Analysis of pectin mutants and natural accessions of Arabidopsis highlights the impact of de-methyl-esterified homogalacturonan on tissue saccharification
- Fedra Francocci†1,
- Elisa Bastianelli†1,
- Vincenzo Lionetti†1,
- Simone Ferrari1,
- Giulia De Lorenzo1,
- Daniela Bellincampi1Email author and
- Felice Cervone1
© Francocci et al.; licensee BioMed Central Ltd. 2013
Received: 31 July 2013
Accepted: 25 October 2013
Published: 18 November 2013
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.
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.
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.
KeywordsSaccharification Plant cell wall Pectin Homogalacturonan Arabidopsis thaliana
Liquid fuels derived from plant biomass are a promising, renewable alternative to fossil fuels. Presently, most bioethanol is produced by fermentation of sucrose or starch deriving from food crops such as sugarcane, corn, and wheat (first generation bioethanol) . However, competition between the increasing needs for food and fuel, discontinuous feedstock availability, and high production costs pose serious socioeconomic and logistical problems that hamper the large-scale adoption of this energy source. A suitable alternative to the use of food crops is the abundant and low cost lignocellulosic biomass derived from dedicated energy crops or from agricultural wastes (second generation bioethanol) . A key step in the bioconversion of lignocellulosic biomass is saccharification, that is, the degradation of the plant cell wall polysaccharides into soluble sugars that can be used by microorganisms for fermentation. Currently, enzymatic hydrolysis is the most promising and environmentally friendly technology available for saccharification . However, a major bottleneck for the industrial implementation of biomass utilization is the natural recalcitrance of the plant cell walls to enzymatic hydrolysis . The use of plants with walls less recalcitrant to hydrolysis may not only improve the saccharification process but also reduce the need of costly pre-treatments. The heterogeneity and complexity of the cell wall and the inter-polymeric interactions of its structural components are major factors contributing to recalcitrance . For example, cellulose microfibrils are tethered by xyloglucan through hydrogen bonds, while hemicelluloses interact with pectin via both non-covalent and covalent bonds . Crystallinity of cellulose as well as branches of xylan also influence enzymatic saccharification [7, 8], and lignification strongly reduces cellulose accessibility to degrading enzymes . The reduction of the level of some lignin precursors in transgenic alfalfa plants significantly increases the efficiency of tissue saccharification. Besides lignin, other cell wall components may be considered as suitable targets for the development of novel varieties more amenable to saccharification . Pectin is one of these components because it influences permeability and adhesive properties of the cell wall, and its position in the wall is crucial for the accessibility of cellulose and other polysaccharides to cell wall-degrading enzymes (CWDEs) [4, 10]. The backbone of pectin is homogalacturonan (HG), which consists of α-1,4-linked galacturonic acid (GalUA) residues partially methyl-esterified in C6. HG is exported to the cell wall in a highly methylated form and undergoes a selective de-methylation by apoplastic pectin methylesterases (PMEs) . The negatively charged carboxyl groups of adjacent HG chains, produced by PME activities, can form rigid calcium-mediated crosslinks that stiffen the cell wall . De-methyl-esterified HG (henceforth HGA) may affect assembly of the cellulose network, influencing the deposition and alignment of microfibrils in the wall . We have generated plants with a reduced content of HGA either by expressing a fungal polygalacturonase (PG plants)  or by overexpressing the PME inhibitors (PMEI plants) . In both cases, transgenic plants exhibit significantly higher efficiency of enzymatic saccharification .
As an alternative to the genetic transformation approach, in the present work we evaluated whether mutations in Arabidopsis genes encoding proteins that influence the level of HGA affect the cell wall recalcitrance to cellulase hydrolysis. In particular, we analyzed the mutant quasimodo2-1 (qua2-1) that carries a mutation in a putative pectin methyltransferase (PMT) and shows defects in cell adhesion and growth , and a knock-out mutant pectin methylesterase 3 (pme3) of a PME isoform that affects the level of pectin methylesterification in leaves . In addition, we characterized, in terms of content of HGA and saccharification efficiency, a polymorphic nested core collection of Arabidopsis accessions . Our study shows a negative correlation between the level of HGA regions and cellulose degradability, indicating that HGA level is a trait that may be exploited for a wide-scale selection and breeding of plants for biofuel production.
