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Ignacio Martín, Berta Dopico, Francisco J. Muñoz, Rocío Esteban, Ronald J. F. J. Oomen, Azeddine Driouich, Jean-Paul Vincken, Richard Visser, Emilia Labrador, In vivo Expression of a Cicer arietinum β-galactosidase in Potato Tubers Leads to a Reduction of the Galactan Side-chains in Cell Wall Pectin, Plant and Cell Physiology, Volume 46, Issue 10, October 2005, Pages 1613–1622, https://doi.org/10.1093/pcp/pci177
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Abstract
We report the generation of Solanum tuberosum transformants expressing Cicer arietinum βIII-Gal. βIII-Gal is a β-galactosidase able to degrade cell wall pectins during cell wall loosening that occurs prior to cell elongation. cDNA corresponding to the gene encoding this protein was identified among several chickpea β-galactosidase cDNAs, and named CanBGal-3. CanBGal-3 cDNA was expressed in potato under the control of the granule-bound starch synthase promoter. Three βIII-Gal transformants with varying levels of expression were chosen for further analysis. The transgenic plants displayed no significant altered phenotype compared to the wild type. However, β-galactanase and β-galactosidase activities were increased in the transgenic tuber cell walls and this affected the potato tuber pectins. A reduction in the galactosyl content of up to 50% compared to the wild type was observed in the most extreme transformant, indicating a reduction of 1,4-β-galactan side-chains, as revealed by analysis with LM5 specific antibodies. Our results confirm the notion that the pectin-degrading activity of chickpea βIII-Gal reported in vitro also occurs in vivo and in other plants, and confirm the involvement of βIII-Gal in the cell wall autolysis process. An increase in the homogalacturonan content of transgenic tuber cell walls was also observed by Fourier transform infrared spectroscopy (FTIR) analysis.
Introduction
Pectic polysaccharides constitute between 30 and 50% of the cell walls of dicotyledonous plants (Carpita and Gibeaut 1993). The pectic matrix of plant cell walls is a complex mixture of homogalacturonan (HGA), rhamnogalacturonan I (RGI) and rhamnogalacturonan II (RGII) polymers. HGA is a linear chain of 1,4-α-galacturonic acid (GalA) residues in which some of the carboxyl groups are methyl-esterified. Depending on the plant source, HGA may also be partially O-acetylated (Ishii 1997, Perrone et al. 2002). RGI is a branched heteropolymer of alternating 1,2-α-rhamnose and 1,4-α-GalA residues that carries neutral side-chains of arabinan, galactan or arabinogalactan attached to the rhamnose residues of the RGI backbone. In most cases, 20–80% of rhamnose residues in RGI are substituted at C-4 with side chains that may vary in size from a single glycosyl residue to 50 or more, resulting in a large and highly variable family of polysaccharides (Albersheim et al. 1996). The RGII molecule has an HGA backbone with side chains containing a diversity of sugars and linkages and is able to dimerize through a borate di-ester cross-link. Although the composition of these molecules is well characterized, their function in cell wall architecture is still unclear. They have been implicated in regulating cell-cell adhesion, cell expansion and wall porosity and have been proposed as a source of signalling molecules (oligosaccharins) in cell differentiation and organogenesis (Baron-Epel et al. 1988, Aldington and Fry 1993, McCann and Roberts 1994, Satoh 1998).
Fujino and Itoh (1998) described a clear difference in cell wall architecture between elongating and non-elongating regions, suggesting a modification in the molecular form of pectin polysaccharides during the elongation of epidermal cells. The change in cell wall porosity could be brought about by structural modifications of pectic molecules, whereas the aggregation of pectin polysaccharides by calcium bridging may not necessarily play an important role in this phenomenon. Those authors suggested that neutral sugar side-chains could be involved in the interaction with other cell wall components. β-galactosidase is one of the enzymes that seem to play a role in the process of modification of pectin structure, removing β-galactosyl linkages on the neutral sugar side-chains (Dopico et al. 1990b, Konno and Tsumuki 1993, Valero and Labrador 1993).
