Aspen pectate lyase Ptxt PL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield
© Biswal et al.; licensee BioMed Central Ltd. 2014
Received: 19 September 2013
Accepted: 7 January 2014
Published: 22 January 2014
Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals.
We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment.
Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass.
KeywordsPopulus Wood development Secondary cell wall Lignocellulose Biofuel Pectin
There is high interest in cultivating Populus species as energy crops because they grow rapidly, producing abundant lignocellulosic biomass that can be used as feedstock for biofuel and biomaterial production . However, a major challenge hindering large-scale commercial use of Populus lignocellulose is its recalcitrance to degradation by bacterial and fungal enzymes, which complicates the separation of non-crystalline carbohydrate polymers and cellulose contents from lignin, and their subsequent saccharification. The constitutive lignocellulose polymers are non-randomly arranged within cell-wall layers that each have a distinct composition and architecture . The outermost thin layer of middle lamella and primary cell wall, frequently referred to as the compound middle lamella, is rich in pectin and xyloglucan (XG) and is heavily lignified, whereas the inner thicker secondary-wall layers are enriched in cellulose and in hemicelluloses such as xylans and mannans, but are thought to contain relatively less lignin. Secondary-wall layers comprise the bulk of wood biomass and are traditionally believed to govern wood properties. However, hydrolysis of XG, a primary wall-layer hemicellulose in Populus, has been unexpectedly shown to affect solid wood traits  and accelerate the lignocellulose saccharification . Moreover, a recent study has shown that alleles of the Eucalyptus genes PME6 and PME7, encoding pectin methyl esterases, are associated with solid-wood quality properties, suggesting that pectin structure significantly influences wood properties .
Pectins in wood cell walls are composed mainly of homogalacturonan (HG) and rhamnogalacturonan I (RGI), which are concentrated in a thin outermost coat of the compound middle lamella, and have not been reported in xylan-containing secondary-wall layers . The backbones of different pectin polymers are thought to be covalently connected, forming a supramolecular network linked to other cell-wall polymers via unknown bonds . A recent study has identified a low abundance proteoglycan covalently linking pectins, arabinogalactan (AG) and arabinoxylan . The final structure of the pectin network with its associated polymers is determined by activities of wall-residing trans-glycosylases, esterases, hydrolases and pectate lyases (PELs) .
Plant PELs (EC 184.108.40.206), which belong to polysaccharide lyase family 1 (PL1) (http://www.cazy.org), cleave the α-1,4 glycosidic bond between the galacturonic acid units of HG by β-elimination and release unsaturated oligogalacturonides. Genome-wide expression analyses in Arabidopsis have suggested that this gene family is involved in growth, cell adhesion and primary cell wall decomposition [9, 10]. A PEL, encoded by ZePel, also appears to be involved in the differentiation of tracheary elements in Zinnia elegans. In Populus, PEL genes have been assigned the CAZy family name PL1 and consecutive numbers PL1-1 to PL1-28. Many of these genes are highly expressed during xylem cell expansion . PtxtPL1-27 has been found to be highly upregulated in transgenic hybrid aspen (Populus tremula L. x tremuloides Michx.; Ptxt) with enhanced secondary growth caused by overexpression of GA20 oxidase . Here we present a biochemical characterization of the recombinant Ptxt PL1-27 protein and an analysis of the effects of its overexpression in hybrid aspen. The results indicate that Ptxt PL1-27 can substantially modify the extractability of polymers from xylem cell walls, enhancing saccharification. Although pectin degradation has been shown previously to enhance saccharification of stems in herbaceous plants , it was surprising to find that it also affects saccharification in the case of aspen wood. Our analyses showed however that these effects in aspen are qualitatively and quantitatively different than those reported for herbaceous plants.
