Journal of Experimental Botany, Vol. 51, No. 344, pp. 529-538,
March 2000
© 2000 Oxford University Press
Endo-ß-mannanase activity increases in the skin and outer pericarp of tomato fruits during ripening
1 Department of Botany, University of Guelph, Guelph, Ontario N1G 2W1, Canada
2 Department of Plant Physiology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
Received 21 June 1999; Accepted 29 October 1999
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Abstract |
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Activity of endo-ß-mannanase increases during ripening of tomato (Lycopersicon esculentum Mill.) fruit of the cultivar Trust. ß-Mannoside mannohydrolase is also present during ripening, but its pattern of activity is different from that of endo-ß-mannanase. The increase in endo-ß-mannanase activity is greatest in the skin, and less in the outer and inner pericarp regions. This enzyme is probably bound to the walls of the outermost cell layers of the fruit during ripening, and it requires a high-salt buffer for effective extraction. The enzyme protein, as detected immunologically on Western blots, is present during the early stages of ripening, before any enzyme activity is detectable. The mRNA for the enzyme is also present at these stages; endo-ß-mannanase may be produced and sequestered in a mature-sized inactive form during early ripening. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-ß-mannanase activity, but a cause-and-effect relationship between the enzyme and ripening is unlikely because some cultivars which ripen normally do not exhibit any endo-ß-mannanase activity in the fruit.
Key words: Tomato, Lycopersicon esculentum, endo-ß-mannanase, softening, ripening.
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Introduction |
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The tomato fruit cell wall is composed of protein and three major polysaccharide components, pectin, hemicelluloses and cellulose (Gross, 1984
). The hemicellulose polymers include xyloglucans, glucomannans and galactomannans which are covalently linked to pectin and hydrogen-bonded to cellulose (Tong and Gross, 1988; Fischer and Bennett, 1991). Polygalacturonase (EC 3.2.1.15) is an enzyme which can solubilize pectin and it increases in activity during tomato fruit ripening (Hobson, 1964). Much is now known about the regulation and expression of this enzyme (Grierson, 1985; Fischer and Bennett, 1991; Hobson and Grierson, 1993) although it may not play a major role in the softening of fruit in situ (Brummell and Labavitch, 1997). Since mannans are a component of tomato fruit cell walls the activity of endo-ß-mannanase (EC 3.2.1.78), the enzyme which hydrolyses these polymers, has been studied and it has been suggested that it plays a role in fruit ripening. There are reports on the presence of this enzyme in ripening tomato fruit (Pressey, 1989; Hong et al., 1996; Sozzi et al., 1996), but the amount of activity extracted, using acidified water, was very low. Two of the studies (Pressey, 1989; Sozzi et al., 1996) reported an increase in endo-ß-mannanase activity during fruit ripening, and one (Hong et al., 1996) a decrease.
Endo-ß-mannanase is very active in tomato seeds during and following germination; it is highly soluble and is present in many isoforms (Voigt and Bewley, 1996; Toorop et al., 1996). A cDNA for the enzyme has now been obtained from germinated seeds (Bewley et al., 1997). Using improved extraction and colorimetric assay techniques (Downie et al., 1994; Dirk et al., 1995), antibodies from the purified seed endo-ß-mannanase (Nonogaki et al., 1995) and the cDNA clone, it is shown in this paper that enzyme activity increases during fruit ripening of cv. Trust, and is concentrated in the outer regions of the fruit. The outer pericarp loses its integrity and becomes very soft during the late stages of ripening. Non-ripening mutants of tomato were also tested for their endo-ß-mannanase, and no consistent pattern of activity was found to link the enzyme to ripening.
