The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.     

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.     

Another JA/COI1-independent role of OPDA detected in tomato embryo development

Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.     

The wound response mutant suppressor of prosystemin-mediated responses6 (spr6) is a weak allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1)

The systemic defense response of tomato plant in response to insect attack and wounding is regulated by the 18 amino acid peptide systemin and the phytohormone jasmonic acid (JA). Recent genetic analyses based mainly on spr (suppressors of prosystemin-mediated responses) mutant screens have led to the hypothesis that systemin acts at, or near, the site of wounding to amplify the production of JA, which in turn functions as a mobile signal to promote the systemic defense response. In order to identify more components involved in the systemin/JA-signaled defense response, we carried out a larger scale screen for new spr mutants in tomato. Here we describe the characterization of spr6, a mutant impaired in wound- and systemin-induced defense gene expression. Using a candidate gene approach based on genetic linkage, we demonstrate that spr6 is allelic to jai1-1, which is a loss-of-function allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. We show several aspects of the spr6 mutant phenotype distinct from that of jai1-1. First, the responsiveness of spr6 plants to exogenous JA shows a dosage dependency, i.e. it is more sensitive to JA than jai1-1 while less sensitive to JA than the wild-type. Secondly, unlike the sterile jai1-1, the spr6 plant displays normal fertility and seed set and thus can be maintained as a pure line and does not require selection. Therefore, spr6 provides a valuable tool, which can complement the limitations of jai1-1, to study JA signaling in tomato. The gene identification process of Spr6 we described herein represents an example showing the convenience of a candidate gene approach, based on genetic linkage, to identify gene functions of genetic loci defined by tomato wound response mutants.     

Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato. The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA). We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants. Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes. These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants.     

The involvement of jasmonates and ethylene in Alternaria alternata f. sp. lycopersici toxin-induced tomato cell death

Previous studies have shown that an ethylene (ET)-dependent pathway is involved in the cell death signalling triggered by Alternaria alternata f. sp. lycopersici (AAL) toxin in detached tomato (Solanum lycopersicum) leaves. In this study, the role of jasmonic acid (JA) signalling in programmed cell death (PCD) induced by AAL toxin was analysed using a 35S::prosystemin transgenic line (35S::prosys), a JA-deficient mutant spr2, and a JA-insensitive mutant jai1. The results indicated that JA biosynthesis and signalling play a positive role in the AAL toxin-induced PCD process. In addition, treatment with the exogenous ET action inhibitor silver thiosulphate (STS) greatly suppressed necrotic lesions in 35S::prosys leaves, although 35S::prosys leaflets co-treated with AAL toxin and STS still have a significant high relative conductivity. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) markedly enhanced the sensitivity of spr2 and jai1 mutants to the toxin. However, compared with AAL toxin treatment alone, exogenous application of JA to the ET-insensitive mutant Never ripe (Nr) did not alter AAL toxin-induced cell death. In addition, the reduced ET-mediated gene expression in jai1 leaves was restored by co-treatment with ACC and AAL toxin. Furthermore, JA treatment restored the decreased expression of ET biosynthetic genes but not ET-responsive genes in the Nr mutant compared with the toxin treatment alone. Based on these results, it is proposed that both JA and ET promote the AAL toxin-induced cell death alone, and the JAI1 receptor-dependent JA pathway also acts upstream of ET biosynthesis in AAL toxin-triggered PCD.     

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.     