Results and discussion
Tissues of mutants with a reduced HGA level have higher cellulose degradability
Analysis of monosaccharides released after enzymatic hydrolysis of leaves from transgenic and mutant plants
0.11 ± 0.05
0.24 ± 0.04
0.99 ± 0.57
0.04 ± 0.02
0.11 ± 0.00
0.28 ± 0.03
2.18 ± 0.32*
0.05 ± 0.01
0.14 ± 0.01
0.21 ± 0.02
1.56. ± 0.05*
0.03 ± 0.0
0.14 ± 0.01
0.30 ± 0.06
2.22 ± 0.07*
0.04 ± 0.01
0.13 ± 0.00
0.21 ± 0.01
1.56 ± 0.09*
0.05 ± 0.01
Arabidopsis natural accessions with a low level of HGA show reduced recalcitrance to enzymatic hydrolysis
Correlation matrix between saccharification efficiency, biomass production, and pectin biochemical traits in Arabidopsis accessions
The results show that a low level of HGA in the cell wall correlates with a high enzymatic degradability of cellulose in Arabidopsis. A positive correlation between low level of HGA and high enzymatic saccharification has also been previously reported in tobacco and wheat plants . This is consistent with the concept that HGA is the domain of pectin that, by forming crosslinks with calcium ions, acts as a ‘glue’ and keeps the wall polysaccharides together . In order to deconstruct the plant cell wall, pathogenic microorganisms have a temporal strategy of producing CWDEs and secrete pectinases before other CWDEs . The hydrolysis of pectin, therefore, is a prerequisite that makes the other cell wall components more accessible to CWDEs. We have shown that tissues of plants with a reduced content of acidic HGA domains are more efficiently digested by cellulase, likely because the accessibility of the enzyme to its substrate is facilitated in these plants. Our analysis of qua2-1 and pme3 mutants, and of Arabidopsis natural accessions, reinforces and validates the concept, and shows that among the pectin-related biochemical parameters the PAM1 epitope level correlates very well to the saccharification potential. The large variability of PAM1-binding epitopes exhibited by the nested core collection may have an adaptive significance. The existence of the variability in the model plant Arabidopsis suggests that the content of HGA may also be variable in other plant species. Therefore, the HGA level may be a suitable marker to identify crop plants with better characteristics of bioconversion. The use of a simple assay to measure the PAM1 epitope by immunodetection potentially makes this trait useful for high-throughput screenings.
Material and methods
Plant material and growth condition
Arabidopsis (Arabidopsis thaliana) Col-0 WT seeds were purchased from Lehle Seeds (Round Rock, TX, USA). The production of transgenic PMEI (line 7) and PG (line 57) plants, and the selection of the pme3 and qua2-1 mutants, have been previously described [17–19]. All mutant lines used in the present work are in the Col-0 background. The 24-accession core collection was provided by the A. thaliana Genomic Resource Centre at National Institute for Agricultural Research (INRA), Versailles, France . All the plants were grown in a growth chamber maintained at 22°C and 70% relative humidity under a 16-hour light/8-hour dark photoperiod (photosynthetic photon flux density; PPFD) of 100 μmoles photons m-2 s-1). Before harvesting, all plants were maintained for 24 hours in darkness to minimize starch accumulation. Fully expanded Arabidopsis rosette leaves were collected at 3.9 growth stage (http://www.arabidopsis.org/portals/education/growth.jsp).
Enzymatic hydrolysis, sugar analysis, and determination of saccharification efficiency
Leaves were sterilized in a 1% sodium hypochlorite solution for 5 minutes and washed twice with sterile water. One hundred mg (wet weight) of leaf tissues from transgenic and ecotypes were cut into 0.25 cm2 square pieces and incubated at 37°C in a 5 mL filter-sterilized solution containing 50 mM sodium acetate buffer (pH 5.5), 0.02% NaN3, and 0.5% (vol/vol) Celluclast 1.5 L (cellulase from Trichoderma reesei ATCC 26921; 60.3 filter paper unit (FPU)/mL; product number C2730; Sigma-Aldrich, St Louis, MO, USA). The FPU for cellulase activity of Celluclast 1.5 L, quantified according to the standard method , was 60.3 FPU/mL. The enzymatic loading was 15 FPU/g leaf tissues. The leaf tissue was infiltrated under vacuum for 5 minutes. The incubation medium from leaves was collected after 24 hours of enzymatic treatment, centrifuged for 2 minutes at 11,000 × g, and the supernatant was then transferred into fresh tubes. The determination of carbohydrates in the enzymatic hydrolysates was performed according to the analytical procedure of the National Renewable Energy Laboratory (NREL) . Enzymatic saccharification efficiency was determined as the percentage of sugars released by enzymatic hydrolysis from the amount of sugars present in the tissue prior to enzymatic hydrolysis. The amount of sugars present in the supernatant and total sugars present in the tissues was determined spectrophotometrically by phenol-sulfuric acid assay  using D-Glc to perform the standard calibration curve. Statistical analyses were performed using the ANOVA procedure of the software package STATISTICA (1999; StatSoft, Tulsa, OK, USA).