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Materials and methods |
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Plant material
Fruits of tomato (Lycopersicon esculentum Mill.) cv. Trust were obtained from The Greenhouse, Brampton, ON. Some experiments were conducted initially on fruits of another market-ready cultivar, Moneymaker, which was grown in environmentally-controlled chambers in Wageningen, The Netherlands. Fruits were staged according to their degree of ripeness. The approximately equivalent stages (denoted by Gillaspy et al., 1993
) are noted in brackets. Full-sized green fruits were designated as stage 1 (mature green); light orange/green coloured, stage 2 (breaker/turning); uniform orange colour, stage 3 (orange); dark orange/red, stage 4; uniform red, stage 5 (red firm); red-ripe/slightly soft, stage 6 (red ripe); very soft red-ripe, stage 7. The smaller, immature hard green fruit was called stage 0 (2.3 cm diameter) since it is at a pre-ripening stage (Fig. 1). Non-ripening mutants of tomato were grown in a greenhouse using natural lighting conditions from seeds kindly supplied by The CM Rick Tomato Genetics Resource Center, UC Davis, CA, USA and Dr Maarten Koornneef, Wageningen Agricultural University, The Netherlands.
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Endo-ß-mannanase extraction and assay
Tomato fruits from all stages were dissected into three regions, and measured fresh weights of skin (exocarp, approximately 250 mg), outer pericarp region (hypodermis and outer mesocarp): the first 2 mm inside the skin (1–1.5 g), and inner pericarp region (mesocarp): 3–10 mm inside the skin (1–1.5 g), were extracted fresh or after freezing in liquid N and storage at -65 °C. Loose tissue was scraped from the inside of the skin following its removal from the fruit, and the skin was thoroughly washed in distilled water. Samples were ground in a mortar and pestle, with washed sea sand, in 1.5–2 ml McIlvaine (pH 5) buffer (0.2 M Na2H PO4.7H2O, 0.1 M citric acid) plus 0.5 M NaCl (McIlvaine, 1921; Halmer and Bewley, 1979). After a clearing spin at full speed in an Eppendorf microfuge at 5 °C the supernatant was adjusted to a known volume (usually 2 ml) and aliquots were used for enzyme assays or gel separation for Western blots or silver staining. Initial procedures to optimize enzyme extraction involved McIlvaine buffer (pH 5) without NaCl added, or 0.1 M HEPES-NaOH buffer (pH 8) with or without 0.5 M NaCl (see Results and discussion). Neither the pH of the extraction buffer, nor the presence of salt affected the activity of the enzyme in the gel assay.
For endo-ß-mannanase assay, a variation developed by Dr M Oluoch, CPRO, Wageningen, The Netherlands (personal communication) of the gel diffusion assay (Downie et al., 1994; Toorop et al., 1996) was used. A 0.5 mm thick gel of 0.1% (w/v) locust bean gum galactomannan (Sigma) in 1.2% (w/v) agarose (Sangon, Scarborough, ON) was poured on Gel-Bond film for agarose (Pharmacia) and 2 mm diameter wells punched out 2 cm apart. Samples (2 µl) were pipetted into the wells, and serial dilutions of Aspergillus niger endo-ß-mannanase used as standard (Megazyme, Bray, Eire). The gel was incubated overnight at 25 °C in a humid atmosphere before placing in McIlvaine buffer (pH 7) for 30 min, 0.5% Congo red (Sigma) for 30 min, water for 2 min, 80% (v/v) ethanol for 10 min, McIlvaine buffer (pH 7) for at least 1 h, and finally several changes of 1 M NaCl until color development was complete (approximately 1–2 h). The gels were computer-scanned into a Bitmap program, the clearing zones on the printout were measured with calipers, and activity expressed in relation to A. niger standards (Downie et al., 1994).
ß-Mannoside mannohydrolase (EC 3.2.1.25) assays
Activity was determined in high- and low-salt-buffer extracts of weighed outer regions of the fruit (skin, approximately 150 mg; pericarp regions, 0.7–0.8 g). The fruit tissue was frozen in liquid N and stored at – 65 °C until extracted. High-salt extracts were made using a HEPES-NaOH buffer (pH 8) or McIlvaine buffer (pH 5) (1.5–2 ml) with 0.5 M NaCl added; low-salt buffer extracts were without NaCl. The assays were conducted in microtitre plates, into the wells of which were pipetted 75 µl McIlvaine buffer (pH 5), 15 µl 10 mM p-nitrophenyl-ß-
-mannopyranoside (Sigma) substrate dissolved in the same buffer and 60 µl enzyme extract. After incubation at 37 °C for 120 min, 75 µl 0.2 M Na2CO3 was added as stop reagent. The yellow colour which developed due to enzymic release of p-nitrophenol was measured using a microtitre plate reader at 405 nm. Control assays consisted of adding substrate after the stop reagent, and these blanks were used for zero calibration. The molar extinction coefficient for p-nitrophenol was taken as 18 400 (Reid and Meier, 1973) to calculate the amount released as pmol min-1 g-1 fr. wt.
