing phenotype. The mutated genes have been cloned by map-based cloning, and all of them encode for JA biosynthesis genes. The most bioactive form of JAs identified so far is the amino acid conjugate jasmonoyl-isoleucin (JA-Ile). In order to conjugate JA to Ile, an enzyme of the GH3 family, JASMONATE RESISTANT 1, is required. We characterized mutants of OsJAR1 on a physiological and biochemical level and found evidence for redundantly active enzymes in rice.     

Octadecanoid and jasmonate signaling in tomato (Lycopersicon esculentum Mill.) leaves: endogenous jasmonates do not induce jasmonate biosynthesis

Jasmonates and their precursors, the octadecanoids, are signals in stress-induced alteration of gene expression. Several mRNAs coding for enzymes of jasmonic acid (JA) biosynthesis are up-regulated upon JA treatment or endogenous increase of the JA level. Here we investigated the positive feedback of endogenous JA on JA formation, as well as its beta-oxidation steps. JA-responsive gene expression was recorded in terms of proteinase inhibitor2 (pin2) mRNA accumulation. JA formed upon treatment of tomato (Lycopersicon esculentum cv. Moneymaker) leaves with JA derivatives carrying different lengths of the carboxylic acid side chain was quantified by gas chromatography-mass spectrometry (GC-MS). The data revealed that beta-oxidation of the side chain occurs up to a butyric acid moiety. The amount of JA formed from side-chain modified JA derivatives correlated with pin2-mRNA accumulation. JA derivatives with a carboxylic side chain of 3, 5 or 7 carbon atoms were unable to form JA and to express on pin2, whereas even-numbered derivatives were active. After treatment of tomato leaves with (10-(2)H)-(-)-12-oxophytoenoic acid, (4-(2)H)-(-)-JA and its methyl ester were formed and could be quantified separately from the endogenously nonlabeled JA pool by GC-MS analysis via isotopic discrimination. The level of 8 nmol per g fresh weight JA and its methyl ester originated exclusively from labeled 12-oxophytoenic acid. This and further data indicate that endogenous synthesis of the JA precursor 12-oxophytodienoic acid, as well as of JA and its methyl ester, are not induced in tomato leaves, suggesting that positive feedback in JA biosynthesis does not function in vivo.     

Jasmonic acid carboxyl methyltransferase regulates development and herbivory‐induced defense response in rice

Jasmonic acid(JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase(JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate(Me JA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene,Os JMT1, whose encoding protein was localized in the cytosol, we found that the recombinant Os JMT1 protein catalyzed JA to Me JA. Os JMT1 is up-regulated in response to infestation with the brown planthopper(BPH; Nilaparvata lugens). Plants in which Os JMT1 had been overexpressed(oeJMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased Me JA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine(JAIle). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs,probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H_2O_2 and Me JA in oe-JMT plants. These results indicate that Os JMT1,by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice.     

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.     

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.     

The levels of jasmonic acid and salicylic acid in a rice-barnyardgrass coexistence system and their relation to rice allelochemicals

Despite increasing knowledge of jasmonic acid (JA) and salicylic acid (SA) as signaling compounds involved in the defense of rice against attacking microbes and insect predators, relatively little is known about their levels in the growth media and their interactions with other plant competitors. In present study we quantified JA and SA in a rice-barnyardgrass coexistence system followed by correlation analysis to access rice allelochemicals. Both rice and barnyardgrass biosynthesized JA and SA, but their contents varied greatly with species, tissues and coexistence. There was a positive correlation in contents between rice allelochemicals and JA in roots or SA in shoots. Endogenous JA was exuded from barnyardgrass roots eliciting the production of rice allelochemicals. SA was not detected in growth media as an exogenous signaling compound in a rice-barnyardgrass coexistence system, but SA content in rice shoots was an indicator for distinguishing the allelopathic rice traits from the non-allelopathic ones.     

A CCCH-type zinc finger nucleic acid-binding protein quantitatively confers resistance against rice bacterial blight disease

Bacterial blight is a devastating disease of rice (Oryza sativa) caused by Xanthomonas oryzae pv oryzae (Xoo). Zinc finger proteins harboring the motif with three conserved cysteine residues and one histidine residue (CCCH) belong to a large family. Although at least 67 CCCH-type zinc finger protein genes have been identified in the rice genome, their functions are poorly understood. Here, we report that one of the rice CCCH-type zinc finger proteins, C3H12, containing five typical CX(8)-CX(5)-CX(3)-H zinc finger motifs, is involved in the rice-Xoo interaction. Activation of C3H12 partially enhanced resistance to Xoo, accompanied by the accumulation of jasmonic acid (JA) and induced expression of JA signaling genes in rice. In contrast, knockout or suppression of C3H12 resulted in partially increased susceptibility to Xoo, accompanied by decreased levels of JA and expression of JA signaling genes in rice. C3H12 colocalized with a minor disease resistance quantitative trait locus to Xoo, and the enhanced resistance of randomly chosen plants in the quantitative trait locus mapping population correlated with an increased expression level of C3H12. The C3H12 protein localized in the nucleus and possessed nucleic acid-binding activity in vitro. These results suggest that C3H12, as a nucleic acid-binding protein, positively and quantitatively regulates rice resistance to Xoo and that its function is likely associated with the JA-dependent pathway.