Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis

Abstract

Salicylic acid (SA) is an important mediator of plant defense response. In Arabidopsis (Arabidopsis thaliana), this compound was proposed to derive mainly from isochorismate, itself produced from chorismate through the activity of ISOCHORISMATE SYNTHASE1 (ICS1). Null ics1 mutants still accumulate some SA, suggesting the existence of an enzymatic activity redundant with ICS1 or of an alternative ICS-independent SA biosynthetic route. Here, we studied the role of ICS2, a second ICS gene of the Arabidopsis genome, in the production of SA. We have shown that ICS2 encodes a functional ICS enzyme and that, similar to ICS1, ICS2 is targeted to the plastids. Comparison of SA accumulation in the ics1, ics2, and ics1 ics2 mutants indicates that ICS2 participates in the synthesis of SA, but in limited amounts that become clearly detectable only when ICS1 is lacking. This unequal redundancy relationship was also observed for phylloquinone, another isochorismate-derived end product. Furthermore, detection of SA in the double ics1 ics2 double mutant that is completely devoid of phylloquinone provides genetic evidence of the existence of an ICS-independent SA biosynthetic pathway in Arabidopsis.

Figure 1.

A, Sequence alignment of Arabidopsis ICS1 (NP_565090) and ICS2 (ACC60228) and C. roseus ICS (CAA06837). The chorismate-binding domain is located in the dashed area and the position of residues conserved in ICS enzymes (Kolappan et al., 2007) is indicated with a star. B, Exon/intron structure of ICS1 and ICS2. Exons are represented by gray boxes and introns by thin lines. Sequence similarity between exon 3 of AtICS1 and 3 and 4 of ICS2, and 4 of AtICS1 and 5 and 6 of ICS2 is highlighted by dashes.

Figure 2.

Subcellular localization of ICS1 and ICS2. GFP fusion constructs were transiently expressed in tobacco cells by agroinfiltration. GFP signal (green) and chlorophyll autofluorescence (magenta) were observed using confocal microscopy. Magenta and green overlay is shown in white. Bars = 10 μm.

Figure 3.

Functional complementation of the PBB8 ICS-deficient strain of E. coli by expression of Arabidopsis ICS1 and ICS2. PBB8 cells were transformed with constructs expressing ICS and ICS-GFP fusions and spread onto CAS medium. Orange coloration indicate iron uptake from the medium and therefore restoration of siderophore production by functional ICS activity. Bacteria were plated either on CAS medium without IPTG (A) or CAS medium containing 0.2 mm of IPTG (B).

Figure 4.

Functional roles of ICS1 and ICS2. A, Phenotype of wild type, ics1, ics2, and ics1 ics2 double mutant when grown in vitro. ics1 ics2 double mutants can be more affected and display a more yellowish pigmentation. B and C, Accumulation of isochorismate-derived compounds in the mutants: phylloquinone (B) and total SA accumulation following UV induction (C).

Figure 5.

Identification of SA in ics1 ics2 by GC-MS. SA in leaf extracts was silylated and separated by GC-MS. o-Anisic acid was used as internal standard. A (wild type), B (ics1 ics2), and C (SA standard) show the ion traces for m/z 209 (dotted line, characteristic for silylated o-anisic acid) and 267 (solid line, silylated SA). D to F, Mass spectra for the peaks eluting at 10.8 min (arrows in A and B) for wild type, ics1 ics2, and the SA standard, respectively. The inset in F shows the fragmentation pattern of silylated SA.