Identification of plant cell wall mutants by means of a forward chemical genetic approach using hydrolases
- aDepartment of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824; and
- bMax Planck-Institute for Molecular Plant Physiology, Am Muehlenberg 1, Golm, Germany
-
Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved July 7, 2009 (received for review May 19, 2009)
Abstract
A previously undescribed forward chemical genetic screen using hydrolases affecting the extracellular matrix is introduced. The developed screen takes advantage of the power of chemical genetics and combines it with the known substrate specificity of glycosylhydrolases, resulting in the selection of conditional mutants that exhibit structural defects in their extracellular matrix. Identification of the responsible genetic locus in those mutants significantly extends our knowledge of genes involved in the biosynthesis, metabolism, signaling, and functionality of components of the extracellular matrix. The method is exemplified by a screen of mutagenized Arabidopsis plants subjected to growth in liquid culture in the presence of a xyloglucanase, an enzyme acting on the major cross-linking glycan found in the extracellular matrix of this plant. Using this hydrolase-based screen, dozens of plant cell wall mutants (xeg mutants) were identified, leading to the identification of 23 genetic loci that affect plant cell walls. One of the identified loci is XEG113, encoding a family 77 glycosyltransferase (GT77). Detailed analysis of the wall of this mutant indicated that its extensins, structural glyocoproteins present in walls, are underarabinosylated. Xeg-113 plants exhibit more elongated hypocotyls than WT, providing genetic evidence that plant O-glycosylation—more specifically, extensin arabinosylation—is important for cell elongation.
The direct contact of cells to the environment is mediated in most organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain more or less complex sugar moieties in the form of glycoproteins, proteoglycans, and/or polysaccharides. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signaling. An extraordinarily complex example of an extracellular matrix in terms of both structure and function is present in the walls of higher plant cells. Walls provide mechanical support, define the shape of the cell, and play vital roles in signaling, including resistance to pathogens (1). Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as division, growth, differentiation, and senescence (2). Plants meet this multitude and diversity of functions through complex and dynamic polymer networks. These networks consist mainly of (i) a cellulose/cross-linking glycan network, (ii) a coextensive heterogeneous pectic polysaccharide network, (iii) glycoproteins (to a lesser extent), and (iv) in nongrowing cells, polyphenols such as lignin.
Structural features of plant cell wall polymers and their diversity have been relatively well described (3), yet elucidation of their corresponding functions in the life cycle of a plant is in its infancy. The few insights that we have come from identifying and characterizing mutants with altered wall structures, particularly in the model species Arabidopsis thaliana using forward and reverse genetic approaches (4). Successful forward genetic screens include wall sugar compositional analysis (5–7), leading to mutants with altered pectic polysaccharides and the major cross-linking glycan in Arabidopsis, xyloglucan (XyG). A screen for mutants with an irregular xylem phenotype set the foundation for mechanistic insights into cellulose synthesis (8) and more recently, the synthesis of glucuronoarabinoxylan, the major cross-linking glycan in secondary walls (9). The completion of the Arabidopsis thaliana genome sequence led to the prediction that about 15% of the plant genes may be involved in the biosynthesis and metabolism of the plant cell wall (10, 11). With the public availability of Arabidopsis lines with T-DNA insertions in specific genes (12), reverse genetic approaches have been successful not only in studying the role of the encoded proteins in wall biosynthesis and metabolism, but also in a functional assessment of the concomitant altered wall structures and their effects on plant growth and developmental phenotypes (13–18). However, because little is known about the regulation of wall biosynthesis and metabolism or molecular mechanisms of wall signaling, the selection of candidate genes for a reverse genetic approach has reached its limits.
The toolbox of biologists has been significantly enhanced by chemical genetic approaches (19). The chemical component of such an approach takes advantage of small organic molecules that interfere with specific biological pathways, resulting in an observable phenotype in organisms (20). The genetic component encompasses the identification of mutants that, when treated with the chemical, display an altered phenotype, usually either hypersensitivity or resistance to the chemical (21). It is then inferred that the mutated gene is either the target of the chemical or a component of the affected biological pathway (22). The major advantage of such chemical genetic approaches is the possibility of modifying the chemical dosage, to address loss-of-function lethality resulting from the tunability of the phenotype. Moreover, genetic redundancy can be overcome because the chemical may target an entire gene family. Using such an approach, important discoveries have been made recently, including the identification of novel components of the auxin signaling pathway (23) and further details of cytoskeleton-cellulose synthesis interactions in plants (24).
