Altered Lignin Biosynthesis Improves Cellulosic Bioethanol Production in Transgenic Maize Plants Down-Regulated for Cinnamyl Alcohol Dehydrogenase

Cinnamyl alcohol dehydrogenase （CAD） is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Trans- genic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alter- ations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and ara- binoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

Changes in Cinnamic Acid Derivatives Associated with the Habituation of Maize Cells to Dichlobenil

The habituation of cell cultures to cellulose biosynthesis inhibitors such as dichlobenil （DCB） represents a valu- able tool to improve our knowledge of the mechanisms involved in plant cell wall structural plasticity. Maize cell lines habituated to lethal concentrations of DCB were able to grow through the acquisition of a modified cell wall in which cellulose was partially replaced by a more extensive network of arabinoxylans. The aim of this work was to investigate the phenolic metabolism of non-habituated and DCB-habituated maize cell cultures. Maize cell cultures were fed [14C]cinnamate and the fate of the radioactivity in different intra-protoplasmic and wall-localized fractions throughout the culture cycle was analyzed by autoradiography and scintillation counting. Non-habituated and habituated cultures did not markedly differ in their ability to uptake exogenous [14C]cinnamic acid. However, interesting differences were found in the radiolabeling of low- and high-Mr metabolites. Habituated cultures displayed a higher number and amount of radiola-beled low-Mr compounds, which could act as reserves later used for polysaccharide feruloylation. DCB-habituated cultures were highly enriched in esterified [14C]dehydrodiferulates and larger coupling products. In conclusion, an extensive and early cross-linking of hydroxycinnamates was observed in DCB-habituated cultures, probably strengthening their cellulose-deficient walls.

Plasticity of Xyloglucan Composition in Bean （Phaseolus vulgaris）-Cultured Cells during Habituation and Dehabituation to Lethal Concentrations of Dichlobenil

Bean cells that have been habituated to grow in a lethal concentration （12 μM） of 2,6-dichlorobenzonitrile （dichlobenil or DCB, a cellulose biosynthesis inhibitor） are known to have decreased cellulose content in their cell walls. Xyloglucan, which is bound to cellulose and together with it forms the main loading network of plant cell walls, has also been described to decrease in habituated cells, but whether the change on cellulose affects the xyloglucan structure besides its abundance has not been analyzed. Fragmentation analysis with xyloglucan-specific endoglucanase （XEG） and endocellulase revealed that habituation to DCB caused a change in the fine structure of xyloglucan, namely a decrease in fucosyl residues attached to the galactosyl-xylosyl residues along the glucan backbone. After the removal of herbicide from the medium （dehabituated cells）, xyloglucan recovered its fucosyl residues. In addition, some cello-oligosaccharides could be detected only in habituated cells＇ xyloglucan digested by XEG and endocellulase, corresponding to a glucan co- valently bound or co-precipitated with the hemicelluloses. These results show that structural flexibility of cell walls relies in part on the plasticity of xyloglucan composition and opens up new perspectives to further research in this field.

Unraveling the Biochemical and Molecular Networks Involved in Maize Cell Habituation to the Cellulose Biosynthesis Inhibitor Dichlobenil

The biochemical and molecular processes involved in the habituation of maize cells to growth in the presence of the cellulose biosynthesis inhibitor dichlobenil （DCB） were investigated. DCB affects the synthesis of cellulose both in active and stationary growth phases and alters the expression of several CesA genes. Of these, ZmCesA5 and ZmCesA7 seem to play a major role in habituating cells to growth in the presence of DCB. As a consequence of the reduction in cellulose, the expression of several genes involved in the synthesis of hydroxycinnamates is increased, resulting in cell walls with higher levels of ferulic and p-coumaric acids. A proteomic analysis revealed that habituation to DCB is linked to modifications in several metabolic pathways. Finally, habituated cells present a reduction in glutathione S-transferase detoxifying activity and antioxidant activities. Plant cell adaptation to the disturbance of such a crucial process as cellulose biosynthesis requires changes in several metabolic networks, in order to modify cell wall architecture and metabolism, and survive in the presence of the inhibitor. Some of these modifications are described in this paper.