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Keywords:

  • Lycopersicon esculentum;
  • cell wall;
  • fruit growth;
  • peroxidase;
  • ripening mutants;
  • tomato

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall-stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent Mr 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non-ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild-type and commercial varieties. It is concluded that the late-appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene-induced. They may act to control fruit growth by cross-linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Tomato is a crop of worldwide economic importance, and the factors controlling its fruit growth have attracted considerable research interest. The rate and extent of fruit growth are central for crop yield; they are also important factors in major disorders such as cracking ( Peet 1992) and blossom end rot ( Ho & Adams 1989). In addition, they determine fruit size, which is a major determinant of quality in tomato, as in many other horticultural products.

In round tomato cultivars grown commercially in the UK, fruit development takes about 7 weeks, depending on temperature. During this time, the fruit grows from virtually zero to some 50–70 mm in diameter and about 70 g in weight (depending on variety). Most of this growth takes place during the period 15–35 d after anthesis ( Pearce, Grange & Hardwick 1993), and virtually all of it occurs by cell expansion rather than division ( Grange 1995). In tomato fruit, as in most plant organs ( Cosgrove 1993), cell expansion is believed to involve a yielding of the cell wall under mechanical stresses arising from turgor pressure. Understanding of the regulation of fruit growth will therefore require analysis of the processes controlling cell wall loosening.

In isolated cells such as those of Nitella, all the stress arising from a cell’s turgor pressure is borne by that cell’s wall. However, in a multi-cellular organ such as a plant stem or tomato fruit, the stresses arising from turgor pressure in one cell can be taken up by walls of distant cells. This leads to strains or ‘tissue tensions’ within the organ ( Kutschera 1989). When the organ is dissected, these strains are betrayed by partial relaxation of the freed parts. In the tomato fruit, such relaxations indicate that, as in growing stems and hypocotyls, most of the resistance to expansion is located within the thin outer skin ( Thompson, Davies & Ho 1998). This finding is supported by observations of cell dimensions: the outer skin (exocarp) comprises a single layer of epidermal cells plus about three layers of small compact thick-walled cells ( Ho & Hewitt 1986). By contrast, the mesocarp (which forms the bulk of the pericarp) is made up of about 30 layers of large thin-walled parenchyma cells. Some of these parenchyma cells are exceptionally large, up to 500 μm in diameter ( Ho & Hewitt 1986), and they have significant turgor pressure throughout most of the period of fruit growth ( Thompson et al. 1998 ). At a given turgor pressure, the stress in the wall of an isolated cell will be proportional to the square of the cell’s radius, and inversely proportional to cell wall thickness. Thus, stress in the thin walls of the large mesocarp parenchyma cells would become extreme unless much of it is exported to the skin.

Since the rate and extent of fruit growth is determined mostly by the skin, it is here that cell wall enzymes controlling fruit growth must be concentrated. Among putative wall-loosening enzymes, xyloglucan-endotransglycosylase (XET) activity shows a general correlation with growth rate in tomato fruit ( Thompson et al. 1998 ). Expansins may also be involved in wall loosening ( McQueen-Mason, Durachko & Cosgrove 1992) and have been reported from tomato fruit ( Rose et al. 1997 ).

With respect to the termination of fruit growth, the enzyme most often linked to decreases in wall extensibility is peroxidase. Correlations between peroxidase activity and the cessation of growth have been reported from a variety of plant organs ( Goldberg et al. 1987 ; Schnabelrauch et al. 1996 ; Bacon, Thompson & Davies 1997; Hohl, Greiner & Schopfer 1995), and peroxidases seem to be involved in the prevention of cell expansion during embryogenesis ( Cella & Carbonera 1997). Peroxidase-catalysed reactions which could stiffen the cell wall have been discussed ( Fry 1987; Everdeen et al. 1988 ; Schopfer 1996; Schnabelrauch et al. 1996 ).