In an attempt to identify previously uncharacterized plant cell wall mutants and genes responsible for wall structure and function, we took advantage of the power of chemical genetics by substituting small organic molecules with cell wall-degrading enzymes. This particular screen involves the germination and growth of mutagenized Arabidopsis seeds in liquid culture containing a cell wall hydrolytic enzyme. The presence of this temporal and spatially controllable “agent” alters the plant morphology specifically. Conceptually, cell wall mutants possessing altered structures attacked by the hydrolase should display a different developmental phenotype and can thus easily be selected from the culture. As a proof of concept, we focused on a screen facilitating a xyloglucanase preparation (25). This enzyme specifically cleaves the most abundant cross-linking glycan in nongraminaceous monocots and dicots, XyG (26). It is thought that XyG metabolism plays a major role during cell elongation (27). Applying the xyloglucanase-based screen led to the identification of many mutants and corresponding genes putatively influencing XyG structure and function. An example is a new cell wall mutant affected in a family 77 glycosyltransferase gene, which we will describe in greater detail.
Results
Effect of XEG on Wild-Type Arabidopsis Plants and Known XyG Mutants in Liquid Culture.
Arabidopsis wild-type (WT) seedlings (ecotype Columbia-0) were grown in liquid culture containing a recombinant fungal xyloglucanase (XEG) preparation from seed to 14-day-old seedling stage. We tested various concentrations of the XEG to monitor its effect on plant growth and development (Fig. 1). Already, low concentrations of the XEG preparation in the medium led to shorter hypocotyls, smaller leaves, and shorter roots as well as discolorations on the cotyledons. These observed effects increased in severity with increasing concentrations of XEG. Rudimentary cell wall analysis of these seedlings (monosaccharide composition of noncellulosic wall polysaccharides) showed a XEG dose-dependent decrease in xylose, galactose, and rhamnose with a concomitant increase in arabinose (see supporting information (SI) Fig. S1). Analysis of the XyG present in these plants using oligosaccharide mass profiling (OLIMP) showed a XEG dose-dependent effect on the composition of the XyG structure (see Fig. S2). To evaluate how deep XEG is able to penetrate into the plant tissue during liquid culture incubation, we labeled sections of stems from these plants with an antibody that recognizes XyG (CCRC-M1) (28) and analyzed them by fluorescence microscopy (see Fig. S3). Plants grown in the presence of XEG showed an overall decrease of fluorescence in the epidermal and cortical cells, whereas the labeling of the vascular cells remained at a WT level. This suggests that the active hydrolase is indeed able to penetrate deep into the plant tissue during the 14-day period.
Arabidopsis thaliana wild-type (Col-0) plants grown in liquid culture with increasing concentrations of XEG. A. thaliana (ecotype Columbia-0) seeds were grown for 14 days in liquid culture containing increasing concentrations of xyloglucanase (XEG). (Scale bar: 1 cm.) Units XEG/mL medium (1 U releases 1 μmol XyG oligosaccharides from polymeric tamarind XyG per min); zero indicates absence of enzyme in the media.
In summary, it is evident that growth of Arabidopsis seedlings in XEG has a dose-dependent effect on seedling growth, morphology, cell wall, and XyG composition. Hence, XEG, as a representative glycosylhydrolase, displays the properties of a chemical agent as defined in chemical genetics (21). Conceptually, these conditions, growth of seedlings in liquid culture containing XEG, could represent a unique approach for a forward genetic screen to identify new XyG mutants altered not only in their structure but also in their functionality. Mutants would be identified by changes in their visual seedling phenotypes when specifically grown in the presence of XEG (21). To ascertain the feasibility of such an approach, known and well-studied Arabidopsis XyG mutants such as mur1 (6), mur2, and mur3 (7) were subjected to growth in XEG and their morphological phenotype compared with that of WT seedlings grown under the same conditions. Indeed, all 3 mutants displayed distinct altered morphological phenotypes (Fig. 2 and Fig. S4). The mur1 mutant exhibited chlorotic cotyledons and smaller leaves (Fig. 2B), whereas mur2 and mur3 showed a brown discoloration of the cotyledons (Fig. S4 B and C), when compared with WT (Fig. 2A). Hence, all 3 XyG mutants could have been easily selected as XyG mutants using this chemical genetic approach facilitating the glycosylhydrolase.