One approach for determining the biological significance of the different structural elements of pectins is to manipulate them in transgenic plants that over-express cell wall-degrading enzymes. Based on this strategy, in this study we report the generation of transgenic plants expressing a β-galactosidase named βIII-Gal isolated from the Cicer arietinum cell wall that displayed significant hydrolytic activity against cell wall pectins (Dopico et al. 1989). This enzyme was characterized as a protein involved in the cell wall autolytic process (Dopico et al. 1990a, Dopico et al. 1990b, Dopico et al. 1990c). The cDNA CanBGal-3, which encodes this protein, has been previously identified among several cDNAs encoding β-galactosidases (Esteban et al. 2003) and here we confirm that CanBGal-3 encodes a pectin-degrading enzyme.
The construction of transgenic plants expressing βIII-Gal also allowed us to study the in vivo function of this protein. Since leguminosae, such as C. arietinum, are very difficult to transform, we chose potato (Solanum tuberosum L.) as a model system for these studies. This plant is easy to transform and provides large quantities of transformed tissue and hence cell walls for characterization. Although similar approaches have been reported previously in potato expressing three fungal enzymes derived from Aspergillus aculeatus—an endo-galactanase (Sørensen et al. 2000), an endo-arabinanase (Skjøt et al. 2002), and a rhamnogalacturonanlyase (Oomen et al. 2002)—this is the first report in which a plant enzyme has been used to modify potato pectin.
Results
Transformation and regeneration of potato plants
A construct was made to introduce the CanBGal-3 clone encoding a β-galactosidase from C. arietinum into S. tuberosum cv. Karnico plants via Agrobacterium tumefaciens-mediated plant transformation. The granule-bound starch synthase (GBSS) promoter was used to drive its expression in potato tubers.
Fifty-two individual transgenic potato plants were generated. Plant transformation was verified by Southern blot analysis (data not shown). Forty-two plants showed a single insertion of the transgene; five plants showed a second transgene insertion, while the other five plants did not contain any insertion. The transformed plants displayed a phenotype similar to wild-type (WT) plants.
RNA isolation from the tubers of the transgenic plants was performed to carry out Northern blot analyses (Fig. 1). RNA from C. arietinum epicotyls was used as a positive control and WT tuber RNA as a negative control. In most transformants (42), transgene transcription could be detected and a number of high and low expressers were identified (Fig. 1). Three βIII-Gal transformants with different levels of expression were chosen for further analysis: L7, with the highest mRNA accumulation; L14, with moderate expression, and L27 as one of the lowest expressers. WT plants were also used for comparative analysis.
A large number of shoots from these three individual transformants were transferred to the greenhouse for tuber production. The tubers of the transformants looked slightly smaller and were more abundant than in the WT, but statistical analysis of the weight of the tubers and the number of tubers per plant indicated that the variations were independent of the level of transgene transcription (data not shown).
Galactosidase and galactanase activities in transgenic tuber cell walls correlates with their transcript levels
Cell wall material was isolated from WT and transgenic tubers and proteins were extracted with 3 M LiCl. Cell wall protein extracts were tested for glycanhydrolytic activities. The amount of purified cell wall was similar in both transformants and WT tubers. However, higher amounts of proteins extracted from transgenic tubers were detected compared with the WT (data not shown), probably due to an increase in the protein extraction capacity. When the β-galactosidase activity was evaluated against p-nitrophenyl substrate, L7, with the highest transgene expression, exhibited, as expected, the highest galactosidase activity: this was almost twice the activity of the WT (Fig. 2A), indicating that the β-galactosidase generated from CanBGal-3 acted in vivo and duplicated the β-galactosidase activity present in the WT tubers. The level of β-galactosidase activity of the transformants correlated well with the mRNA accumulation levels as determined by Northern blot analysis.
Since activity towards p-nitrophenyl galactoside does not necessarily reflect activity towards 1,4-β-galactan, we also checked the galactanase activity of the transgenic plants using lupin 1,4-β-galactan as the substrate. Fig. 2B shows that the β-galactanase activity of the transformants increased the corresponding activity in wild type tubers to a considerable extent, which confirms that the protein encoded by CanBGal-3 exhibited 1,4-β-galactanase activity and acted in vivo. Also in this case, the levels of the β-galactanase activity of the transformants correlated with the mRNA accumulation levels, the highest activity being observed in L7 transformants (6-fold higher than the WT).
