Overaccumulation of γ-Glutamylcysteine in a Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition.

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and having only 27% of wild-type GSH level, was not jasmonate hypersensitive. Growth defects for jah2, but not pad2, were also seen in plants grown to maturity. Surprisingly, all phenotypes in the jah2 pad2-1 double mutant were weaker than in jah2. Quantification of γ-EC indicated these defects result from hyperaccumulation of this GSH precursor by 294- and 65-fold in jah2 and the double mutant, respectively. γ-EC reportedly partially substitutes for loss of GSH, but growth inhibition seen here was likely not due to an excess of total glutathione plus γ-EC because their sum in jah2 pad2-1 was only 16% greater than in the wild type. Further, the jah2 phenotypes were lost in a jasmonic acid biosynthesis mutant background, indicating the effect of γ-EC is mediated through jasmonate signaling and not as a direct result of perturbed redox status.

A tomato enzyme synthesizes (+)-7-iso-jasmonoyl-L-isoleucine in wounded leaves.

Jasmonoyl-L-isoleucine (JA-Ile) is a key jasmonate signal that probably functions in all plant species. The JASMONATE RESISTANT 1 (JAR1) enzyme synthesizes JA-Ile in Arabidopsis [Arabidopsis thaliana (L.) Heynh.], but a similar enzyme from tomato [Solanum lycopersicum (L.)] was not previously described. Tomato SlJAR1 has 66% sequence identity with Arabidopsis JAR1 and the SlJAR1-GST fusion protein purified from Escherichia coli catalyzed the formation of JA-amino acid conjugates in vitro. Kinetic analysis showed the enzyme has a strong preference for Ile over Leu and Val and it was about 10-fold more active with (+)-7-iso-JA than with its epimer (-)-JA. Leaf wounding rapidly increased JA-Ile 50-fold to about 450 pmol g(-1) FW at 30 min after wounding, while conjugates with Leu, Phe, Val and Met were only marginally increased or not detected. Nearly all of the endogenous JA-Ile was the bioactive epimer (+)-7-iso-JA-Ile and there was no evidence for its conversion to (-)-JA-Ile up to 6 h after wounding. A transgenic RNAi approach was used to suppress SlJAR1 transcript that reduced JA-Ile accumulation by 50-75%, suggesting that other JA conjugating enzymes may be present. These results show that SlJAR1 synthesizes the bioactive conjugate (+)-7-iso-JA-Ile and this is the predominant isomer accumulated in wounded tomato leaves.

Jasmonoyl-L-Tryptophan Disrupts IAA Activity through the AUX1 Auxin Permease.

Amide-linked conjugates between tryptophan (Trp) and jasmonic (JA) or indole-3-acetic (IAA) acids interfered with gravitropism and other auxin-dependent activities in Arabidopsis, but the mechanism was unclear. To identify structural features necessary for activity several additional Trp conjugates were synthesized. The phenylacetic acid (PAA) conjugate was active, while several others were not. Common features of active conjugates is that they have ring structures that are linked to Trp through an acetic acid side chain, while longer or shorter linkages are inactive or less active. A dominant mutant, called tryptophan conjugate response1-D that is insensitive to JA-Trp, but still sensitive to other active conjugates, was identified and the defect was found to be a substitution of Asn for Asp456 in the C-terminal domain of the IAA cellular permease AUX1. Mutant seedling primary root growth in the absence of added conjugate was 15% less than WT, but otherwise plant phenotype appeared normal. These results suggest that JA-Trp disrupts AUX1 activity, but that endogenous JA-Trp has only a minor role in regulating plant growth. In contrast with IAA- and JA-Trp, which are present at <2 pmole g-1 FW, PAA-Trp was found at about 30 pmole g-1 FW. The latter, or other undiscovered Trp conjugates, may still have important endogenous roles, possibly helping to coordinate other pathways with auxin response.

Jasmonic acid-amino acid conjugation enzyme assays.

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid.

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.

Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation.

Jasmonic acid (JA) and related cyclopentanones are critical plant signaling molecules, but their mode of action at the molecular level is unclear. A map-based approach was used to identify the defective gene in the Arabidopsis JA response mutant jar1-1. JAR1 is 1 of 19 closely related Arabidopsis genes that are similar to the auxin-induced soybean GH3 gene. Analysis of fold predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates for their subsequent biochemical modification. An ATP-PPi isotope exchange assay was used to demonstrate adenylation activity in a glutathione S-transferase-JAR1 fusion protein. Activity was specific for JA, suggesting that covalent modification of JA is important for its function. Six other Arabidopsis genes were specifically active on indole-3-acetic acid (IAA), and one was active on both IAA and salicylic acid. These findings suggest that the JAR1 gene family is involved in multiple important plant signaling pathways.