A. thaliana wild-type (WT) and mutant seedlings after growth in liquid culture containing XEG. (A) Wild-type (Col-0), (B) mur1, and (C and D) examples for Arabidopsis mutants identified in the XEG liquid culture screen. (Scale bar: 3 mm.)
XEG-Based Liquid Culture Screen of EMS and T-DNA Insertion Mutants.
Based on these findings, a 2-staged XEG liquid culture mutant screen was developed to identify novel conditional XyG mutants in a population of randomly chemical and T-DNA activation-tagged mutagenized Arabidopsis thaliana seed populations (Fig. 3). In the first phase of the screen, pools of mutagenized seeds (≈300 per flask) were grown in liquid culture containing XEG. After 14 days, the seedlings were visually inspected and plants exhibiting morphological differences compared with the WT seedlings were transferred from the liquid medium to solid MS agar plates followed by transfer to soil for the plants to recover and produce seeds. In total, ≈25,000 T-DNA activation-tagged and 16,000 EMS-mutagenized Arabidopsis seeds were screened. During the first phase, ≈1,700 T-DNA and 460 EMS mutants were selected as displaying distinct changes in plant morphology. Among the observed alterations were mutant seedlings that were not obviously affected by XEG displaying larger leaves (Fig. 2C), plants where XEG had even more detrimental effects on overall seedling development (Fig. 2D), plants with necrotic lesions on stems and leaves (Fig. S4 F and I), black spots on leaves (Fig. S4I), brown discoloration of cotyledons (Fig. S4F), chlorotic cotyledons (Fig. 2D), elongated hypocotyls and/or leaves (Fig. S4 G and H), and crippled roots and leaves (Fig. S4 E and F). Of the selected T-DNA mutants, only 775 plants (46%), and of the EMS mutants, only 260 plants (56%) fully recovered and produced seeds. These seeds were subjected to the second stage of the screen, in which the seeds of individual mutants were split. One portion was subjected again to the XEG liquid culture conditions to confirm the phenotype results of the first stage. If the morphological alteration was not reproducible, the mutant was dismissed. Another portion of the mutant seeds was grown in liquid culture without the enzyme so that we could identify mutant phenotypes specifically arising from the presence of XEG, and thus exclude general developmental mutants. To date, of 137 tested T-DNA mutants, 50 passed the second-stage selection criteria (36%); of 64 tested EMS mutants, 23 (40%) passed. These remaining mutants were termed xeg mutants based on the enzyme used in the screen.
Principle of the 2-staged XEG-based liquid culture forward genetics screen. In the first stage of the screen, mutagenized Arabidopsis seeds were subjected to XEG in liquid culture. After 14 days, the morphology of the seedlings was assessed. Seedlings exhibiting a phenotype different from WT were selected and placed on soil for seed production. The resulting seed population was split and subjected to growth in liquid medium either with or without XEG to select only mutants with an XEG-dependent phenotype.
We subjected the selected T-DNA insertion mutants to TAIL-PCR to identify the location of the T-DNA insertion in the genome. To date, the location of T-DNAs in 23 xeg mutants in or close to coding regions of the Arabidopsis genome has been identified. In addition to 6 genes encoding proteins of unknown functions, genes encoding proteins involved in the circadian cycle, sugar conversion pathways, proteins with kinase activities, transferases, hydrolases, and DNA-binding proteins were tagged (Table S1).
xeg113: Identification and Characterization of a New Cell Wall Mutant.