SDS electrophoresis of tuber cell wall proteins was carried out to determine whether there was an increase in βIII-Gal in tuber cell walls. The cell wall protein extracts from transformants contained a protein with a molecular mass of 77.8 kDa, corresponding to the molecular mass of βIII-Gal (Fig. 3). A clear band corresponding to this molecular mass could be seen in L7. This band appeared with lower intensity in L14 and was not detected at all in L27 nor in the WT potato tubers.
Monosaccharide analysis reveals a lower galactose content in the cell walls of transformants
Monosaccharide compositional analysis of the isolated cell walls of the transgenic and WT tubers showed clear differences. Fig. 4 shows a dramatic reduction of up to 50% of the galactose content in transformant L7 compared with the WT. The sugar composition of L14 showed similar, but less pronounced, changes when compared with the WT. The percentage of galactose in L27 was similar to the WT, which shows that although we detected a very low level of expression in this βIII-Gal transformant it was not producing enough enzyme to induce changes in the cell wall. No significant modifications were observed in other cell wall neutral sugars, except for glucosyl residues, which increased in L7 and L14 (Fig. 4). Analysis of the amount of uronic acids in the cell walls revealed that cell walls from L7 had the highest content, increasing by more than 28% compared with the WT (Table 1). A lower increase in the uronic acids content was observed in L14, while the amount of uronic acids in L27 was similar to the WT.
Cell wall autolytic activity of potato WT and βIII-Gal transformants
We decided to check whether transformation with the βIII-Gal transgene would result in the acquisition of autolytic activity in potato cell walls. When tuber cell walls from βIII-Gal transformants were incubated in 10 mM Na-citrate-phosphate buffer they displayed autolytic activity, releasing galactose into the incubation medium, this sugar reaching values of 9.38 µg per mg of cell wall after 24 h of incubation (Table 2). The amount of galactose released was higher in L7 and decreased in L14 and L27, in agreement with the transgene expression level. The WT was not able to release any sugars in an autolysis-like way. This release of sugars was carried out enzymatically in the transformants, since control cell walls incubated at 4°C did not display such activity.
In order to demonstrate that the autolytic activity in potato cell walls was similar to the autolysis carried out by the chickpea cell wall, we checked whether C. arietinum cell wall protein was able to hydrolyse potato cell walls whose enzymatic system had been inactivated by heat treatment. Chickpea protein released up to 56 µg of galactose per mg of WT tuber cell wall after 24 h of incubation (Table 2). The amount of galactose released from the βIII-Gal transformants was higher in L27 and lower in L7 and L14, in which the galactose content was very reduced.
Galactan epitopes are reduced in abundance in the tuber walls of transformants
Galactosyl residues are present in different wall polysaccharides (hemicellulose, arabinogalactan proteins and RGI), but only RGI is known to contain 1,4-β-linked galactan. Accordingly our subsequent analysis of cell wall material focused on confirming the reduction of this polymer.
To determine whether, in particular 1,4-β-d-galactan was hydrolysed by the βIII-galactosidase, we used the monoclonal antibody LM5, which recognizes tetramers of 1,4-β-d-galactan. By using epi-fluorescence microscopy (Fig. 5A), it became clear that the galactan epitope was restricted to the primary wall of parenchymal cells and absent from the middle lamella and from the expanded middle lamella at the corners.
In the high βIII-Gal expressing tubers, L7, galactan epitopes were greatly reduced throughout parenchymal walls, indicating that the reduction in galactose of transformant cell walls corresponded to a reduction in 1,4-β-d-galactan side chains (Fig. 5). The perimedullary parenchymal cell walls of the WT were strongly labelled, indicating a high abundance of the epitope recognized by LM5, using both epi-fluorescence microscopy (Fig. 5B) and reflectance confocal laser scanning microscopy (Fig. 5C), whereas similar walls in transformed tubers of L7 were only weakly stained at some cell corners.
Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy is established as a rapid method for screening altered cell wall phenotypes. Fig. 6 shows the region of pectins with an absorption band between 900 and 1,200 cm–1. Comparison of average spectra from WT and βIII-Gal-expressing transformants revealed a different small peak at 1,100 cm–1 (marked with an arrow in Fig. 6), which according to the literature corresponds to polygalacturonic acid. This peak appeared clearly in the high and medium expressors L7 and L14, where it was more pronounced than in the WT or in the transformant with very low transgene expression, L27.
