Accumulation of isochorismate-derived 2,3-dihydroxybenzoic 3-O-beta-D-xyloside in arabidopsis resistance to pathogens and ageing of leaves

Abstract

An intricate network of hormone signals regulates plant development and responses to biotic and abiotic stress. Salicylic acid (SA), derived from the shikimate/isochorismate pathway, is a key hormone in resistance to biotrophic pathogens. Several SA derivatives and associated modifying enzymes have been identified and implicated in the storage and channeling of benzoic acid intermediates or as bioactive molecules. However, the range and modes of action of SA-related metabolites remain elusive. In Arabidopsis, Enhanced Disease Susceptibility 1 (EDS1) promotes SA-dependent and SA-independent responses in resistance against pathogens. Here, we used metabolite profiling of Arabidopsis wild type and eds1 mutant leaf extracts to identify molecules, other than SA, whose accumulation requires EDS1 signaling. Nuclear magnetic resonance and mass spectrometry of isolated and purified compounds revealed 2,3-dihydroxybenzoic acid (2,3-DHBA) as an isochorismate-derived secondary metabolite whose accumulation depends on EDS1 in resistance responses and during ageing of plants. 2,3-DHBA exists predominantly as a xylose-conjugated form (2-hydroxy-3-beta-O-D-xylopyranosyloxy benzoic acid) that is structurally distinct from known SA-glucose conjugates. Analysis of DHBA accumulation profiles in various Arabidopsis mutants suggests an enzymatic route to 2,3-DHBA synthesis that is under the control of EDS1. We propose that components of the EDS1 pathway direct the generation or stabilization of 2,3-DHBA, which as a potentially bioactive molecule is sequestered as a xylose conjugate.

FIGURE 1.

Accumulation of SA, 2,3-DHBA, and 2,5-DHBA after challenge with avirulent pathogens. The data represent the averages ± S.D. of three replicate samples. Metabolite levels were significantly different in Col-0 samples compared with corresponding samples from the mutants at p < 0.05 (*) and p < 0.01 (**). The experiments were repeated twice with similar results. Total (A) and free (B) levels of phenolic metabolites were measured in leaves of 4-week-old plants that were syringe-infiltrated with 10 mm MgCl₂ (mock) or with 5 × 10⁶ colony-forming units/ml Pst DC3000 avrRpm1 (Pst avrRpm1). C, accumulation of total phenolic metabolites was determined in 3-week-old plants 4 days after treatment with water (mock) or spray inoculation with 10⁵ spores/ml of avirulent H. arabidopsidis isolate Cala2 (recognized by R gene RPP2 in Col-0).

FIGURE 2.

Structural elucidation of 2,3-DHB3X by LC-MS. The molecular structure was determined by NMR spectroscopy (supplemental Fig. S3) and confirmed by LC-MS. A, LC-electrospray ionization-MS analysis of the peak representing 2,3-DHB3X. LC-electrospray ionization-MS analysis in negative ion mode revealed an ion of m/z 285.061 as [M-H], suggesting a molecular formula of M = C₁₂H₁₄O₈. B and C, the insource fragment at m/z 153.018 and subsequent CID experiments of the parent ion (285.06) (B) and the daughter ion (153.02) (C) showed a neutral loss of a pentose moiety indicating that 2,3-DHBA is linked to the pentose xylose. D, structure of 2,3-DHB3X.

FIGURE 3.

2,3-DHBA accumulation increases with age in wild type but not eds1 mutant plants. A, total phenolic metabolite content was determined in healthy plants at the indicated age in weeks. B, relative total levels of 2,3-DHBA and 2,5-DHBA compared with total SA levels (%) were calculated from the data in Fig. 3A. C, content of free SA, 2,3-DHBA, and 2,5-DHBA. The data points are the averages ± S.D. of three replicate samples. The metabolite levels were significantly different in Col-0 samples compared with corresponding samples from the mutants at p < 0.05 (*) and p < 0.01 (**).

FIGURE 4.

Exogenously applied deuteriated SA ([²H]SA) is transformed into [²H]SAG and [²H]DHBAs including [²H]2,3-DHB3X in planta. A and B, DEX-treated (A) and mock treated (B) plants of the DEX-inducible transgenic line AvrRpm1-HA infiltrated with [²H]SA show a high level of a [²H]SA-hexoside m/z 303.101 (calculated) (blue line). An increase of ∼3-fold was evident for the nondeuteriated natural form of the SA-hexoside at m/z 299.076 (calculated) (red line) in DEX-triggered (A) compared with mock treated plants (B). Infiltration of [²H]SA into DEX-elicited (C) Arabidopsis produced low amounts of [²H]DHBA pentosides m/z 288.079 (calculated) (orange line) compared with high concentrations of the natural DHBA pentosides m/z 285.060 (calculated) (green line). D, [²H]DHBA conjugates were not detected in mock treated leaves. Using a biosynthetic standard, the second peak at a retention time of 3.75 min was identified as [²H]2,3-DHB3X.

FIGURE 5.

2,3-DHBA levels correlate with the onset of senescence in different genetic backgrounds. A, total levels of phenolic compounds were measured in 8-week-old, untreated wild type and mutant plants, as indicated. The data represent the averages ± S.D. of three replicate samples. The metabolite levels were significantly different in Col-0 samples compared with corresponding samples from the mutants at p < 0.05 (*) and p < 0.01 (**). B, relative transcript levels of SAG13 determined by quantitative real time PCR and displayed after normalization to the internal control ACTIN and relative to the expression in Col-0 5-week-old sample (set at 1). The data bars represent the mean levels of transcripts ± S.D. C, leaves (third and fourth emerging after the cotyledons) from mutants displaying different degrees of senescence-induced leaf necrosis. The experiment was repeated twice with similar results, and representative leaves are shown. The bar represents 2 cm.

FIGURE 6.

Exogenously applied 2,3-DHBA is a poor inducer of PR1 expression compared with SA. A, Arabidopsis seedlings harboring the SA-responsive PR1p::GUS reporter gene, grown for 2 weeks in liquid culture, were treated with increasing concentrations of SA or 2,3-DHBA for 24 h. GUS activity was determined in total extracts. The data represent the averages ± S.D. of four replicate samples normalized to total extractable protein. GUS activity was significantly different between SA and 2,3-DHBA at p < 0.05 (*). B, appearance of seedlings 24 h after treatment with SA or 2,3-DHBA. SA concentrations exceeding 100 μm are toxic (visible as tissue bleaching), whereas detrimental effects of 2,3-DHBA are apparent only at concentrations exceeding 300 μm.

FIGURE 7.

A model for SA-metabolizing pathways leading to 2,3- and 2,5-DHBA and its various sugar conjugates in Arabidopsis. SA synthesis is promoted during leaf senescence and plant defense, forming a positive feedback loop. This feedback loop is under the positive control of EDS1, FMO1, and PAD2 and under the negative control of both NUDT7 and VTC1. Free cellular SA is removed by SA conjugation with glucose and by SA hydroxylation leading to 2,5-DHBA and subsequent glucose conjugation to 2,5-DHBA glucosides. SA hydroxylation to 2,3-DHBA and subsequent xylose conjugation to 2,3-DHB3X is a newly discovered pathway, representing a detoxification mechanism for SA or 2,3-DHBA or a means of channeling bioactive benzoic acid molecules in cells and tissues (see “Discussion”). The dotted arrows represent uncertainty regarding whether 2,3-DHBA is derived from SA or from isochorismate under normal physiological conditions.