Thomas MooreZ and Alan B. Bennett*. Mann Laboratory ... and the B subunit during the extraction of tomato fruit tissue when .... colloidal gold (Stoscheck, 1990).
Plant Physiol. (1994) 106: 1461-1469
Tomato Fruit Polygalacturonase Isozyme 1' Characterization of the /3 Subunit and Its State of Assembly in Vivo Thomas MooreZ and Alan B. Bennett*
Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616
~
that has been referred to as the @ subunit (Moshrefi and Luh, 1983; Pogson et al., 1991). These isozymes can be distinguished by their enzymatic characteristics, migration during nondenaturing PAGE, heat stability, and polypeptide composition (Pressey and Avants, 1973; Ali and Brady, 1982; Moshrefi and Luh, 1983). Perhaps most importantly, PGl and PGZA-PG2B differ in their appearance during fruit ripening; PGl appears first, but PG2A and PG2B predominate in terms of relative abundance in fully ripe fruit (Tucker et al., 1980; Brady et al., 1982). Tucker et al. (1981) first described a heat-stable factor from green fruit tissue that could convert PG2 to a heat-stable PG1-like activity. Pressey (1984a) later partially purified and characterized this activity, which he termed the "converter," from ripe fruit and observed a much lower level of this activity in tomato leaf tissue. Knegt et al. (1988) have also partially characterized a converter activity isolated from fruit extracts and from heat-denatured PG1 preparations. The precise relationship between the /3 subunit of PGl and the converter activity observed in green fruit, vegetative tissue, or from heated extracts of PG1 has remained unclear (Knegt et al., 1988, 1991). This may be due in part to problems associated with the use of an impure activity. Knegt et al. (1988) have proposed that PG1 may be the During tomato (Lycopersicon esculentum) fruit ripening, a physiologically relevant isozyme with respect to pectin degnumber of changes occur in the protein and polymer comradation during tomato fruit development. At least four obposition of cell walls (Fischer and Bennett, 1992). One of the servations support this hypothesis: (a) During the earliest best-characterized changes is the appearance of the enstage of tomato fruit ripening, at a time when PGl is predomzyme PG (po~y[~,4-cu-~-galacturonide]g~ycanohydro~ase, EC inantly or exclusively present, the majority of pectin degra1.2.1.15). PG appears to be responsible for the degradation dation that is observed to occur over the course of developof the pectin-rich component of the middle lamella of cell ment has occurred. This observation was made both in normal walls (Crookes and Grierson, 1983). Three isozymes of totomato fruit and in a ripening-inhibited mutant of tomato mato fruit PG have been characterized: PG1, PGZA, and fruit transformed with a PG catalytic subunit gene under the PGZB. PG2A and PG2B are single polypeptides and differ control of an ethylene-inducible promoter (DellaPenna et al., from each other primarily in their degree of glycosylation 1990). (b) In studies comparing the activities of PG1 and PG2 (Ali and Brady, 1982; DellaPenna and Bennett, 1988; Pogson with different pectic substrates, PGl appeared to have greater et al., 1991). PGl appears to contain either PG2A or PG2B activity at lower ionic strength (Pressey and Avants, 1973; glycoforms, and another polypeptide of unknown function Pogson et al., 1991). Since low ionic strength would be expected in the apoplast, this suggested that PG1 would be This research was supported by United States Department of the more active isozyme in this compartment (Pogson et al., Agriculture-National Research Initiative Competitive Grants Pro1991). (c) In transgenic tomato plants in which the expression gram grant No. 91-37304-6508 and by a research gift from Urdever/ of PG has been suppressed using antisense technology, residVan den Bergh Foods. ual PG activity in fruit was recovered as PGI, and the degree Present address: Center for Engineering Plants for Resistance of pectin solubilization observed was still substantial (Smith Against Pathogens (CEPRAP), University of California, 1930 Fifth
Polygalacturonaseisozyme 1 (PC1) is a heterodimer comprising a catalytic and noncatalytic or B subunit, whereas polygalacturonase isozyme 2 (PC2) comprises only the catalytic subunit. To assess the state of assembly of PCI in vivo, both subunits were purified to homogeneity and used to study assembly of the heterodimer. PC1 could be reconstituted in vitro from purified B subunit and purified PC2 under a wide range of salt and pH conditions, and PC1 reconstituted in vitro was indistinguishablefrom PC1 isolated from tomato (Lycopersicon esculentum) fruit. Specific antibodies indicated that the B subunit was present in fruit of all developmental stages, but absent in vegetative tissue. l h e state of assembly of PC1 in vivo was tested based on the differential thermal stability of PC1 and PC2 by heating segments of ripe fruit pericarp tissue. Temperatures well below those required to inactivate PC1 in vitro caused the loss of activity of both PC1 and PC2, suggesting that only heat-labile PC2 i s present in vivo. In addition, when extracts of ripe fruit were rigorously maintained and analyzed at 4"C, PC1 was absent or barely detectable. These results are consistent with the hypothesis that PG1 can assemble spontaneously and i s essentially absent in intact tomato fruit but forms artifactually from PG2 and the B subunit during the extraction of tomato fruit tissue when low temperatures are not rigorously maintained.
Street, Davis, CA 95616. * Corresponding author; fax 1-916-752-4554.
