Redox Regulation of Arabidopsis 3-Deoxy-<scp>d</scp>-<i>arabino</i>-Heptulosonate 7-Phosphate Synthase

Entus, Robert; Poling, Michael D.; Herrmann, Klaus M.

doi:10.1104/pp.002626

Cited by 87 publications

(52 citation statements)

References 40 publications

(26 reference statements)

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“…Ref. 72). Taken together, our findings suggest that the light-dependent regulation of AtICS1 activity due to changes in stromal pH, Mg 2ϩ , and/or redox status do not significantly contribute to the light dependence observed for SA-dependent defense responses.…”

Section: Discussionsupporting

confidence: 55%

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

Strawn

Marr

Inoue

et al. 2007

Journal of Biological Chemistry

219

146

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Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (K eq ‫؍‬ 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent K m of 41.5 M and k cat ‫؍‬ 38.7 min ؊1 for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg 2؉ , or redox and that it is remarkably active at 4°C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.

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Section: Discussionsupporting

confidence: 55%

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

Strawn

Marr

Inoue

et al. 2007

Journal of Biological Chemistry

219

146

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“…A redox regulation of amino acid biosynthesis in chloroplast has been suggested (30), and the involvement of TRX in this process has been proposed recently, when the first enzyme in the biosynthesis of aromatic amino acids was shown to be regulated by TRX f in higher plants (31). In C. reinhardtii we found six potential targets involved in biosynthesis of arginine (argininosuccinate synthase), threonine (threonine synthase), lysine (diaminopimelate epimerase), branched amino acids valine and isoleucine (dihydroxyacid dehydratase and ketolacid reductoisomerase), and leucine (isopropylmalate dehydrogenase).…”

Section: Resultsmentioning

confidence: 99%

New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii

Lemaire

Guillon

Maréchal

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

235

189

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Proteomics were used to identify the proteins from the eukaryotic unicellular green alga Chlamydomonas reinhardtii that can be reduced by thioredoxin. These proteins were retained specifically on a thioredoxin affinity column made of a monocysteinic thioredoxin mutant able to form mixed disulfides with its targets. Of a total of 55 identified targets, 29 had been found previously in higher plants or Synechocystis, but 26 were new targets. Biochemical tests were performed on three of them, showing a thioredoxindependent activation of isocitrate lyase and isopropylmalate dehydrogenase and a thioredoxin-dependent deactivation of catalase that is redox insensitive in Arabidopsis. In addition, we identified a Ran protein, a previously uncharacterized nuclear target in a photosynthetic organism. The metabolic and evolutionary implications of these findings are discussed.O ur knowledge on redox regulation of various physiological processes through thiol-disulfide interchange with proteins of the thioredoxin (TRX) superfamily is rapidly progressing as genome-wide approaches and functional genomics studies are expanding. In the plant biology domain, thioredoxin-dependent regulation has been first discovered for chloroplastic enzymes involved in carbon assimilation (1). The completion of the sequencing of the Arabidopsis genome revealed that TRXs constituted a multigene family and led to the assumption that numerous TRX targets were still to be discovered (2-4). Thus, proteomic approaches aimed at identifying the largest possible number of TRX targets have been developed. The first attempts took advantage of the reaction mechanism of TRXs with their targets, in which a transient heterodisulfide forms between the reduced TRX and the oxidized protein, followed by the release of the reduced target due to the attack of the mixed disulfide by the second cysteine of TRX. Model studies showed that when this second cysteine was mutated, the heterodisulfide was stabilized (5, 6). This property was applied in vivo by transforming a TRX-null mutant yeast with a monocysteinic TRX and allowed isolating a peroxiredoxin (PRX) (7). Subsequently, monocysteinic TRX mutants were used in affinity chromatography to trap interacting proteins (8-11). Native TRX affinity columns also allowed retaining some targets by electrostatic interaction (12). Another technique was also developed that consists of a reduction of a crude soluble protein extract with reduced TRX followed by a separation of the proteins on a 2D gel after derivatization of the newly appeared thiols either with a fluorescent (13) or with a radioactive (14) reagent. In one case, both techniques have been applied in parallel (15), yielding similar results.Most of the disulfide proteomics have been led with higher plants: spinach (8, 10, 12), Arabidopsis (11, 14), or cereal grains (12). A single study was led with cyanobacteria and revealed targets mostly different from those of higher plants (16). The unicellular eukaryotic green alga Chlamydomonas reinhardtii possesses a TRX syste...

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“…In bacteria, regulation of carbon flow into the shikimate pathway is largely under transcriptional control and feedback inhibition of the first enzyme in the pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; the aromatic amino acids mediate both processes. Although there is no evidence for a feedback-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in plants, a recent study suggests that reduced thioredoxin plays a role in activation of this enzyme (Entus et al, 2002). Generally speaking, it is believed that all of the plant shikimate pathway enzymes are principally regulated at the transcriptional level and are rapidly induced by wounding, elicitors, and various environmental assaults (Schmid and Amrhein, 1994).…”

Section: Discussionmentioning

confidence: 99%

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

et al. 2004

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p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5# untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T 1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T 1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nucleartransformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.All plants normally produce p-hydroxybenzoic acid (pHBA), albeit usually in small quantities. Radioisotope studies with Lithospermum erythrorhizon suggest that this compound is derived from the CoA ester of p-hydroxycinnamic acid (pHCA-CoA) through a b-oxidation-like mechanism (Loscher and Heide, 1994). However, earlier studies with the same plant species (Yazaki et al., 1991) and carrot (Daucus carota) cell cultures (Schnitzler et al., 1992) supported a cleavage mechanism that occurs via intermediacy of p-hydroxybenzaldehyde. Notwithstanding our current ignorance of the detailed plant biosynthetic pathway, a number of studies have shown that it is possible to dramatically elevate pHBA levels in plants through metabolic engineering. Indeed, two different ''singleenzyme'' pathways have been described, both involving microbial proteins that have no known plant counterparts. One of these enzymes is chorismate pyruvate-lyase (CPL). Its substrate is chorismate, an important branch po...

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Redox Regulation of Arabidopsis 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase

Cited by 87 publications

References 40 publications

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

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