SDS-PAGE, Western blots and tissue prints
A polyclonal rabbit antibody to the purified M1 form of tomato seed endo-ß-mannanase (Nonogaki et al., 1995) was generously provided by Dr Yukio Morohashi, Saitama University, Japan. Equal amounts of protein (16 µg) were loaded in each lane and separated on SDS-PAGE (12%T) gels (Laemmli, 1970) and transferred to nitrocellulose membranes by Western blotting (Towbin et al., 1979) before being challenged with a 10-4 dilution of primary antibody. Detection of binding of the antibody to endo-ß-mannanase was achieved using an anti-rabbit HRP-labelled secondary antibody and the enhanced chemiluminescence (ECL) method (Amersham).
Proteins from the combined skin and pericarp regions of the various stages of fruit development were separated by SDS-PAGE (16 µg protein per lane) and silver stained using Silver-Stain Plus (BioRad).
Localization of endo-ß-mannanase within tomato fruits was determined by placing freshly-cut surfaces of the fruit on a 0.5 mm thick polyacrylamide (10% T, 0.9%C) activity gel containing 0.1% locust bean gum in McIlvaine buffer (pH 5) and 0.5 M NaCl. After incubation for 15 min in a humid atmosphere at 37 °C the gel was stained with Congo red and developed as above.
Laser-scanning confocal microscopy
A small cube of tissue (8 mm3) was cut from ripening tomatoes to include the skin and pericarp and fixed for 1 h in 25 mM phosphate buffer (pH 7.2) and formaldehyde (3%, v/v). Thin slices were then made with a razor blade and washed with phosphate buffer saline (PBS: 137 mM NaCl, 5.4 mM Na2HPO4, pH 7.4). The sections were blocked with 2% (w/v) skim milk in PBS for 30 min, with several changes, and then incubated in the same solution plus 0.3% (v/v) Triton X-100 and primary endo-ß-mannanase antibody (diluted 500-fold) overnight at 4 °C. The sections were rinsed five times for 5 min with PBS and then incubated with a fluorescent-labelled secondary antibody (anti-rabbit IgG-Rhodamine, Sigma) diluted 200-fold in PBS containing 2% skim milk and 0.3% Triton X-100 for 3 h in darkness at room temperature. Finally, the sections were rinsed three times with PBS for 5 min each and mounted on a microscope slide in PBS and glycerol (1 : 2, v/v) for viewing with a Laser Scanning Confocal microscope (MRC 600, BioRad). Control sections were made in PBS buffer, primary or secondary antibody alone, or pre-immune serum (diluted 500-fold) and secondary antibody. Only pre-immune serum controls are shown (Fig. 5) since the other controls were less reactive.
Analysis of tomato fruit texture
This was analysed and described in terms of firmness, which is the amount of force applied (N) per unit of tissue deformation (mm) when the fruit is penetrated with a probe. The raw data, in the form of force-deformation curves, was obtained using a Model 1122 Instron Universal Testing machine equipped with a 5 cm long, 2 mm diameter cylindrical, flat-tipped probe in the chuck which was attached to the crosshead of the machine. The crosshead speed was set at 20 mm min-1. For each puncture, the slope (N mm-1) was determined from the point of probe-fruit contact to the point of exocarp puncture (Adegoroye et al., 1989; Jackman et al., 1990).
For each mutant or wild-type tomato, three fruits from three ripening stages were analysed: mature green, turning and ripe (although this was not complete for most mutant lines). All fruits were punctured four times around the equitorial region, avoiding any locular ridges. The average of these values was taken as the firmness of that particular fruit; this and the standard deviation of the three fruits tested at each ripening stage were then plotted.