In the tomato fruit, growth slows and ceases at 40–50 d postanthesis (DPA). No marked change in cell turgor pressure or in wall-loosening enzymes has been found at this time, but a marked increase in the activity of wall-bound peroxidase in the fruit skin was recently reported ( Thompson et al. 1998 ). This increase is here investigated using native gel electrophoresis, and its relationship with fruit growth is assessed with the aid of non-ripening mutants.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant material

Tomato plants (Lycopersicon esculentum [L] Mill cv Counter) of stem length up to 8 m were grown in rockwool in a glasshouse under semi-commercial conditions ( van de Vooren, Welles & Hayman 1986). Fruit of all stages were harvested as required, taking only the first (most proximal) fruit from any truss. For temperature treatments, plants of cv Espero were grown to the seven-truss stage in controlled environment rooms with lighting levels of 400 μmol m–2 s–1 applied as 12 h light/12 h dark. The plants were held at constant temperature of 26 or 18 °C. A nutrient film system was installed for these plants ( Winsor & Massey 1978).

Several non-ripening mutants, together with their parental line (cv Ailsa Craig) were grown in pots of peat-based compost under glasshouse conditions. The mutants were non-ripening (nor), ripening inhibited (rin), never ripe (Nr), colourless never ripe (Cnr) and greenripe (Gr) ( Darby, Ritchie & Taylor 1978; Grierson et al. 1987 ; Thompson et al. 1999 ). All are near-isogenic lines. The mutant fruit were kindly provided by Drs A. Thompson & G. Seymour of HRI Wellesbourne.

Peroxidase extraction

Pieces of mesocarp and strips of skin (exocarp) were cut from the equatorial region of washed fruit using a razor blade. About 30 mg was taken from each fruit. This, together with an equal weight of acid-washed sand, was ground thoroughly in a pestle and mortar in the presence of liquid N2. The powdered sample was suspended in ice-cold 10 m M Na-acetate/citric acid, pH 6·0, using 100 μL of buffer per mg of original fresh weight of tissue (OFW). This suspension was mixed and centrifuged at 3000 g for 15 min at 3 °C. The pellet was resuspended in the same buffer and again centrifuged. This process was repeated up to eight times to ensure that all the soluble peroxidase had been washed out. Supernatant from the final wash was assayed to confirm that soluble peroxidase had been reduced to a negligible level.

The washed pellet was resuspended in 100 μL mg–1 OFW of 100 m M Na-acetate/citric acid buffer, pH 6·0, containing 1 M NaCl. The suspension was mixed thoroughly and incubated on ice for 60 min with periodic shaking. The sample was then centrifuged at 3 °C for 15 min at 3000 g. The resulting supernatant contains the salt-extractable cell wall peroxidase; this is assumed to represents that fraction of peroxidase that is ionically bound to the cell wall in vivo ( Goldberg et al. 1987 ; Bacon et al. 1997 ).

Peroxidase assay

Soluble peroxidase was assayed in microtitre plates. The substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was preferred because of its low mutagenicity ( Bos et al. 1981 ). TMB was made up at 20 mg mL–1 in DMSO, and stored in aliquots at − 20 °C. An aliquot of 5 μL of sample was added to each assay well followed by 100 μL of 100 m M Na-acetate/citric acid buffer, pH 6·0, containing 0·1 mg mL–1 TMB and 0·5 μL mL–1 of 6% (w/v) H2O2. After 60 min, the reaction was stopped by 100 μL 0·6 M H2SO4 and absorbance was read at 450 nm. There was no reaction in the absence of H2O2. Enzyme activity was expressed by reference to a standard curve made with horseradish peroxidase (P6782, Sigma Chemical Co, Poole, UK). For localization of peroxidase (in gels and tissues), chloronaphthol is more appropriate than TMB because of the former’s insoluble reaction product (see below).