Discussion
Potato plants expressing a plant pectin-degrading enzyme—βIII-Galactosidase (βIII-Gal) from C. arietinum—under the control of the tuber-specific GBSS promoter have been successfully generated. As expected, the cell walls from the transgenic tubers displayed more that twice the β-galactosidase activity and more than 6 times the β-galactanase activity of WT plant tubers. These increased activities confirm the idea that the protein generated in potato from the chickpea CanBGal-3 is able to act in vivo in cell wall potato tubers. The level of increased activity in each transformant line was correlated with its transgene expression. As a consequence of these increased enzymatic activities, the cell wall material of these transgenic tubers was modified, a significant reduction in the amount of galactose being observed: up to 50% in the highest βIII-Gal expresser compared with the WT (Fig. 4). This result allows us to confirm that the pectin-degrading activity of the chickpea βIII-Gal reported in vitro against epicotyl cell walls (Esteban et al. 2003) also occurs in vivo and in other dicotyledoneous cell walls.
The use of LM5 antibodies to probe the presence of 1,4-β-galactan hairs revealed that the βIII-Gal-expressing tubers had far fewer galactan epitopes recognized by LM5 in their primary walls than WT tubers (Fig. 5). This shows that, in vivo, chickpea βIII-Gal effectively removes 1,4-β-galactan from the side chains of RGI in potato tuber cell walls (Fig. 5), since although several wall polysaccharides contain galactosyl residues, only RGI is known to contain 1,4-β-galactan side chains. A similar reduction in 1,4-β-galactan in potato cell walls was reported by Sørensen et al. (2000) after an endo-1,4-β-d-galactanase from Aspergillus aculeatus had been expressed. In both cases, no altered phenotype was found in transgenic tubers, indicating that potato tuber cell walls seem to tolerate low levels of 1,4-β-galactan side chains without plant or tuber viability being compromised. The residual galactose content may be attributable to the 1,4-β-galactose not available for the action of the enzyme or to galactose from wall components other than 1,4-β-galactan, such as xyloglucan, known to contain 1,2-β-galactosyl residues (York et al. 1985) or traces of 1,3-β- and or 1,6-β-galactosyl residues, since such residues have been found to be present in potato wall RGI (Jarvis et al. 1981). Additionally, the similar arabinose content of both WT and transgenic cell walls (Fig. 4) indicates that the hydrolyzed galactan contains few, if any, arabinosyl residues.
The generation of these transgenic potatoes also confirmed the involvement of βIII-Gal in the cell wall autolytic processs (Dopico et al. 1989), since βIII-Gal transformant tuber cell walls displayed an autolytic activity, similar to the autolysis carried out by chickpea, releasing galactose into the incubation medium (Table 1), while WT potatoes did not exhibit such a cell wall autolysis.
Pectin models do not suggest specific roles for pectic side-chains. Neutral side chains, such as arabinans, are very flexible molecules in aqueous solution. Cros et al. (1994) and Renard and Jarvis (1999) demonstrated that they are also very mobile molecules in muro. One hypothesis to explain the function of galactan side chains is that the reduced galactan content of RGI results in a more porous wall architecture. In this sense the involvement of neutral side chains in interactions with other cell wall components in the cell wall architecture has been suggested (Fujino and Itoh 1998, Vincken et al. 2003). The modified pectic polymers in the transformants point either to a particular role of galactan in wall architecture opening wall pores, or to other structural changes in the wall, compensating for the reduced level of galactan. The absence of an altered phenotype in the transformed tubers suggests that homeostatic mechanisms compensate the loss of galactans and produce a functional wall architecture. In contrast to the side chain function, RGI plays a more important role in cell wall assembly. Thus, fragmentation of the RGI backbone leads to wrinkled tubers with abnormal periderm development and large intercellular spaces in the cortex, as referred to by Oomen et al. (2002) in transgenic tubers expressing a fungal rhamnogalacturonanlyase.