Abbreviation: PG, polygalacturanase. 1461
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et al., 1990). (d) Pectin degradation was not observed in tobacco plants that were transformed with the PG catalytic subunit gene and that were expressing only PG2A and PG2B glycoforms and did not contain the PGl isozyme (Osteryoung et al., 1990). Models describing how the ,8 subunit and PG2 might interact to form PGI during the course of fruit development, or how they might function to localize or regulate other cell wall proteins, have been proposed (Bruisma et al., 1989; Zheng et al., 1992; Pogson and Brady, 1993a). In contrast, Pressey has argued that PGI is formed as an artifact of the extraction process (Pressey, 1986b, 1988). Under conditions that did not disrupt PGI in vitro (pH 1.6), PG2 and then PG converter could be sequentially extracted from tomato fruit. PGl itself, however, was only a minor component of these extracts. Direct extraction of the same tissue at pH 6.0 and with 1.0 M NaCl led to the detection of a large amount of both PG2 and PG1, suggesting that PGI formed under these extraction conditions (Pressey, 1988). A potentially useful approach in reconciling these observations has included purifying and better characterizing the PGI ,8 subunit (Pogson et al., 1991). We have extended this approach, addressing whether the ,8 subunit of PGl is identical to the previously described converter activity and describing the nature of the PGl isozyme reconstituted in vitro using purified ,8 subunit and PG2. In addition, we have assessed whether PG1 exists in tomato fruit tissue prior to the extraction or whether it is an artifact of extraction. MATERIALS A N D METHODS Purification of PG1 and the
B Subunit
Tomatoes were obtained from both field-grown and greenhouse-grown plants or from local markets. Chemicals were purchased from Sigma unless otherwise noted. Tissue was homogenized in 1.0 mL/1.0 g fresh weight of a low-salt extraction buffer of 10 m Na citrate, pH 5.5, 5 m ,8mercaptoethanol, and 0.5% (w/v) PVP. The homogenate was centrifuged at 16,OOOg for 20 min and the pellet was resuspended in 0.5 to 1.0 mL/g fresh weight of starting material in a high-salt extraction buffer of 50 m Na citrate, pH 5.5, 1.7 M NaCl, 15 m EDTA, and 5 m ,8-mercaptoethanol.The homogenate was shaken vigorously for 1 h then centrifuged at 16,OOOg for 20 min. The supernatant, designated the highsalt extract, was filtered through Miracloth, and through a 0.45-pm polyvinylidene difluoride filter, and finally concentrated approximately 10-fold by ultrafiltration (Pressey, 1986a) through a YM-30 membrane (Amicon, Beverly, MA). This extract was diluted with an equal volume of Con A column buffer (1 M NaCl, 1 m MnC12, 1 m MgC12, 1 m CaC12, 20 m Na Mes, pH 6.0) and reconcentrated two times. The concentrate was applied to a Con A-Sepharose 4B column (Sigma) and elution was performed with column buffer containing 0.2 M a-methyl mannoside. After 10-fold concentration of the Con A column eluant, it was diluted with an equal volume of 0.1 M NaCl, 20 m Na Mes, pH 6.0, buffer, reconcentrated, and then this step was repeated. PG1 was purified from the Con A eluant as described by Pressey (1984b) and subsequently chromatographed on Superose 6 (Pharmacia) in 1.0 M NaC1, 50 mM Na acetate, pH 4.5. PG1
Plant Physiol. Vol. 106, 1994
was dissociated in 8 M urea, 20 mM Na formate, pH 3.8, and the 0 subunit was isolated by chromatography 07 a Mono-S column using a linear gradient of 0.1 to 1.0 M NiiC1 in 8.0 M urea (United States Biochemical), 20 m Na formate, pH 3.8. Fractions were dialyzed against 1.0 M NaC1, 10.0 m Na formate, pH 3.8, to maintain solubility of the /3 subunit (Pressey, 1984a). The use of silanized pipette tips and tubes was found to be essential to prevent nonspecific losses of purified 0 subunit. Protein determinations were routinely performed with either Coomassie blue (Bradford, 1976) or bicinchoninic acid (Smith et al., 1985). Quantitalion of purified ,8 subunit from column fractions was performed with colloidal gold (Stoscheck, 1990). Antibody to the B Subunit
Approximately 100 fig of purified ,8 subunit was dialyzed overnight against 20 m ammonium bicarbonate buffer and subsequently concentrated by lyophilization. The protein was resuspended in water, emulsified in Freund's complete adjuvant, and injected subcutaneously at multiple sites into a rabbit as described (Harlow and Lane, 1988). Enzyme Assays
PG assays were performed as described by Gross (1982). Incubations were performed for 10 to 30 min ai: 37OC with dilutions of enzyme to ensure that the determinations were within the linear range of the assay. A standxd enzyme assay contained 0.2 M NaCl, 0.1 M sodium acetate buffer, pH 4.0,0.01% (w/v) BSA, and enzyme, and was initiated by the addition of polygalacturonic acid (Sigma, degree of polymerization 20-40) to 0.1% (w/v). The inclusion of BSA was found to increase the total activity and reproducibility in enzymatic assays of PG and conversion of PG2 to PG1 by the ,8 subunit, presumably by reducing nonspecifil: adsorption of these proteins to surfaces. Conversion of PG2 to PG1 was assayed as previously described (Knegt et al., 19El8). Samples were boiled and substrate was subsequently added before incubation at 37OC to constitute the enzyme blanks. Nondenaturing PAGE and Activity Staining