Separation of peroxidase isozymes by PAGE

Native polyacrylamide gel electrophoresis (PAGE) was performed using a BioRad Mini Protean II apparatus according to the manufacturer’s instructions. Stacking gels were made using 4% (w/v) acrylamide. Aliquots of 50 μL of test solution were mixed with 30 μL 80% glycerol and 30 μL 0·5% bromophenol blue, and 10 μL of the resulting solution were loaded onto each lane. Separating gels were made using 12% (w/v) acrylamide (Sigma A8887) in 375 m M Tris–HCl buffer, pH 8·8. These were run at 4 °C for about 90 min at 150 V. On completion, the gels were stained for peroxidase activity by immersion in 25 mL 100 m M Na-acetate/citric acid buffer, pH 6·0, containing 0·06% H2O2 and 0·06 mg mL–1 of chloronaphthol (freshly prepared at 0·3 mg mL–1 in methanol). Isozyme patterns shown here have each been repeated in at least five independent experiments.

Estimation of molecular mass

Salt-extractable wall proteins were prepared from a batch of mature fruit skin (cv Counter). They were de-salted by dialysis against 0·05 m M CaCl2 for 24 h at 4 °C, freeze dried, and stored at − 20 °C. This material was resuspended in 100 m M Na-acetate/citric acid buffer, pH 6·0, at 0·15 mg mL–1, and given a mild treatment with sodium dodecylsulphate (SDS) as follows: 20 μL of protein solution were added to 60 μL of 60 m M Tris buffer, pH 6·8, containing 0·05% mercaptoethanol and 1% SDS. This solution was incubated at 0 °C for 4 min. Some samples were also treated with boiling SDS (2% in the same buffer as above) for 4 min prior to electrophoresis.

PAGE was carried out as above, except that the gels and running buffer contained 2% SDS. A set of molecular mass marker proteins was included (Sigma SDS-7B). After running, the gels were immersed for 30 min in 100 m M Tris buffer, pH 6·0, containing 0·1% Triton X-100. This solution removes or neutralizes much of the SDS and should allow the isoenzymes to renature to some extent ( Akins, Levin & Tuan 1992). The gel was then rinsed in several changes of cold buffer for a further 30 min to remove the Triton. It was stained for peroxidase activity by immersion for 15 min in 25 mL Na-acetate/citric acid buffer, pH 6, containing 0·6 mg mL–1 of freshly prepared chloronaphthol and 0·06% H2O2. The gel was then placed in a solution of 0·1% Coomassie blue R-250 in fixative (40% v/v methanol in water plus 10% v/v acetic acid) for 30 min, and finally destained for 3 h in fixative.

Tissue printing

Washed fruit of a range of developmental stages were halved vertically (i.e. by a cut in a radial plane passing through the calyx insertion). The cut surfaces were rinsed repeatedly in 10 m M Na-acetate/citric acid buffer, pH 6·0, to remove soluble material. They were then weighted down lightly onto nitrocellulose paper for 3 min. The tissue was then removed and the nitrocellulose paper was stained for peroxidase activity using chloronaphthol, as above. Best results were achieved if the paper was first soaked in buffer containing 1 M NaCl. This is presumably because the salt aids desorption of ionically bound enzyme from the cell wall.

Localization under the light microscope

Strips of fruit exocarp were excised, frozen and thawed, and then rinsed extensively in 10 m M Na-acetate/citric acid buffer, pH 6·0, to remove soluble material. The strips were blotted on filter paper to remove excess moisture, mounted in embedding compound (Tissue-Tek; Agar Scientific, Stansted, UK) and sectioned at − 40 °C in a cryostat (Bright Ltd, Huntingdon, Cambridgeshire, UK). Sections were mounted on glass slides and stained for peroxidase activity using chloronaphthol, as above. Control sections were heated in the presence of dithiothreitol (DTT) to eliminate peroxidase activity prior to staining.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

PAGE on native gels showed that one or more peroxidase isozymes were present in most fractions from the mesocarp and skin of tomato fruit throughout their development ( Fig. 1). On a fresh-weight basis ( Fig. 1), as well as on a wall-weight basis (not shown), the skin contained more peroxidase activity than the mesocarp. Wall-bound peroxidase activity was negligible in the mesocarp. The skin of mature fruit contained three additional peroxidase isoforms which were absent from that of the immature fruit.

image

Figure 1. Native gels stained for peroxidase activity. Soluble (upper) and corresponding wall bound (lower) fractions are shown from the mesocarp and skin of tomato fruit at six stages of development from immature green through to pink. Apparent molecular weights are indicated (right). Values in brackets along the top row indicate approximate fruit age (DPA).

