iTRAQ-Based Quantitative Proteomic Analysis of the Arabidopsis Mutant opr3-1 in Response to Exogenous MeJA.

Jasmonates (JAs) regulate the defense of biotic and abiotic stresses, growth, development, and many other important biological processes in plants. The comprehensive proteomic profiling of plants under JAs treatment provides insights into the regulation mechanism of JAs. Isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis was performed on the Arabidopsis wild type (Ws) and JA synthesis deficiency mutant opr3-1. The effects of exogenous MeJA treatment on the proteome of opr3-1, which lacks endogenous JAs, were investigated. A total of 3683 proteins were identified and 126 proteins were differentially regulated between different genotypes and treatment groups. The functional classification of these differentially regulated proteins showed that they were involved in metabolic processes, responses to abiotic stress or biotic stress, the defense against pathogens and wounds, photosynthesis, protein synthesis, and developmental processes. Exogenous MeJA treatment induced the up-regulation of a large number of defense-related proteins and photosynthesis-related proteins, it also induced the down-regulation of many ribosomal proteins in opr3-1. These results were further verified by a quantitative real-time PCR (qRT-PCR) analysis of 15 selected genes. Our research provides the basis for further understanding the molecular mechanism of JAs' regulation of plant defense, photosynthesis, protein synthesis, and development.

Identification of Jasmonic Acid and Jasmonoyl-Isoleucine, and Characterization of AOS, AOC, OPR and JAR1 in the Model Lycophyte Selaginella moellendorffii.

Jasmonic acid (JA) is involved in a variety of physiological responses in seed plants. However, the detection and role of JA in lycophytes, a group of seedless vascular plants, have remained elusive until recently. This study provides the first evidence of 12-oxo-phytodienoic acid (OPDA), JA and jasmonoyl-isoleucine (JA-Ile) in the model lycophyte Selaginella moellendorffii. Mechanical wounding stimulated the accumulation of OPDA, JA and JA-Ile. These data were corroborated by the detection of enzymatically active allene oxide synthase (AOS), allene oxide cyclase (AOC), 12-oxo-phytodienoic acid reductase 3 (OPR3) and JA-Ile synthase (JAR1) in S. moellendorffii. SmAOS2 is involved in the first committed step of JA biosynthesis. SmAOC1 is a crucial enzyme for generating the basic structure of jasmonates and is actively involved in the formation of OPDA. SmOPR5, a functionally active OPR3-like enzyme, is also vital for the reduction of (+)-cis-OPDA, the only isomer of the JA precursor. The conjugation of JA to Ile by SmJAR1 demonstrates that S. moellendorffii produces JA-Ile. Thus, the four active enzymes have characteristics similar to those in seed plants. Wounding and JA treatment induced the expression of SmAOC1 and SmOPR5. Furthermore, JA inhibited the growth of shoots in S. moellendorffii, which suggests that JA functions as a signaling molecule in S. moellendorffii. This study proposes that JA evolved as a plant hormone for stress adaptation, beginning with the emergence of vascular plants.

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea.

Cis-(+)-12-oxo-phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12-oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3-1 and SiOPR3-2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA-Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen-induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3-1 and SiOPR3-2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.

Activated expression of AtEDT1/HDG11 promotes lateral root formation in Arabidopsis mutant edt1 by upregulating jasmonate biosynthesis.

Root architecture is crucial for plants to absorb water and nutrients. We previously reported edt1 (edt1D) mutant with altered root architecture that contributes significantly to drought resistance. However, the underlying molecular mechanisms are not well understood. Here we report one of the mechanisms underlying EDT1/HDG11-conferred altered root architecture. Root transcriptome comparison between the wild type and edt1D revealed that the upregulated genes involved in jasmonate biosynthesis and signaling pathway were enriched in edt1D root, which were confirmed by quantitative RT-PCR. Further analysis showed that EDT1/HDG11, as a transcription factor, bound directly to the HD binding sites in the promoters of AOS, AOC3, OPR3, and OPCL1, which encode four key enzymes in JA biosynthesis. We found that the jasmonic acid level was significantly elevated in edt1D root compared with that in the wild type subsequently. In addition, more auxin accumulation was observed in the lateral root primordium of edt1D compared with that of wild type. Genetic analysis of edt1D opcl1 double mutant also showed that HDG11 was partially dependent on JA in regulating LR formation. Taken together, overexpression of EDT1/HDG11 increases JA level in the root of edt1D by directly upregulating the expressions of several genes encoding JA biosynthesis enzymes to activate auxin signaling and promote lateral root formation.