Nondenaturing PAGE was performed at 4OC in 7.5% (w/ v) polyacrylamide gels essentially as described (Reisfeld et al., 1962), except that the stacking gel was polymerized with O. 1% (w/v) ammonium persulfate instead of ribol'lavin. Nondenaturing PAGE analysis of conversion of PG2 to PGI was performed without camer protein and after samples were dialyzed against 10 m NaCl, 1.0 m Na formt3te, pH 4.0, for 5 h at 4OC in a microdialysis apparatus (Pierce, Rockford, IL). After electrophoresis, gels were equilibrated twice for 15 min in 0.2 M NaCl, 0.1 M Na acetate buffer, pH 4..0,at 37OC. The gels were then incubated for 30 to 60 min at 37OC in 1.0% (w/v) polygalacturonic acid, 0.2 M NaC1, pH 4.0. The gel was briefly rinsed with water before staining; for 10 min in 0.5% (w/v) methylene blue and destaining in water. lmmunoblotting
High-salt extracts of tomato tissues were prepared as described above and 5.0 pg of protein was separated by SDS-
Tomato Polygalacturonase Isozyme 1
PAGE (Laemmli, 1970). Proteins were detected by silver staining (Oakley, et al., 1980). Proteins were electroblotted to polyvinylidene difluoride (Millipore, Marlborough, MA) for immunodetection (Matsudaira, 1987). Nondenaturing gels were preincubated in 10 mM 3-(cyclohexylarnino)-l-propanesulfonic acid, pH 11.0,10% (v/v)ethanol containing 0.01% (w/v) SDS three times for 10 min each before electroblotting. Blots were incubated in 3.0 to 5.0% (w/v) Blotto (Johnson et al., 1984) in IX Tris-buffered saline (25 nw Tris [tris(hydroxymethyl)-aminomethane]-HCl, pH 7.5, 0.9% [w/ v] NaCl) for 1 h. Blots were next incubated for 1 h in rabbit serum diluted 1000-fold in Tris-buffered saline with 0.1% Tween-20 (Sigma). Blots were washed in Tris-buffered saline with 0.1% Tween-20 three times for 10 min each and then incubated for 1 h with alkaline phosphatase-conjugated goat anti-rabbit antibodies (Bio-Rad), washed, and developed with chromogenic or chemiluminescent substrates according to the manufacturer's suggestions (Bio-Rad, or Tropix, Bedford, MA, respectively). Blots were stripped with acetic acid (Mirendorf et al., 1987) and were reblocked and probed for chemiluminescent detection of proteins according to the manufacturer's suggestions. Heat Treatment of Pericarp Tissue
Ripe fruit pericarp was divided into approximately 1-cm square sections and 5 g was sealed in plastic bags for immersion in water baths. After immersion at the indicated temperature and for the indicated time, the tissue was removed and placed on ice for 10 min before extraction. The treated tissue was homogenized in 1.0 mL/g fresh weight of low-salt extraction buffer in a blender as described above but containing 5% j3-mercaptoethanol. The samples were then cenrrifuged at 27,000g for 20 min and the pellet was resuspended in 1.0 mL/g fresh weight of high-salt extraction buffer as described above, and shaken vigorously at 4°C for 1 h. The extract was again centrifuged as before and the supernatants were snap frozen in liquid N2 until desalting and analysis by nondenaturing PAGE.
mately 2 min while remaining in an ice-water bath. This method minimized heating of the sample, and homogenization with a blender, even in a cold (4°C) room, allowed the sample to reach a temperature of 20°C (T. Moore and A. Bennett, unpublished observations). The sample was centrifuged at 16,000g for 10 min. The pellet was resuspended in low-salt extraction buffer and recentrifuged. The resulting pellet was resuspended in high-salt extraction buffer with brief homogenization. An aliquot was removed immediately and snap frozen in liquid N2. The extract was shaken vigorously and aliquots were removed at 10-min intervals and snap frozen. After 1 h the extract was centrifuged as described above and the supernatant was aliquoted to tubes held at 22 or 37°C while the remainder was held on ice. Aliquots were removed immediately, and subsequently at 10-min intervals. Before analysis by nondenaturing PAGE, all samples were thawed in ice water and recentrifuged at 16,000g for 10 min at 4°C, and the supernatants were desalted as described above. RESULTS Purification of the & Subunit and Reconstitution of PG1 in Vitro
PG1 was purified from a high-salt extract of ripe tomato fruit using Con A lectin affinity chromatography, Mono-S cation-exchange chromatography, and gel filtration. PG1 was then dissociated into PG2 and the /? subunit using 8 M urea and acidic formate buffer. The protein subunits were separated by cation-exchange chromatography under these conditions (Pogson et al., 1991). Figure la shows SDS-PAGEresolved proteins from aliquots of the denaturing ionexchange chromatographic separation. Dialyzed protein from
xPG2b
(a)
Desalting of Tissue Extracts Rapid desalting of tissue extracts by centrifugation through gel-filtration media (Helmerhorst and Stokes, 1980) was used to prepare samples for analysis by nondenaturing PAGE. BioRad P6DG resin was resuspended in a buffer of 10 mM NaCl, 1.0 min Na acetate, pH 4.0, and 0.025% Cyt c as a protein carrier to prevent nonspecific losses of PG isozymes to the resin. A volume of extract representing 20% or less of the bed volume of the column was desalted by centrifugation at 500g for 4 min, then snap frozen in liquid N2. Preparation and Incubation of Ripe Fruit Extracts Fruit pericarp tissue was frozen in liquid nitrogen, pulverized to a fine powder, and stored at —80°C until use. All subsequent steps, including final analysis by nondenaturing PAGE, were performed at 4°C. Pulverized tissue was thoroughly homogenized in low-salt extraction buffer with a Polytron probe (Brinkmann, Westbury, NY) for approxi-
