Absence Of Jasmonic Acid Research Articles

Alternative splicing of CsJAZ1 negatively regulates flavan-3-ol biosynthesis in tea plants.

Flavan-3-ols are abundant in the tea plant (Camellia sinensis) and confer tea with flavor and health benefits. We recently found that alternative splicing of genes is likely involved in the regulation of flavan-3-ol biosynthesis; however, the underlying regulatory mechanisms remain unknown. Here, we integrated metabolomics and transcriptomics to construct metabolite-gene networks in tea leaves, collected over five different months and from five spatial positions, and found positive correlations between endogenous jasmonic acid (JA), flavan-3-ols, and numerous transcripts. Transcriptome mining further identified CsJAZ1, which is negatively associated with flavan-3-ols formation and has three CsJAZ1 transcripts, one full-length (CsJAZ1-1), and two splice variants (CsJAZ1-2 and -3) that lacked 3' coding sequences, with CsJAZ1-3 also lacking the coding region for the Jas domain. Confocal microscopy showed that CsJAZ1-1 was localized to the nucleus, while CsJAZ1-2 and CsJAZ1-3 were present in both the nucleus and the cytosol. In the absence of JA, CsJAZ1-1 was bound to CsMYC2, a positive regulator of flavan-3-ol biosynthesis; CsJAZ1-2 functioned as an alternative enhancer of CsJAZ1-1 and an antagonist of CsJAZ1-1 in binding to CsMYC2; and CsJAZ1-3 did not interact with CsMYC2. In the presence of JA, CsJAZ1-3 interacted with CsJAZ1-1 and CsJAZ1-2 to form heterodimers that stabilized the CsJAZ1-1-CsMYC2 and CsJAZ1-2-CsMYC2 complexes, thereby repressing the transcription of four genes that act late in the flavan-3-ol biosynthetic pathway. These data indicate that the alternative splicing variants of CsJAZ1 coordinately regulate flavan-3-ol biosynthesis in the tea plant and improve our understanding of JA-mediated flavan-3-ol biosynthesis.

Cloning, characterization, and subcellular localization of a novel JAZ repressor from Eleusine coracana

Jasmonate ZIM domain (JAZ) proteins are key regulators of the jasmonic acid (JA) signaling pathway. Repressors of JAZ remain bound to the myelocytomatosis 2 (MYC2) or MYC3/MYC4 transcription factors in the absence of JA and negatively regulate transcription of the JA responsive genes. In the presence of JA, JAZ proteins interact with coronative insensitive 1 (COI1), the recognition molecule of E3 ubiquitin ligase SCFCOI1 (COI1 stabilized by Skp, cullin, F-box containing complex), get ubiquitinated, and subsequently degraded by the 26S proteasome. However, there is a dearth of knowledge about this gene family in monocot cereals, specifically its role in finger millet is unknown till date. Here we present the isolation and characterization of a novel JAZ family repressor gene from nonsequenced Eleusine coracana (EcJAZ) utilizing available genome information of Oryza, Sorghum, and Setaria. The EcJAZ sequence showed the presence of a conserved ZIM domain, the Jas motif, and N-terminal motif 7 like other Group1 TIFY sequence containing proteins. We observed coronatine (an analog of JA-Ile) dependent and time dependent degradation of recombinant EcJAZ that thereby fulfilled the basic characteristic of the JAZ proteins. We found a proteasome inhibitor N-(phenylmethoxy) carbonyl-L-leucyl-N-[(1R)-1-formyl-3-methylbutyl]-L-leucinamide) (MG132) mediated degradation inhibition of EcJAZ that supported its 26S proteasome mediated degradation. Our study shows the nuclear localization of GFP-EcJAZ by Agrobacterium mediated transient transformation of onion scale epidermal cells. In Eleusine leaves, transcription of EcJAZ increased 4.2-fold by salt stress and 5.5-fold by coronatine application; thus ascertained its inducibility by the abiotic stress as well as by bioactive JA-Ile. Taken together, all these results contribute to our understanding of the JA signaling pathway in Eleusine coracana.

The Pokeweed Leaf mRNA Transcriptome and Its Regulation by Jasmonic Acid.

The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP), a ribosome inactivating protein (RIP) that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA), a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

BHLH05 is an interaction partner of MYB51 and a novel regulator of glucosinolate biosynthesis in Arabidopsis.