Functional diversification of acyl-coenzyme A oxidases in jasmonic acid biosynthesis and action

The biosynthesis of jasmonic acid (JA) in plant peroxisomes requires the action of acyl-coenzyme A oxidase (ACX). Among the five expressed members (ACX1-5) of the ACX gene family in Arabidopsis (Arabidopsis thaliana), only ACX1 is known to serve a role in JA production. Here, we used transgenic promoter-reporter lines to show that ACX1 is highly expressed in mature and germinating pollen, stem epidermal cells, and other tissues in which jasmonate-signaled processes occur. Wound-induced JA accumulation was reduced in a mutant that is defective in ACX1 and was abolished in a mutant that is impaired in both ACX1 and its closely related paralog, ACX5. The severe JA deficiency in acx1/5 double mutants was accompanied by decreased resistance to the leaf-eating insect Trichoplusia ni. The double mutant also showed reduced pollen viability and fecundity. Treatment of acx1/5 plants with JA restored both protection against T. ni larvae and normal seed set. Unexpectedly, acx1/5 plants accumulated JA in response to infection by the necrotrophic fungal pathogen Alternaria brassicicola. In contrast to mutants that are impaired in jasmonate perception or early steps of the JA biosynthetic pathway, acx1/5 plants maintained resistance to A. brassicicola infection. These results indicate that ACX1/5-mediated JA synthesis is essential for resistance to chewing insects and male reproductive function and further suggest that other ACX isozymes contribute to JA production in response to A. brassicicola challenge. Thus, different types of biotic stress may induce JA synthesis via distinct enzymatic routes.     

Jasmonates in flower and seed development

Jasmonates are ubiquitously occurring lipid-derived signaling compounds active in plant development and plant responses to biotic and abiotic stresses. Upon environmental stimuli jasmonates are formed and accumulate transiently. During flower and seed development, jasmonic acid (JA) and a remarkable number of different metabolites accumulate organ- and tissue specifically. The accumulation is accompanied with expression of jasmonate-inducible genes. Among these genes there are defense genes and developmentally regulated genes. The profile of jasmonate compounds in flowers and seeds covers active signaling molecules such as JA, its precursor 12-oxophytodienoic acid (OPDA) and amino acid conjugates such as JA-Ile, but also inactive signaling molecules occur such as 12-hydroxy-JA and its sulfated derivative. These latter compounds can occur at several orders of magnitude higher level than JA. Metabolic conversion of JA and JA-Ile to hydroxylated compounds seems to inactivate JA signaling, but also specific functions of jasmonates in flower and seed development were detected. In tomato OPDA is involved in embryo development. Occurrence of jasmonates, expression of JA-inducible genes and JA-dependent processes in flower and seed development will be discussed.     

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.     

Gibberellins and Light-Stimulated Seed Germination

Bioactive gibberellins (GAs) promote seed germination in a number of plant species. In dicots, such as tomato and Arabidopsis, de novo GA biosynthesis after seed imbibition is essential for germination. Light is a crucial environmental cue determining seed germination in some species. The red (R) and far-red light photoreceptor phytochrome regulates GA biosynthesis in germinating lettuce and Arabidopsis seeds. This effect of light is, at least in part, targeted to mRNA abundance of GA 3-oxidase, which catalyzes the final biosynthetic step to produce bioactive GAs. The R-inducible GA 3-oxidase genes are predominantly expressed in the hypocotyl of Arabidopsis embryos. This predicted location of GA biosynthesis appears to correlate with the photosensitive site determined by using R micro-beam in lettuce seeds. The GA-deficient non-germinating mutants have been useful for studying how GA stimulates seed germination. In tomato, GA promotes the growth potential of the embryo and weakens the structures surrounding the embryo. Endo-b-mannanase, which is produced specifically in the micropylar endosperm in a GA-dependent manner, may be responsible for breaking down the endosperm cell walls to assist germination. Recently, a role for GA in overcoming the resistance imposed by the seed coat was also suggested in Arabidopsis from work with a range of seed coat mutants. Towards understanding the GA signaling pathway, GA response mutants have been isolated and characterized, some of which are affected in GA-stimulated seed germination.     

Loss of function of FATTY ACID DESATURASE7 in tomato enhances basal aphid resistance in a salicylate-dependent manner

We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families.     

