Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β‐subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β‐subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β‐subunit protein. Cell walls prepared from mature‐green fruit were employed for binding and assay of PG2. Walls were provided with rate‐limiting quantities of purified PG2 and incubated at 100 m M KCl, pH 4.5, or 25 m M KCl, pH 6.0. Cell walls of both β‐subunit antisense and wild‐type fruit retained comparable quantities of added PG2. The release of pectin from PG2‐loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β‐Subunit‐antisense cell walls released 2‐ to 3‐fold higher levels of pectin in response to PG2 than did wild‐type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2‐mediated hydrolysis of β‐subunit‐antisense compared with wild‐type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG‐mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 m M CDTA ( trans ‐1,2‐cyclohexanediamine‐N,N,N′,N′‐tetraacetic acid), and were more extensive for the β‐subunit‐antisense cell walls compared with the wild‐type walls. Pectic polymers derived from ethanol insoluble‐powders showed molecular mass downshifts during ripening but differences between the β‐subunit‐antisense and wild‐type fruits were not observed.