By means of yeast (Saccharomyces cerevisiae) two-hybrid screening, we identified basic helix-loop-helix transcription factor05 (bHLH05) as an interacting partner of MYB51, the key regulator of indolic glucosinolates (GSLs) in Arabidopsis (Arabidopsis thaliana). Furthermore, we show that bHLH04, bHLH05, and bHLH06/MYC2 also interact with other R2R3-MYBs regulating GSL biosynthesis. Analysis of bhlh loss-of-function mutants revealed that the single bhlh mutants retained GSL levels that were similar to those in wild-type plants, whereas the triple bhlh04/05/06 mutant was depleted in the production of GSL. Unlike bhlh04/06 and bhlh05/06 mutants, the double bhlh04/05 mutant was strongly affected in the production of GSL, pointing to a special role of bHLH04 and bHLH05 in the control of GSL levels in the absence of jasmonic acid. The combination of two specific gain-of-function alleles of MYB and bHLH proteins had an additive effect on GSL levels, as demonstrated by the analysis of the double MYB34-1D bHLH05D94N mutant, which produces 20-fold more indolic GSLs than bHLH05D94N and ecotype Columbia-0 of Arabidopsis. The amino acid substitution D94N in bHLH05D94N negatively affects the interaction with JASMONATE-ZIM DOMAIN protein, thereby resulting in constitutive activation of bHLH05 and mimicking jasmonic acid treatment. Our study revealed the bHLH04, bHLH05, and bHLH06/MYC2 factors as novel regulators of GSL biosynthesis in Arabidopsis.

Evidences for structural basis of altered ascorbate peroxidase activity in cadmium-stressed rice plants exposed to jasmonate

Binding interactions of cadmium (Cd) with rice ascorbate peroxidase (OsAPX) in presence or absence of jasmonate was examined in-silico. OsAPX is a 250 amino acid long protein with 90 % sequence similarity to soybean-APX. The 3D model of OsAPX obtained by homology modeling using soybean APX (PDBID:1OAF) as template was associated with -15975.85 kJ/mol energy, 100 % residues in favoured region, verify score of 0.85, ERRAT score 89.625 and a negative ProSA graph, suggesting OsAPX model to be of good quality, robust and reliable which was submitted with Protein Model Database with PMDBID: PM0078091. The rice ascorbate peroxidase ascorbate [OsAPX-Asc] complex had a substrate binding cavity involving residues at position (30)KSCAPL(35), (167)RCH(169) and (172)R wherein ascorbate accommodated via three H-bonds involving (30)Lys at the γ-edge of heme. (169)His served as a bridge between heme-porphyrin of OsAPX and ascorbate creating a charge relay system. Cd bound in [OsAPX-Asc-Cd] complex at (29)EKSCAPL(35), a site similar to ascorbate binding site. The binding of Cd caused breaking of (169)His bridge shifting the protein conformation. Cadmium exhibited four electrostatic interactions via (29)Glu of OsAPX backbone. Docking of [OsAPX-Asc] with jasmonic acid (JA) resulted in [OsAPX-Asc-JA] complex where 4-H-bonds held JA to OsAPX in a cavity at γ-edge on the distal side of heme. The binding of [OsAPX-Asc-JA] to Cd show the metal to bind at a position other than that involved in binding of OsAPX with Cd alone. Results indicate that Cd does not replace iron or ascorbate or JA but binds to OsAPX on the surface at a separate site electrostatically. In presence of JA the interactions involved in formation of [OsAPXAsc] are restored which is otherwise altered by the presence of Cd. The formation and reformation of H-bond take place between the [OsAPX-Asc] and Cd/JA. It is the interaction between heme and ascorbate which is modulated differently in presence of Cd/JA. In absence of JA, Cd-binds to the [OsAPX-Asc] complex at the proximal end of APX near Asc-binding site, whereas in presence of JA, Cd-binds on the opposite site of the Asc-binding site involving (30)Lys and (29)Glu residues. In-silico binding studies well correlate with the wet-lab results where exogenous application of JA increased the activity of OsAPX in rice grown under Cd-stress. Therefore it is concluded that the activity of OsAPX in rice roots and shoots are compromised under Cd-stress alone.

