Journal of Experimental Botany, Vol. 52, No. 354, pp. 105-111,
January 2001
© 2001 Oxford University Press
Original Papers |
Endo-ß-mannanase is present in an inactive form in ripening tomato fruits of the cultivar Walter
Department of Botany, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Received 6 June 2000; Accepted 21 August 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Fruits of the tomato cultivar Walter undergo normal development to the red-ripe stage but, unlike those of the cultivar Trust, they do not produce any active endo-ß-mannanase. Reasons for this failure to produce the enzyme were sought. The cv. Walter contains genes for endo-ß-mannanase, as shown by Southern blot analysis, and transcripts for the enzyme are present in ripening fruits, as revealed using Northern hybridization. Moreover, the enzyme protein is detectable by Western blots using an endo-ß-mannanase-specific antibody from tomato. In addition, the inactive enzyme is localized appropriately in the wall regions of the outer layers of the fruit (skin and outer pericarp). Mixing inactive fruit extracts of cv. Walter, in excess, with extracts from cv. Trust fruits, which contain active enzyme, leads to an increase rather than a reduction in enzyme activity, showing that there are no inhibitors of endo-ß-mannanase in cv. Walter fruits. Similar results were obtained with fruits of the tomato cv. Heinz 1439. In contrast to the situation in fruits, the seeds of both cvs Walter and Heinz 1439 produce active enzyme, especially following germination.
Key words: Endo-ß-mannanase, fruit, inactive enzyme, Lycopersicon esculentum, tomato.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Fruit ripening is a complex process that involves the softening of cell walls by a variety of hydrolases (Fischer and Bennett, 1991
). The tomato fruit cell wall is composed of pectins, hemicellulose, cellulose, and protein (Gross, 1984). Among the hemicellulose components are xyloglucans, glucomannanas and galactomannans, which are covalently linked to pectin and hydrogen-bonded to cellulose (Tong and Gross, 1988; Fischer and Bennett, 1991). The most studied of the cell wall hydrolases is polygalacturonase (Grierson, 1985; Hobson and Grierson, 1993) although its role in softening of tomato fruit in situ has been questioned (Brummell and Labavitch, 1997).
The presence of other cell wall-hydrolysing enzymes in ripening tomato fruits has also been reported, e.g. pectinesterase (Tucker et al., 1982), ß-galactosidase (Pressey, 1983) and endo-ß-(1
3) glucanase (Wallner and Walker, 1974). Some isoforms of these enzymes are strongly adsorbed into the cell wall matrix.
Mannans are a component of tomato fruit cell walls, and mannosyl-linked residues increase during ripening (Tong and Gross, 1988; Huysamer et al., 1997). Mannosyl residues may replace arabinosyl side chains during ripening, concurrently with the synthesis of glucomannans (Tong and Gross, 1988). While they are a minor component of the tomato fruit cell wall, such changes in mannan residues may contribute to texture changes, or ripening-related softening as the fruit matures.
The presence during ripening of endo-ß-mannanase, the enzyme which hydrolyses mannan polymers, has been reported (Pressey, 1989; Hong et al., 1996; Sozzi et al., 1996). Two 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. All three studies, however, employed acidified water to extract the enzyme, which was subsequently shown to be an inefficient method, since the enzyme is cell-wall associated and requires salt-containing buffers for extraction (Bewley et al., 2000; Bourgault et al., 2000). The enzyme indeed increases during ripening, and is concentrated in the skin and outer pericarp of the fruit. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-ß-mannanase activity, although a cause-and-effect relationship has not been established between ripening and enzyme activity (Bewley et al., 2000). Indeed, one ripening cultivar, Walter, which undergoes normal reddening and softening, does so without any detectable endo-ß-mannanase activity in the fruit (Bewley et al., 2000). The study reported here was designed to determine why activity of this enzyme is absent from this cultivar.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Plant material
Fruits of tomato (Lycopersicon esculentum Mill.) cvs Walter, Trust and Heinz 1439 were grown in the greenhouse of the Department of Botany, University of Guelph under natural daylight conditions following self-pollination. Stages of fruit development were detailed previously (Bewley et al., 2000
).
