Cell wall modifications of bean (Phaseolus vulgaris) cell suspensions during habituation and dehabituation to dichlobenil

Bean (Phaseolus vulgaris L.) cell suspensions were adapted for growth in 12 muM dichlobenil (2,6-dichlorobenzonitrile or DCB) by a stepwise increase in the concentration of the inhibitor in each subculture. Non-tolerant suspensions (I-50 = 0.3 muM) gave rise to single cells or small clusters while tolerant cell suspensions (150 = 30 muM) grown in DCB formed large clusters. The cells in these clusters were surrounded by a thick and irregular cell wall with a lamellate structure and lacking a differentiated middle lamella. Analysis of habituated cell walls by Fourier transform infrared spectroscopy and cell wall fractionation revealed: (1) a reduced amount of cellulose and hemicelluloses, mainly xyloglucan (2) qualitative and quantitative differences in pectin levels, and (3) a non-crystalline and soluble beta-1,4-glucan. When tolerant cells were returned to medium lacking DCB, the size of the cell clusters was reduced; the middle lamella was only partly formed, and the composition of the cell wall gradually reverted to that obtained with non-tolerant cells. However, dehabituated cells (150 = 12 muM) were 40-fold more tolerant to DCB than nontolerant cells and were only 2.5-fold more sensitive than tolerant cells.