Authors such as Shedletzky et al. (1992) and Burton et al. (2000) have postulated that the biosynthetic routes of pectins and cellulose could be connected. Accordingly we expected that the reduction in galactans could be compensated by an increase in the amount of cellulose. However, our analysis indicated that the cellulose level was similar (around 25%) in both WT and transgenic tuber cell walls, with no relationship between the level of transgene expression and the amount of cellulose. Our results also revealed that the reduction in galactans due to the transformation of potato with a chickpea β-galactosidase was accompanied by an increase in uronic acids (Table 1), which is in agreement with the results reported by Sørensen et al. (2000) after transformation of potato with a fungal endogalactanase. This higher uronic acid content in transgenic tuber cell walls suggests variations in acid polysaccharides, and so we decided to study the organization of the different pectic polysacharides by using FTIR microspectroscopy. This spectroscopic method can quantitatively detect a range of functional groups including carboxyl esters, phenolic esters, protein amides and carboxylic acids, and can provide a complex ‘fingerprint’ of carbohydrate constituents and their organisation (McCann et al. 1992, McCann et al. 1997). Thus, the major classes of cell wall pectic polysaccharides—HGA, RGI, AG—each have distinctive absorption bands (Kacuráková et al. 2000). Our study revealed that the band corresponding to HGA appeared to have increased in cell walls from βIII-Gal transgenic potato tubers (Fig. 6), suggesting that the reduction of galactan in RGI side chains in transgenic tuber cell walls was compensated by an increase in the HGA content. This may indicate a biosynthetic switch from one type of galacturonan backbone to another, as suggested by Willats et al. (2001).
In conclusion, the approach of generating plants with modified cell walls by expressing polysaccharide hydrolases in the plant complements other mutant approaches. In the present report, by using βIII-Gal—a chickpea cell wall-degrading enzyme—wall structural changes such as the removal of galactan and increased homogalacturonan levels were observed. This provides us with experimental tools for assigning particular physiological and structural roles to these wall components, although more work remains to be done. This work also allowed us to establish the function in vivo of the chickpea βIII-Gal protein.
In addition to this physiological point of view, the manipulation of cell wall polymers in plants to generate, as in the present case, modified pectins to suit a specific commercial use should represent a significant advance in biotechnology. During the process of starch isolation the potato starch industry generates large volumes of residues relatively rich in pectins. However, potato pectin is less suitable for many industrial food uses than pectins from other sources, such as apple or citrus, in part because of its high content of neutral galactans. The abundance of neutral side chains in potato and in sugar beet pulp is believed to impair the gelling ability of the pectin (Hwang and Kokini 1991, Hwang et al. 1993). Our transgenic approaches, decreasing the amount of galactans, may allow improvements to be made in potato pectin quality.
Materials and Methods
Vector construction and potato transformation
To accomplish vector construction for potato transformation, the granule-bound starch synthase (GBSS) promoter was excised from the vector pPGB121s as described by Visser et al. (1991) by digestion with HindIII and BamHI, and was cloned into the pBluescript SK-vector (Stratagene, La Jolla, CA, USA) giving pGBSS. Subsequently, the GBSS promoter was digested with HindIII and SpeI and cloned into the pBIN20 binary vector (Hennegan and Danna 1998), creating the DNA construct pBIN20-GBSS. The complete coding region of the chickpea CanBGal-3 cDNA was cloned into the SpeI and KpnI sites of the pBIN20-GBSS, generating the pBIN20-GBSS-CanBGal-3 construct.
Internodal cuttings from in vitro-grown plants of Solanum tuberosum cv. Karnico were used for Agrobacterium tumefaciens-mediated transformation according to Visser et al. (1989). After regeneration of in vitro shoots on selective kanamycin medium, 62 shoots were transferred to a greenhouse to generate mature plants. Fifty-two individual transgenic potato plant were generated. Wild-type (WT) and CanBGal-3 transformant potato tubers were collected.
Northern analysis
Northern experiments were performed as described in Muñoz et al. (1997). A cDNA fragment from the 3′ non-coding end of the CanBGal-3 (362 bp from nt 2386 to nt 2747) clone was used as specific probe. cDNA specific probes were amplified by PCR in a GeneAmp PCR system 9700 thermocycler (PE Biosystems, Norwalk, CT, USA), cleaned by the High Pure PCR product purification kit (Roche, Mannheim, Germany), and labelled with 32[P] using the Random primed kit (Roche, Mannheim, Germany). Total RNA (20 µg per lane) was electrophoresed, transferred onto Hybond N nylon membranes (Amersham Pharmacia Biotech, Manchester, UK), and hybridized using the radiolabelled specific probes. After hybridization, membranes were washed twice with 2× SSC, 0.1% SDS at 42°C for 5 min each and twice with 0.1× SSC, 0.1% SDS at 42°C for 20 min each.