Results and discussion
Lignin, hydrolyzable sugar anhydrate content and crystalline cellulose content of aspen wood
16.8 ± 0.2 1 *
17.7 ± 0.3**
17.1 ± 0.3
Acid soluble lignin
2.1 ± 0.0
2.1 ± 0.0
2.0 ± 0.1
364 ± 28
346 ± 62
363 ± 28
11.7 ± 1.2
9.2 ± 0.6
9.0 ± 1.2
5.6 ± 0.1
6.8 ± 0.4
6.1 ± 0.5
312.4 ± 5.7
304.1 ± 6.4
317.8 ± 2.1
20.5 ± 0.4**
18.8 ± 0.6
16.1 ± 0.9**
11.5 ± 0.9
13.3 ± 0.5
13.1 ± 1.0
178.8 ± 0.4*
187.2 ± 2.3
191.9 ± 4.8
540.4 ± 7.4
539.4 ± 5.3
554.1 ± 6.8
31.8 ± 2.4
30.4 ± 2.0
27.9 ± 1.5
In conclusion, the above cell-wall analyses revealed that PEL overexpression induced no major changes in the composition of cell walls in the wood, but substantially altered extractabilities of pectins, AGs and xylans in the wood. These results concerning the cells with secondary cell-walls are reminiscent of the previously published data on primary-walled tissues. In strawberry fruits, the downregulation of PEL expression was found to reduce the solubility of UAs and neutral sugars . Thus, in both primary and secondary walls, the integrity of an enzyme-accessible HG domain appears to influence the mobilization of major cell-wall matrix components (HG, RGI, xylan and AG). Several previous studies have suggested that pectins and hemicelluloses are cross-linked in primary walls [28–34]. A covalent linkage between RGI, HG, and arabinoxylan involving an AG protein has been identified recently in the Arabidopsis cell suspension culture medium . It is possible that similar covalent linkages bind HG to AG and glucuronoxylan in the wood cell walls of aspen, and therefore the fragmentation of HG by PEL increases solubility of xylan and AG (Figure 7). Another possibility is that PEL activity alters pectin structure in a way that influences lignin polymerization, either by affecting lignin nucleation sites or activities of laccases or peroxidases [35, 36]. Slightly increased Klason lignin contents were seen in the transgenic lines (Table 1) and might have affected polysaccharide extractability. Yet another possibility is that PEL increases cell wall porosity, thus facilitating access of native cell-wall endohydrolases to their substrates , thereby making these polymers more soluble.
Total sugar yields for transgenic aspen lines and wild-type after acidic pretreatment
Total sugar yields (pretreatment liquid and enzymatic hydrolysate)
0.390 ± 0.01 (100%)**
0.126 ± 0.01 (100%)***
0.517 ± 0.01 (100%)***
0.421 ± 0.01 (108%)**
0.144 ± 0.01 (114%)**
0.565 ± 0.01 (109%)**
0.418 ± 0.03 (107%)
0.157 ± 0.01 (124%)**
0.574 ± 0.01 (111%)**
Cellulose crystallite structure in transgenic lines and wild-type determined by x-ray diffraction
Crystallite width Å
28.7 ± 1.5
29.9 ± 0.1
29.4 ± 1.8
30.0 ± 0.2
30.7 ± 1.7
30.2 ± 0.1
In overall conclusion, we have shown that Ptxt PL1-27 from clade Ib of the PL1 family is a PEL, which is expressed in differentiating xylem at the onset of secondary wall formation. When overexpressed, it is capable of loosening several matrix components from secondary-walled xylem cells without significantly affecting their overall cell wall composition. PEL activity is beneficial for lignocellulose saccharification, increasing yields of monosaccharides, especially xylose, with and without pretreatment. Thus, pectin structure may affect wood saccharification even though it is a minor wood component. These findings could be exploited in several ways. First, our results could be used to design more efficient saccharification cocktails for deconstruction of woody biomass. Second, our results suggest that targeting pectin in the genetic improvement programs for woody feedstocks might prove beneficial for subsequent processing and deconstruction of woody biomass. Third, genetic engineering targeted toward pectin-modification in woody plants might improve their suitability as feedstock for biofuel production. They also show that the genetic engineering strategy would need to be targeted to mature woody tissues in order to avoid negative effects on plant growth and development that have been observed in this and other studies [15, 42].
Materials and methods
Cloning of PtxtPL1-27
Full-length cDNA was cloned from the hybrid aspen cDNA from elongating shoots by 5′ RACE using Marathon (Clontech Laboratories Inc., Mountain View, CA, USA), and the gene-specific primer Race1 (Tab. S3) based on AI164054. The complete cDNA was reconstructed by PCR using Advantage PCR Cloning Kit (Clontech) with F1 and R1 primers that included BamHI restriction sites (Additional file 7) and hybrid aspen cDNA. The PCR product was sub-cloned, sequenced and named PtxtPL1-27 [GenBank: EU379971.1].