One of the T-DNA mutants identified by the XEG liquid culture screen was xeg113, hereafter termed xeg113–1. When subjected to growth in the presence of XEG, xeg113–1 displays an elongated hypocotyl and longer petioles of the rosette leaves (Fig. S4G) compared with WT grown under the same conditions (Fig. S4A). However, when the mutant is grown in liquid culture without the enzyme, no alteration in plant morphology compared with WT can be observed (Fig. S5D). The mutant thus passed our 2-stage screen criteria. TAIL-PCR of isolated genomic DNA of xeg113–1 revealed a T-DNA insertion 228 bp upstream of the start codon of At2g35610 (Fig. S5A). This locus encodes a protein of unknown function with a length of 644 aa and a calculated molecular weight of 73.2 kDa; it is classified as a family 77 glycosyltransferase (GT77) protein (www.cazy.org/). The expressed protein is predicted to contain a single transmembrane domain within the first 50 aa in the N terminus (ARAMEMNON database) (29) and conserved domain known as reticulon (RTN; PF02453 and IPR003388), suggesting a subcellular localization in the secretory pathway, likely in the Golgi apparatus, where the protein was localized in a proteomic study by Dunkley et al. (30).
To confirm the link between the discovered genetic disturbance and the observed XEG-dependent phenotype, additional T-DNA lines with insertions in the At2g35610 locus were acquired. Indeed, both lines, SALK_066991 (xeg113–2) and SALK_058092 (xeg113–3), displayed an elongated hypocotyl and long petioles, again only when grown in the presence of XEG (Fig. 4A). Also, genetic complementation was achieved by cloning the genomic At2g35610 locus, including a 2-kbp promoter sequence upstream of the start codon, into a binary vector and transformation into xeg113–2. This line was termed 113comp. When 113comp was grown in liquid culture with or without XEG, only a WT-like phenotype in both culture conditions was observed (Fig. 4A and Fig. S5D). Taken together, these findings confirm that a loss of transcript of this GT77 gene is responsible for the observed phenotype.
Phenotypes of A. thaliana WT, xeg113–2, xeg113–3, and 113comp. (A) Phenotype after growth in liquid culture with XEG. (Scale bar: 3 mm.) (B) Seven-day-old etiolated seedlings. (Scale bar: 5 mm.) (C) Length of 7-day-old etiolated seedlings, n = 10. (D) Time course of hypocotyl growth of etiolated seedlings, n = 10.
Interestingly, etiolated seedlings of both xeg113–2 and xeg113–3 grown on agar plates exhibited higher growth rates leading to longer hypocotyls when compared with WT (Fig. 4 B–D). The rescued line, 113comp, did not display any significant difference in hypocotyl length (Fig. 4 B–D). Moreover, when grown on soil in light, both knockout mutants had a larger rosette leaf diameter and displayed earlier inflorescence bolting compared with WT and 113comp (Fig. S6). No other change in plant morphology was observed.
The overall wall structure of xeg113 was characterized. Analyses of the monosaccharide composition of the noncellulosic polymers of etiolated seedlings revealed an up to 18% decrease in arabinose and a concomitant increase in mannose and glucose in xeg113–2 and xeg113–3, when compared with WT and xeg113comp (Table 1), suggesting a change in the composition of an arabinosylated matrix component, such as pectic polysaccharides, arabinogalactan proteins (AGP), or the glycoprotein extensin. For a more detailed analysis, we sequentially extracted whole etiolated seedlings from WT, xeg113–2, and xeg113–3 using specific enzymes and salt solutions; the resulting extracts were subsequently analyzed for their monosaccharide composition (Table 1 and Table S2). No consistent significant changes in the monosaccharide composition between the 2 xeg113 knockout lines and WT were observed in the AGP/soluble components, the pectic fraction, and the EGII (cross-linking glycans and noncristalline cellulose) (Table S2). The remaining residue was treated with Ba(OH)2 at high temperature to release cross-linked glycosylated extensins from the wall. In this extensin-enriched fraction, a decrease of up to 33% in arabinose was observed (Table 1), suggesting an underarabinosylation of extensins in xeg113. The remaining material was hydrolyzed under strong acidic conditions and high temperature to solubilize all remaining cell wall components, including crystalline cellulose. No significant changes in the monosaccharide composition of this residue were observed (Table S2).
