Cell wall remodelling of cellulose deficient maize cellsgoing through the key role of phenolics

Resumen

The cell wall is a dynamic structure that provides important mechanical and physiological functions for the plant. Previous studies in our group have demonstrated that a reduction in the cellulose content in maize primary cell walls leads to a modified cell wall architecture and composition, causing changes in the amount and structure of other components. Therefore, the overall aim of the present study was to have a deep insight into the knowledge of the compensation mechanisms associated to the structural plasticity of the cell wall, paying special attention to the role of the phenolic compounds. In order to achieve this goal, maize suspension-cultured cells with a reduction in the cellulose content by habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor, were used. Cellulose impairment involved changes in the metabolism and structure of hemicelluloses. By using a radiochemical approach, it was demonstrated that cell wall bound hemicelluloses and sloughed polymers were more homogeneously sized and had a lower weight-average relative molecular mass in DCB-habituated cells when compared to non-habituated ones. Despite their lower molecular mass, hemicelluloses underwent massive cross-linking mediated by hydroxycinnamates, mainly ferulic and p-coumaric acids, as a part of their strategy to compensate the lack of cellulose. As previous results pointed out, it is expected for DCB-habituated cells to find a modified phenolic metabolism. The comparative study between the hydroxycinnamates esterified into protoplasmic and cell wall polysaccharides through the cell culture cycle revealed that DCB-habituated cell walls showed an enrichment in ferulic acid, p-coumaric acid and dehydrodiferulates indicating an extra dimerization in muro. Furthermore, the 8-O-4’-diferulic acid emerged as a diagnostic feature of DCB habituation process. These results support the idea that cell wall hydroxycinnamates play a key role in the remodelling of cellulose deficient cell walls. In order to explain the molecular basis for a cell wall enriched in hydroxycinnamates that underwent a more extensive crosslinking, a study on peroxidases (POX) and genes related with cell wall feruloylation/coumaroylation was carried out. A comparative study of POX activity and isoforms associated with cell wall remodelling was addressed. POX activity was higher in DCB-habituated cells than in control ones in all cell wall compartments assayed. Moreover, the comparison of the isoPOX profile obtained from the ionically bound cell wall proteins and spent medium proteins revealed a distinctive higher activity for certain POX isoforms in DCB-habituated cells. It is hypothesized that some of these isoPOXs could be involved in the cross-linking of arabinoxylans in DCB-habituated cells. In order to gain a further insight into the genes that could be involved in the feruloylation/coumaroylation of the cell wall, a preliminary survey into the maize BAHD genes was undertaken. Thirteen maize homologues to BAHD genes previously found in rice and Arabidopsis were identified by bioinformatics approaches. All the protein sequences shared the transferase domain HXXXDG characteristic of BAHD proteins involved in the acylation of arabinoxylans. From the BAHD genes analysed, ZmBAHD1, ZmBAHD2 and ZmBAHD12 were overexpressed in DCB-habituated cells. Interestingly, ZmBAHD1 and ZmBAHD12 showed a high coexpression level with genes related to the synthesis and remodelling of the cell wall. These results point to ZmBAHD1 and ZmBAHD12 as good candidates as putative feruloyl and/or coumaroyl transferases in maize cell walls. Finally, as maize cultured cells exposed to DCB have been reported to induce the synthesis of ectopic lignin, an investigation was conducted in order to verify if a cellulose biosynthesis inhibitor with a different mode of action, such as cobtorin, also induce ectopic lignification. The negative phloroglucinol staining of cells after cobtorin treatment indicated that this compound was not inducing ectopic lignification in our experimental system. In contrast, DCB-treated cells showed positive staining for phloroglucinol, a high cell wall autofluorescence and ammonium induced fluorescence related to the accumulation of phenolics. All these three findings showed the presence of a lignin-like polymer in DCB-treated cells. Furthermore, the overexpression of ZmBAHD5, ZmBAHD6, ZmBAHD9 and ZmBAHD12 together with an enhancement of peroxidase activity in DCB-treated cells were found. Therefore, the induction of ectopic lignification promoted by a restriction in the synthesis of cellulose seems to be a spatiotemporal process in which changes in the expression of selected BAHD genes and in the activity of POX enzymes, together with structural modifications, contribute to reinforce the weakened cell wall entanglement. The results obtained in this Thesis project afford new data that shed light into the key contribution of phenolics in the remodelling of cell walls with a low cellulose content. Moreover, additional information on the biochemical and genetic control of this process are provided.