Heterologous expression and enzyme assays
Mature Ptxt PL1-27 peptide (Δ1-26Ptxt PL1-27) was expressed in E. coli (ER2566) using the pTYB11 vector (IMPACT™-CN System, New England Biolabs Inc., Ipswich, MA, USA) carrying the PCR product obtained with the primers MF1 and MR1 (Additional file 7). Cells containing the recombinant plasmid were grown in LB medium with 100 μg/ml carbenicillin to an OD of A600 = 0.5. Expression of the recombinant gene was then induced by incubating the cells with 0.3 mM isopropyl β-D-thiogalactoside (IPTG) at 14°C for 15 h. The cells were resuspended in 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 500 mM NaCl and 1 mM EDTA, pH 8.0, lysed by sonication on ice, and centrifuged at 20,000 g for 30 minutes at 4°C. The pellet was dissolved in the same buffer and purified using an IMPACT™-CN chitin beads column (New England Biolabs). PEL activity (EC 220.127.116.11) was assayed in the column-purified protein extract according to Collmer et al. using 0.24% w/v of polygalacturonic acid (Sigma-P3889) in 60 mM Tris–HCl, pH 8.5 (unless otherwise indicated), 0.5 mM CaCl2, and 0.3 ml of protein extract in 1.5 ml reaction mixtures. The unsaturated uronide products were measured by absorbance at 235 nm over 180 minutes at 30°C using the molar extinction coefficient 4.6 × 103 M-1 cm-1. Specific activity was expressed per mg of protein as determined by a Bio-Rad Assay kit (Life Science, Sunbyberg, Sweden) using BSA as a protein standard.
Generation of transgenic aspen lines
The full-length PtxtPL1-27 cDNA clone lacking 5′ and 3′ untranslated regions (UTRs) was released by digestion with XbaI and SalI and ligated in sense orientation into the plant binary vector pMH1.kana (a gift from Mattias Holmlund, SLU, Umeå, Sweden) under control of the CaMV 35S promoter. The construct was transformed into hybrid aspen clone T89 by Agrobacterium-mediated transformation, and plants were regenerated, planted in the greenhouse and grown for approximately three months, as previously described . Briefly, the growing conditions were: 18-h photoperiod, a temperature regime of 22°C/17°C (day/night), and a relative humidity of 70%.
Analysis of RNA in the transgenic and WT lines
RNA was isolated from developing xylem scraped from the wood surface after peeling the bark, cambium and phloem scraped from the exposed bark surface, root tips approximately 0.5 cm long, internodes up to 50%, 50 to 80%, and 100% of their final length, designated internodes 1, 2 and 3, leaves subtending these internodes (leaf 3 was fully expanded), the apical bud and petioles from internodes 1 and 2, pooled. In addition, we extracted RNA from a series of samples of tangentially sectioned developing wood. Total RNA was extracted by hexadecyl-trimethylammonium bromide (CTAB) and purified as previously described .
Protein extraction and PEL activity in aspen
Proteins were extracted from scraped xylem from internodes 44 to 60: 5 g of tissue were ground in liquid N2 to a fine powder, stirred for 30 minutes at 4°C in 25 ml of buffer A (50 mM sodium phosphate, pH 7.0, 2 mM EDTA, 4% polyvinylpyrolidone (PVP) mw 360 000, 1 mM dithiothreitol (DTT), and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected as the soluble fraction and the pellet was re-suspended in 25 ml of buffer A supplemented with 1 M NaCl, stirred for 30 minutes at 4°C, centrifuged as above, and the supernatant was collected as the wall-bound fraction. Saturated ammonium sulfate (AS) was added to reach 20% and 19% of saturation, to the soluble- and wall-bound fractions, respectively, stirred for 30 minutes at 4°C, and centrifuged as above to remove PVP from solution. Solid AS was added to the supernatants, increasing the AS saturation to 90% and 80% for the soluble- and wall-bound fractions, respectively, and stirred for 30 minutes at 4°C to precipitate proteins. Following the centrifugation as above, the pellets were dissolved in 1 ml TE buffer, pH 8.0. The dissolved proteins were desalted with PD10 columns and protein concentrations were determined by the Bradford method using the Bio-Rad Assay kit (Life Science). Pectate lyase activity was assayed as described above using 0.3 ml of extracted proteins.
Wood chemical analyses
Wood of internodes 40 to 44 was freeze-dried and ground using an A11 Basic Analytical Mill (IKA, Staufen, Germany) and then using an Ultra Centrifugal Mill ZM 200 equipped with a 0.5 mm ring sieve (Retsch) at 30 Hz for 150 sec. The contents of acid-resistant lignin (Klason lignin) and acid-soluble lignin were determined according to Theander and Westerlund .
