Methods of Isolation and Characterization of Oligogalacturonide Elicitors.

Oligogalacturonides (OGs) are pectic fragments derived from the partial degradation of homogalacturonan in the plant cell wall and able to elicit plant defence responses. Recent methodological advances in the isolation of OGs from plant tissues and their characterization have confirmed their role as bona fide plant Damage-Associated Molecular Patterns. Here, we describe the methods for the isolation of OGs from Arabidopsis leaf tissues and for the characterization of OG structure and biological activity.

Sensitive detection and measurement of oligogalacturonides in Arabidopsis.

Oligogalacturonides (OGs) are pectin fragments derived from the partial hydrolysis of the plant cell wall pectin; they are elicitors of various defense responses. While their activity is well documented, the detection of OGs produced in planta is still a challenging task. A protocol has been developed for the extraction and analysis of OGs from small samples of Arabidopsis tissues by using fluorescent labeled OGs, which allowed to monitor the efficiency of extraction. An efficient recovery was obtained by using a combination of calcium chelating agents at acidic pH. Off-line coupling of high performance anion exchange chromatography with matrix assisted laser desorption ionization- time of flight-mass spectrometryor nanoESI-Orbitrap-MS/MS was used for the identification and characterization of oligosaccharides. The protocol was successfully applied to detect OGs by using low amounts (50 mg) of Arabidopsis leaves and very low amounts (30 mg) of senescent leaves. The protocol was also successfully used to detect OGs in Arabidopsis cell wall material digested with pectinases. The proposed extraction protocol followed by sensitive and high-resolution analysis methods allowed detection of OGs released from the cell wall in Arabidopsis tissues by using minimal sample material. The protocol may be useful to study OG-triggered plant immunity and cell wall remodeling during Arabidopsis growth and development.

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.