Final Report Summary - FUEL-PATH (Exploiting the saccharification potential of pathogenic microorganisms to improve biofuel production from plants.) Plant biomass has been the main source of energy for most part of human history and, due to the increasing demand for renewable materials to produce energy as well as industrial products, is reconsidered today as one of the possible strategic resources. A key process for the utilization of plant biomass is the initial degradation of the complex polysaccharides of the plant cell wall into simple and fermentable sugars, a process which is called saccharification. The major bottleneck for the industrial scale-up of this process is the recalcitrance of the plant polysaccharides to hydrolysis. We have investigated whether saccharification can be improved by engineering or selecting plants with an altered pectin structure and/or composition. Homogalacturonan (HGA), a major component of pectin in plant cell walls, is synthesized and secreted in a highly methylesterified form and is subsequently de-esterified by apoplastic pectin methylesterases (PMEs) to its acidic form characterized by free carboxylic residues. Calcium ions interact with the acidic form of HGA to form rigid cross-linked structures called “egg-boxes”. Since the egg-box structure is involved in cell adhesion, its genetic variation or its manipulation may determine major changes on saccharification of the plant tissue as well as on plant growth and development. In FUEL-PATH we have demonstrated that de-esterified homogalacturonan (HGA) negatively affects saccharification, since plants with reduced levels of HGA, as a consequence of the expression of either a polygalacturonase (PG) or an inhibitor of pectin methylesterases (PMEI), show increased saccharification efficiency. This indicates that pectin composition affects cellulose degradability and that plants improved for biomass conversion can be obtained using pectin-modifying proteins. Arabidopsis mutants and ecotypes with reduced HGA levels also show increased saccharification, indicating that HGA esterification can be a marker for the selection of genotypes with better biofuel yield. Plants overexpressing a PMEI are also more resistant to fungal, bacterial and viral infections, suggesting that plants with altered pectin composition have additional desirable features. However, elevated levels of expression of pectin-degrading enzymes cause reduced growth, possibly as a consequence of the accumulation of oligogalacturonides (OGs), pectin fragments that elicit defense responses. But plants expressing a pectic enzyme under the control of a senescence-specific promoter display normal growth and increased saccharification efficiency at late stages of development. To understand the molecular mechanisms underlying the reduced growth of plants with altered pectin composition, we have studied Arabidopsis genes that either show increased expression in plants expressing a PG, or that are involved in OG signalling. We have discovered molecular elements of the OG-mediated signalling pathways. We have obtained evidence that plants cope with an altered pectin composition by activating multiple mechanisms that reduce the damage caused by loss of cell wall integrity. These mechanisms can restrict plant growth and may also influence saccharification efficiency, and must therefore be taken into account when developing novel varieties with cell walls tailored for biofuel production.