Xenobiotic- and Jasmonic Acid-Inducible Signal Transduction Pathways Have Become Interdependent at the Arabidopsis CYP81D11 Promoter

Plants modify harmful substances through an inducible detoxification system. In Arabidopsis (Arabidopsis thaliana), chemical induction of the cytochrome P450 gene CYP81D11 and other genes linked to the detoxification program depends on class II TGA transcription factors. CYP81D11 expression is also induced by the phytohormone jasmonic acid (JA) through the established pathway requiring the JA receptor CORONATINE INSENSITIVE1 (COI1) and the JA-regulated transcription factor MYC2. Here, we report that the xenobiotic- and the JA-dependent signal cascades have become interdependent at the CYP81D11 promoter. On the one hand, MYC2 can only activate the expression of CYP81D11 when both the MYC2- and the TGA-binding sites are present in the promoter. On the other hand, the xenobiotic-regulated class II TGA transcription factors can only mediate maximal promoter activity if TGA and MYC2 binding motifs, MYC2, and the JA-isoleucine biosynthesis enzymes DDE2/AOS and JAR1 are functional. Since JA levels and degradation of JAZ1, a repressor of the JA response, are not affected by reactive chemicals, we hypothesize that basal JA signaling amplifies the response to chemical stress. Remarkably, stress-induced expression levels were 3-fold lower in coi1 than in the JA biosynthesis mutant dde2-2, [corrected] revealing that COI1 can contribute to the activation of the promoter in the absence of JA. Moreover, we show that deletion of the MYC2 binding motifs abolishes the JA responsiveness of the promoter but not the responsiveness to COI1. These findings suggest that yet unknown cis-element(s) can mediate COI1-dependent transcriptional activation in the absence of JA.

Tobacco Transcription Factors NtMYC2a and NtMYC2b Form Nuclear Complexes with the NtJAZ1 Repressor and Regulate Multiple Jasmonate-Inducible Steps in Nicotine Biosynthesis

Biotic and abiotic stress lead to elevated levels of jasmonic acid (JA) and its derivatives and activation of the biosynthesis of nicotine and related pyridine alkaloids in cultivated tobacco (Nicotiana tabacum L.). Among the JA-responsive genes is NtPMT1a, encoding putrescine N-methyl transferase, a key regulatory enzyme in nicotine formation. We have characterized three genes (NtMYC2a, b, c) encoding basic helix-loop-helix (bHLH) transcription factors (TFs) whose expression is rapidly induced by JA and that specifically activate JA-inducible NtPMT1a expression by binding a G-box motif within the NtPMT1a promoter in in vivo and in vitro assays. Using split-YFP assays, we further show that, in the absence of JA, NtMYC2a and NtMYC2b are present as nuclear complexes with the NtJAZ1 repressor. RNA interference (RNAi)-mediated knockdown of NtMYC2a and NtMYC2b expression results in significant decreases in JA-inducible NtPMT1a transcript levels, as well as reduced levels of transcripts encoding other enzymes involved in nicotine and minor alkaloid biosynthesis, including an 80-90% reduction in the level of transcripts encoding the putative nicotine synthase gene NtA662. In contrast, ectopic overexpression of NtMYC2a and NtMYC2b had no effect on NtPMT1a expression in the presence or absence of exogenously added JA. These data suggest that NtMYC2a, b, c are required components of JA-inducible expression of multiple genes in the nicotine biosynthetic pathway and may act additively in the activation of JA responses.

Jasmonate Controls Polypeptide Patterning in Undamaged Tissue in Wounded Arabidopsis Leaves

Wounding initiates a strong and largely jasmonate-dependent remodelling of the transcriptome in the leaf blades of Arabidopsis (Arabidopsis thaliana). How much control do jasmonates exert on wound-induced protein repatterning in leaves? Replicated shotgun proteomic analyses of 2.5-mm-wide leaf strips adjacent to wounds revealed 106 differentially regulated proteins. Many of these gene products have not emerged as being wound regulated in transcriptomic studies. From experiments using the jasmonic acid (JA)-deficient allene oxide synthase mutant we estimated that approximately 95% of wound-stimulated changes in protein levels were deregulated in the absence of JA. The levels of two tonoplast proteins already implicated in defense response regulation, TWO-PORE CHANNEL1 and the calcium-V-ATPase ACA4 increased on wounding, but their transcripts were not wound inducible. The data suggest new roles for jasmonate in controlling the levels of calcium-regulated pumps and transporters, proteins involved in targeted proteolysis, a putative bacterial virulence factor target, a light-dependent catalyst, and a key redox-controlled enzyme in glutathione synthesis. Extending the latter observation we found that wounding increased the proportion of oxidized glutathione in leaves, but only in plants able to synthesize JA. The oxidizing conditions generated through JA signaling near wounds help to define the cellular environment in which proteome remodelling occurs.