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-PG2a — p-sub
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32 33
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35 36 37
38
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Fraction Number
Figure 1. The (3 subunit has converter activity, a, PC1 purified from ripe tomato fruit was dissociated into the j3 subunit, PC2a, and PC2b. The subunits were separated by ion-exchange chromatography and gradient fractions were analyzed by SDS-PACE on a 12.5% gel and visualized by Coomassie blue staining, b, Dialyzed aliquots from the Mono-S gradient were analyzed for the ability to convert PC2 to heat stability (65°C, 5 min, see "Materials and Methods"). PC activity was determined by reducing sugar assay, and the amount of heat-resistant activity resulting from incubation of purified PC2 with /3 subunit from the corresponding fractions in (a) is plotted as a percent of input PG activity (percent conversion).
Moore and Bennett
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the chromatographic fractions was assayed for the ability to convert pure PG2 from a thermolabile to a thermostable form (Tucker et al., 1981). The ability to convert PG2 to thermostable PGl was found only in those fractions of the Mono-S column that contained the ft subunit (Fig. Ib). Purified PG2 was incubated with increasing amounts of purified ft subunit and assembly of PGl was monitored by nondenaturing PAGE (Reisfeld et al., 1962) and activity staining (Lisker and Retig, 1974). In the presence of the /3 subunit a new band of PG activity was formed from PG2, which co-migrated with PGl isolated from ripe tomato fruit (Fig. 2a). At the lowest amount of ft subunit added, new activity formed that was diffuse and slowly migrating. The PG activity formed in vitro was heat stabile and co-migrated with PGl after heat treatment (Fig. 2a, lanes 8-10). Polyclonal antibodies to purified /3 subunit were prepared and used to assess the presence of the ft subunit in fruit PG2 and PGl and PGl formed in vitro (Fig. 2b). The ft subunit was detected only in PGl from fruit or PGl formed in vitro (Fig. 2b). This result indicates that the antibody does not cross-react with PG2 and that the ft subunit is a part of the PGl isozyme. Chromatofocusing of the ft subunit indicated an isoelectric point of approximately 5.0 (data not shown), a value similar to that reported for fruit converter activity (Pressey, 1984a). This result accounted for the failure to
(a)
Plant Physiol. Vol. 106, 1994
detect free ft subunit in this immunoblot, because under these elecrrophoresis conditions it would not migrate into the gel. Further characterization of the in vitro PG activity reconstituted in vitro from purified PG2 and the ft subunit was performed with respect to pH and salt optima and thermostability. The pH optimum for fruit PGl and PG2 were found to be similar to previously reported values (pH 3.5-4.5), although PG2 was found to have a slightly more acidic pH optimum and PGl a more basic optimum than generally reported (Pressey and Avants, 1973; Knegt et al., 1988). However, Pressey and Avants (1973) have noted that the pH optima of these enzymes is highly dependent on the substrate size as well as the NaCl concentration. As shown in Figure 3, PGl formed in vitro and PGl isolated from ripe tomato fruit were essentially indistinguishable by these three criteria. Because both assembly and activity were carried out under the indicated conditions of pH and salt, these results also demonstrate that conversion of PG2 by the ft subunit can occur over a wide range of conditions. Based on these results, the ft subunit appears to be both necessary and sufficient for the conversion of PG2 to PGl, and in this respect it fulfills at least one previously described criterion for PG converter (Tucker et al., 1981). These results confirm and extend previous observations on the relationship between converter and the ft subunit (Pressey, 1984a; Pogson and Brady, 1993a) and contrast with the results of Knegt et al. (1988), who observed differences between PGl and a PGl-like activity reconstituted in vitro. Distribution of the 0 Subunit in Tomato
heat
-PGl
PG2-
9
10
(b)
*** -PG1
Figure 2. PC1 isolated from tomato fruit and PCI produced in vitro co-migrate on nondenaturing gels both before and after heat treatment and contain the 0 subunit. a, Nondenaturing PACE and activity staining were performed on PG and PG1 isolated from ripe tomato fruit and on PC1 produced in vitro from the conversion of PC2 by the 0 subunit as described in "Materials and Methods." Lane 1, PC2 (0.2 jig) from ripe tomato fruit; lane 2, as in lane 1, but after 5 min at 65°C; lane 3, PG1 from ripe tomato fruit; lane 4, as in lane 3, but after 5 min at 65°C; lane 5, PG2 (0.2 Mg) and 0 subunit (0.05 Mg); lane 6, PG2 (0.2 Mg) and /3 subunit (0.1 Mg); lane 7, PC2 (0.2 Mg) and /3 subunit (0.2 Mg). Lanes 8 to 10, as in lanes 5 to 7, after 5 min at 65°C. b, A duplicate gel of that shown in (a) was run, electroblotted, and probed with anti-/3 subunit antibody. Cross-reaction was visualized with chemiluminescence and a 10-min exposure to x-ray film.