Endo-ß-mannanase (EC 3.2.1.78) and ß-mannoside mannohydrolase (EC 3.2.1.25) extraction and assay
Tomato fruits were picked when green (stage 1), orange (stage 3) or red-ripe (stage 6) and the skin (exocarp) and the first 2 mm inside the skin, the outer pericarp (hypodermis and outer mesocarp), were used for extraction of the enzymes in McIlvaine (McIlvaine, 1921) buffer pH 5 plus 0.5 M NaCl (Bewley et al., 2000). A modified gel diffusion assay was used (Bewley, 1997; Bewley et al., 2000) and activity expressed in relation to the activities of a serial dilution of Aspergillus niger endo-ß-mannanase (Megazyme, Bray, Eire) as standard (Downie et al., 1994). Seeds were extracted for endo-ß-mannanase activity during and following germination with 0.1 M HEPES-NaOH buffer (pH 8) without added salt.
ß-Mannoside mannohydrolase assays were conducted on the high salt-soluble endo-ß-mannanase extracts from the outer regions of the fruit, in microtitre plates using p-nitrophenyl-ß-D-mannopyranoside (Sigma) as substrate. The molar extinction coefficient for p-nitrophenyl was taken as 18 400 (Reid and Meier, 1973) to calculate the amount released as pmol min-1 g-1 fresh weight.
SDS-PAGE and Western blots
A polyclonal antibody to the purified M1 form of tomato seed endo-ß-mannanase (Nonogaki et al., 1995) was kindly provided by Dr Y Morohashi, Saitama University, Japan. Equal amounts of protein (16 µg) were loaded in each lane and separated on a 12% SDS-PAGE gel (Laemmli, 1970), transferred to nitrocellulose membrane, and 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/Pharmacia Biotech.).
Laser-scanning confocal microscropy
Thin 1–2 mm sections of the outer skin and pericarp from red-ripe tomatoes were fixed for 1 h in 25 mM phosphate buffer (pH 7.2) and formaldehyde (3% v/v). The sections were washed with phosphate buffered saline (PBS: 137 mM NaCl, 5.4 mM Na2HPO4, pH 7.4), blocked with 2% w/v skim milk (Carnation) 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 the 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 five 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.
RNA isolation and Northern hybridization
Total RNA was prepared from 10 g skin and outer pericarp of different stages of ripening cv. Walter fruit using the hot phenol/LiCl method (Verwoerd et al., 1989) following initial grinding in liquid N2. Poly A+ RNA was purified from this using the poly AT Tract mRNA Isolation System IV (Promega, Madison, WI). For fractionation on 1.2% (w/v) agarose gels in 2.2 M formaldehyde, either 2.5 µg fruit poly A+ RNA or 10 µg seed total RNA were used. The RNA was transferred to Zeta-probe nylon membrane (Bio-Rad) (Sambrook et al., 1989). The membrane was prehybridized and probed with 
-32P-labelled tomato seedling cDNA (LeMan1, Bewley et al., 1997) using a random-primed DNA labelling kit (Roche Diagnostics). Following hybridization for 24 h the membrane was washed with 2xSSC, 0.1% (w/v) SDS at 42 °C for 20 min and then 1xSSC, 0.5xSSC and 0.1xSSC, all with 0.1% w/v SDS, at 42 °C for 20 min each. Finally, the membrane was dried and exposed to Kodak X-OMAT X-ray film.