RNA isolation and Northern hybridization
Total RNA was prepared using the hot phenol/LiCl method (Verwoerd et al., 1989) from stage 0–7 skin and underlying pericarp following initial grinding of the tissue in liquid N2. Poly A+ RNA was purified from the total RNA using the PolyAT Tract mRNA Isolation System IV (Promega, Madison, WI), and 72 h germinated tomato seeds was used as a positive control. The poly A+ and total RNA samples were fractionated in 1.2% (w/v) agarose gels in 2.2 M formaldehyde and transferred to Zeta-probe nylon membrane (Bio-Rad) (as described by Sambrook et al., 1989). The membrane was prehybridized and then probed with 
-32P-labelled tomato seedling cDNA (Bewley et al., 1997), labelled with [-32P] ATP (ICN, Costa Mesa, CA) using a random-primed DNA labelling kit (Boehringer, Mannheim). Following hybridization for at least 24 h at 42 °C, the membrane was washed with 2x SSC, 0.1% (w/v) SDS at 42 °C, for 20 min (Sambrook et al., 1989) and then 1x SSC, 0.5x SSC and 0.1x SSC, all with 0.1% (w/v) SDS, at 42 °C for 20 min each. The final wash was in 0.1x SSC, 0.1% (w/v) SDS at 55 °C for 20 min before drying and exposure to Kodak X-OMAT X-ray film.
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Results and discussion |
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Endo-ß-mannanase requires high-salt buffers for extraction from tomato fruits
In the first report on endo-ß-mannanase activity in tomato fruits, 1 kg of pericarp tissue extracted in acidified water was used (Pressey, 1989
). Very low amounts of activity were obtained; subsequent reports on the enzyme in ripening tomato fruits also used the same extraction technique. Using this extraction technique on gram quantities of red-ripe tissue from the outer regions of the fruit it was not possible to detect any activity. McIlvaine buffer (pH 5) and HEPES buffer (pH 8) were both effective in extracting some enzyme activity at the red-ripe stage, but even this was low (1–15%, depending on the region of the fruit extracted) compared to the amount extracted when salt (0.5 M NaCl) was added to either extraction buffer. McIlvaine buffer (pH 5) with salt extracted approximately 25% more enzyme activity than did the HEPES buffer (pH 8) with salt. The distribution of extractable enzyme activity in the fruit was uneven, with most being obtained from the skin, from which the attached outer pericarp tissue was scraped, and the skin washed. The outer pericarp (approximately the first 2 mm of tissue immediately beneath the skin) contained less activity, with the inner pericarp (from 3 mm up to 10 mm from the skin depending on the thickness of the pericarp in a particular fruit) containing much less again. The inner firm locular tissue, the gel-like locular tissue and the sheath surrounding the seed were assayed for endo-ß-mannanase activity after high-salt extraction, and none was found. In addition to the cv. Trust, two other red-ripe market-ready tomato cultivars (one greenhouse-grown and another field-grown, cvs unknown) were extracted for endo-ß-mannanase activity, and one processing cultivar (cv. Heinz 902). All showed the same enhanced extraction of enzyme by salt, and the same distribution of activity within the fruit. Higher salt concentrations in the buffer did not yield more endo-ß-mannanase activity. The requirement for high salt in the extraction medium suggests that the enzyme is tightly associated with, or bound to cell walls. This low solubility of endo-ß-mannanase in fruit is in contrast to the highly soluble nature of the seed enzyme, which is readily extractable in low-salt buffers (Voigt and Bewley, 1996; Toorop et al., 1996).