Extracts of tomato tissues, including fruit at four different developmental stages, were prepared, separated by SDSPAGE, and immunoblotted to determine the tissue distribution of the ft subunit. In Figure 4a total proteins from these extracts, as well as samples from the purification of PGl, were visualized by silver staining. Silver staining readily detects PG2 as the prominent 45-kD protein in ripening fruit and in purified PGl; however, the ft subunit is poorly stained and not detectable after silver staining. As shown in Figure 4b, and in agreement with previous results (Pogson and Brady, 1993a), anti-ft subunit antibody detected the 40-kD protein in all four stages of fruit tissue examined, with the ft subunit most abundant in partially ripe (turning) fruit. A dilution of purified ft subunit indicated that the limit of detection was less than 10 ng, or about 0.2% of the protein present in each lane. No cross-reactive protein was observed in either leaf or stem tissue extracts. The prominent 45-kD ripening-induced protein is confirmed as PG2 by its crossreactivity with anti-PG2 antibody (Fig. 4c). PG2 was detected in ripening fruit tissues but not in leaves or stems. As previously described, PG2 antibody does not recognize the ft subunit (Pogson et al., 1991). Therefore, the ft subunit appears to be present in the cell-wall fraction prior to the appearance of PG2, which suggests that it could be the source of converter activity in green fruit (Tucker et al., 1981). Thermostability of PG1 in Vivo
The relative abundance of PGl and PG2 appears to be highly dependent on extraction conditions, which has been
Tomato Polygalacturonase Isozyme 1
._ x
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1O0
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.-
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1 O0
Temperature ("C)
Comparison of pH and salt optima and thermostability for PG2 and PG1 isolated trom tomato fruit and PCl produced in vitro. All samples were preincubated for 30 min at 37°C to allow conversion of PG2 to PG1. Activity was determined by reducing sugar assay. Activity is plotted as a percent of maximal activity at each optima. A, pH optima. Assays contained 0.2 M NaCl and 0.1 M buffer (sodium formate, pH 3.0-4.4; sodium acetate, pH 4.0-5.4). For PC1, samples were heated for 5 min at 65°C before determination by reducing sugar assays. B, Salt optima. Assays contained 0.1 M sodium acetate buffer, pH 4.0, and increasing concentrations of NaCl as indicated. C, Thermostability of PC activity. Assays contained 0.2 M NaCI, 0.1 M sodium acetate and were incubated at the indicated temperature for 5 min and cooled to room temperature before assay. W, PC2; O, PC1; A, PG1 made in vitro from purified PC2 and 0 subunit.
Figure 3.
suggested as evidence for the absence of PG1 in vivo and its formation during extraction (Pressey, 1986b, 1988). Based on the results shown in Figure 3, as well as the observations of Pogson and Brady (1993a), PG2 can undergo conversion in vitro under a variety of pH or salt conditions, serving to underscore concems regarding the formation of PGl during extraction. Therefore, we sought to analyze the state of assembly of PGl in vivo by a criterion that could be applied prior to extraction. PGl exhibits a far greater thermostability than PG2 in vitro (Tucker et al., 1980), and if this differential thermostability is expressed in vivo it should be possible to test for the presence of heat-stable PG1 by heating intact tomato tissue to temperatures that would inactivate PG2 but not PG1 (Knegt et al., 1991; Pogson and Brady, 1993b). If PG1 forms artifactually from PG2 during extraction, both isozymes should exhibit similar kinetics of inactivation at low temperatures in vivo. Ripe tomato fruit pericarp was used for this experiment, because at this stage in fruit development the ratio of PG2 to PGl is typically greatest (Tucker et al., 1980), providing a basis on which to assess the efficacy of the heat treatment in vivo by determining the extent of PG2 inactivation. Analysis of the isoenzymes present after heating was performed by nondenaturing PAGE and activity staining, rather than with chromatography and enzyme analysis by reducing sugar formation (Knegt et al., 1991; Pogson and Brady, 1993b). Ripe fruit pericarp sections were incubated at 50 to 7OoC for 5 to 20 min and a high-salt extract was prepared from the tissue immediately after treatment. In parallel experiments, purified PG2 and PG1 were heated at either 50 or 7OoC and the results of the experiments are shown in Figure 5. Both isozymes appeared to be stable at 5OoC for at least 20 min in
vivo (Fig. 5a, lane 6) and 30 min in vitro (Fig. 5b). However, after incubation of pericarp tissue for 10 min at 6OoC, almost no PG1 activity was recovered, whereas a considerable proportion of PG2 activity remained (Fig. 5a, lane 8). This is in contrast to the situation in vitro, where PGl remained active after 30 min at 7OoC (Fig. 5b) or after 5 min at 85OC (Fig. 3). Interestingly, PG2 activity was still detectable after incubation of the tissue at 7OoC for 5 min (Fig. 5a, lane 10). The apparently greater heat stability of PG2 in vivo than in vitro could represent PG2 isolated from a part of the tissue that had not yet reached the incubation temperature. The considerably lower thermostability of PGl in vivo than in vitro is consistent with, but does not prove, the hypothesis that PGl is absent in vivo and forms by assembly of the p subunit and PG2 during the extraction process. Densitometric scanning of the results of the activity gel shown in Figure 5a was performed. At 6OoC, the rates of decay of PGl and PG2 were similar, suggesting that they could represent the loss of a single, rather than two, active PG species (data not shown). Alternatively, the time- and temperature-dependent generation or release of an inhibitor of PGl activity could account for the apparent loss of PG1 activity during heating in vivo. High-salt extracts from pericarp sections heated to 7OoC for either 5 or 30 min were incubated with fresh PG2 and the formation of thermostable (PG1) activity was determined. Both extracts efficiently converted PG2 to PG1, since heatstable PG activity was observed (data not shown). This result suggeststhat there were no specific inhibitors of PGl present in these extracts. It is still possible that inhibitors formed within the heated tissue but were removed or decayed during fractionation.