Cell wall and protein extraction
Cell walls were isolated from WT and CanBGal-3-transformant potato tubers. For each isolation of cell wall material, 100 g (FW) of frozen potato tubers slices were homogenized at 4°C in 50 mM NaCl using an Omni-mixer. The homogenate was filtered through double Miracloth (Calbiochem, Darmstadt, Germany) and washed with 50 mM NaCl, acetone at –20°C, 50 mM NaCl and distilled water. The cell wall material obtained was suspended in 3 M LiCl in 10 mM Na-citrate phosphate buffer, pH 5.5 at 4°C for protein extraction, according to Huber and Nevins (1981). After 48 h, the suspension was filtered through Miracloth and centrifuged (10 min, 2200×g), and the supernatant was diafiltered using the Ultralab System (Pall Filtron, Cortland, NY, USA) with a Ultrasette membrane (5K tangential flux) to carry out LiCl removal and salt exchange. A volume of 2.5 liters of 20 mM Na-acetate buffer, pH 5.0 was used in the diafiltration. The protein extract was concentrated in an Amicon device, using a 3K Pall Filtron membrane, to a final volume of 3–4 ml. Both the protein diafiltration and the subsequent concentration were carried out at 4°C.
After protein extraction, the cell wall material retained in the Miracloth was washed with distilled water, frozen dried, and ground to a fine powder. Starch was removed according to the procedure reported by Sørensen et al. (2000) by incubation with α-amylase and pullulanase. The starch content was evaluated using the Starch Kit (Roche, Mannheim, Germany), for which solubilized starch samples were used, following the manufacturer’s instructions.
Cell wall monosaccharide composition
The neutral monosaccharide composition of the cell walls was analyzed by gas phase chromatography after hydrolysis with 2 N trifluoroacetic acid for 90 min at 121°C. The hydrolyzed sugars were converted into alditol acetates according to Albersheim et al. (1967). A Konik 3000-HRGC device equipped with a column of 3% ECNSS-M on Gas Chrom P (Konik Instruments S.A., Barcelona, Spain) was employed. Inositol was used as an internal standard. The non-hydrolyzed cell wall residue was considered as cellulose.
For the analysis of uronic acids, 1 mg of cell wall material was hydrolyzed in 2 N trifluoroacetic acid for 120 min at 121°C. The hydrolyzed products were air-dried at 45°C and incubated overnight in 80% ethanol at –20°C. The ethanol-soluble sugars were dried and 3 ml of water was added. The uronic acid content was evaluated according to the Filisetti-Cozzi and Carpita method (Filisetti-Cozzi and Carpita 1991), using galacturonic acid for the standard curve.
Protein and enzymatic activity measurements
The total amount of protein was assayed by the Protein Assay (Bio-Rad, Baltimore, MD, USA), based on the Bradford assay (Bradford 1976). β-d-Galactosidase activity was assayed using the corresponding p-nitrophenylglycoside substrate, as described in Dopico et al. (1989); the free p-nitrophenol concentration was determined by absorbance at 400 nm. β-galactanase activity was measured against a lupin galactan pretreated with α-l-arabinofuranosidase (Megazyme, Wicklow, Ireland). The reaction mixture consisted of 40 µl 0.1 M sodium acetate/acetic acid buffer, pH 4.0, 40 µl enzyme solution containing 50 µg total protein and 20 µl 1% polysaccharide substrate. Samples were incubated at 37°C for up to 4 hours. The galactose released was analyzed by thin layer chromatography on silicagel plates (Merck, Darmstadt, Germany) according to the method described by Bosch-Reig et al. (1992). The plates were sprayed with the detection reagent (0.1% orcinol in 50% sulphuric acid) and heated to 100°C for 5 min. Galactose was quantified in a CS-9000 Dual-wavelength Flying-spot scanner densitometer (Shimadzu, Tokyo, Japan), using commercial galactose (Sigma, St Louis, USA) as standard.
SDS-PAGE
Electrophoresis in polyacrylamide gels containing SDS was carried out according to the method of Laemmli (1970), using the Protean II xi (Bio-Rad, Baltimore, MD, USA) system. The length and thickness of the running gel were 20 cm and 1.5 mm, respectively, using approximately 17 µg of cell wall protein. Molecular weight markers (Bio-Rad) were employed as standards for calibration. The separating gel contained 10% acrylamide (acrylamide : bis-acrylamide 30 : 0.8). A current of 25 mA per gel was employed for the electrophoresis. Protein bands were visualized using the silver staining method described by Morrissey (1981). Once stained, the gels were dried under vacuum at 80°C for 1 h.