Endo-ß-mannanase activity increases in the fruit during ripening
Enzyme activity increased greatly in the skin of tomato fruits, cv. Trust, following stage 2, the breaker/turning stage, peaking when the fruit was firm and orange-red, at stage 5 (Fig. 1). Increases in activity also occurred in the outer pericarp tissue, reaching a maximum one stage later, and the inner pericarp tissue where the increase started last (stage 4) and reached a lower peak of activity at stage 6. Because the two pericarp layers have only an approximate demarcation line separating them, and both contain mesocarp tissue, some overlap in activity due to inconsistent dissection of the tissue probably occurred. Nevertheless, it is clear that most activity was associated with the skin and the region immediately beneath it throughout fruit ripening. The high activity of the enzyme in the skin to some extent could be due to the larger amount of cell wall material with which it is associated, compared to the two other tissues, i.e. there are more and smaller cells in this region that are densely packed together. Dry weight of the skin accounts for about 21% of fresh weight, whereas in the two pericarp regions only about 10%; it is reasonably assumed that cell walls account for most of the dry weight. Thus, on a per-cell basis, the maximum activities (Fig. 1) of endo-ß-mannanase in the three regions are closer than indicated by the fresh-weight measurements, although the skin of the ripening fruit clearly has more total activity than the outer pericarp per unit volume, which in turn has more than the inner pericarp.
Stage 0 fruits exhibited no activity, nor did any of the inner fruit tissues or seeds at any stage of development (not shown). Using the similar, but less sensitive Petri-dish assay (Downie et al., 1994) for endo-ß-mannanase, an increase in activity in the fruit of cv. Moneymaker during ripening was also found, with activity peaking at stages 5 and 6. Activity in the skin of this cultivar was approximately six times greater than in the combined pericarp regions at its peak.
Variation in enzyme activity occurred within the same fruit if it ripened unevenly. Segments of skin and pericarp together were cut from a pale green, green-orange and red region of the same fruit and assayed for endo-ß-mannanase activity. The amount of activity was greater in the riper regions than in the green regions (not shown). Hence the presence of endo-ß-mannanase was related to the ripeness of the fruit, and not solely to its age. Thus for experiments it was important to use uniformly ripening fruits.
ß-Mannoside mannohydrolase activity does not follow the endo-ß-mannanase pattern
Following initial hydrolysis of the galactomannan backbone by endo-ß-mannanase, further modifications are effected by ß-mannoside mannohydrolase (exo-ß-mannanase) to cleave the small mannose oligomers. Despite the fact that the two enzymes might be expected to work co-operatively to mobilize galactomannans during ripening, their activity patterns were considerably different.
In lettuce seeds, ß-mannoside mannohydrolase requires a high-salt buffer for extraction (Ouellete and Bewley, 1986). In the tomato fruit skin and pericarp regions this enzyme is also most soluble in a high-pH, high salt buffer (Fig. 2) with optimum activity at an assay pH of 5. Endo-ß-mannanase has also been determined to be most extractable in high-salt buffer, and thus it is likely that both this enzyme and the mannohydrolase are present in a bound form in the cell, perhaps in the same compartment, the cell wall. However, their activity patterns during fruit ripening were very different, with mannohydrolase activity declining sharply in the skin, the region of most activity, as endo-ß-mannanase increased (Figs 1, 2). Whether these enzymes can and do work co-operatively is therefore debatable. While the mannohydrolase exhibited a steady decline in activity in the skin during ripening, following stage 3 (Fig. 2), both pericarp regions contained about the same low and steady amount of activity.
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Protein changes during ripening, and detection of endo-ß-mannanase
The pattern of high-salt buffer-soluble proteins in the combined skin and pericarp regions changed in the fruit during ripening, with a band of approximate molecular mass of 40 kDa becoming more prominent from stage 2 onwards, and one of approximately 50 kDa increasing at stage 4 (Fig. 3A). A Western blot of proteins extracted from the outer regions of fresh tomato fruits, using an antibody to tomato seed endo-ß-mannanase, showed a faint reaction with a band at approximately 40 kDa at stages 2 and 3 (barely visible at stage 2), strong reaction at stages 4–6 and a weaker one at stage 7 (Fig. 3B). The presence of the enzyme, as detected by the Western blot, thus coincided well with the increase and decline in its activity within the fruit. Other bands also reacted with the antibody, probably spuriously, at all stages of ripening; the 45 kDa band was the only one to react with the pre-immune serum (not shown). There is no coincidence between the bands reacting with the endo-ß-mannanase antibody and those most predominantly silver-stained; this is consistent with the observation in several seeds that the enzyme is a very minor fraction of the total cell proteins, even though its activity in crude extracts can readily be measured (Dulson and Bewley, 1989).
