The biochemical properties of the twoArabidopsis thalianaisochorismate synthases

Abstract

The important plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) regulates several key plant responses including, most notably, defence against pathogens. A key enzyme for SA biosynthesis is isochorismate synthase (ICS), which converts chorismate into isochorismate, and for which there are two genes in Arabidopsis thaliana. One (AtICS1) has been shown to be required for increased SA biosynthesis in response to pathogens and its expression can be stimulated throughout the leaf by virus infection and exogenous SA. The other (AtICS2) appears to be expressed constitutively, predominantly in the plant vasculature. Here, we characterise the enzymatic activity of both isozymes expressed as hexahistidine fusion proteins in Escherichia coli. We show for the first time that recombinant AtICS2 is enzymatically active. Both isozymes are Mg2+-dependent with similar temperature optima (ca. 33°C) and similar Km values for chorismate of 34.3 ± 3.7 and 28.8 ± 6.9 µM for ICS1 and ICS2, respectively, but reaction rates were greater for ICS1 than for ICS2, with respective values for Vmax of 63.5 ± 2.4 and 28.3 ± 2.0 nM s−1 and for kcat of 38.1 ± 1.5 and 17.0 ± 1.2 min−1. However, neither enzyme displayed isochorismate pyruvate lyase (IPL) activity, which would enable these proteins to act as bifunctional SA synthases, i.e. to convert chorismate into SA. These results show that although Arabidopsis has two functional ICS enzymes, it must possess one or more IPL enzymes to complete biosynthesis of SA starting from chorismate.

Inositol hexakisphosphate biosynthesis underpins PAMP‐triggered immunity to Pseudomonas syringae pv. tomato in Arabidopsis thaliana but is dispensable for establishment of systemic acquired resistance

“…We examined the responses of ips3 plants to virulent Pst and found that they were significantly more susceptible to the bacterial pathogen than transformation control plants (Figure ). The susceptibility of ips3 to Pst was similar to that of ips2 , previously shown to be hypersusceptible to Pst (Murphy et al ., ), and the sid2 mutant (Wildermuth et al ., ; Macaulay et al ., ), which is impaired in salicylic acid (SA) biosynthesis and highly susceptible to pathogen attack (Figure ). As would be anticipated, plants carrying both the ips2 and ips3 mutations showed increased susceptibility to virulent Pst (Figure ).…”

“…Hydathodes are open pores on the leaf margin that provide an entry point for bacterial pathogens such as Pst into the apoplast (Rufián et al, 2018). Elevated levels of defence-related gene expression in cells surrounding hydathodes has been previously reported indicating a role of localized constitutive defence around these vulnerable openings (Macaulay et al, 2017;van den Burg et al, 2010).…”

Conservation of N-hydroxy-pipecolic acid-mediated systemic acquired resistance in crop plants

“…Specifically, we analyzed isochorismate synthase 1 (ICS1; ) for SA, OPDA reductase (OPR3; (Stintzi and Browse, 2000)) and jasmonate resistance 1 (JAR1; (Staswick and Tiryaki, 2004)) for JA, and S-alkyl thiohydroximate lyase (SUR1; (Mikkelsen et al, 2004)) and cytochrome P450 monooxygenase CYP83B1 (SUR2; (Bak et al, 2001)) for GSL. For the SA biosynthetic enzyme ICS1, reverse BLASTs returning A. thaliana ICS1 and isochorismate synthase 2 (ICS2; a functionally redundant enzyme (Macaulay et al, 2017)) were considered best reciprocal BLASTs. The analysis predicts that SA and JA pathways are highly conserved throughout the plant kingdom, while the GSL pathway is only conserved within the Brassicaceae.…”

Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties

“…For example, the tertiary structures of EntC and MenF have been determined, and many amino acid residues essential or important for ICS activity have been identified [ 22 , 23 ]. In contrast, reports on the biochemical analysis of plant ICSs are limited to that of Catharanthus roseus (CrICS) purified from elicited cell cultures [ 24 ], and those of AtICS1 and AtICS2 expressed in E. coli showed very similar catalytic characteristics [ 25 , 26 ]. In the present study, we analyzed and compared the biochemical properties of AtICS1, NtICS, NbICS, and OsICS expressed in E. coli and N. benthamiana leaves.…”