Constitutive overexpression of allene oxide cyclase in tomato (Lycopersicon esculentum cv. Lukullus) elevates levels of some jasmonates and octadecanoids in flower organs but not in leaves

The allene oxide cyclase (AOC), an enzyme in jasmonate biosynthesis, occurs in vascular bundles and ovules of tomato flowers which exhibit a tissue-specific oxylipin signature (Plant J. 24, 113-126, 2000). Constitutive overexpression of the AOC did not led to altered levels of jasmonates in leaves, but these levels increased upon wounding or other stresses suggesting regulation of jasmonate biosynthesis by substrate availability (Plant J. 33, 577-589, 2003). Here, we show dramatic changes in levels of jasmonic acid (JA), of 12-oxo-phytodienoic acid (OPDA), their methyl esters (JAME, OPDAME), and of dinor-OPDA in most flower organs upon constitutive overexpression of AOC. Beside a dominant occurrence of OPDAME and JA in most flower organs, the ratio among the various compounds was altered differentially in the organs of transgenic flowers, e.g. OPDAME increased up to 53-fold in stamen, and JA increased about 51-fold in buds and 7.5-fold in sepals. The increase in jasmonates and octadecanoids was accompanied by decreased levels of free lipid hydro(per)oxy compounds. Except for 16:2, the AOC overexpression led to a significant increase in free but not esterified polyunsaturated fatty acids in all flower organs. The data suggest different regulation of JA biosynthesis in leaves and flowers of tomato.     

Bestatin, an inhibitor of aminopeptidases, provides a chemical genetics approach to dissect jasmonate signaling in Arabidopsis

Bestatin, a potent inhibitor of some aminopeptidases, was shown previously to be a powerful inducer of wound-response genes in tomato (Lycopersicon esculentum). Here, we present several lines of evidence showing that bestatin specifically activates jasmonic acid (JA) signaling in plants. First, bestatin specifically activates the expression of JA-inducible genes in tomato and Arabidopsis (Arabidopsis thaliana). Second, the induction of JA-responsive genes by bestatin requires the COI1-dependent JA-signaling pathway, but does not depend strictly on JA biosynthesis. Third, microarray analysis using Arabidopsis whole-genome chip demonstrates that the gene expression profile of bestatin-treated plants is similar to that of JA-treated plants. Fourth, bestatin promotes a series of JA-related developmental phenotypes. Taken together, the unique action mode of bestatin in regulating JA-signaled processes leads us to the hypothesis that bestatin exerts its effects through the modulation of some key regulators in JA signaling. We have employed bestatin as an experimental tool to dissect JA signaling through a chemical genetic screening, which yielded a collection of Arabidopsis bestatin-resistant (ber) mutants that are insensitive to the inhibitory effects of bestatin on root elongation. Further characterization efforts demonstrate that some ber mutants are defective in various JA-induced responses, which allowed us to classify the ber mutants into three phenotypic groups: JA-insensitive ber mutants, JA-hypersensitive ber mutants, and mutants insensitive to bestatin but showing normal response to JA. Genetic and phenotypic analyses of the ber mutants with altered JA responses indicate that we have identified several novel loci involved in JA signaling.     

Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling

The allene oxide cyclase (AOC)-catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue-specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i). activation of systemin-dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii). the tissue-specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii). the tissue-specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.     

Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato

The phytohormone jasmonic acid (JA) regulates the synthesis of secondary metabolites in a wide range of plant species. Here, we show that exogenous methyl-JA (MeJA) elicits massive accumulation of caffeoylputrescine (CP) in tomato leaves. A mutant (jai1) that is defective in jasmonate perception failed to accumulate CP in flowers and MeJA-treated leaves. Conversely, a transgenic tomato line (called 35S::PS) that exhibits constitutive JA signaling accumulated high levels of leaf CP in the absence of jasmonate treatment. RNA blot analysis showed that genes encoding enzymes in the phenylpropanoid and polyamine pathways for CP biosynthesis are upregulated in MeJA-treated wild-type plants and in untreated 35S::PS plants. These results indicate that CP accumulation in tomato is tightly controlled by the jasmonate signaling pathway, and provide proof-of-concept that the production of some plant secondary metabolites can be enhanced by transgenic manipulation of endogenous JA levels.