kDa 68-
(a)
Plant Physiol. Vol. 106, 1994
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1
2
—**
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~~
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——
-p-sub
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'Discussion"). This procedure provided the basis for the examination of the variables of time and temperature of incubation of pericarp high-salt extracts to determine their effect on the appearance of PG1. Aliquots from the high-salt extract of ripe pericarp were removed after 10-min intervals. These samples were analyzed and the results are shown in Figure 6a. Interestingly, PG2 activity was recovered as soon as the pericarp tissue was resuspended in the high-salt extraction buffer, which suggests that extended extraction of the cell walls may be unnecessary. After 60 min of extraction, cell walls were removed by centrifugation and the extract was divided into three portions for subsequent incubation. One portion was maintained at 4°C, and samples were removed at 10-min intervals (Fig. 6b). The other two portions were maintained at 22 or 37°C, and samples were removed as described above (Fig. 6, c and d, respectively). PG1 activity was not observed in the initial high-salt extract but was weakly detectable in extracts maintained at 4°C and was
-PG2
(a)
50 °C
Tem
P
PG1-
PG2-
1 (b)
Appearance of PC1 during Incubation of Extracts
If PG1 does not exist in assembled form in vivo, then its assembly during extraction or analysis must be sufficient to account for its previously reported presence in tissue extracts. For example, this could explain the presence of PG1 in lanes 4 to 8 of Figure 5. Until a point at which most of PG2 was inactivated, PG1 could still form during the extraction process in spite of precautions taken to minimize PG1 formation during analysis by rapid desalting and nondenaturing PAGE. Given the ability of PG2 and the 0 subunit to assemble under a range of conditions, the possibility of conversion of PG2 to PG1 in the high-salt extraction buffer during the course of extraction seemed reasonable. Knegt et al. (1988) described the kinetics of formation of a PGl-like activity from purified PG2 by a converter activity at 37°C over a time course of 30 min. Therefore, we reduced as much as possible the formation of PG1 during sample analysis by the rigorous maintenance of 4°C (see 'Materials and Methods"), the use of nondenaturing PAGE for rapid separation of PG1 and PG2, and the use of spin columns for rapid desalting of high-salt extracts in preparation of the samples for nondenaturing PAGE (see
70 °C
5 10 20 5 10 20 5 10 20
Time (min) Figure 4. The /3 subunit is present in green fruit tissue prior to the appearance of PC. a, Silver-stained proteins resolved by SDS-PACE on a 10% gel. Lane 1, Molecular mass markers; lane 2, high-salt extract of tomato leaves; lane 3, high-salt extract of tomato stems; lane 4, high-salt extract of green fruit; lane 5, high-salt extract of turning fruit; lane 6, high-salt extract of pink-light red fruit; lane 7, high-salt extract of red fruit; lane 8, purified Con A-bound fraction of ripe fruit high-salt extract; lane 9, Mono-S-purified PG1; lane 10, Superose 6-purified PCL b, Immunoblot of proteins with anti-/3 subunit antibody. Lanes 1 to 10 are as described in (a); lanes 11 to 15, purified 0 subunit: 200, 100, 50, 25, and 10 ng, respectively, c, Immunoblot as described in (b), stripped and reprobed with antiPC antibody.
60 "C
Temp
2
50 "C
3 4 5 6 70 °C
Time (min) 5 10 30 5 10 30
7 8 9 10 11 12
50 °C
70 °C
5 10 30 5 10 30
PG1-
PG2-
Figure 5. a, PC1 is much less thermostabile in vivo than in vitro. Five grams of ripe tomato pericarp was sectioned and sealed in plastic bags. The tissue was submerged in a water bath for the time and at the temperatures indicated, processed as described in "Materials and Methods," and analyzed by nondenaturing PAGE and activity staining. Lane 1, Purified PC1; lane 2, purified PG2; lane 3, extract from tissue maintained at 22 °C; lanes 4 to 6, extract from tissue heated to 50°C for 5, 10, or 20 min, respectively; lanes 7 to 9, extract from tissue heated to 60°C for 5, 10, or 20 min; lanes 10 to 12, extract from tissue heated to 70°C for 5, 10, or 20 min. b, Purified PG2 and PG1 were heated in vitro at the temperature and for the time indicated and analyzed as in (a). Approximately 0.5 /ig of protein was applied to each lane.