Genomic DNA preparation
Genomic DNA from tomato leaf was extracted by a modified CTAB method (Murray and Thomson, 1980). About 5 g tomato leaf tissue was ground to a fine powder in liquid N2 with a mortar and pestle. The powder was lyophilized overnight and the dried powder was rehydrated with an equal volume of extraction buffer (50 mM TRIS-HCl, pH 8.0, 700 mM NaCl, 10 mM EDTA, 1% w/v CTAB and 1% v/v ß-mercaptoethanol) by mixing gently. Following incubation at 56 °C for 15 min, 1 vol of chloroform:isoamyl alcohol (24:1) were added and mixed well. After centrifugation for 5 min at 21 000 g the supernatant was removed to a fresh tube, 0.1 vol of 10% CTAB v/v was added, and re-extracted with 1 vol of chloroform: isoamylalcohol (24:1). The upper aqueous phase was removed to a fresh tube and 1 ml precipitation buffer (50 mM TRIS-HCl, pH 8.0, 10 mM EDTA, 1% w/v CTAB) was added and mixed well. Following incubation at room temperature for 20 min the sample was pelleted by centrifugation for 10 min at 21 000 g. The pellet was dissolved in 0.5 vol of 1 M NaCl, and 2 µl RNase (10 mg ml-1) were added and incubated at 37 °C for 30 min. To precipitate DNA from the aqueous phase 1 ml ethanol was added and incubated at -70 °C for 30 min. DNA was collected by centrifugation for 10 min at 4 °C at 21 000 g. The pellet was washed with 70% ethanol, dried and dissolved in 50 µl sterile distilled water.
Southern blot analysis
Genomic DNA (25 µg) was digested with HindIII and BamHI restriction enzymes and separated on a 1% agarose gel. Agarose gel electrophoresis, Southern blotting on to Zeta-probe nylon membrane (Bio-Rad) and hybridization were essentially as described previously (Sambrook et al., 1989). The membrane was probed with -32P-labelled full-length LeMan1 cDNA (Bewley et al., 1997). Following hybridization the membrane was washed down to 0.1xSSC and 1% SDS at 65 °C for 20 min, dried and exposed to Kodak X-OMAT X-ray film.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Southern blot analysis of the cv. Walter tomato genome
Since no endo-ß-mannanase activity was detected during ripening of the fruits of cv. Walter, it was prudent to determine if this was due to the deletion of a gene for this enzyme. Endo-ß-mannanase increases in the fruit of cv. Trust during ripening (Bewley et al., 2000
) and a restriction digest of its DNA is indicative of at least three genes being present (Fig. 1). This is in line with the conclusion reached following a similar digestion of cv. Dynamo F1 seedling DNA (Bewley et al., 1997). A very similar restriction pattern was obtained following digestion of DNA from cv. Walter (Fig. 1), showing this cultivar at least contains three genes for endo-ß-mannanase.
|
Northern blot analysis of cv. Walter during fruit ripening
The next question is whether the gene for endo-ß-mannanase in cv. Walter is transcribed during ripening. In cv. Trust the mRNA for this enzyme is present most abundantly at stage 1 (green) of ripening and yet no activity is detectable until two stages of ripening later (Bewley et al., 2000). Likewise, in cv. Walter, the mRNA for endo-ß-mannanase is present in the green stage fruit, and throughout ripening (Fig. 2) even though it is never active. The cDNA probe used for the Northern blot was derived from a seed endo-ß-mannanase, which may account for the weaker signal observed for the fruit mRNA. Even so, the location on an agarose gel of cv. Walter fruit mRNA (approximately 1.3 kb) is identical to that from the cv. Trust seed (Fig. 2), which in turn is identical in size to the mRNA from the cv. Trust fruit (Bewley et al., 2000). Thus endo-ß-mannanase gene transcription proceeds during ripening of cv. Walter fruit.
|
Western blot analysis of cv. Walter fruit during ripening
In the cv. Trust fruit there is endo-ß-mannanase protein detectable on a Western blot at the early stages of ripening when it is not active (Bewley et al., 2000). Since the mRNA for the enzyme is present in cv. Walter fruits during ripening, but there is no endo-ß-mannanase activity, it was determined if there is translation of the message into protein. This, indeed, is the case (Fig. 3). At the green, orange and red stages of fruit ripening an immunoreactive band is present on the Western blot, which is identical in molecular mass (approximately 39 kDa) to the immunoreactive band for the enzyme extracted from germinated seed of cv. Walter (in which it is active, see later). This is also the same molecular mass as obtained for endo-ß-mannanase using protein extracts from cv. Trust seeds (Bewley et al., 2000). Thus endo-ß-mannanase is translated during cv. Walter ripening, and the inactive form is the same mass as the mature active enzyme.