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Tentative molecular weights are assigned to the peroxidase isozymes in Fig. 1. These were estimated by SDS–PAGE after partial denaturation in SDS. Proteins are usually boiled with SDS prior to SDS–PAGE, but this inactivates the enzyme and prevents subsequent localization of activity on gels. The mild procedure used here, involving dilute SDS, without boiling, has been shown to permit separation on the basis of molecular mass, whilst preserving sufficient activity for localization on gels ( Heussen & Dowdle 1980; Akins et al. 1992 ). In our gels, the molecular-weight marker proteins, as well as sample proteins stained with Coomassie blue, ran to very similar patterns whether treated with cold or boiling SDS. This suggests that mild SDS–PAGE gives a good estimate of molecular mass with our material. The major peroxidase isozyme of immature fruit had an apparent Mr of 58 kDa, while the additional bands appearing towards fruit maturity had apparent Mr values of 44, 48 and 53 kDa. Values of about 42 kDa have been reported previously for tomato fruit peroxidases ( Marangoni et al. 1989 ). Trials on iso-electric focusing gels revealed that the wall-bound peroxidase from mature fruit comprised of only a single band at pI 4·6. This indicates that the various isoforms all have similar charge properties.

The relatively high activity of wall-bound peroxidase activity in the skin, especially in mature fruit, is clear from ‘tissue prints’ of salt-extractable peroxidase activity ( Fig. 2, upper panel). These prints also show significant activity associated with vascular tissue towards the calyx end, and with individual vascular bundles distributed regularly within the mesocarp. In Fig. 2, such vascular bundles are mostly cut transversely. In young fruit, much of the activity is associated with the developing seeds.

image

Figure 2. Wall-bound peroxidase isozymes in the skin of fruit at several stages of development. Upper panel: tissue prints of peroxidase. These were made by pressing the washed, cut surface of a halved fruit onto nitrocellulose paper impregnated with 1 M NaCl. Lower panel: isozymes on native PAGE. Isozymes in the wall-bound fraction are shown. Each lane is from fruit of the developmental stage depicted in the tissue print directly above it. These ranged from immature green (left) to red ripe (right).

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The additional, higher-mobility isoforms of wall-bound peroxidase (apparent Mr 42–53 kDa) develop progressively in the fruit skin during the ‘mature green’ stage of development ( Fig. 2, lower panel). This is the stage at which the fruit normally achieves its final size and stops growing ( Andrews 1995). Towards full red ripeness, there is also a decrease in the 58 kDa band in cell walls of the fruit skin.

Peroxidase patterns in the skin of mature fruit of several non-ripening mutants were compared with those of the wild type (cv Ailsa Craig) and the commercial variety (cv Counter). The mutants used (nor, rin, Nr, Cnr and Gr) are all believed to be non-allelic single-gene mutants ( Darby, Ritchie, & Taylor 1978; Grierson et al. 1987 ; Thompson et al. 1999 ). The fruit of these mutants grows in an apparently normal manner and to a near-normal size, but they show greatly delayed or reduced ripening; some fruit fail to ripen even after many weeks or months. In each of the mutants, the additional isoforms of wall-bound peroxidase were apparent in the skin by the time of fruit maturity ( Fig. 3, upper panel), as in the wild type and commercial varieties ( Fig. 1). In addition, wall-bound peroxidase in the skin of the rin mutant showed a progressive development of isozymes towards fruit maturity ( Fig. 3, lower panel) similar to that in the commercial material ( Fig. 1).

image

Figure 3. Wall-bound peroxidase isozymes in fruit of non-ripening mutants. Upper panel: wall-bound isozymes from the skin of mature fruit of various mutants. Lower panel: wall-bound isozymes from the skin of rin fruit at various stages of development from immature (left) to mature (right).