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Endo-ß-mannanase from the tomato seed is composed of 346 amino acid residues with a calculated Mr of 38 950, and a pI of 5.4 (Bewley et al., 1997
). Thus the immunologically-reactive, approximately 40 kDa band is most likely to be the active form of endo-ß-mannanase, although the fruit enzyme is somewhat different from the seed enzyme in that the major form has a pI of about 9 (Pressey, 1989). The bands which cross-reacted with the antibody in fruit extracts were of identical molecular mass to the forms extracted from germinated seed (Fig. 3C). When proteins extracted from fruit tissues that had been frozen in liquid N2 and stored at -65 °C were used for SDS-PAGE and Western blots, a different pattern of immunoreactivity was obtained (Fig. 3C). Now, the antibody bound to the ~40 kDa protein band from stages 1–7 of ripening (but not at stage 0, not shown), as well as to the higher molecular mass bands. At stages 6 and 7 some bands of lower molecular mass were detected, but very faintly, and these could have been degraded enzyme. Following extraction after freezing of the tissue, two immunoreactive bands of ~40 kDa became apparent; this may be due to the release of a second isoform by the extraction procedure. Endo-ß-mannanase in the tomato fruit was only extractable in a high-salt buffer, which is indicative of its association with a macromolecular structure, namely cell walls, by hydrogen bonding. Its resistance to high-salt extraction was decreased by rapidly freezing the fruit tissues, which presumably disrupted the structural integrity of the cell wall and rendered associated proteins more soluble. Although there were differences on the Western blots between enzyme immunoreactivity in extracts from fresh or frozen tissues, there were no differences in enzyme activities between these extracts at any stage of ripening. Thus endo-ß-mannanase protein was present in the outer tomato fruit tissues from the full-size green stage of development (stage 1) to the over-ripe stage (stage 7), but it was only active from stage 3 onwards. At the earliest stages the enzyme was presumably sequestered in a processed (~40 kDa), but inactive or inactivated form.
Localization of endo-ß-mannanase the outer region of the fruit by tissue printing and microscopy
A tissue print of a stage 6 tomato fruit was made by placing a cut fruit on an acrylamide gel containing locust bean gum substrate (Fig. 4). Enzyme activity, as detected by dissolution of the substrate was clearly visible in the peripheral regions. Incorporation of 0.5 M NaCl into the gel was necessary to solubilize the endo-ß-mannanase to detect its activity, but a short exposure of the fruit was necessary to prevent diffusion of the solubilized enzyme over the gel in the fruit sap. While placing the fruit for a longer time in contact with the substrate gel, or pressing the fruit harder into the gel, resulted in a complete circle of activity around the periphery of the fruit, diffusion of the enzyme resulted in a much wider band.
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Using confocal microscopy and a fluorescent tag to identify the endo-ß-mannanase antibody it was possible to localize this enzyme within fixed sections of the outer regions of the fruit. At stage 1 of ripening, the green stage (Fig. 1), endo-ß-mannanase could not be localized because of autofluorescence, due to the presence of chloroplasts in the skin and outer pericarp. Similar pictures were obtained using either the pre-immune serum or the tagged antibody to the enzyme (Fig. 5a
, b). By stage 4 of ripening, however, autofluorescence was low, and mostly confined to the skin (Fig. 5c). Considerable reactivity with endo-ß-mannanase was detectable in the skin and outer pericarp region with less in the inner pericarp (Fig. 5d). That the fluorescence was in the outer region of the cell, presumably in the cell walls, was confirmed using bright-field microscopy of the same sections (Fig. 6a, b). A higher magnification of some cells of the outer pericarp region showed that the endo-ß-mannanase was mostly associated with the periphery of the cells (Fig. 5e), as was also apparent at the lower magnification (Fig. 5d). This location of the enzyme was also clear in the pericarp at stage 6 (Fig. 5f), when there was only minor autofluorescence (Fig. 5i), and there was a strong reaction of the endo-ß-mannanase antibody with the skin and outermost pericarp cells (Fig. 5g). In contrast, there was much less enzyme detectable in the inner pericarp (Fig. 5h) and that which was, also appeared to be peripherally localized. Hence these data confirm the concentrated activity of wall-associated endo-ß-mannanase within the skin of the fruit, at least partly because of the high density of small cells with proportionately more cell walls, less activity in the few cell layers of the outer pericarp and the most diffuse activity in the inner pericarp beneath.
