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

Abstract

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.

Figure 1

Saccharification efficiency of WT, pme3, PMEI, qua2-1, and PG leaves. Saccharification efficiency was measured after 24 hours of treatment with 0.5% Celluclast. Bars represent average of saccharification efficiency ± SE (n ≥4). Different letters indicate statistically significant differences, according to ANOVA followed by Tukey’s test (P <0.05). The experiments were repeated at least three times with similar results. PG, polygalacturonase; pme3, pectin methylesterase 3; PMEI, pectin methylesterase inhibitor; qua2-1, quasimodo2-1; SE, standard error; WT, wild type.

Figure 2

Variability of content of HGA regions in Arabidopsis mutants affected in pectin composition. The levels of HGA domains were determined by immunodot assay on ChASS fraction extracted from leaves of 4-week-old Arabidopsis WT (Col-0), mutants (qua2-1 and pme3), and transgenic plants (PG and PMEI). Equal amounts of ChASS were hybridized with the monoclonal antibody PAM1. PAM1 epitope levels were normalized with respect to the standard polygalacturonic acid (PGA). Bars represent means of PAM1 level ± SD (n = 3). Different letters indicate statistically significant differences, according to ANOVA followed by Tukey’s test (P <0.05). Similar results were obtained in at least three other experiments. ChASS, chelating agent-soluble solids; Col-0, Columbia-0; HGA, de-methyl-esterified homogalacturonan; PG, polygalacturonase; PGA, polygalacturonic acid; pme3, pectin methylesterase 3; PMEI, pectin methylesterase inhibitor; qua2-1, quasimodo2-1; WT, wild type.

Figure 3

Scatter plot between PAM1 epitope level and enzymatic saccharification efficiency. Correlation between PAM1 level and saccharification efficiency was examined based on the Pearson correlation efficiency. A strong negative correlation was found (r = -0.96) among the genotypes analyzed. PG, polygalacturonase; pme3, pectin methylesterase 3; PMEI, pectin methylesterase inhibitor; qua2-1, quasimodo2-1; WT, wild type.

Figure 4

Analysis of the levels of HGA regions and saccharification efficiency in the Arabidopsis nested core collection. (A) PAM1 epitope content. Bars represent average of PAM1 epitope level ± SD (n = 3) express with respect to 1 μg PGA signal. (B) Saccharification efficiency of leaves. Bars represent average of saccharification efficiency ± SD (n ≥10). Different letters indicate statistically significant differences, according to ANOVA followed by Tukey’s test (P <0.05). The experiments were repeated three times with similar results. HGA, de-methyl-esterified homogalacturonan; PGA, polygalacturonic acid.

Figure 5

Scatter plot of correlation analysis between enzymatic saccharification efficiency and PAM1 level in Arabidopsis natural accessions. PAM1 epitope levels were determined by immunodot assay (express with respect to 1 μg PGA signal). Enzymatic saccharification efficiency was measured after 24 hours of treatment with 0.5% Celluclast. Pearson correlation analysis revealed a high negative correlation (r = -0.72) between saccharification efficiency and PAM1 content among the accessions analyzed. PGA, polygalacturonic acid.