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Elevated temperatures are known to promote fruit growth rate in the short term ( Pearce et al. 1993 ). However, the final fruit size may be unaffected or even reduced at elevated temperatures, because the duration of the growing period is reduced ( de Koning 1994). This means that fruit growth will cease earlier at high temperature. Native PAGE revealed that the new, more mobile isoforms also appear in the fruit skin earlier when fruit are exposed to elevated temperatures (not shown).

In some organs, such as the hypocotyl of Lupinus, peroxidase in the outer layers appears to be associated with the cuticle rather than with the cell wall ( Ferrer, Munoz & Ros Barcelo 1991). This was not the case in tomato fruit. Freeze–thawed preparations of the skin of mature fruit were washed thoroughly and stained with chloronaphthol. Light microscopy revealed that peroxidase activity in this tissue was associated with the thick hypodermal cell walls, and that there was negligible activity in the cuticle itself ( Fig. 4).

image

Figure 4. Localization of wall-bound peroxidase in tomato skin. Freeze–thawed sections were rinsed thoroughly, stained for peroxidase, and viewed under the light microscope. To serve as a control, the section in (b) was boiled briefly with DTT to inactivate peroxidases prior to addition of the chloronaphthol substrate. Staining is apparent as dark regions over the cell wall region in (a) but not the cuticle. No staining is apparent in (b). Scale: the epidermal cells in (a) are about 20 μm wide.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

In common with many previous workers ( Goldberg et al. 1987 ; Thompson et al. 1998 ; Barcelo, Munoz & Sabater 1987), we infer that the salt-extractable ‘wall-bound’ peroxidase is located within the cell wall in vivo. An alternative possibility is that some of the salt-extractable enzyme becomes associated with the cell wall by non-specific binding during homogenization ( Schloss, Walter & Mader 1987). This possibility cannot be excluded at present; however, it seems unlikely given the extensive washing procedures employed here. In addition, cell wall and apoplastic fluids certainly can contain substantial peroxidase activity in vivo, as revealed by histochemical and infiltration techniques ( Goldberg et al. 1987 ; Parish 1975). Also, a number of consistent differences were observed between the pattern of isozymes in the wall-bound and soluble fractions: for example, the 58 kDa band diminishes substantially in the wall-bound fraction, but not in the soluble fraction of the skin towards full fruit maturity ( Fig. 2). Likewise, the cell walls of the mesophyll show negligible salt-extractable peroxidase even when the soluble activity in that tissue is high ( Fig. 1).

Barcelo et al. (1987) employed a similar extraction procedure with lupin hypocotyls, and showed that cell wall material contained salt-extractable peroxidases but no significant quantity of cytoplasmic marker enzymes. Similarly, preliminary studies in our laboratory failed to find any significant rebinding of soluble peroxidase from mature fruit onto salt-extracted cell walls from skin or mesocarp of fruit of a range of developmental stages. These various lines of evidence indicate that the salt-extractable peroxidases are present in the wall in vivo.

The extraction procedures used here probably measure the appearance of new isoforms rather than changes in the extractability of existing forms. This is because tests on the residual cell wall material remaining after extraction showed little or no peroxidase activity. Furthermore, no additional isoforms could be released by extraction with stronger salt solutions (up to 3 M NaCl was tried, as was 200 m M CaCl2). A solution of 1 M NaCl was used routinely for consistency with previous work. In many organs, peroxidase is associated with sites of lignification. It may be involved in polymerization of lignin components ( Lagrimini, Bradford & Rothstein 1990). This may account for the concentrations of peroxidase found over the vascular tissue in older fruit, and over the developing seed coats in young fruit ( Fig. 2). Presumably, both will be sites of lignification. By contrast, very little lignification occurs in the fruit skin, and peroxidases there must play a different role. The results presented here demonstrate that new peroxidase isozymes of apparent Mr 44, 48 and 53 kDa appear in cell walls of the fruit skin coincident with the period of cessation of fruit growth ( Fig. 2, lower panel). No such bands appear in cell walls of the mesocarp ( Fig. 1). Given that fruit growth depends mainly on the mechanical properties of the skin, and that peroxidases may stiffen cell walls ( Fry 1987), these isozymes could be causally involved in the cessation of fruit growth. This possibility is supported by the finding that high temperatures, which advance the cessation of fruit growth ( de Koning 1994), also hasten the appearance of the new isoforms.

