Deepening into the knowledge of maize cell wall plasticityproteomic and biochemical approaches

Laburpena

Plant cells are surrounded by a complex and dynamic compartment that defines the outer limits of the cell, the cell wall. This structure comprises three interconnected and independent structural networks (cellulose, hemicellulose and/or lignin) that constitute an essential scaffold to protect the cell and therefore play a crucial role in plant cell biology. Although widely regarded as a static compartment, the cell wall shows a remarkable plasticity in structure and composition. The study of cell wall components and the interaction among different structural networks allows us to gain knowledge about the synthesis, assembly of the cell wall components and molecular basis of cell wall plasticity. Maize is an important crop worldwide due to its uses for food, forage and bioethanol production. For this reason, it is a suitable plant model for studying the cell wall plasticity in response to deficiencies in at least one of the three main structural networks of the cell wall. The overall aim of the present study is to elucidate the mechanisms associated with the structural plasticity of the cell wall, paying special attention to the biosynthesis of the different polysaccharides that form plant cell wall. For this purpose, two strategies were used. On the one hand, DCB – a well known cellulose biosynthesis inhibitor - was used to induce changes in the cell wall composition and structure. Maize suspension-cultured cells habituated to DCB have a reduced content of cellulose in their walls but an extensive and more cross-linked arabinoxylan network together with lignin-like material accumulation. These cells reflect a high cell wall plasticity in response to structural alterations and therefore they are a good material for the study of the compensation mechanism. A total comparative proteomic profile between DCB-habituated cells and control ones revealed that the capacity of cells to cope with stress conditions produced an altered proteomic profile with 596 highly misregulated proteins, mainly located in organelles and cytoplasm. Most of the altered proteins are related to cell wall metabolism, antioxidant systems and stress-responses, which reflect the general metabolic alteration of cells as a result of the reduction in cellulose content. Moreover, a group of six up-regulated proteins associated with cell wall remodelling, Class III Peroxidases (CIII-PRX), have been identified by an exhaustive bioinformatics study. These CIII-PRX were putatively located in the apoplast and related to lignification process. The sequence analysis of primary, secondary and 3D structure of our proteins in comparison to previously characterized CIII-PRX allowed us to detect important changes in key residues involved in catalytic domains which determined the putative role of some CIII-PRX in lignification process. In order to gain on insight further xylan synthesis, a protocol for the isolation of maize microsomal membranes and xylosyltransferase (XylT) activity optimization was carried out. It was possible to determine high XylT activity in CHAPS solubilized fraction especially when low molecular weight polysaccharide acceptors were added to the reaction. However, the presence of cold UDP-Xylose did not increase the XylT activity. On the other hand, loss-of-function mutants and transformed maize plants in enzymes of the lignin biosynthesis pathway, such as 4-coumarate 3-hydroxylase (C3H) or two O-methyltransferases (COMT and CCoAOMT) have been used for better understanding of the cell wall plasticity when lignin network is altered. In general, maize plants analyzed did not show quantitative changes in lignin content. However, a compositional analysis revealed changes in the lignin monomer composition. In this way, C3H1-RNAi plants were enriched in H-subunits, tricin and lignin-bound ferulates whereas double-mutants ccaomt1xcomt had an increment in G-subunits. Moreover, higher hemicelluloses content was detected in most of the mutant lines. These results reveal the interaction among polymeric cell wall networks and the influence of lignin modification on the other components of the cell wall. Interestingly, some of the modifications in lignin biosynthesis analysed affected biotechnology properties of the cell wall such as enzymatic degradability.