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.

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.

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.