Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion.

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.

Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis mlo2 mutant.

Arabidopsis (Arabidopsis thaliana) genes MILDEW RESISTANCE LOCUS O2 (MLO2), MLO6, and MLO12 exhibit unequal genetic redundancy with respect to the modulation of defense responses against powdery mildew fungi and the control of developmental phenotypes such as premature leaf decay. We show that early chlorosis and necrosis of rosette leaves in mlo2 mlo6 mlo12 mutants reflects an authentic but untimely leaf senescence program. Comparative transcriptional profiling revealed that transcripts of several genes encoding tryptophan biosynthetic and metabolic enzymes hyperaccumulate during vegetative development in the mlo2 mlo6 mlo12 mutant. Elevated expression levels of these genes correlate with altered steady-state levels of several indolic metabolites, including the phytoalexin camalexin and indolic glucosinolates, during development in the mlo2 single mutant and the mlo2 mlo6 mlo12 triple mutant. Results of genetic epistasis analysis suggest a decisive role for indolic metabolites in mlo2-conditioned antifungal defense against both biotrophic powdery mildews and a camalexin-sensitive strain of the necrotrophic fungus Botrytis cinerea. The wound- and pathogen-responsive callose synthase POWDERY MILDEW RESISTANCE4/GLUCAN SYNTHASE-LIKE5 was found to be responsible for the spontaneous callose deposits in mlo2 mutant plants but dispensable for mlo2-conditioned penetration resistance. Our data strengthen the notion that powdery mildew resistance of mlo2 genotypes is based on the same defense execution machinery as innate antifungal immune responses that restrict the invasion of nonadapted fungal pathogens.

Impaired Cuticle Functionality and Robust Resistance to Botrytis cinerea in Arabidopsis thaliana Plants With Altered Homogalacturonan Integrity Are Dependent on the Class III Peroxidase AtPRX71.

Pectin is a major cell wall component that plays important roles in plant development and response to environmental stresses. Arabidopsis thaliana plants expressing a fungal polygalacturonase (PG plants) that degrades homogalacturonan (HG), a major pectin component, as well as loss-of-function mutants for QUASIMODO2 (QUA2), encoding a putative pectin methyltransferase important for HG biosynthesis, show accumulation of reactive oxygen species (ROS), reduced growth and almost complete resistance to the fungal pathogen Botrytis cinerea. Both PG and qua2 plants show increased expression of the class III peroxidase AtPRX71 that contributes to their elevated ROS levels and reduced growth. In this work, we show that leaves of PG and qua2 plants display greatly increased cuticle permeability. Both increased cuticle permeability and resistance to B. cinerea in qua2 are suppressed by loss of AtPRX71. Increased cuticle permeability in qua2, rather than on defects in cuticle ultrastructure or cutin composition, appears to be dependent on reduced epidermal cell adhesion, which is exacerbated by AtPRX71, and is suppressed by the esmeralda1 mutation, which also reverts the adhesion defect and the resistant phenotype. Increased cuticle permeability, accumulation of ROS, and resistance to B. cinerea are also observed in mutants lacking a functional FERONIA, a receptor-like kinase thought to monitor pectin integrity. In contrast, mutants with defects in other structural components of primary cell wall do not have a defective cuticle and are normally susceptible to the fungus. Our results suggest that disrupted cuticle integrity, mediated by peroxidase-dependent ROS accumulation, plays a major role in the robust resistance to B. cinerea of plants with altered HG integrity.

Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth.

Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion.

Analysis of pectin mutants and natural accessions of Arabidopsis highlights the impact of de-methyl-esterified homogalacturonan on tissue saccharification.

BACKGROUND: Plant biomass is a potentially important renewable source of energy and industrial products. The natural recalcitrance of the cell walls to enzymatic degradation (saccharification), which plants have evolved to defend themselves from biotic stresses, represents a major bottleneck for the industrial bioconversion of lignocellulosic biomasses. The identification of factors that influence the cell wall recalcitrance to saccharification may help to overcome the existing limitations that hamper the utilization of biomass. RESULTS: Here we have investigated in Arabidopsis thaliana the impact of homogalacturonan (HG) content and structure on tissue saccharification. We characterized mutants affected in genes encoding proteins involved in HG biosynthesis (quasimodo2-1; qua2-1) and methylesterification (pectin methylesterase 3; pme3). We also analyzed the natural variation of Arabidopsis through the characterization of a nested core collection of 24 accessions generated to maximize genetic variability. We found a negative correlation between the level of de-methyl-esterified HG (HGA) and cellulose degradability. CONCLUSIONS: We propose to use the level of HGA domains as a biochemical marker of the cell wall recalcitrance to saccharification. This may be utilized for selecting, on a large scale, natural variants or mutants with improved bioconversion features.