Tomato Polygalacturonase Isozyme 1 (a)
(b) 4°C
Time(min)
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o 3040
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37 °C
0 10 20 3040
-PG1
•PG2
Figures. PC1 activity is not found in 4°C extracts but is observed after extended incubation or with increased temperature of incubation, a, Aliquots from the initial HSE of ripe fruit pericarp prepared at 4°C were removed at the times indicated, snap frozen in liquid N2, and processed for nondenaturing PACE and activity analysis, b, After 1 h of extraction at 4°C, cell walls were removed by centrifugation, a sample of the supernatant of the high-salt extract was held at 4°C, and aliquots were removed at the indicated times, c, High-salt extract as in (b) was held at 22°C, and aliquots were removed at the indicated times, d, As in (b) and (c), but the sample was held at 37°C. Approximately 0.1 \i% of protein was applied to each lane.
slightly greater in extracts held at 22°C. However, after as little as 20 min at 37°C, PG1 was readily detectable and continued to increase in activity during further incubation. This increase could not represent increased solubilization of PG1 from cell walls because of their removal by centrifugation at the end of the extraction period. The absence of PG1 from the initial extract and the increase in PG1 activity with increasing time or temperature of incubation suggests that PG1 assembled in the extract at temperatures above 4°C. DISCUSSION Tomato Fruit Converter Activity and Characterization of the /? Subunit
Previous efforts to characterize a tomato fruit converter activity that 'converts' PG2 to PG1 focused on the isolation of a converter activity from heat-treated (100°C, 5-7 min) tissue or crude protein extracts (Pressey, 1984a; Knegt et al., 1988). The activity formed has not been completely characterized, possibly because of breakdown of the protein by the requisite high-heat treatment. This treatment appears to render a portion of the activity able to pass through a 10-kD membrane filter (Knegt et al., 1988), whereas molecular mass estimates of the converter based on gel filtration chromatography range from 81 to 102 kD (Pressey, 1984a; Knegt et al., 1991). This estimate is in contrast to the 38- to 40-kD ft subunit observed with SDS-PAGE (Moshrefi and Luh, 1983; Pogson et al., 1991). This discrepancy could be due to poor solubility of this protein (Pressey, 1984a) or interactions of its glycosylated groups with column matrices (Knegt et al., 1991). Because of its apparent heterogeneity and incomplete characterization, previous results from these converter preparations must be regarded with some caution. More recently, purification and characterization of the ft subunit from PG1 has been reported under conditions that avoid heat treatment and so may minimize protein degradation or denaturation (Pogson et al., 1991, 1993a). We have taken this approach to more completely characterize the ft subunit of PG1 and to determine if it has converter activity. Purified ft subunit was unequivocally shown to convert PG2
in vitro to a PG activity that is indistinguishable from PG1 isolated from fruit, in terms of thermostability, pH and salt optima, and migration and activity after nondenaturing PAGE. Pogson et al. (1991) reported that antibodies to PG2 do not recognize the ft subunit. Subsequently, Pogson and Brady (1993a) prepared antibodies to the ft subunit and to PG1 but found these antibodies to have extensive cross-reaction with PG2, which necessitated preadsorption to PG2 before their use. We prepared antibodies to the ft subunit, but our antiserum did not appear to cross-react with PG2, which is consistent with the lack of homology between the ft subunit gene (Zheng et al., 1992) and PG2 (Grierson et al., 1986; Bennett and DellaPenna, 1987). Anti-0 subunit antibodies detected a protein in green fruit, consistent with the presence of a converter activity in this tissue (Tucker et al., 1981) and consistent with the presence of the mRNA for this protein at this stage in fruit development (Zheng et al., 1992). The ft subunit protein appears to be most abundant during the turning stage of fruit development. However, it is still readily detected in ripe fruit even though the mRNA for this protein has severely declined by this stage (Zheng et al., 1992). Our detection by western blotting of the ft subunit only in fruit is in agreement with previous observations on the distribution of this protein and its mRNA (Zheng et al., 1992; Pogson and Brady, 1993a). Taken together, the characteristics of the ft subunit described here and elsewhere fulfill at least three of the characteristics associated with converter activity previously described: it is capable of rendering PG2 thermostable, it is present in green fruit tissue prior to the appearance of PG2, and it is an acidic protein with an isoelectric point of 5.1 (Pressey, 1984a). Based on this correspondence between the ft subunit and converter activity found in fruit, it appears likely that this polypeptide is the converter described by Tucker et al. (1981) in tomato fruit tissue. However, we did not observe cross-reaction of anti-j3 subunit antibodies to extracts from either tomato stem or leaf tissues, leaving unresolved the question of what constitutes converter activity previously described in these tissues (Pressey, 1984a).