|
Immunolocalization of endo-ß-mannanase in cv. Walter fruit
The active enzyme in cv. Trust fruits is localized in the skin and outer pericarp regions, in association with the cell walls (Bewley et al., 2000). The possibility was tested that the enzyme is produced, but not transported to its site of action in the cell wall. However, it is clear that in the red-ripe fruit cv. Walter, the inactive enzyme is located in the outermost layers of the fruit, in association with the periphery of the cells (Fig. 4d). In the absence of antibody (Fig. 4a) or in the presence of only secondary antibody (Fig. 4c) there is no immunoreaction, and in the presence of preimmune serum there are some minor reactions, but not confined to cell walls or the outer region of the fruit (Fig. 4b). A faint autofluorescence is evident in control sections, especially in the skin (Fig. 4a–c).
|
ß-Mannoside mannohydrolase activity during fruit ripening
ß-Mannoside mannohydrolase (ß-mannosidase) hydrolyses the mannobiose and mannotriose oligomers resulting from the degradation of mannan-containing polymers by endo-ß-mannanase. It may also exhibit exo-mannanase activity, however, cleaving terminal mannose residues from a polymeric chain (McCleary, 1982). Activity of this enzyme is high in the skin of cv. Trust fruits during the early stages (1–4) of ripening, but after the orange stage it declines sharply, in contrast to the increase in endo-ß-mannanase activity (Bewley et al., 2000). The possibility was tested that, in the absence of endo-ß-mannanase from the fruits of cv. Walter, there is a compensating increase in activity of ß-mannosidase, possibly acting as an exo-ß-mannanase, was tested. This is not the case, however, for there is a marked decline in activity as the fruit changes from green to red-ripe (Fig. 5), as in the cv. Trust fruits.
|
Is there an inhibitor of endo-ß-mannanase activity in cv. Walter fruits?
The possibility was tested that the lack of endo-ß-mannanase activity in ripening cv. Walter fruits is due to the presence of an enzyme inhibitor. Thus inactive extracts from the combined skin and outer pericarp regions of cv. Walter at the red-ripe stage were mixed with similar, but active extracts from cv. Trust in various proportions (Fig. 6). When cv. Trust extracts were mixed with extraction buffer only, there was the expected decline in endo-ß-mannanase activity due to dilution. In marked contrast, when cv. Trust extracts were mixed with cv. Walter extracts, considerable activity was retained even at an 8:1 ratio of inactive:active cv. Walter:cv. Trust extracts, and well in excess of that present following dilution of cv. Trust extracts with extraction buffer (Fig. 6). Clearly, then, the cv. Walter extracts did not inhibit the endo-ß-mannanase activity present in the cv. Trust extracts, even when there was an 8-fold excess. Rather, there was a proportional increase in activity at all ratios from 1:1 to 8:1, with the observed decline at the higher ratios presumably being due to the relatively diluted amounts of cv. Trust extract in the mixture as the amount of cv. Walter extract was increased.
|
The reasons for this increase in activity when the extracts were mixed is unknown, but two obvious possibilities were considered: (i) that the inactive enzyme in the cv. Walter extract is being activated by some component of the cv. Trust extract, or (ii) the already active enzyme in the cv. Trust extract is being enhanced by some component of the cv. Walter extract. Consequently, an appropriate control was run in which a heat-denatured cv. Walter extract was added to an unheated cv. Trust extract. Heat-inactivation of the cv. Walter extract was for 5 min at 85 °C; heating active extracts (e.g. from cv. Trust) at 85 °C for only 1 min destroys all endo-ß-mannanase activity (unpublished data). Even heat-denatured extracts of cv. Walter, when added to cv. Trust extracts caused an increase in enzyme activity (Fig. 6
), thus making either possibility difficult to support.
Thus a more likely explanation of the results is that some component of the cv. Walter extract (heated or unheated) is protecting the enzyme in the active cv. Trust extract from degradation during the assay. That there is protection against proteases is supported by the observation that the addition of bovine serum albumin (BSA) to the extracts of cv. Trust enhances their activity (Fig. 6). The plate diffusion assay (Downie et al., 1994), the modified method of Still et al. (Still et al., 1997), and the multi-well gel diffusion assay (Toorop et al., 1996; Bewley et al., 2000) used here all require up to 24 h incubation times at 25–35 °C. Hence enzyme degradation could be occurring during the course of the assay resulting in an underestimation of total activity. It is possible that the fruit extracts added to active ones of the cv. Trust are increasing the overall protein content, and thus helping protect endo-ß-mannanase against degradation by endogenous proteases. The addition of a Protease Inhibitor Cocktail For Plant Cell Extracts (concentration according to manufacturer's instructions, Sigma) to active cv. Trust extracts did not interfere with the enzyme assay and thus could be used routinely.
