The influence of extensin cross-linking on biomass recalcitrance

Abstract

Plant cell walls are under investigation as a source for biofuel production, yet conversion of cell walls (biomass) into biofuel is currently too expensive to be competitive with gasoline. Biomass is recalcitrant; that is, it resists enzymatic degradation by cellulases into monosaccharides such as glucose. One source of recalcitrance may be the presence of extensins, covalently bound cell wall proteins that are extremely insoluble. To determine what influence, if any, extensins have on biomass recalcitrance, I performed several experiments. I first turned to poplar biomass, which is a model source for biofuels. I found that protease treatment of poplar biomass after liquid hot water pretreatment reduced the hydroxyproline content (a proxy for extensins). The reduction in hydroxyproline content correlated with reduced recalcitrance, seen as an increase in glucose release after cellulase digestion of poplar biomass. I also tested whether Arabidopsis T-DNA insertional mutations in the genes encoding enzymes that perform extensin post-translational modifications could reduce extensin content or cross-linking, and whether this reduction was associated with reduced biomass recalcitrance. I found that although these mutants were hypothesized to have reduced incorporation of extensin in cell walls, no significant effects on extensin content in inflorescence stem cell walls (an analog for woody biomass), nor on glucose release from biomass, were found in any mutant line. Finally, I looked at the effects of extensin overexpression on glucose release in transgenic Arabidopsis lines containing synthetic genes encoding the complete extensin domain from SlLRX1 or a short C-terminal region of 20 amino acids of SlLRX1, fused to the red fluorescent reporter protein tdTomato. Observation of the tdTomato fluorescence in transgenic biomass after various chemical and enzymatic treatments indicated that the C-terminal 20 amino acids of SlLRX1 are sufficient to allow a strong association with the cell wall, while the complete SlLRX1 extensin domain leads to an even stronger, perhaps covalent linkage. Lines transformed with the complete SlLRX1 extensin domain had more than twice the hydroxyproline content in their stems than wild-type, but this increase in hydroxyproline did not affect the amount of glucose released from stems upon cellulase digestion. Since protease treatment reduced both hydroxyproline content and recalcitrance in poplar biomass, further experiments to assess the nature of the association between extensins and cell walls are warranted to attempt to further reduce recalcitrance. In the experiments I performed, the stems of extensin modification mutant Arabidopsis lines showed no change in extensin modification, and therefore no effect on recalcitrance was observed; stems of transgenic overexpression Arabidopsis lines showed increased extensin content, but again, no effect on recalcitrance was observed. My investigations in Arabidopsis focused on stem tissue, as this is analogous to material used in biofuel production. However, extensins are most abundantly expressed in roots in many plants, particularly in Arabidopsis. Examination of roots of both mutant and transgenic Arabidopsis may be more revealing of the interactions between extensins, cell walls, and recalcitrance.