Phytochrome Chromophore Deficiency Leads to Overproduction of Jasmonic Acid and Elevated Expression of Jasmonate-Responsive Genes in Arabidopsis

An Arabidopsis mutant line named hy1-101 was isolated because it shows stunted root growth on medium containing low concentrations of jasmonic acid (JA). Subsequent investigation indicated that even in the absence of JA, hy1-101 plants exhibit shorter roots and express higher levels of a group of JA-inducible defense genes. Here, we show that the hy1-101 mutant has increased production of JA and its jasmonate-related phenotype is suppressed by the coi1-1 mutation that interrupts JA signaling. Gene cloning and genetic complementation analyses revealed that the hy1-101 mutant contains a mutation in the HY1 gene, which encodes a heme oxygenase essential for phytochrome chromophore biosynthesis. These results support a hypothesis that phytochrome chromophore deficiency leads to overproduction of JA and activates COI1-dependent JA responses. Indeed, we show that, like hy1-101, independent alleles of the phytochrome chromophore-deficient mutants, including hy1-100 and hy2 (CS68), also show elevated JA levels and constant expression of JA-inducible defense genes. We further provide evidence showing that, on the other hand, JA inhibits the expression of a group of light-inducible and photosynthesis-related genes. Together, these data imply that the JA-signaled defense pathway and phytochrome chromophore-mediated light signaling might have antagonistic effects on each other.

Plant defense in the absence of jasmonic acid

Abundant research suggests that jasmonic acid can regulate a plant's responses to wounding, insects and some pathogens. Following exposure to such stimuli, jasmonic acid concentrations are known to increase. Exogenous applications of jasmonic acid upregulate the expression of several defense-related genes. In addition, mutations in genes that are crucial for jasmonic acid biosynthesis or sensing cause reduced resistance to some biotic stresses. However, Annick Stintzi et al. [1] now provide evidence that this is not true for all jasmonic acid-related mutants, suggesting that jasmonic acid is not the only jasmonate family member with the capability to activate plant defenses in vivo. They show that jasmonic acid has partially overlapping resistance functions with an upstream precursor – the cyclopentenone, 12-oxo-phytodienoic acid (OPDA) – and propose that other oxylipins could have similar effects.

Plant defense in the absence of jasmonic acid: the role of cyclopentenones.

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxo-phytodienoate (OPDA) reductase required for jasmonic acid (JA) biosynthesis. Oxylipin signatures of wounded opr3 leaves revealed the absence of detectable 3R,7S-JA as well as altered levels of its cyclopentenone precursors OPDA and dinor OPDA. In contrast to JA-insensitive coi1 plants and to the fad3 fad7 fad8 mutant lacking the fatty acid precursors of JA synthesis, opr3 plants exhibited strong resistance to the dipteran Bradysia impatiens and the fungus Alternaria brassicicola. Analysis of transcript profiles in opr3 showed the wound induction of genes previously known to be JA-dependent, suggesting that cyclopentenones could fulfill some JA roles in vivo. Treating opr3 plants with exogenous OPDA powerfully up-regulated several genes and disclosed two distinct downstream signal pathways, one through COI1, the other via an electrophile effect of the cyclopentenones. We conclude that the jasmonate family cyclopentenone OPDA (most likely together with dinor OPDA) regulates gene expression in concert with JA to fine-tune the expression of defense genes. More generally, resistance to insect and fungal attack can be observed in the absence of JA.

Jasmonic acid-dependent increase in vegetative storage protein in soybean tissue cultures

Adding jasmonic acid (JA) to autotrophic, photomixotrophic, or heterotrophic suspension cultures of soybean specifically increased the level of the Mr 30,000 subunit of soybean vegetative storage protein (VSP-30) and a polypeptide at Mr 18,000 that interacted with antibody raised against VSP. Using photomixotrophic cells, the increase was observed at concentrations as low as 10 nM JA and the increase was evident within 2 h following treatment. Below 10 μM, JA did not inhibit growth of the cells but did cause browning at higher concentrations. Other plant growth regulators, including abscisic acid (ABA), gibberellic acid, and benzyl adenine, did not alter the level of VSP-30 either in the presence or absence of JA. Methyl jasmonate (JA-Me), 3-oxo-2-butyl-cyclopentane-1-acetate, and 3-oxo-2-pentyl-cyclopentane-1-acetate also increased VSP-30 but at higher concentrations than JA. Altering the level of reduced nitrogen or sucrose in the medium did not alter VSP-30 levels in the cells, but at higher sucrose concentrations, sensitivity to JA was reduced. The dramatic increase in VSP-30 elicited by JA appears to be a specific response to the phytohormone.