For the purposes of this study, what is clear is that cv. Walter extracts do not contain inhibitors of endo-ß-mannanase activity, and hence they cannot account for the lack of active enzyme in this fruit.
Inactive endo-ß-mannanase is also present in the fruits of cv. Heinz 1439
A limited number of ripening fruits of the cv. Heinz 1439 were available to us, which also do not exhibit endo-ß-mannanase activity during ripening. It was possible to repeat some of the experiments conducted on the Trust cultivar to determine if the enzyme properties in the two cvs were comparable.
Restriction digestion and gel separation of genomic DNA from Heinz 1439 revealed a somewhat different Southern hybridization pattern from that of cvs Trust and Walter. Two bands resulted from Hind III digestion, one clear band of about 7 kb following BamHI digestion, and one which migrated only a little into the gel (Fig. 1). Nevertheless, at least two genes for endo-ß-mannanase are present in the Heinz 1439 cultivar. More importantly, the red-ripe fruit contains the inactive enzyme, recognized on a Western blot using the same specific antibody that recognizes the inactive endo-ß-mannanase in cv. Walter (Fig. 3) and the inactive enzyme in the green fruits of cv. Trust (Bewley et al., 2000). When heated or unheated extracts of cv. Heinz 1439 fruits were mixed and incubated with those of red-ripe cv. Trust there was a similar (slightly greater) relative increase in activity as when cv. Walter extracts were added (not shown), confirming the absence of an inhibitor of enzyme activity, and pointing towards the presence of protecting proteins during the assay.
Endo-ß-mannanase activity in germinated seeds of cvs Walter and Heinz 1439
The final experiment was to determine if the absence of active enzyme from the fruits of cvs Walter and Heinz 1439 is also manifest in the seeds. Mature seeds from these two cvs, and cv. Trust, were imbibed in water at 25 °C and assayed for enzyme activity during and following germination. Radicle emergence from about 5% of the seeds of all cvs occurred by 36 h, 50% the seeds had germinated by 48 h and 85% by 84 h. Seeds of all three cvs showed a marked increase in endo-ß-mannanase activity following germination (Fig. 7). The differences in enzyme activity at 84 h, expressed on a per seed basis were probably related to the sizes of the seeds; cv. Heinz seeds are smaller than those of cv. Walter, which in turn are smaller than those of cv. Trust. As might be expected, there is mRNA for endo-ß-mannanase in seeds of cv. Walter, as revealed by a Northern blot (Fig. 2), and the fruit of both cvs Walter and Heinz 1439 contain enzyme that is recognized by endo-ß-mannanase-specific antibodies on a Western blot (Fig. 3). The sizes of the seed mRNA and protein are identical to those of the fruit.
|
In conclusion, while cvs Walter and Heinz 1439 contain inactive endo-ß-mannanase throughout fruit ripening, the reason does not appear to lie at the genomic, transcriptional or translational level nor is it due to improper localization. There is no inhibitor in the fruits of these cultivars which suppresses enzyme activity. Inactive enzyme is present in green fruits (stages 1 and 2) of cv. Trust, but active enzyme is present by stage 3 (Bewley et al., 2000
). That the ripening fruit of the cv. Walter produces and sequesters an inactive enzyme is a novel observation for a so-called ‘wild-type’ line. Mutant lines of soybean are known that produce an inactive lipoxygenase 2 in the seed due to an amino acid substitution (Wang et al., 1994), and a mutant of Arabidopsis that lacks an appropriate activator has an inactive form of RuBP carboxylase (Somerville et al., 1982). A substitution of one or a few amino acids in the endo-ß-mannanase in the tomato fruit of cvs Walter and Heinz 1439, presumably in the active site or a key binding site, could account for its lack of activity. In cv. Walter fruits there is a decline in firmness as ripening proceeds, which is comparable to that which occurs in other cultivars in which endo-ß-mannanase activity is present (Bewley et al., 2000). Hence the presence of the enzyme is not vital for the ripening process. It has been suggested that the enzyme plays a role in defence against pathogens (Bewley et al., 2000); while no data are available, it is possible that cultivars lacking endo-ß-mannanase are more susceptible to certain diseases. Alternatively, the properties of the protein, rather than of the enzyme may be important, in that, like the lectins, it is an anti-nutritional agent and hence its presence rather than its activity is important.
