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

Abstract

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.

Figure 1.

Comparison of the Sensitivity of Arabidopsis Mutants to MeJA. Seedlings were grown for 5 days on vertical agar plates containing 50 μM MeJA. Bars indicate mean root length of 19 seedlings with standard errors. WT, wild type.

Figure 2.

Positional Cloning of JAR1. (A) The location of JAR1 on the right end of chromosome II is shown relative to phenotypic markers AS1 and CER8. Fractions indicate number of recombinant chromosomes between JAR1 and the respective markers. (B) Molecular mapping of JAR1. Numbers in parentheses above markers indicate number of recombinant chromosomes between JAR1 and the respective markers. Dashed lines indicate BAC clones, and short lines with labels depict subclones used to test for complementation of jar1-1. Restriction endonucleases used to generate subclones (B, BamHI; E, EcoRI; K, KpnI; P, Pst1; S, SacI; Sp, SpeI) and their sizes (in kb) are shown. Complementing subclones are indicated by (+). (C) Structure of the single ORF on complementing subclone B5.1. Black and white bars represent predicted coding and noncoding transcribed regions, respectively, and intervals between bars represent introns (www.tigr.org/tdb/ath1/htmls/index.html).

Figure 3.

The JAR1 Sequence and Its Similarity to 18 Closely Related Arabidopsis Proteins. The positions of mutations in various jar1 alleles are indicated below the sequence by single letters (amino acid substitutions), dots (premature stop codons), or triangles (deletions). Residues highlighted in black are invariant, and those highlighted in gray are similar among at least 16 of the 19 Arabidopsis proteins. Gaps introduced by PileUp (Genetics Computer Group, Madison, WI) to optimize alignment are not shown. Open boxes identify conserved motifs in the firefly luciferase–like adenylate-forming enzyme superfamily.

Figure 4.

Chromosomal Distribution of the Arabidopsis JAR1-Like Gene Family. Genes are designated by the BAC open reading frame numbers assigned by the Arabidopsis Genome Initiative (www.arabidopsis.org). Chromosome numbers are shown at left. No homologs exist on chromosome III.

Figure 5.

Biochemical Assay for JAR1 and Related Enzyme Activities. (A) Analysis of purified GST-JAR1 fusion protein on a Coomassie blue–stained polyacrylamide gel. Positions of molecular mass markers (kD) are shown at left. (B) Substrate specificity of JAR1 determined by ATP-³²P-PPi exchange. The positions of PPi and ATP after chromatography are indicated. Lanes are labeled with the substrates assayed. (C) The first lane contains the complete reaction mixture with JA as in (B). Other lanes are the same as the first minus the component indicated. (D) Representative example of a substrate specificity assay for proteins related to JAR1. The top chromatograph shows T20D16.20; the bottom chromatograph shows M4I22.70. Reaction conditions are the same as those described above. ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; GA, gibberellic acid; PDA, 12-oxophytodienoic acid.

Figure 6.

Summary of Sequence Homology and Substrate Specificity Relationships among Arabidopsis JAR1-Like Proteins. Pair-wise similarity tree constructed with PileUp (Genetics Computer Group) using the blosum 62 scoring matrix. Numbers in parentheses indicate chromosomes. Substrate specificity for each protein and sequence homology subgroups are indicated at left and right, respectively. Four genes not tested (nt) were not obtained by reverse transcriptase–mediated PCR or were nearly identical to others. Inactivity on substrates tested (see Fig. 5) is indicated by (−).

Figure 7.

Response of jar1 Alleles to Far-Red Light. Seedlings were grown for 5 days at room temperature under far-red light. Bars indicate mean hypocotyl length of 25 seedlings with standard errors. WT, wild type.