Monosaccharide composition and hydroxyproline content of total AIR (μg mg−1 AIR) and Ba(OH)2 extract after sequential cell wall extraction (μg mg−1 dry weight); for analyses of other extracts see Table S2
We determined the abundance of hydroxyproline (Hyp), a prominent component of the glycoprotein extensin (Table 1 and Table S2). The highest amount of Hyp was detected in the Ba(OH)2 fraction (Table 1), confiming that the largest amount of extensin is released with this treatment. No significant changes in the Hyp content released from the cell wall were observed. For the Ba(OH)2 fraction, the molar ratios between Hyp and arabinose in WT and both mutants were calculated. For the WT, this ratio was calculated to be 1:3.2 (Hyp:Ara), for 113comp, 1:2.97. For the knockout lines this ratio was lower (xeg113–2, 1:2.3; xeg113–3, 1:2.2). Taken together, these data show that xeg113 exhibits a significant underarabinosylation of the extensins in the wall.
The Ba(OH)2 fraction was also subjected to glycosidic linkage analysis (Table 2). Here, both mutants showed a decrease of up to 38% in 2-linked arabinofuranose (2-Araf) and up to 80% in 3-linked arabinofuranose (3-Araf), 2 of the major constituents of extensins. Terminal-linked arabinofuranose (t-Araf) and terminal-linked galactopyranose (t-Galp) remained at WT levels. All other detected glycosyl residues showed no significant changes when compared with WT (Table 2), providing strong evidence for an altered extensin arabinosylation in xeg113 (see model Fig. S7).
Linkage analysis of Ba(OH)2 extracted extensins (percent of total peak area)
Discussion
A forward chemical genetic approach facilitating glycosylhydrolases was developed to identify previously uncharacterized conditional plant cell wall mutants and thus genes responsible for wall structure and function. When growing an Arabidopsis plant in liquid culture medium containing an XEG (25) (Fig. 1), for example, the plant's growth and morphology changes. XEG hydrolyzes XyG, the dominant cross-linking glycan in nongraminaceous monocots and dicots, which can make up to 20% of the cell wall dry weight (26). The wall hydrolase acts in the apoplast on the extracellular matrix and appears to be freely diffusible throughout the epidermis and cortex tissue, but does not penetrate the central cylinder (Fig. S3). In addition to a reduction of XyG represented by xylose, galactose, and fucose, other monosaccharides derived from nonxyloglucan polysaccharides were reduced in the XEG-treated seedlings. One example is rhamnose, probably representing a concomitant reduction in the abundance of pectic rhamnogalacturanan I (RGI). There are several possible reasons for an RGI reduction, including an altered cell-type composition of the XEG-treated seedlings. In addition, the removal of XyG through the XEG or the other minor glycosidase activities present in the XEG preparation (25) could have facilitated the solubilization of other wall components, such as RG-I. Moreover, the plants try to compensate for the wall “injury” with an altered pattern of biosynthesis, rendering the reduction in RGI not a direct effect of the enzyme preparation.
We then took advantage of this hydrolase-based mutant screen to analyze an Arabidopsis mutant population for plants that show resistance or hypersensitivity to the enzyme preparation. Mutants were selected based on phenotypic alterations only in the presence of the wall hydrolase in the medium to eliminate general plant developmental mutants, and can thus be considered conditional mutants. Examples of the observed phenotypes of the xeg mutants are described (Fig. 2); they display phenotypes extending well beyond hypersensitivity/resistance.
There are many possible explanations for an observed mutant phenotype when exposing the plant seedlings to a wall hydrolase. Explanations include enzyme accessibility to wall structures or up- or downregulated wall hydrolase inhibitor proteins (31). Hydrolyzing a particular wall polysaccharide can lead to mechanical stress. XyG, for example, is known to form a network with cellulose microfibrils (4), and compromising the stability of this network by XEG in a mutant would lead to the identification of other wall components that compensated for XyG in such a stress situation. In addition, a change in polysaccharide abundance or structure (in this case XyG) due to changes in biosynthesis or metabolism could render the enzyme (XEG) more or less effective. Also, oligosaccharides are generated as a result of hydrolase action. XyG oligosaccharides have been shown to act as wall-remodeling molecules (32) or even signal molecules (33). Hence, a mutation in the oligosaccharide perception and signaling pathways could lead to a change in observed mutant phenotype. Also, treatment of seedlings with the XEG preparation resulted in a change in the abundance and possibly structure of other polysaccharides, such as RGI. Hence, the hydrolase-based liquid culture screen might not only identify gene products directly involved in the metabolism of the polysaccharide being digested, it could also point to gene products that affect the functionality of this wall polysaccharide or other wall components.