|
Acknowledgments |
|---|
Seeds of the cultivar Walter were kindly provided by the CM Rick Tomato Genetic Resources Center, UC Davis, CA and the cultivar Heinz 1439 by Dr KP Pauls, Department of Plant Agriculture, University of Guelph. Ms Beixin Mo, Department of Botany, University of Guelph generously conducted the ß-mannoside mannohydrolase assays. This work was supported by the Natural Sciences and Engineering Research Council of Canada grant A2210 to JDB and by grants from the Ontario Tomato Research Institute and the Ontario Ministry of Agriculture, Food and Rural Affairs.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +1 519 767 1991. E-mail: dbewley{at}uoguelph.ca
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Bewley JD.1997. Breaking down the walls—a role for endo-ß-mannanase in release from seed dormancy? Trends in Plant Science2, 464–469.
Bewley JD, Burton RA, Morohashi Y, Fincher GB.1997. Molecular cloning of a cDNA encoding a (14)-ß-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum). Planta203, 454–459.[Web of Science][Medline]
Bewley JD, Banik M, Bourgault R, Feurtado JA, Toorop P, Hilhorst HWM.2000. Endo-ß-mannanase activity increases in the skin and outer pericarp of tomato fruits during ripening. Journal of Experimental Botany51, 529–538.
Bourgault R, Bewley JD, Alberici A, Decker D.2000. Endo-ß-mannanase activity in tomato and other ripening fruits. HortScience (in press).
Brummell DA, Labavitch JM.1997. Effect of antisense suppression of endopolygalacturonase activity on polyuronide molecular weight in ripening tomato fruit and in fruit homogenates. Plant Physiology115, 717–725.[Abstract]
Downie B, Hilhorst HWM, Bewley JD.1994. A new assay for quantifying endo-ß-mannanase activity using Congo red dye. Phytochemistry36, 829–835.[Web of Science]
Fischer RL, Bennett AB.1991. Role of cell wall hydrolases in fruit ripening. Annual Review of Plant Physiology and Plant Molecular Biology42, 67–73.
Grierson D.1985. Gene expression in ripening tomato fruit. Critical Reviews in Plant Science3, 113–132.
Gross KC.1984. Fractionation and partial characterization of cell walls from normal and non-ripening mutant tomato fruit. Physiologia Plantarum62, 25–32.
Hobson G, Grierson D.1993. Tomato. In: Seymour G, Taylor J, Tucker G, eds. Biochemistry of fruit ripening. London: Chapman and Hall, 405–442.
Hong SJ, Lee SK, Kim J-K.1996. Changes in cell wall carbohydrates and glycanase activity, and their relationship to the ripening of tomato fruits. Journal of the Korean Society of Horticultural Science37, 380–385.
Huysamer M, Greve C, Labavitch JM.1997. Cell wall metabolism in ripening fruit. VIII. Cell wall composition and synthetic capacity of two regions of the outer pericarp of mature green and red ripe cv. Jackpot tomatoes. Physiologia Plantarum101, 314–322.
Laemmli UK.1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685.[Medline]
McCleary BV.1982. Purification and properties of ß-mannosidase mannohydrolase from guar. Carbohydrate Research101, 75–92.
McIlvaine TC.1921. A buffer solution for colorimetric comparison. Journal of Biological Chemistry49, 183–186.
Murray MG, Thompson WF.1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research8, 4321–4325.
Nonogaki H, Nomaguchi M, Morohashi Y.1995. Endo-ß-mannanases in the endosperm of germinated tomato seeds. Physiologia Plantarum94, 328–334.
