Increased Chorismate Mutase Levels as a Response to Wounding in Solanum tuberosum L. Tubers

Abstract

Discs excised from Solanum tuberosum L. cv White Rose tubers demonstrated a 4.5-fold increase in chorismate mutase activity 48 hours after excision. Incubation in the presence of cycloheximide (25 micromolar Chorismic acid is located at the branchpoint between tryptophan biosynthesis on one hand, and phenylalanine and tyrosine biosynthesis on the other. CM is involved in the regulation of flux among the three aromatic amino acids. CM was reported to be present in multiple forms in a number of plant species (8,9,21,22 Disc Incubation Conditions. Tubers were removed from cold storage 24 h prior to disc preparation and placed in the dark at room temperature. Tubers were sliced into 2 to 3 mm sections. Discs (approximately 1.2 gfw each) were excised from the center tissue with a number 15 cork borer and rinsed with distilled water followed by streptomycin sulfate (25 mg/L). Three discs were placed in sterile Petri dishes (10 x 1.5 cm) containing sterile Whatman No. 3 filter paper (7.0 cm diameter), 9 ml distilled water, and streptomycin sulfate (25 mg/L). Wound response inhibition studies were performed essentially as described above with the inclusion of either cycloheximide (25 ,um) or actinomycin D (100 ,um). The Petri dishes were covered with aluminum foil and placed in the dark at room temperature. Enzyme activities were monitored 12 h and every 24 h after excision for 10 d. Studies of light activation were performed by incubating discs under white fluorescent lights (156 uE m-2 s-1), with assays being performed every 8 h after excision for 48 h.Enzyme Assays. Unless otherwise noted, all steps were carried out at 4°C. Discs were homogenized in a mortar containing 5.0 ml of 0.5 M citrate-phosphate buffer (pH 8.0), 1.5% (v/v),-mercaptoethanol, 1.0 mM tryptophan, 7.5 g (w/v) hydrated PVPP, 30 mm thiourea, and 2.5 g quartz sand. Higher buffer concentrations were necessary since homogenization typically resulted in a five-fold dilution. The homogenate was filtered through Miracloth moistened with homogenization buffer and centrifuged for 20 min at 10,000g. The supernatant was decanted and aliquots (2.5 ml) applied onto a Sephadex G-25 column (Pharmacia PD-10) equilibrated with either 0.01 M Tris-HCI buffer (pH 8.0) (for CM assays) or 0.1 M borate-NaOH buffer (pH 8.8), containing 0.1% (v/v), 3-mercaptoethanol (for PAL assays). Proteins were eluted with 3.0 ml of the respective buffer.The standard assay for CM activity contained 0.317 mm chorismic acid, 0.1 M citrate-phosphate buffer (pH 7.0) and up to 40 ,ul of enzyme in a final volume of 0.167 ml. Assays were performed either in the presence or absence of 0.1 mM tryptophan. After incubation for 10 min at 25°C, reactions were terminated by adding 0.

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

“…We have reported an increase in the activity ofa tryptophan sensitive form of CM following wounding of Solanum tuberosum tubers (9). Studies with transcriptional and translational inhibitors suggested this increase was a result of de novo synthesis of CM-1.…”

“…Studies with transcriptional and translational inhibitors suggested this increase was a result of de novo synthesis of CM-1. The apparent absence of CM-2 reported earlier (9) was based, in part, on the observation that, in a crude extract, the tryptophan activation ratio remained constant during aging of wounded discs. The resolution of CM-2 in potato tubers indicates that the tryptophan activation ratio is not a reliable measure for the presence of CM isozymes if the fractional amount of CM-2 is small.…”

“…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.…”

“…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).…”

“…Enzyme Assays CM activity was determined essentially as previously described (9). Column fractions were assayed at a final chorismate concentration of 66 AiM.…”