The screening of an Arabidopsis T-DNA insertional line population made possible the rapid identification of the affected genes (Table S1). Although some of these genes can be conceptually linked to cell wall biosynthesis and/or metabolism (such as enzymes of sugar conversion pathways, transferases, and hydrolases), others are not so obviously involved in cell wall metabolism (such as genes encoding protein kinases or transcription factors). For example, 2 of the identified genes are involved in regulation of the circadian cycle, which has previously been shown to control and regulate cell wall biosynthesis (34). However, it will be necessary to confirm that these particular genes are responsible for the observed XEG-dependent phenotype, by analyzing additional independent insertional alleles, and/or genetically complement these lines. The precise roles of many of these genes on cell wall structure and function, in particular on XyG, will be part of future studies.
We identified one mutant, xeg113 (Figs. 2K and 4). The genetic locus was identified as XEG113 (At2g35610), classified as a family 77 glycosyltransferase (35). Based on the wall analytical data obtained from xeg113, the glycoprotein extensin found in its wall is underarabinosylated. Based on the presented biochemical evidence and the fact that other members of the GT77 family have been proposed to be involved in the glycosylation of extensins (35), we suggest that XEG113 could indeed encode an extensin arabinosyltransferase (see SI Discussion).
Xeg113, a mutant with altered extensin arabinosylation, was identified in a screen using XEG, which hydrolyses exclusively the cross-linking glycan XyG, but not extensins. Thus, we need to learn how XyG and extensins are related. Extensins are believed to be secreted as glycosylated monomers into the apoplast, where they become insoluble by creating a highly cross-linked network through isodityrosine linkages (36, 37). Arabinosylation of the peptide backbone seems to be crucial for the structural role of extensins in the cell wall (38). The extensin network has been proposed to be involved in the control and termination of cell elongation and to be one of the load-bearing polymers of the cell wall (39). Xeg113 provides the first genetic evidence of that role of extensin in the process of cell elongation and the importance of extensin arabinosylation in normal plant growth and development (Fig. S6), as xeg113 etiolated hypocotyls are more elongated compared with WT (Fig. 4 B–D). When xeg113 is grown in liquid culture they also exhibit an elongated hypocotyl, when compared with WT plants, but only in the presence of XEG (Fig. 4A and Fig. S5D). In the presence of XEG, the XyG network is clearly compromised, but an additional compromised extensin network (brought about by underarabinosylation in xeg113) apparently leads to a further extended tissue. Thus, we hypothesize that both networks—extensin and XyG—may have not only similar, but redundant, functions in the control of cell elongation.
In conclusion, a previously uncharacterized hydrolase-based liquid culture screen was developed that resulted in the identification of plant cell wall mutants. The methodology presented here complements other genetic approaches and, as exemplified with xeg113, has an enormous potential to identify other essential components of the wall polymer biosynthetic, metabolic, and signaling machinery, including functional assessment of wall polymers. Based on the findings presented, this kind of mutant screen should not be restricted to Arabidopsis or plant systems. For identification of wall mutants of bacterial, fungal, or animal cells, other extracellular matrix hydrolases or effectors could easily be used instead.
Materials and Methods
Plant Material.
The EMS-mutagenized M2 A. thaliana seed population used in the primary screen was provided by T. Altmann, Max Planck-Institute, Golm, Germany (40). An activation-tagged Arabidopsis mutant population collection transformed with pSKI015 was provided by W. Scheible (Max Planck-Institute). SALK_066991 and SALK_058092 were obtained from the ABRC stock center at Ohio State University (12). The mutant populations as well as the T-DNA insertion lines from the stock center were in the ecotype Columbia (Col-0) background.
Arabidopsis thaliana Grown in Liquid Culture.