Pressey R.1983. ß-Galactosidases in ripening tomatoes. Plant Physiology71, 132–135.
Pressey R.1989. Endo-ß-mannanase in tomato fruit. Phytochemistry28, 3277–3280.
Reid JSG, Meier H.1973. Enzymic activities and galactomannan mobilization in germinating seeds of fenugreek Trigonella foenum-graecum L. Leguminosae. Secretion of -galactosidase and ß-mannosidase by the aleurone layer. Planta122, 319–222.
Sambrook J, Fritsch EF, Maniatis T.1989. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press.
Somerville CR, Portis AR Jr, Ogren WL.1982. A mutant of Arabidopsis thaliana which lacks activation of RuBP carboxylase in vivo. Plant Physiology70, 381–387.
Sozzi GO, Cascone O, Fraschina AA.1996. Effect of a high-temperature stress on endo-ß-mannanase and -and-ß-galactosidase activities during tomato fruit ripening. Postharvest Biology and Technology9, 49–63.
Still DW, Dahal P, Bradford KJ.1997. A single-seed assay for endo-ß-mannanase activity from tomato endosperm and radicle tissues. Plant Physiology113, 13–20.[Abstract]
Tong CBS, Gross KC.1988. Glycosyl-linkage composition of tomato fruit cell wall hemicellulosic fractions during ripening. Physiologia Plantarum74, 365–370.
Toorop P, Hilhorst HWM, Bewley JD.1996. Endo-ß-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not necessarily linked to the completion of germination. Planta200, 153–158.
Tucker GA, Robertson NG, Grierson D.1982. Purification and changes in activities of tomato pectinesterase isozymes. Journal of the Science of Food and Agriculture33, 396–400.
Verwoerd TC, Dekker BMM, Hoekma A.1989. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research17,2362.
Wallner SJ, Walker JE.1975. Glycosidases in cell wall-degrading extracts of ripening tomato fruits. Plant Physiology55, 94–98.
Wang WH, Takano T, Shibata D, Kitamura, Takeda G.1994. Molecular basis of a null mutation in soybean lipoxygenase. 2. Substitution of glutamine for an iron-ligand histidine. Proceedings of the National Academy of Sciences, USA91, 5828–5832.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Schroder, R. G. Atkinson, and R. J. Redgwell Re-interpreting the role of endo-{beta}-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall Ann. Bot., May 19, 2009; (2009) mcp120v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, J. Ju, H. Peng, F. Gao, C. Zhou, Y. Zeng, Y. Xue, Y. Li, B. Henrissat, G. F. Gao, et al. Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A J. Biol. Chem., November 14, 2008; 283(46): 31551 - 31558. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Saladie, A. J. Matas, T. Isaacson, M. A. Jenks, S. M. Goodwin, K. J. Niklas, R. Xiaolin, J. M. Labavitch, K. A. Shackel, A. R. Fernie, et al. A Reevaluation of the Key Factors That Influence Tomato Fruit Softening and Integrity Plant Physiology, June 1, 2007; 144(2): 1012 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Filichkin, J. M. Leonard, A. Monteros, P.-P. Liu, and H. Nonogaki A Novel Endo-{beta}-Mannanase Gene in Tomato LeMAN5 Is Associated with Anther and Pollen Development Plant Physiology, March 1, 2004; 134(3): 1080 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Itai, K. Ishihara, and J. D. Bewley Characterization of expression, and cloning, of {beta}-D-xylosidase and {alpha}-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit J. Exp. Bot., December 1, 2003; 54(393): 2615 - 2622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-C. Li, C.-T. Chang, E. S. L. Hsiao, J. S. F. Hsu, J.-W. Huang, and J. T. C. Tzen Purification and Characterization of an Antifungal Chitinase in Jelly Fig (Ficus awkeotsang) Achenes Plant Cell Physiol., November 15, 2003; 44(11): 1162 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Bourgault and J. D. Bewley Variation in Its C-Terminal Amino Acids Determines Whether Endo-beta -Mannanase Is Active or Inactive in Ripening Tomato Fruits of Different Cultivars Plant Physiology, November 1, 2002; 130(3): 1254 - 1262. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||