Role of Jasmonic Acid Pathway in Tomato Plant-Pseudomonas syringae Interaction.

The jasmonic acid pathway has been considered as the backbone of the response against necrotrophic pathogens. However, a hemi-biotrophic pathogen, such as Pseudomonas syringae, has taken advantage of the crosstalk between the different plant hormones in order to manipulate the responses for its own interest. Despite that, the way in which Pseudomonas syringae releases coronatine to activate jasmonic acid-derived responses and block the activation of salicylic acid-mediated responses is widely known. However, the implication of the jasmonic intermediates in the plant-Pseudomonas interaction is not studied yet. In this work, we analyzed the response of both, plant and bacteria using SiOPR3 tomato plants. Interestingly, SiOPR3 plants are more resistant to infection with Pseudomonas. The gene expression of bacteria showed that, in SiOPR3 plants, the activation of pathogenicity is repressed in comparison to wild type plants, suggesting that the jasmonic acid pathway might play a role in the pathogenicity of the bacteria. Moreover, treatments with JA restore the susceptibility as well as activate the expression of bacterial pathogenicity genes. The observed results suggest that a complete jasmonic acid pathway is necessary for the susceptibility of tomato plants to Pseudomonas syringae.

Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory.

Feeding by chewing insects induces chemical defenses in plants that are regulated by the jasmonic acid (JA) pathway. Jasmonates are usually quantified by liquid chromatography-mass spectrometry (LC-MS) analysis of precursors and products in the biosynthetic pathway or inferred from the extraction and expression of genes known to respond to elevated levels of JA. Both approaches are costly and time consuming. To address these limitations, we developed a rapid reporter for the synthesis of JA based on the OPR3promoter:YFP-PTS1. Yellow fluorescent protein (YFP) fluorescence was increased by mechanical wounding and methyl jasmonate (MeJA) treatment and by caterpillar feeding. To develop an optimal sampling time for a quantitative bioassay, OPR3promoter:YFP-PTS1 plants were sampled at 1, 2, 3, and 24 h after treatment with 115 µM MeJA. The first increase in YFP fluorescence was detected at 2 h and remained elevated 3 and 24 h later; as a result, 3 h was chosen as the sampling time for a quantitative bioassay of jasmonate response to insect attack. Feeding by Pieris rapae caterpillars induced a 1.8-fold increase in YFP fluorescence, consistent with the known induction of JA production by this insect. We also assessed the utility of this reporter in studies of plant responses to caterpillar feeding vibrations, which are known to potentiate the JA-dependent production of chemical defenses. Pretreatment with feeding vibrations increased expression of the OPR3promoter:YFP-PTS1 in response to 14 µM MeJA. Feeding vibrations did not potentiate responses at higher MeJA concentrations, suggesting that potentiating effects of prior treatments can only be detected when plants are below a response threshold to the elicitor. The expression of OPR3 does not indicate levels of specific downstream jasmonates and quantification of specific jasmonates still requires detailed analysis by LC-MS. However, OPR3 expression does provide a rapid and inexpensive way to screen large numbers of plants for the involvement of jasmonate signaling in their response to a wide variety of treatments, and to study the induction and expression of AtOPR3.

Arabidopsis MYB24 Regulates Jasmonate-Mediated Stamen Development.

