Abstract
Fluorescence microscopy of rice (Oryza sativa L.) callus sections showed that all of the walls fluoresced blue in water (pH 5.8) and green in ammonia (pH 10.0), both characteristics of feruloyl esters. Such fluorescence in the walls of cells cultured in Gamborg's B5 medium was much stronger than that in amino acid (AA) medium. Laser scanning microscopy showed that the level of fluorescence was higher in the intercellular layer, especially at corner junctions between cells, suggesting that ferulic acid ester derivatives are located in the middle lamella as well as in the wall. Extracellular polysaccharides appearing during cultivation in AA medium were more highly feruloylated than those in B5 medium during cultivation. Both the levels of ferulic and diferulic acid and the relative proportion of diferulic acid in the walls of cells increased on transfer of the cells cultured in AA medium to B5 medium. The walls of cells cultured in B5 medium maintained constant levels and proportions of the phenolic acids. Removal of phenolic acids from wall preparations by carboxylesterase facilitated the solubilization of noncellulosic polysaccharides. Treatment of the cell aggregates grown in AA medium with an enzyme that hydrolyzes feruloyl esters decreased the size of the aggregates to between 20 and 500 [mu]m, compared with an original size between 200 and 1000 [mu]m. These findings suggest that feruloyl and diferuloyl esters between polysaccharides are involved in the aggregation of cultured rice cells.