Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner

Highlight Allantoin, a stress-related purine metabolite, can activate JA responses via ABA in Arabidopsis, suggesting its possible involvement in the homeostasis of these phytohormones and their interplay in stress signaling.

. The purine catabolism pathway and metabolites derived therefrom.       Table S1. Primers used in this study. Table S2. LC-ESI-MS/MS parameters for jasmonate determination. Table S3. Genes with significantly increased expression in the aln-1 mutant. Table S4. Genes with significantly reduced expression in the aln-1 mutant.

Quantification of jasmonic acid (JA) and JA-Ile
Extraction and quantification of JA and JA-Ile were performed following the method of Preston et al. (2009) with minor modifications. The stable isotope-labeled compounds used as internal standards were: [ 2 H 2 ]JA (Tokyo Chemical industry Co., Ltd., Tokyo, Japan) and [ 13 C 6 ]JA-Ile, which was synthesized with [ 13 C 6 ]Ile (Cambridge Isotope Laboratories, Andover, MA, USA) as described in Jikumaru et al. (2004). Aerial parts (500 mg) from 2-week-old seedlings grown aseptically on solid half-strength MS medium were frozen in liquid N 2 , ground, and extracted with 80% (v/v) methanol containing 1% (v/v) acetic acid at 4°C for 1 h.
After removal of cell debris by centrifugation, the supernatants were condensed and dried in vacuo, and the resultant residues were extracted twice with methanol. The extracts were evaporated to dryness and resuspended with 80% methanol containing 1% acetic acid and internal standards, which were again evaporated and then extracted with water acidified with 1% (v/v) acetic acid. The extracts were loaded onto pre-equilibrated Oasis HLB column cartridge (Waters Corporation, Milford, MA, USA). After washed with water acidified with 1% acetic acid, the column was eluted with 80% (v/v) acetonitrile containing 1% (v/v) acetic acid. The eluted samples were evaporated to obtain extracts in water acidified with 1% acetic acid, and loaded onto a pre-equilibrated Oasis MCX column cartridge (Waters). The cartridge was washed with water acidified with 1% acetic acid and eluted with 80% acetonitrile containing 1% acetic acid. The eluate was loaded onto pre-equilibrated Oasis WAX column cartridges (Waters) followed by washing, first with water acidified 1% acetic acid and then with 80% acetonitrile, and the fraction containing JA and JA-Ile was eluted with 80% acetonitrile containing 1% acetic acid. The obtained fraction was dried and reconstituted in ultra-pure water acidified with 1% (v/v) acetic acid for quantification of JA and JA-Ile by      RNA was extracted from aerial parts of 2-week-old seedlings of WT and aln-1 mutants grown under normal aseptic conditions. Relative mRNA levels were determined by real-time reverse transcription-quantitative PCR using ACTIN2 expression as reference and presented as values relative to the WT level. The sequences of primers are listed in Supplementary Table S1. Data are means ± SEM from three independent experiments (*P < 0.05 by Student's t-test comparison to the WT level). Figure S5. Characterization of the aln-1 jar1-1 double mutant.
The homozygous mutants of aln-1 (SALK_000325; Yang and Han, 2004;Watanabe et al., 2014) and jar1-1 (CS8072;Staswick et al., 1992)  The nucleotide sequence of the wild-type JAR1 allele (a) from TAIR (At2g46370; https://www.arabidopsis.org/) was compared to that of the double mutant, as determined by dideoxy sequencing with primers F2 (b) and R2 (c), to confirm the single nucleotide missense mutation that occurs in exon 3 (Staswick et al., 2002). The sequences of primers are listed in Supplementary Table S1. (D) Typical root growth of 8-day-old seedlings of WT, jar1-1, and aln-1 jar1-1 genotypes in the presence of 10 µM methyl jasmonate (MeJA). Note that the root growth of the jar1-1 mutant is moderately insensitive to MeJA (Staswick et al., 1992, 2002. Horizontal bars indicate a scale of 10 mm in length (white) and the position of a primary root tip (yellow). Sterile seedlings of WT and the aah mutant were grown for 8 days on standard medium supplemented with 10 µM MeJA and examined for anthocyanin accumulation as described in the main text. FW, fresh weight. Data are means ± SEM (n = 8; *P < 0.001 by Student's t-test comparison to the WT levels).  Supplementary Table S1. Table Legends   Table S1. Primers used in this study. a Arabidopsis thaliana gene identifier code assigned by the Arabidopsis Genome Initiatives (AGI; https://www.arabidopsis.org/portals/nomenclature/). b Gene symbol as provided by The Arabidopsis Information Resource (TAIR; release 10; https://www.arabidopsis.org/) except for T-DNA of Agrobacterium tumefaciens. Table S2. LC-ESI-MS/MS parameters for jasmonate determination. Table S3. Genes with significantly increased expression in the aln-1 mutant.