Cell wall autolysis and hydrolysis of cell walls by extracted cell wall proteins
The autolysis experiments were performed with freshly isolated cell walls from 7.5 g of potato tubers as indicated above. Cell walls were suspended in 7.5 ml 10 mM Na-citrate phosphate buffer, pH 5.5 at 34°C. In experiments designed to measure the hydrolytic capacity of chickpea wall proteins, heat-inactivated cell walls extracted from 7.5 g of potato tubers were suspended in 7.5 ml 10 mM Na-citrate phosphate buffer, pH 5.5, and incubated with 500 µg of extracted chickpea cell wall proteins. In both types of experiments, incubation was carried out at 34°C for 24 h. The amount of galactose released by autolysis or cell wall hydrolysis was analysed by gas phase chromatography as indicated above.
Immunofluorescence labelling of potato tubers
Sections of potato tubers (5–7 µm) were labelled with monoclonal antibody LM5, specific for 1,4-β-galactan (Jones et al. 1997). Sections were washed and hydrated for 5 min in Tris-buffered saline [TBS: 50 mM Tris, 140 mM NaCl (pH 7.4)]. Sections were then incubated for 45 min in blocking solution [50 mM TBS (pH 7.4), 0.2% BSA], followed by treatment with primary antibodies LM5 (diluted 1 : 50 or 1 : 100) in TBST (50 mM TBS containing 0.1% Tween 20) for 3h at 37°C in a humid chamber. After washing with TBST, the sections were incubated with the secondary antibody (Goat anti rat-IgG conjugated with FITC) diluted 1 : 25 for 1–2 h at 37°C in the dark. The sections were rinsed in TBST and in distilled water and mounted with citifluor reagent. Sections were examined either with a Leica DMLB (Leica, Wetzlar, Germany), Camera Nikon coolpix 995 digital (Nikon, Herndon, VA, USA) for epi-fluorescence or with a Confocal Leica TCS-SP2 for confocal fluorescence microscopy.
Fourier transform infrared (FTIR) spectroscopy
After starch removal, potato tuber cell walls were freeze-dried, mixed with KBr (1 : 300 w/w), and pressed in a Beckman press under a pressure of 8 Tm m–2 for 5 min. The samples were analyzed with a spectrophotometer (Perkin-Elmer System 1600 FTIR, Perkin-Elmer, Foster City, CA, USA). Spectra were collected in transmission mode at 4 cm–1 resolution. The spectra were baseline-corrected.
Acknowledgements
We would like thank Marie Laure-Follet Gueye for her help in the microscopy analysis. This work was supported by a grant from the EC (CT97 2224) and a grant from the Dirección General de Investigación, Spain (BOS2002–01900).
Line | UA content (µg per mg cell wall) | UA increase (%) |
WT | 87.15 ± 19.71 | |
L7 | 112.10 ± 17.18 | 28.6 ± 10.5 |
L14 | 100.75 ± 16.33 | 15.6 ± 5.6 |
L27 | 87.61 ± 15.55 | 0.53 ± 0.18 |
Line | UA content (µg per mg cell wall) | UA increase (%) |
WT | 87.15 ± 19.71 | |
L7 | 112.10 ± 17.18 | 28.6 ± 10.5 |
L14 | 100.75 ± 16.33 | 15.6 ± 5.6 |
L27 | 87.61 ± 15.55 | 0.53 ± 0.18 |
Numbers refer to µg of uronic acis per mg of cell wall and percentage of increase in uronic acid content compared with the wild type. Data are means of three replicates of three independent isolations for each line ± SE.
Line | UA content (µg per mg cell wall) | UA increase (%) |
WT | 87.15 ± 19.71 | |
L7 | 112.10 ± 17.18 | 28.6 ± 10.5 |
L14 | 100.75 ± 16.33 | 15.6 ± 5.6 |
L27 | 87.61 ± 15.55 | 0.53 ± 0.18 |
Line | UA content (µg per mg cell wall) | UA increase (%) |
WT | 87.15 ± 19.71 | |
L7 | 112.10 ± 17.18 | 28.6 ± 10.5 |
L14 | 100.75 ± 16.33 | 15.6 ± 5.6 |
L27 | 87.61 ± 15.55 | 0.53 ± 0.18 |
Numbers refer to µg of uronic acis per mg of cell wall and percentage of increase in uronic acid content compared with the wild type. Data are means of three replicates of three independent isolations for each line ± SE.