A. thaliana seeds were surface sterilized by washing in 70% ethanol, followed by treatment with 3% sodium hypochloride + 0.1% Triton X-100 and 4 rinses with sterile water. Approximately 50–100 seeds were added to 20 mL MS (41) liquid medium containing 0.5% sucrose and grown under sterile conditions (16 h light, 8 h dark) at 22 °C, shaking at 80 rpm. For the mutant screen, 140 U/mL sterile-filtered recombinant monocomponent xyloglucanase preparation (XEG; EC 3.2.1.151; Novozymes [25]; gift from Kirk Schnorr; 1 U releases 1 μmol of XyG oligosaccharides per min), not further purified, was added to the cultures. Seedlings were grown for 14 days before visual analysis.
Thermal Asymmetric Interlaced (TAIL) PCR for Mapping T-DNA Insertion Flanking Sequences.
The location of the T-DNA insertion in xeg mutants from the activation-tagged mutant populations was determined by thermal asymmetric interlaced (TAIL) PCR. We adapted and modified the method previously described by Liu et al. (42) (see SI Materials and Methods).
Preparation and Analysis of Cell Wall Material.
Sterilized A. thaliana seeds were grown for 7 days in the dark on MS medium containing 1% agar. Etiolated seedlings were harvested, pooled, and frozen in liquid nitrogen. The alcohol-insoluble residue (AIR) of the etiolated seedlings was prepared by the adapted method previously described by Fry (43) and Lerouxel et al. (13). The tissue was ground to a fine powder using a beat mill (Retsch). The powder was extracted with 1 mL 70% aqueous ethanol. The supernatant was removed by centrifugation for 10 min at 14,000 rpm. A 1:1 (v:v) chloroform/methanol mixture (1 mL) was added. The supernatant was again removed by centrifugation and the resulting residue air-dried.
Freeze-dried WT and mutant plant material was sequentially extracted using specific polysaccharide-degrading enzymes and salt solutions. In brief, 7-day-old etiolated whole seedlings were freeze-dried, ground to a fine powder, and aliquoted into 3–5 mg dry weight. After the extraction of water-soluble components, pectic polysaccharides, cross-linking glycans, and partially cellulose (for details, see SI Materials and Methods), the remaining pellet was resolubilized in 1 mL of saturated barium hydroxide solution (≈0.22 M) and incubated for 6 h at 105 °C (44). The supernatant containing solubilized extensins was separated by centrifugation, neutralized with 1 M sulfuric acid, and the resulting precipitated salts removed by centrifugation.
The monosaccharide composition of 1 mg total cell wall material and sequentially extracted cell wall components was determined by hydrolyzing cell wall material in trifluoroacetic acid (TFA) followed by alditol acetate derivatization and GC-MS analysis as described (45). Glycosidic linkage analysis on the Ba(OH)2 fraction was performed by GC-MS analysis of partially methylated alditol acetates as described by Ciucanu and Kerek (46) with modifications by Ciucanu (47).
The hydroxyproline (Hyp) content of sequentially extracted cell wall components was determined by the colorimetric assay described by Kivirikko and Liesmaa (48) after the extracts were hydrolyzed in 6N hydrochloric acid for 18 h at 110 °C.
Acknowledgments
We thank Kirk Schnorr (Novozymes) for the generous gift of the XEG enzyme; Michael Hahn (Complex Carbohydrate Center, University of Georgia) for providing the CCRC-M1 antibody, Marcia Kieliszewski for protocol discussions of extensin extractions, and Karen Bird for editing. This work was funded by the Max-Planck Society and by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (Award No. DE-FG02–91ER20021).
Footnotes
- 3To whom correspondence should be addressed. E-mail: paulymar{at}msu.edu
-
Author contributions: S.G. and M.P. designed research; S.G., U.H., and M.Z. performed research; S.G., U.H., M.Z., and M.P. analyzed data; and S.G. and M.P. wrote the paper.
-
The authors declare no conflict of interest.
-
This article is a PNAS Direct Submission.
-
This article contains supporting information online at www.pnas.org/cgi/content/full/0905434106/DCSupplemental.
-
Freely available online through the PNAS open access option.