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mRNA for endo-ß-mannanase is present throughout ripening
Northern hybridization was carried out on poly A+ RNA extracted from the skin and pericarp region of ripening tomato fruit using the cDNA probe of endo-ß-mannanase obtained from germinated tomato seed (Bewley et al., 1997). The message for fruit endo-ß-mannanase was present at all stages, with the exception of stage 0 (Fig. 7). This observation is consistent with there being enzyme protein in the fruit from stage 1, even though enzyme activity itself was only manifest later. At stage 1, prior to the increase in endo-ß-mannanase activity, its mRNA was most predominant. The mRNA from germinated tomato seeds ran at the same location on an agarose gel as the fruit messages, confirming the similarity between the enzyme messages from the two sources.
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Fruit firmness and endo-ß-mannanase in ripening mutants
The firmness of tomato fruits of three ‘wild-type’ cultivars and their non-ripening mutant lines (Tigchelar et al., 1978) was measured during ripening. All wild-type fruits of Ailsa Craig, Rutgers and Walter became less firm during ripening (Fig. 8a). The non-ripening mutants of cv. Ailsa Craig (Nr, nor and rin) softened to different extents, with rin and nor remaining much firmer at their stage of maximum ripeness. Of these mutants, only the fruits of Nr became red-ripe, but much more slowly than those of the wild type; the ‘ripe’ fruits of rin were yellowish, while the nor fruits were yellowish-orange, indicating a greater degree of ripeness and hence softness. Endo-ß-mannanase activity increased in the wild type and the Nr mutant fruits, while in those of the firmer nor and rin mutants there was only a very slight increase in enzyme activity (Fig. 8b). Hence some relationship between enzyme activity and decline in firmness seems to be evident. Previously it was reported that the rin mutant of tomato cv. Naebyungjangsu has very little endo-ß-mannanase activity during ripening (Hong et al., 1996). In the Rutgers cultivar, the wild-type fruit softened more than that of the non-ripening mutants (the Nr mutants of this cultivar did not ripen beyond yellow and the alc mutants were orange-red), with the former producing large amounts of endo-ß-mannanase activity, while the mutants produced none (Fig. 8a, b). In sharp contrast to these observations, neither the wild type nor the non-ripening dg mutant fruits of cv. Walter produced any endo-ß-mannanase during ripening (Fig. 8a, b), although even the fruit of the non-ripening mutant turn red-ripe, with many dark-green flecks, at maturity. Similarly, another wild-type fruit was found, cv. Heinz 1439, a plum-type tomato, which ripened normally, but failed to exhibit enzyme activity at any stage. Hence, while some cultivars and mutants of tomato do show a temporal correlation between loss of firmness and endo-ß-mannanase during ripening, this is clearly not a universal characteristic.
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Further discussion
Endo-ß-mannanase is a high-salt-soluble enzyme which is most active in the very outermost layers of the tomato fruit of several cultivars and types during ripening. The reason for its low solubility is probably its association with cell walls. Several other cell-wall-hydrolysing enzymes, e.g. polygalacturonase, PG (Grierson and Tucker, 1983), pectinesterase, PE (Tucker et al., 1982), ß-galactosidase (Pressey, 1983) and endo-ß-(1
3) glucanase (Wallner and Walker, 1975) have been reported to be present in ripening tomato fruits. These enzymes are strongly adsorbed onto cell wall material and low pH or high-salt buffers have been used to extract them also (Tucker et al., 1980, 1982; Pressey, 1988; Wallner and Walker, 1975). There is no indication that the concentration of activity of any of the other hydrolases is as narrowly localized in the fruit as endo-ß-mannanase, although they were extracted from pericarp tissue.