Monosaccharide composition of the enzymatic hydrolysates was determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Ion Chromatography System ICS-3000, Dionex, Sunnyvale, CA, USA) with a 3 × 150 mm CarboPac PA20 column (Dionex). Before injection of each sample, the column was washed with 200 mM NaOH for 10 minutes and equilibrated with 6 mM NaOH for 10 minutes. Samples were subjected to an isocratic elution with 6 mM NaOH at a flow rate of 0.32 mL min-1 for 20 minutes. Monosaccharides were detected using a gold pulsed amperometric detector set on waveform A, according to the manufacturer’s instructions. Peaks were identified and quantified by comparison to a standard mixture of arabinose (Ara), galactose (Gal), glucose (Glc), and xylose (Xyl) (Sigma-Aldrich).
Isolation of alcohol-insoluble solids (AIS) and degree of methylesterification (DM)
Extraction of AIS and DM analysis were performed as previously described  with minor modifications. Starch was removed by treating the AIS with the porcine Type I-A α-amylase (100 U g-1 AIS; product number A4268; Sigma-Aldrich) in a 100 mM potassium phosphate buffer pH 7., 5 mM NaCl, and 0.02% (w/v) NaN3 for 24 hours at 37°C. The suspension was centrifuged at 25,000 × g for 20 minutes, then washed with distilled water and 80% acetone.
Extraction of chelating agent-soluble solids (ChASS) and immunodot assay
ChASS fractions were extracted from 10 mg of AIS for 4 hours at 70°C in a buffer containing 50 mM Tris-HCl, 50 mM ammonium oxalate, and 50 mM trans-1,2-cyclohexanediaminetetraacetic acid (CDTA) (pH 7.2). Pectin was precipitated by adding absolute ethanol to a final concentration of 30% to the supernatant and incubated at 4°C overnight. The samples were centrifuged at 25,000 × g for 20 minutes and suspended in water. The concentration of sugars in the ChASS fractions was spectrophotometrically determined with the phenol-sulfuric acid assay using D-GalUA as standard . Extraction of ChASS was performed from two fully expanded leaves per plant. For each genotype three independent replicates were analyzed. For each experiment, ChASS fractions were applied as 1 μL aliquots to nitrocellulose membrane (0.45 μm pore size; Bio-Rad, Hercules, CA, USA) in a threefold dilution series (10, 3, and 1 μg/μL). Polygalacturonic acid (PGA; P-3889; lot number 68 F08231; Sigma-Aldrich) was used as standard and also applied as 1 μL aliquots in a dilution series (3, 1, and 0.3 μg/μL). Arrays were incubated for 1 hour in 5% (w/v) milk protein (MP; Bio-Rad) in PBS pH 7.8 (MP-PBS), and probed for 1.5 hours with primary PAM1ScFv (PAM1) monoclonal antibodies (purchased from PlantProbes, Paul Knox Cell Wall Lab, University of Leeds, Leeds, UK)  diluted 1:50 in 3% MP-PBS. After extensive washes in PBS, arrays were incubated with anti-His conjugated to horseradish peroxidase (A7058; Sigma-Aldrich) diluted 1:500 in MP-PBS buffer. After washing in PBS, arrays were developed using 4-chloro-1-naphthol as previously described . A representative antibody array is shown in Additional file 6: Figure S4. Densitometric analyses of the dot signals were obtained using ImageJ software (http://rsbweb.nih.gov/ij/). The PAM1 signal per μg of samples was normalized with respect to PAM1 signal per μg of PGA. For each experiment, the PAM1 signals relative to the nine individual spots relative to the single genotype were averaged and expressed as the level of PAM1 epitope. Statistical analyses were performed using the ANOVA procedure using the software package STATISTICA.
Monosaccharide composition of ChASS
ChASS was extracted as described above from AIS saponified with 80 μL of 0.25 M NaOH and neutralized with HCl after 1 hour of incubation at room temperature. ChASS was then hydrolyzed with 2 M trifluoroacetic acid (TFA) for 90 minutes at 120°C and dried under N2 gas stream. The monosaccharide composition of the pectin-enriched fraction was determined by HPAEC-PAD as described above. Peaks were identified and quantified by comparison to a standard mixture of rhamnose (Rha), Ara, fucose (Fuc), Gal, Glc, mannose (Man), Xyl, GalUA, and glucuronic acid (GlcUA) (Sigma-Aldrich).
Analysis of variance
Chelating agent-soluble solids
Coefficient of variation
Cell wall-degrading enzyme
Degree of methylesterification
Filter paper unit
High-performance anion-exchange chromatography with pulsed amperometric detection
National Institute for Agricultural Research
National Renewable Energy Laboratory
pectin methylesterase 3
Pectin methylesterase inhibitor
Photosynthetic photon flux density
This work was supported by the European Research Council (ERC) (Advanced Grant 233083), the Ministero delle Politiche Agricole e Forestali (grant BIOMASSVAL), Ministero dell’Università e della Ricerca (MIUR grant 2010T7247Z), and the Institute Pasteur Fondazione Cenci Bolognetti. The authors thank JP Knox for providing PAM1 antibodies.
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