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Moore and Bennett
Assembly State of PG lsozymes in Vivo
Based on the ability to differentially extract PG2 and converter activity, Pressey (1988) proposed that PG1 is an artifact formed during extraction by reaction of solubilized PG2 and converter. Recently, Pogson and Brady (1993b) have presented evidence that indicates that PG2, soluble in relatively moderate salt buffer, can react with /3 subunit still localized in the wall. Thus, it was suggested that differential extraction conditions are not sufficient to determine whether PG1 exists prior to extraction. Knegt et al. (1988) have provided evidence that reaction of converter and PG2 form a unique enzymatic activity, which they termed PGx. After further study, Knegt et al. (1991) concluded that PG1 consists of PG2 and converter protein in a 1:l ratio, whereas PGx, formed in vitro from excess PG2 and converter, probably has a ratio of PG2 to converter of 2:l. Thus, because PGx and not PG1 was formed from conversion of PG2, they concluded that PG1 is unliely to be an artifact. In contrast, our results suggest that PG1 produced in vitro by conversion of PG2 with the /3 subunit is identical to PGl isolated from fruit, supporting the possibility that PG1 could arise as an artifact of extraction. Knegt et al. (1991) employed temperature pretreatment of pericarp extracts in an effort to determine whether a pool of thermostable activity (PGI) exists in vivo. Almost 80% of the IJGl activity extractable from untreated tissue was lost upon preheating tissue to 65OC for 5 min. They concluded that the remaining heat-stable activity must have existed in vivo, prior to extraction. Pogson and Brady (1993b) have also examined the effects on PG activity of prior heat treatment of pericarp tissue at 6OoC for up to 1 h. They observed that the loss of PG activity in vivo appeared to have biphasic kinetics, with between 65 and 80% of total activity lost after 10 min and the remaining activity declining more gradually over the next 50 min. This pattem was similar to that they observed when thermal inactivation curves of PG2 and PGI, performed independently in vitro, were superimposed. They concluded that PG1 was present in vivo and represented the more thermostable activity. Based in part on the relative ease with which an enzymatic activity indistinguishable from PGl seemed to form in vitro from PG2 and the /3 subunit (Figs. 2a and 3), we re-examined the previous results of Knegt et al. (1991) and Pogson and Brady (1993b). Replotting and analysis of the data of Pogson and Brady (1993b; fig. 1)for the inactivation of PG in pericarp suggested that these data could be fit equally well by a firstorder decay or biphasic decay curve. First-order decay of PG activity would be consistent with the loss of a single active PG species in vivo. We employed a different analytical method, i.e. nondenaturing PAGE and activity staining, in an attempt to minimize the time before physical separation of PG2 and the /3 subunit after extraction of tissue segments. PG2 activity was found even after 5 min at 7OoC and PG1 activity was essentially lost after 10 min at 6OoC, even though PG1 heated in vitro is completely active after 30 min at 7OoC and retains activity after exposure to 85OC for 5 min. These results are consistent with the hypothesis that PG1 is formed by conversion of PG2 during extraction. The method of desalting samples for nondenaturing PAGE after tissue heat
Plant Physiol. Vol. 106, 1994
treatment was found to be critical. If the saniples were extensively dialyzed to remove salt, even in the cold, then activity was recovered as a diffuse zone approxlmately comigrating with PGl (data not shown) and probably represented conversion of residual PG2 by the p subunit. If PGl does not exist in vivo, then the rate of itl; formation during extraction must be significant enough to xcount for its observation. When extracts of ripe pericarp íissue were rigorously maintained at 4OC, only PG2 was observed in the extract. Incubation of this extract after remova1 of cell walls indicated that PG1 activity dramatically increased at 37OC within 20 min. This result, in addition to the obseilration that pericarp sections did not appear to contain thermostable PG activity, suggests that PGl does not occur in vivo. An alternative possibility is that PG1 exists in vivo but is dissociated by the high-salt buffer typically used in its extraction. This seems unlikely based on the relatively vigorous conditions needed to dissociate PGl(6-8 M urea, low pH) for purification of the p subunit, and the fact that PG1 is completely stable to incubation at extremes of pH (pH 11, Pressey, 1984a; pH 1.6, Pressey, 1986a). If PG1 is indeed an artifact of extraction, the chiiracteristics attributed to PG1, such as its appearance earlier than PG2 during ripening, and its lower relative abundance to PG2 are most likely due to relative differences in abundarice of the /3 subunit and PG2. Early in fruit development /3 sutanit would be more abundant than PG2, and extraction of j'ruit at this stage would lead to the appearance primarily of PGI, by conversion of PG2 with excess /3 subunit. Later in development, when PG2 becomes more abundant, the level of PG1 would appear to plateau, whereas the level of l'G2 would appear to increase, probably due to the titration of the /3 subunit. Reports on relative differences of PGI and PG2 between cultivars (Tucker et al., 1980; Knegt ei: al., 1988; Pressey, 1988) could also be due to differences in t he absolute amount of /3 subunit present in these cultivars, which could lead to different amounts of PG2 converted during extraction. Finally, procedural differences in the preparation of extracts and their analysis could lead to different levels of conversion of PG2 with concomitant differences in levels of PGI. ACKNOWLEDCMENT The helpful discussions with John Gardner are gratefully acknowledged.
Received June 22, 1994; accepted September 16, 1994. Copyright Clearance Center: 0032-0889/94/106/1461/l19 LITERATURE CITED
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