The effect of pH on cell wall composition and its relationship to cell aggregation in carrot cell cultures

Plant cell suspension cultures were used to investigate the effects of medium pH on cell aggregation in wild carrot cells. Two hypotheses were examined: 1) the origin of aggregates or the lack thereof in cell culture is a result of pH controlled, differential growth of aggregating or disaggregating genotypes, and 2) aggregate formation or dissociation is the result of the direct effect of H⁺ ions on one or more components of the cell wall. Changes in aggregate size distribution of clones and cultures composed of a single size aggregate demonstrated that selection of an aggregating or disaggregating genotype was not involved in the formation of aggregates in culture. Cell walls were then analyzed for differences between the two phenotypes. Electron microscopy revealed less middle lamella in disaggregated cells grown at pH 4.5 and three times less wall bound Ca²⁺ than cells grown at pH 5.5. However, removal of Ca2+ from the tissue grown at pH 5.5 did not cause disaggregation nor did excess Ca²⁺ in cultures grown at pH 4.5 cause aggregation. It was concluded that changing the cell wall Ca²⁺ content was not sufficient to produce the changes in carrot cell aggregation. Cell wall fractionation showed the carbohydrate composition between the two phenotypes to be very similar while the total amount of cell wall material per unit volume of culture was 26% higher in the aggregates grown at pH 5.5. The amount of uronic acid was significantly higher in high molecular weight polymeric wall material released into the media of disaggregated cultures. All fractions of cell wall from aggregated tissue grown at pH 5.5 had significantly greater amounts of hydroxyproline than did fractions from non- aggregated tissue grown at pH 4.5. In addition, the β-galactosidase activity was lower in non-aggregated tissue suggesting that cell wall degradation by this enzyme is not involved in the aggregation/disaggregation of cells seen in this system.

The results support a hypothesis that the effect of pH on carrot cell aggregation does not involve large changes in the existing cell wall structure alone, but arises from a pH controlled inhibition or activation of synthesis or incorporation of wall material into the existing wall. Prolonged growth at either pH 4.5 or pH 5.5 results in structural weakening or strengthening respectively and leads to the disaggregation or aggregation of the carrot cells in culture.