Polygalacturonase-mediated solubilization and depolymerization of pectic polymers in tomato fruit cell walls . Regulation By ph and ionic conditions

Abstract

The hydrolysis of cell wall pectins by tomato (Lycopersicon esculentum) polygalacturonase (PG) in vitro is more extensive than the degradation affecting these polymers during ripening. We examined the hydrolysis of polygalacturonic acid and cell walls by PG isozyme 2 (PG2) under conditions widely adopted in the literature (pH 4.5 and containing Na+) and under conditions approximating the apoplastic environment of tomato fruit (pH 6.0 and K+ as the predominate cation). The pH optima for PG2 in the presence of K+ were 1.5 and 0.5 units higher for the hydrolysis of polygalacturonic acid and cell walls, respectively, compared with activity in the presence of Na+. Increasing K+ concentration stimulated pectin solubilization at pH 4.5 but had little influence at pH 6.0. Pectin depolymerization by PG2 was extensive at pH values from 4.0 to 5.0 and was further enhanced at high K+ levels. Oligomers were abundant products in in vitro reactions at pH 4.0 to 5.0, decreased sharply at pH 5.5, and were negligible at pH 6.0. EDTA stimulated PG-mediated pectin solubilization at pH 6.0 but did not promote oligomer production. Ca2+ suppressed PG-mediated pectin release at pH 4.5 yet had minimal influence on the proportional recovery of oligomers. Extensive pectin breakdown in processed tomato might be explained in part by cation- and low-pH-induced stimulation of PG and other wall-associated enzymes.

Figure 1

PG2 hydrolysis of PGA in response to pH. Reaction mixtures contained 0.5 mL of PGA (4 mg mL⁻¹) in 20 mm NaOAc and 150 mm NaCl or 20 mm KOAc and 100 mm KCl, adjusted to the desired pH with dilute HCl or NaOH. After the addition of 50 μL of purified PG2 (approximately 2 μg of protein), the samples were incubated for 1 h at 34°C. Activity was measured reductometrically using galacturonic acid as the standard. •, 150 mm NaCl; ○, 100 mm KCl.

Figure 2

PG2 hydrolysis of cell walls from mature-green cv Ailsa Craig tomato fruit. Cell walls (5 mg) preloaded with purified PG2 (0.44 μg protein mg⁻¹ of cell wall) were incubated in 4 mL of 20 mm NaOAc and 150 mm NaCl or 20 mm KOAc and 100 mm KCl for 2 h at 34°C. Reaction mixtures were filtered and soluble uronic acids measured as described in Methods. Activity is expressed as micrograms of galacturonic acid equivalents released per milligram of cell wall. •, 150 mm NaCl; ○, 100 mm KCl.

Figure 3

PG2 hydrolysis of cell walls from mature-green cv Ailsa Craig tomato fruit at pH 4.5 and 6.0 in response to increasing [K⁺]. Cell walls (5 mg) in 4 mL of 20 mm KOAc at the indicated pH and KCl concentrations were incubated for 2 h at 34°C. Reaction mixtures were filtered and soluble uronic acids determined. Activity is expressed as micrograms of galacturonic acid equivalents released per milligram of cell wall. ○, pH 4.5; •, pH 6.0.

Figure 4

Sepharose CL-6B-100 profiles of uronic acids released from mature-green cv Ailsa Craig cell walls by PG2 at pH 6.0 or 4.5 in response to increasing [K⁺]. Cell walls (5 mg) in 4 mL of 20 mm KOAc, pH 4.5 or 6.0, containing from 5 to 200 mm KCl were incubated for 2 h at 34°C. Two-milliliter fractions were collected and 0.5-mL aliquots measured for soluble uronic acids. Uronic acid levels in each fraction are expressed as a percentage of total uronic acids recovered from the column. Vo, Void volume; Vt, total volume.

Figure 5

Effect of pH on uronic acid release from mature-green cv Ailsa Craig cell walls in response to PG2 at 25 or 100 mm KCl. Activity is expressed as micrograms of galacturonic acid equivalents released per milligram of cell wall.

Figure 6

Sepharose CL-6B-100 profiles of uronic acids released from mature-green cv Ailsa Craig cell walls by PG2 at 25 or 100 mm KCl over the pH range from 4.0 to 6.0. Cell walls (5 mg) in 2 mL of 20 mm KOAc at the indicated pH and KCl concentrations were incubated with the enzyme for 2 h at 34°C. Vo, Void volume; Vt, total volume.

Figure 7

Bio-Gel P-4 profiles of uronic acids released from mature-green cv Ailsa Craig cell walls in response to PG2. Cell walls (5 mg) in 2 mL of 20 mm KOAc and 100 mm KCl were incubated for 2 h at 34°C. Shown are uronic acids released at pH 4.5 (A), pH 5.5 (B), and pH 6.0 (C). Total uronic acids in each fraction were determined at A₅₂₀ (Abs 520nm). Vertical ticks at the top of each profile show the elution positions of uronic acid oligomers generated from PG2 hydrolysis of PGA and galacturonic acid.

Figure 8

Bio-Gel P-4 profiles of uronic acids released from mature-green cv Ailsa Craig cell walls by PG2 in the presence of EDTA or Ca²⁺. Cell walls (5 mg) in 2 mL of 20 mm KOAc and 100 mm KCl were incubated for 2 h at 34°C. Shown are uronic acids released at pH 4.5 and 1.5 mm CaCl₂ (A) and at pH 6.0 and 2 mm EDTA (B). Total uronic acids in each fraction were determined at A₅₂₀ (Abs 520nm).