Line | Galactose released (µg per mg cell wall) | |||
Control 1 a | Control 2 b | Autolysis c | +Chickpea proteins d | |
WT | 0 ± 0 | 0 ± 0 | 0.25 ± 0.12 | 56.51 ± 24.22 |
L7 | 0 ± 0 | 0.37 ± 0.11 | 9.38 ± 1.67 | 6.28 ± 8.81 |
L14 | 0 ± 0 | 0.12 ± 0.01 | 7.69 ± 2.05 | 16.61 ± 8.79 |
L27 | 0.37 ± 0.28 | 0.08 ± 0.06 | 1.66 ± 0.22 | 45.59 ± 28.32 |
Line | Galactose released (µg per mg cell wall) | |||
Control 1 a | Control 2 b | Autolysis c | +Chickpea proteins d | |
WT | 0 ± 0 | 0 ± 0 | 0.25 ± 0.12 | 56.51 ± 24.22 |
L7 | 0 ± 0 | 0.37 ± 0.11 | 9.38 ± 1.67 | 6.28 ± 8.81 |
L14 | 0 ± 0 | 0.12 ± 0.01 | 7.69 ± 2.05 | 16.61 ± 8.79 |
L27 | 0.37 ± 0.28 | 0.08 ± 0.06 | 1.66 ± 0.22 | 45.59 ± 28.32 |
Incubations were carried out for 24 h in 10 mM Na-citrate phosphate buffer, pH 5.5. Numbers refer to µg of galactose per mg of cell wall. The data are means of three independent isolations ± SE.
a Heat-inactivated tuber cell walls.
b Active tuber cell walls incubated at 4°C.
c Autolytic process of active tuber cell walls at 37°C as indicated in Materials and Methods.
d Inactivated tuber cell walls incubated at 37°C with 500 µg of chickpea cell wall proteins.
Line | Galactose released (µg per mg cell wall) | |||
Control 1 a | Control 2 b | Autolysis c | +Chickpea proteins d | |
WT | 0 ± 0 | 0 ± 0 | 0.25 ± 0.12 | 56.51 ± 24.22 |
L7 | 0 ± 0 | 0.37 ± 0.11 | 9.38 ± 1.67 | 6.28 ± 8.81 |
L14 | 0 ± 0 | 0.12 ± 0.01 | 7.69 ± 2.05 | 16.61 ± 8.79 |
L27 | 0.37 ± 0.28 | 0.08 ± 0.06 | 1.66 ± 0.22 | 45.59 ± 28.32 |
Line | Galactose released (µg per mg cell wall) | |||
Control 1 a | Control 2 b | Autolysis c | +Chickpea proteins d | |
WT | 0 ± 0 | 0 ± 0 | 0.25 ± 0.12 | 56.51 ± 24.22 |
L7 | 0 ± 0 | 0.37 ± 0.11 | 9.38 ± 1.67 | 6.28 ± 8.81 |
L14 | 0 ± 0 | 0.12 ± 0.01 | 7.69 ± 2.05 | 16.61 ± 8.79 |
L27 | 0.37 ± 0.28 | 0.08 ± 0.06 | 1.66 ± 0.22 | 45.59 ± 28.32 |
Incubations were carried out for 24 h in 10 mM Na-citrate phosphate buffer, pH 5.5. Numbers refer to µg of galactose per mg of cell wall. The data are means of three independent isolations ± SE.
a Heat-inactivated tuber cell walls.
b Active tuber cell walls incubated at 4°C.
c Autolytic process of active tuber cell walls at 37°C as indicated in Materials and Methods.
d Inactivated tuber cell walls incubated at 37°C with 500 µg of chickpea cell wall proteins.
Abbreviations
- FTIR
Fourier transform infrared
- GalA
1,4-α-galacturonic acid
- GBSS
granule-bound starch synthase promoter
- HGA
homogalacturonan
- RG
rhamnogalacturonan
- TBS
Tris-buffered saline
- WT
wild-type
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