Proteomic analysis reveals a novel set of cell wall proteins in a transformed tobacco cell culture that synthesises secondary walls as determined by biochemical and morphological parameters

A cell suspension culture of a tobacco (Nicotiana tabacum L. cv. Petit Hava na) cell line derived from a cultivar transformed with the Tcyt gene from A grobacterium, which leads to high endogenous levels of cytokinin, has been established. This cell line shows increased cell aggregation, elongated cel ls and a 5-fold increase in wall thickness. If allowed to carry on growing it can form a single mass without shedding cells into the medium. When anal ysed at an earlier growth stage, these cultures were found to produce impro ved levels of vascular nodule formation than in other systems that employ e xogenous cytokinin. This differentiation was optimised with respect to sucr ose and auxin signals in order to induce maximum production of cells with t hickened walls and a morphology characteristic of fibre cells and tracheids , in addition to cells that remain meristematic. In order to establish the validity of this system for studying secondary wall formation, the walls an d associated biosynthetic changes were analysed in these cells by chemical analysis of the walls, changes in activities of enzymes of xylan and monoli gnol synthesis, and expression of mRNAs coding for enzymes of lignin biosyn thesis. The wall composition of the transformed cells was compared with tha t determined for primary walls from a typical untransformed tobacco cell li ne. Recovery of wall material was 50% greater in the transformed culture. I n this material a major difference was found in the pectin fraction where t here was a distinct difference in size distribution together with a lower l evel of methylation for the transformed line, which may be related to incre ased adhesiveness. There were increased amounts of xylan, although the rati o of xyloglucan to xylan content was not substantially different due to the mixture of cell types. There was also an increase in cellulose and phenoli c components. Increased activity of enzymes involved in the synthesis of xy lan as a marker for the secondary wall occurred around the time of tracheid differentiation and coincided with a broad peak of cinnamyl alcohol dehydr ogenase activity. The expression of mRNAs coding for enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase, cinnamate 4-hydroxyl ase, catechol O-methyl transferase was relatively constitutive in the cultu res while transcripts of ferulate 5-hydroxylase, cinnamoyl CoA-reductase, c innamyl alcohol dehydrogenase and lignin peroxidase were induced. The walls of the transformed cells also showed considerable differences in the subse t of extractable proteins from that found in primary walls of tobacco when these were subjected to proteomic analysis. Many of these proteins appear t o be novel and not present in primary walls. However an M-r-32,000 chitinas e, an M-r-34,000 peroxidase, an M-r-65,000 polyphenoloxidase/laccase and po ssibly an M-r-68,000 xylanase could be identified as well as structural pro teins.