Ultrastructural and biochemical effects of endopectate lyase on cell walls from cell suspension cultures of bean and rice

Abstract

Cell walls prepared from suspension cell cultures of bean and rice in log-phase growth were used to examine the effects of endopectate lyase (PL) on the solubilization of cell wall carbohydrates and concomitant ultrastructural alterations. Cell wall preparations from both plant sources were heterogeneous and contained a range of wall types from primary walls to xylem elements with spiral, secondary wall thickenings. Marked differences in wall thickness and number of wall laminations typified both preparations.Bean cell walls were more susceptible to degradation by PL than were those of rice. Upon treatment of the former with 2.3 × 10⁻³ units/mL of PL (1 unit released 1 μmol of unsaturated uronide/min at 30 °C from polygalacturonic acid at pH 8.5), 27% of the noncellulosic wall carbohydrate was solubilized in 1 h. This represented 50% of the PL susceptible carbohydrate in the preparation. Only 3% of the noncellulosic carbohydrate was released from rice cell walls in 1 h when treated with 115 × 10⁻³ units of PL/mL. This accounted for 60% of the PL susceptible wall fractions. Only uronic acid, rhamnose, galactose, and arabinose were solubilized from both preparations by PL.Cell walls in the bean and rice preparations were affected differentially by the PL. Those walls with secondary thickenings did not appear to be degraded, while the distinct fibrillar appearance of both bean and rice walls tended to fade or disappear. The middle lamella tended to dissolve to varying degrees in the presence of PL. Bean walls were more severely degraded than were the rice walls and many exhibited swelling, separation of wall layers, markedly reduced staining intensity, and (or) a granular ultrastructure.This study has demonstrated that as PL acts on susceptible cell walls there are major changes evoked in cell wall structure which suggest that the rhamnogalacturonan fraction of the higher plant cell wall contributes significantly to cell wall integrity. This study also emphasizes the need for cell wall material of proven uniformity for investigations of both cell wall composition and effects of specific polysaccharide degrading enzymes on cell wall integrity. Preliminary studies indicate that tobacco pith may provide more uniform cell walls than do cell suspension cultures.