Endo-ß-mannanase activity increases within the fruits of cvs Trust, Heinz 902, Ailsa Craig, and Rutgers with ripening and peaks when the fruit is uniformly red. Softening of the pericarp immediately under the skin occurs during over-ripening (starting at stage 6, and prevalent at stage 7), when there is a progressive loss of cell wall integrity in this region (Fischer and Bennett, 1991). Endo-ß-mannanase activity in cv. Trust is at a maximum in the pericarp at the commencement of this softening. Its subsequent decline in activity could be a consequence of the increasing degeneration of the cells in the outer pericarp region which could also involve proteolysis, and hence endo-ß-mannanase degradation. Western blots indicate the presence of degraded forms of endo-ß-mannanase at the late ripening stages. The skin (exocarp) itself does not undergo any noticeable change in firmness during ripening, and hence the reason for the large increase in endo-ß-mannanase in this region is difficult to explain. Also, there is no change in total cell-wall mannose content of the inner or outer pericarp (including skin) region during ripening, nor are there differences between the mannose contents of the walls in these two regions (Huysamer et al., 1997), in contrast to the large differences in the mannan degrading activity present.
The skin is unlikely to be the sole site of synthesis of the enzyme, from where it is secreted into the outer pericarp, because the enzyme is tightly associated with the cell walls. A secreted enzyme should be soluble, at least until it reaches its destination, and no water-soluble enzyme was obtained. Endo-ß-mannanase is a secreted enzyme in the endosperms of several seeds, e.g. fenugreek (Reid et al., 1997), lettuce (Halmer and Bewley, 1979) and tomato (Nonogaki et al., 1995), and is highly soluble.
An increase occurs in two major proteins in the salt-soluble extracts of the outer tissues of cv. Trust tomato fruits during ripening, but these proteins are of a different molecular mass from those which react with a tomato seed endo-ß-mannanase antibody. Major PG isoforms have molecular masses of 43, 45 and 84–115 kDa, the major PE is 23 kDa, ß-galactosidases 62–144 kDa and ß(13) glucanase, 12 kDa; hence they are also not the major bands in the high-salt protein extracts separated on the SDS-PAGE gel. The active endo-ß-mannanase in tomato seeds has a molecular mass close to 40 kDa (Bewley et al., 1997), and there is an antigenically-related protein of the same molecular mass in the fruit. Two antigenically coincident higher molecular mass forms of 50 and 55 kDa also occur in seeds and fruits; these might be inactive and unprocessed forms of the enzyme. Surprisingly, an approximately 40 kDa form of the enzyme is present within the fruit at times when it is not active, and endo-ß-mannanase mRNA is present also at these times. It has been proposed that in fenugreek seeds there is also an inactive form of endo-ß-mannanase (Malek and Bewley, 1991) under conditions where enzyme activity is suppressed. Conversion of PG from an inactive to an active form in ripening tomato fruits has been proposed, perhaps by interaction with other proteins (Hobson and Grierson, 1993).
Because of its absence of activity from some cultivars and lines, endo-ß-mannanase cannot have a universal role in tomato fruit ripening, as previously suggested (Sozzi et al., 1996). The enzyme in the tomato skin, when present, might be involved in the ripening or softening process, although mannose content of the cell wall does not change appreciably during this event (Sakurai and Nevins, 1993), which is suggestive of a reorganization of hemicelluloses within the wall, rather than mobilization therefrom. Alternatively, the enzyme might be used in the defence against pathogens. While there is no precedent for such a role for endo-ß-mannanase in plants, some plant pathogenic filamentous fungi have mannan-rich surface polysaccharides, and mannan-degrading enzymes are required to lyse their mycelia (Watanabe and Ogasawara, 1990).
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This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Agriculture, Food and Rural Affairs, and the Ontario Tomato Research Institute. JDB is grateful for support from Wageningen Agricultural University, The Netherlands and the opportunity to conduct research there. We are grateful to Drs John S Greenwood and Melissa Farquhar for their help and advice with the confocal microscopy, and to Dr David Stanley for his advice and instruction on the operation of the Instron.
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3 To whom correspondence should be addressed. Fax: +1 519 767 1991. E-mail:dbewley{at}uoguelph.ca
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