Supplementary
Listed are those whose changes in transcript levels increased by equal to or greater than 3-fold, with the statistical significance level of 0.001 by a two-way analysis of variance (ANOVA).
These genes were selected from the revised microarray data (Gene Expression Omnibus accession number GSE73841) that had been parametrically renormalized using the SuperNORM data service (Skylight Biotech Inc, Akita, Japan), according to the three-parameter lognormal distribution method (Konishi, 2004). a Arabidopsis thaliana gene identifier code assigned by the Arabidopsis Genome Initiatives (AGI; https://www.arabidopsis.org/portals/nomenclature/). b Mean of two independent biological experiments (aln-1 versus wild-type).
c Gene symbol and description as provided by The Arabidopsis Information Resource (TAIR; release 10; https://www.arabidopsis.org/). d P-value determined by a two-way ANOVA. Table S4. Genes with significantly reduced expression in the aln-1 mutant.
Listed are those whose changes in transcript levels decreased by equal to or greater than 3-fold with the statistical significance level of 0.001 by a two-way ANOVA. These genes were selected from the revised microarray data (Gene Expression Omnibus accession number GSE73841) that had been parametrically renormalized using the SuperNORM data service (Skylight Biotech Inc, Akita, Japan), according to the three-parameter lognormal distribution method (Konishi, 2004 Figure S1. The purine catabolism pathway and metabolites derived therefrom. The pathway is schematically illustrated starting from the common intermediate xanthine, at which all purine nucleotides converge when they are subjected to degradation. The abbreviated names of the genes encoding the enzymes that catalyze each step are shown in italics and knockout mutants used in this study are indicated in parentheses under the gene names: XDH, xanthine dehydrogenase; UOX, urate oxidase; AS, allantoin synthase; ALN, allantoinase (allantoin amidohydrolase); AAH, allantoate amidohydrolase; UGAH, ureidoglycine aminohydrolase; UAH, ureidoglycolate amidohydrolase.  Figure S2. Hierarchical tree graph of over-represented GO terms for genes with significantly increased expression in the aln-1 mutant. A total of 47 enriched GO terms under Biological Process were found for 211 genes with significantly increased expression (≥ 3fold changes, Supplementary  1e-1 1e-2 1e-3 1e-4 1e-5 1e-6 1e-7 1e-8 1e-9 1e-10 <1e-10 P-value Figure S3. Hierarchical tree graph of over-represented GO terms for genes with significantly reduced expression in the aln-1 mutant. A total of 33 enriched GO terms under Biological Process were found for 113 genes with significantly reduced expression (≥ 3-fold changes, Supplementary Table S4) using the BioMaps tool of VirtualPlant version 1.3 (Katari et al., 2010; http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/) in the default-setting mode (Fisher's exact test with false discovery rate correction, P < 0.01). Each box shows the name of the GO term and, where appropriate, the P value (< 0.01) for the significance of enrichment. The P values are also indicated by the frame color of each box according to the gradient scale. The rank direction of the graph runs from top to bottom. Figure S4. Basal level expression of PR-1 as a canonical SA marker. RNA was extracted from aerial parts of 2-week-old seedlings of WT and aln-1 mutants grown under normal aseptic conditions. Relative mRNA levels were determined by real-time reverse transcription-quantitative PCR using ACTIN2 expression as reference and presented as values relative to the WT level. The sequences of primers are listed in Supplementary Table S1. Data are means ± SEM from three independent experiments (*P < 0.05 by Student's t-test comparison to the WT level).  Table S1. Primers used in this study   Table S3. Genes with significantly increased expression in the aln-1 mutant.

Supplementary
Listed are those whose changes in transcript levels increased by equal to or greater than 3-fold, with the statistical significance level of 0.001 by a two-way analysis of variance (ANOVA). These genes were selected from the revised microarray data (Gene Expression Omnibus accession number GSE73841) that had been parametrically renormalized using the SuperNORM data service (Skylight Biotech Inc, Akita, Japan), according to the three-parameter lognormal distribution method (Konishi 2004).
Listed are those whose changes in transcript levels decreased by equal to or greater than 3-fold with the statistical significance level of 0.001 by a two-way analysis of variance (ANOVA). These genes were selected from the revised microarray data (Gene Expression Omnibus accession number GSE73841) that had been parametrically renormalized using the SuperNORM data service (Skylight Biotech Inc, Akita, Japan), according to the three-parameter lognormal distribution method (Konishi 2004).