The phytohormone jasmonates (JAs) regulate various defense responses and diverse developmental processes including stamen development and fertility. Previous studies showed that JA induces CORONATINE INSENSITIVE 1-mediated degradation of JA ZIM-domain (JAZ) proteins, and activates the MYB transcription factors (such as MYB21 and MYB24) to regulate stamen development. In this study, we further uncover the mechanism underlying how MYB24 interacts with JAZs to control JA-regulated stamen development. We show that N-terminus of MYB21/24 interacts with 10 out of 12 JAZ proteins while both N-terminus and C-terminus of MYB24 are involved in dimerization of MYB21 and MYB24. Interestingly, male sterility of the JA-deficient mutant opr3 can be rescued by suitable level of the MYB24 overexpression but not by excessive high level of MYB24. Surprisingly, overexpression of MYB24NT, but not MYB24CT, could cause male sterility. These results provide new insights on MYB factors in JA-regulated stamen development.

Inbreeding compromises host plant defense gene expression and improves herbivore survival.

Inbreeding commonly occurs in flowering plants and often results in a decline in the plant's defense response. Insects prefer to feed and oviposit on inbred plants more than outbred plants--suggesting that selecting inbred host plants offers them fitness benefits. Until recently, no studies have examined the effects of host plant inbreeding on insect fitness traits such as growth and dispersal ability. In a recent article, we documented that tobacco hornworm (Manduca sexta L.) larvae that fed on inbred horsenettle (Solanum carolinense L.) plants exhibited accelerated larval growth and increased adult flight capacity compared to larvae that fed on outbred plants. Here we report that M. sexta mortality decreased by 38.2% when larvae were reared on inbred horsenettle plants compared to larvae reared on outbreds. Additionally, inbred plants showed a notable reduction in the average relative expression levels of lipoxygenease-D (LoxD) and 12-oxophytodienoate reductase-3 (OPR3), two genes in the jasmonic acid signaling pathway that are upregulated in response to herbivore damage. Our study presents evidence that furthers our understanding of the biochemical mechanism responsible for differences in insect performance on inbred vs. outbred host plants.

Four shades of detachment: regulation of floral organ abscission.

Abscission of floral organs from the main body of a plant is a dynamic process that is developmentally and environmentally regulated. In the past decade, genetic studies in Arabidopsis have identified key signaling components and revealed their interactions in the regulation of floral organ abscission. The phytohormones jasmonic acid (JA) and ethylene play critical roles in flower development and floral organ abscission. These hormones regulate the timing of floral organ abscission both independently and inter-dependently. Although significant progress has been made in understanding abscission signaling, there are still many unanswered questions. These include considering abscission in the context of reproductive development and interplay between hormones embedded in the developmental processes. This review summarizes recent advances in the identification of molecular components in Arabidopsis and discusses their relationship with reproductive development. The emerging roles of hormones in the regulation of floral organ abscission, particularly by JA and ethylene, are examined.

Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit.

To investigate the role of jasmonates (JAs) in the ripening of Fragaria chiloensis fruit, two concentrations of methyl jasmonate (MeJA, 10 and 100 µM) were evaluated at 2, 5 and 9 d using an in vitro ripening system. Fruit quality parameters; the contents of anthocyanin, lignin and cell wall polymers; and the transcriptional profiles of several ripening-related genes were analyzed. MeJA accelerated fruit ripening by means of a transitory increase in the soluble solid content/titratable acidity ratio, anthocyanin accumulation and an increase in softening at day 5. The expression of several phenylpropanoid-related genes, primarily those associated with anthocyanin biosynthesis, was increased under MeJA treatment, which correlated with an increased accumulation of anthocyanin. MeJA also altered the expression profiles of some cell wall-modifying genes, namely, EG1 and XTH1, and these changes correlated with a transient reduction in the firmness of MeJA-treated fruits. MeJA-responsive elements were observed in the promoter region of the EG1 gene. MeJA also increased the expression of LOX, AOS and OPR3, genes involved in the biosynthesis of JAs, and these changes correlated with the transient activation of fruit ripening observed. Conversely, the expression of ethylene and lignin biosynthesis genes (ACS, ACO, CAD and POD27) increased in MeJA-treated fruits at day 9. The present findings suggest that JAs promote the ripening of non-climacteric fruits through their involvement in anthocyanin accumulation, cell wall modification and the biosynthesis of ethylene and JAs.