Alcohol-insoluble residue (AIR) was obtained by washing the ground wood sequentially in 70% ethanol, methanol:chloroform 1:1 (v/v) and acetone, then drying overnight under vacuum. Starch was removed by treatment with Bacillus α-amylase (Sigma - A6380) at 5,000 units per g of AIR, 16 h at 37°C. Crystalline cellulose was purified by hydrolysing non-cellulosic polysaccharides with acetic acid:nitric acid:water (8:1:2, v/v/v) and removing the supernatant. The resulting pellet was washed four times with acetone, dried, hydrolysed in 72% sulphuric acid, and the glucose content was determined with the anthrone method .
Sequential extraction of the wood cell walls followed a previously published procedure . The neutral glycosyl residue composition of non-fractionated and fractionated cell wall polysaccharides was analyzed by the alditol acetate method . Briefly, after preparing alditol acetates, 1 μL samples were injected, splitless, into a Hewlett-Packard chromatograph 5890 (Ramsey, MI, USA) equipped with an SP 2330 (30 m × 0.25 mm, 0.25 μm film thickness, 24019 Supelco) column using helium as carrier gas, and the analytes were detected by a coupled mass spectrometer. UA contents of the cell wall polysaccharides were determined according to Filisetti-Cozzi and Carpita .
Cell wall extracts were diluted to 60 μg mL - 1 and 50 μL samples were used for glycome profiling, as previously described . α-Amylase (type II-A from Bacillus) was obtained from Sigma (A6380), and EPG (I and II from Aspergillus niger)  and PME (from A. niger)  were donated by Carl Bergmann (Complex Carbohydrate Research Center, University of Georgia), and used at approximately 1.0 units/100 mg AIR in 50 mM sodium acetate, pH 5.0. Plant cell wall glycan-directed monoclonal antibodies  were obtained as hybridoma cell-culture supernatants either from laboratory stocks maintained by the Complex Carbohydrate Research Center (CCRC, JIM and MAC series; available from CarboSource Services (http://www.carbosource.net)) or from Fabienne Guillon (AX1  (INRA, Nantes)).
Freeze-dried wood from internodes 21 to 39 was milled as above, followed by sieving with an Analytical Sieve Shaker AS 200 (Retsch) to obtain 0.1- to 0.5-mm particles. Pre-pretreatment (if applied) consisted of incubation of 50 mg milled and sieved wood in 1% (w/w) sulfuric acid at 165˚C for 10 minutes under stirring in a single-mode microwave system (Initiator Exp, Biotage, Uppsala, Sweden). After acid pretreatment, the suspension was centrifuged, the liquid phase (the pretreatment liquid) was removed, and the pellet was washed. The remaining solid (pretreated wood), and the untreated wood powder (50 mg), were treated with 50 mg of a liquid enzyme mixture consisting of equal proportions of Celluclast 1.5 L (a liquid enzyme preparation from Trichoderma reesei and the main source of cellulase) and Novozyme 188 (a liquid enzyme preparation from A. niger and the main source of β-glucosidase) (both from Sigma-Aldrich, St. Louis, MO, USA) in sodium citrate buffer (50 mM, pH 5.2), using 1,000 mg as the total size of the reaction mixture, at 45°C for 72 h in an Ecotron orbital shaker (Infors, Bottmingen, Switzerland) at 170 rpm.
Concentrations of monosaccharides (Ara, Gal, Glc, Man, and Xyl) in the samples were then determined using an HPAEC system (ICS3000, Dionex, Sunnyvale, CA, USA) equipped with a Pulsed Amperometric Detection system and a CarboPac PA20 column (3 × 150 mm) with a CarboPac PA20 guard column (3 × 30 mm) (Dionex).
analysis of variance
enzyme-linked immunosorbent assay
We thank Mattias Holmlund for a gift of pMH1.kana vector and Jesper Harholt for fruitful discussions. The research was funded by FORMAS, the Swedish Research Council, UPSC Berzelii Center of Forest Biotechnology (funded by VR and Vinnova), Biomime Center (funded by the Swedish Foundation for Strategic Research, the Knut & Alice Wallenberg Foundation), Bio4Energy and SNS. The glycome profiling was supported by the BioEnergy Science Center administered by Oak Ridge National Laboratory and funded by a grant (DE-AC05-00OR22725) from the United States Department of Energy. Generation of the CCRC series of plant glycan-directed monoclonal antibodies was supported by the NSF Plant Genome Program (DBI-0421683).
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