Around the time of cessation of fruit growth or shortly thereafter, various developmental changes associated with fruit ripening will begin. It is possible that enzymes appearing at this time are associated with these changes rather than with the control of growth. Ripening is associated with changes in the mechanical properties of the cell wall, and it is likely to involve a range of new wall hydrolytic enzymes ( Hobson 1964; Maclachlan & Brady 1992; Rose et al. 1997 ; Smith, Starrett & Gross 1998). To determine whether the late-appearing peroxidase isoforms are associated with ripening, a range of non-ripening mutants were examined. These mutants are non-allelic and have lesions which compromise fruit ripening in different ways ( Thompson et al. 1999 ). The mutants have near-normal patterns of fruit growth. In each of the mutants, the mature fruit skin was found to contain a series of wall-bound isoforms very similar to those of the wild-type and commercial varieties ( Fig. 3, upper panel; compare with the mature stage in Fig. 2, lower panel). In at least one of the mutants (rin, probably the least ripening of all these mutants), there was a progressive appearance of the more mobile isoforms towards fruit maturity ( Fig. 3, lower panel) as in the commercial, ripening material. Since fruit softening and ripening are greatly reduced and delayed in the mutants, these findings indicate that the new wall-bound peroxidase isozymes are unlikely to be associated with softening or ripening.

Similarly, increased ethylene production is a common feature in the ripening of climacteric fruit, including tomato ( Andrews 1995), and ethylene can induce peroxidases in a range of tissues ( Ridge & Osborne 1970). It is therefore conceivable that the new isoperoxidases are induced by ethylene concomitant with ripening. However, some of the mutants tested here (particularly rin and nor) lack ethylene receptors and/or ethylene synthesis, yet the new wall isozymes appear in these as in normal fruit. This indicates that the new isoforms are probably not induced by ethylene.

These results support the conclusion of Thompson et al. (1998) that a correlation can be drawn between the cessation of fruit growth and the activity of wall-bound peroxidase in the fruit skin. They go further in showing that several new isoforms of peroxidase appear in the cell walls of the skin at around the time that fruit growth is terminating. Ku et al. (1970) also used electrophoresis to demonstrate changes in the pattern of peroxidase isozymes in tomato fruit towards maturity. However, their measurements did not distinguish between cytoplasmic and wall-bound enzyme, nor between mesocarp and skin. They interpreted their results in terms of changes associated with ethylene synthesis and ripening, rather than with control of fruit growth.

The late-appearing isozymes in the skin cell walls might develop as a result of changes in gene expression, but it is perhaps more likely that they arise through post-translational modifications. Such modifications are common among peroxidases ( Lagrimini et al. 1990 ; Bestwick, Brown & Mansfield 1998).

If the late-appearing isozymes are involved in stiffening of the cell walls, they must have different substrate specificity in muro from the major isozyme (Mr 58 kDa) which is present throughout most stages of development. It may seem unlikely that these isozymes could have such different substrate specificities. However, marked differences in substrate specificity are often recorded among peroxidase isozymes ( Schopfer 1996), especially towards macromolecular substrates such as those of the cell wall ( Schnabelrauch et al. 1996 ). These different specificities may depend on subtle changes in folding of the enzyme, and can occur even when there is no difference in specificity towards substrates of low molecular weight ( Schnabelrauch et al. 1996 ).

It is concluded that new peroxidase isozymes appear in cell walls of the skin towards fruit maturity. These are not involved in fruit ripening or senescence, but they could, by altering the mechanical properties of the fruit skin, be responsible for the cessation of fruit growth. They may offer new approaches for the control of fruit size in tomato.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This work was funded by MAFF grant HH1323. The authors are grateful to Drs A. Thompson, G. Seymour, S. Adams, R. Napier, and Ms. C. Cave, of HRI Wellesbourne, for provision of plant material and for helpful discussion.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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