Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

d'Amato, T. A.; Ganson, Robert J.; Gaines, C G; Jensen, Roy A.

doi:10.1007/bf00410205

Cited by 71 publications

(27 citation statements)

References 23 publications

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“…This data indicates a physiologically significant role for CM-2 in green potato leaves. A similar change in the expression of CM isozymes was observed in cell suspension cultures and green leaves of N. silvestris where CM-1 comprised most of the activity in cell cultures and CM-2 was the predominant isozyme in green leaves (1,6). This shift in the ratio of isozyme activities was suggested to reflect increased secondary metabolism in leaves versus cell cultures in exponential growth (6).…”

Section: Resultssupporting

confidence: 55%

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L.

Kuroki

Conn²

1989

Plant Physiol.

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Chromatography on DEAE cellulose equilibrated with Pipes buffer resolved three forms of chorismate mutase (CM) from tubers and leaves of Solanum tuberosum: CM-IA and CM-1B were activated by tryptophan and inhibited by phenylalanine and tyrosine; CM-2 was unaffected by these aromatic amino acids. When compared to freshly excised discs, 3 day old tuber discs demonstrated a 4.5-fold increase in CM-1 activity following wounding. By contrast, CM-2 activity levels were not affected by this treatment. In aged tuber discs the CM-1:CM-2 activity ratio was 9:1. However, in green leaves the CM-1:CM-2 activity ratio was 1:4 suggesting organ specific regulation for the expression of these isozymes. The CM-I isozymes isolated from both tubers and leaves shared similar native molecular weight values of 55,000, Km values of 40 to 56 micromolar, and inhibition by phenylalanine (110-145 micromolar concentrations required for 50% inhibition) and tyrosine (50-70 micromolar concentrations required for 50% inhibition). The resolution of CM-1 into two forms occurred only in the presence of Pipes buffer. When this buffer was replaced with Aces, Bes, imidazole or Tris, only a single peak of CM-1 activity was observed. In these buffers CM-2 eluted as a shoulder on the CM-1 peak. Analytical isoelectric focusing of the CM-1 fraction followed by assay of the gel yielded only one form of CM-1 with an isoelectric point of 5.0. Gel filtration studies with Pipes buffer yielded molecular weights of 60,000 for both CM-1A and CM-lB indicating these forms are not the result of aggregation. The two forms of CM-1 may be artifacts generated by Pipes buffer.Chorismate mutase (EC 5.4.99.5) isozymes have been well resolved by chromatography on DEAE cellulose in a number of plants (5,6,14,15). Isozyme CM2-I has been shown to be activated by tryptophan and inhibited by phenylalanine and tyrosine, whereas CM-2 is not regulated by these aromatic amino acids. However, in other studies, only CM-1 activity could be detected following anion exchange chromatography (9, 15). In a previous investigation we detected only CM-1 activity in tubers of Solanum tuberosum following DEAE cellulose chromatography in citrate-phosphate buffer (9). Recent studies with parsley cell cultures (KF McCue, EE Conn, unpublished data) indicated that CM-1 and CM-2 may coelute on DEAE cellulose. This suggested that a similar phenom- enon might be occurring in the potato system. Utilizing DEAE cellulose equilibrated with Pipes buffer, Morris et al. (12) have recently detected CM-2 in potato tubers. We also report the resolution of CM-1 and CM-2 isozymes from tubers and green leaves of S. tuberosum. The data presented in this study revealed that ofthe five buffers employed, complete resolution of tuber CM isozymes on DEAE cellulose occurred only with Pipes buffer.The separation of CM-I and CM-2 by chromatography on DEAE cellulose allowed us to observe the effects of wounding and organogenesis on the expression of these shikimate pathway enzymes. Similar studies have been reported f...

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Section: Resultssupporting

confidence: 55%

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L.

Kuroki

Conn²

1989

Plant Physiol.

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“…2). In various plant species, chorismate mutase activity is found in both the plastid and cytosol (17)(18)(19)(20)(21)(22). In petunia, a chorismate mutase homolog may be involved in the synthesis of phenylalanine-derived volatile phenylpropanoids and benzenoids (23).…”

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confidence: 99%

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases

Westfall

Jez

2014

Journal of Biological Chemistry

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Background: Chorismate mutase is essential for aromatic amino acid biosynthesis. Results: Structural and biochemical studies of three chorismate mutases from Arabidopsis reveal distinct sets of effector molecules. Conclusion: Key residues in the effector site modulate the regulatory effects of ligands. Significance: Evolution of effector control may lead to specialized regulation of this enzyme in plants.

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“…1), which catalyzes the first committed step in the Phe and Tyr biosynthesis pathway, is the only one known to be present in both the plastid and the cytosol. The presence of a cytosolic form of CM has been clearly detected both at the enzymatic and molecular levels in several plant species, such as Arabidopsis (Arabidopsis thaliana), Nicotiana sylvestris, and Papaver somniferum (Benesova and Bode, 1992;d'Amato et al, 1992;Eberhard et al, 1996). The physiological significance of this cytosolic CM is still an enigma.…”

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confidence: 99%

Tyrosine and Phenylalanine Are Synthesized within the Plastids in Arabidopsis

et al. 2009

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While the presence of a complete shikimate pathway within plant plastids is definitively established, the existence of a cytosolic postchorismate portion of the pathway is still debated. This question is alimented by the presence of a chorismate mutase (CM) within the cytosol. Until now, the only known destiny of prephenate, the product of CM, is incorporation into tyrosine (Tyr) and/or phenylalanine (Phe). Therefore, the presence of a cytosolic CM suggests that enzymes involved downstream of CM in Tyr or Phe biosynthesis could be present within the cytosol of plant cells. It was thus of particular interest to clarify the subcellular localization of arogenate dehydrogenases (TYRAs) and arogenate dehydratases (ADTs), which catalyze the ultimate steps in Tyr and Phe biosynthesis, respectively. The aim of this study was to address this question in Arabidopsis (Arabidopsis thaliana) by analysis of the subcellular localization of the two TYRAAts and the six AtADTs. This article excludes the occurrence of a spliced TYRAAt1 transcript encoding a cytosolic TYRA protein. Transient expression analyses of TYRA-and ADT-green fluorescent protein fusions reveal that the two Arabidopsis TYRA proteins and the six ADT proteins are all targeted within the plastid. Accordingly, TYRA and ADT proteins were both immunodetected in the chloroplast soluble protein fraction (stroma) of Arabidopsis. No TYRA or ADT proteins were immunodetected in the cytosol of Arabidopsis cells. Taken together, all our data exclude the possibility of Tyr and/or Phe synthesis within the cytosol, at least in green leaves and Arabidopsis cultured cells.

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Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

Cited by 71 publications

References 23 publications

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L.

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L.

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases

Tyrosine and Phenylalanine Are Synthesized within the Plastids in Arabidopsis

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