JXB Advance Access originally published online on September 25, 2003
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Journal of Experimental Botany, Vol. 54, No. 392, pp. 2503-2510,
November 1, 2003
© 2003 Oxford University Press
The relationship between ß-mannosidase and endo-ß-mannanase activities in tomato seeds during and following germination: a comparison of seed populations and individual seeds
Received 27 February 2003; Accepted 13 July 2003
Department of Botany, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
* Present address: Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region, China. To whom correspondence should be addressed: Fax: +1 519 767 1991. E-mail: dbewley{at}uoguelph.ca
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Abstract |
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ß-Mannosidase and endo-ß-mannanase are involved in the mobilization of the mannan-containing cell walls of the tomato seed endosperm. The activities of both enzymes increase in a similar temporal manner in the micropylar and lateral endosperm during and following germination. This increase in enzyme activities in the micropylar endosperm is not markedly reduced in seeds imbibed in abscisic acid although, in the lateral endosperm, endo-ß-mannanase activity is more suppressed by this inhibitor than is the activity of ß-mannosidase. Gibberellin-deficient (gib-1) mutants of tomato do not germinate unless imbibed in gibberellin; low ß-mannosidase activity, and no endo-ß-mannanase activity is present in seeds imbibed in water, but both enzymes increase strongly in activity in the seeds imbibed in the growth regulator. For production of full activity of both ß-mannosidase and endo-ß-mannanase in the endosperm, this tissue must be in contact with the embryo for at least the first 6 h of imbibition, which is indicative of a stimulus diffusing from the embryo to the endosperm during this time. These results suggest some correlation between the activities of ß-mannosidase and endo-ß-mannanase, particularly in the micropylar endosperm, in populations of tomato seeds imbibed in water, abscisic acid and gibberellin. However, when individual micropylar endosperm parts are used to examine the effect of the growth regulators and of imbibition in water on the production of the two enzymes, it is apparent that within these individual seed parts there may be large differences in the amount of enzyme activity present. Micropylar endosperms with high endo-ß-mannanase activity do not necessarily have high ß-mannosidase activity, and vice versa, which is indicative of a lack of co-ordination of the activities of these two enzymes within individuals of a population.
Key words: Abscisic acid, ß-mannosidase, endo-ß-mannanase, endosperm, gib-1 mutant, tomato seed germination.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The endosperm in the mature tomato seed completely surrounds the embryo and, for germination to be completed, this structure must be penetrated in the micropylar region by the emerging radicle. The cell walls of the endosperm are composed largely of mannose, with lesser amounts of glucose, galactose and arabinose (Groot et al., 1988). Thus it has been proposed that for the breakdown of the mannan-rich walls in the micropylar region of the endosperm, there must be the production of endo-ß-mannanase, a hemicellulase that cleaves the ß-1,4 links between the mannose residues in the mannan backbone. Indeed, there are several reports that the enzyme is synthesized in the endosperm during and following germination and this is under the control of gibberellin released from the embryo (Groot et al., 1988; Nomaguchi et al., 1995; Toorop et al., 1996; Still and Bradford, 1997; Nonogaki et al., 1998). There has been controversy about whether the increase in endo-ß-mannanase in the micropylar endosperm during germination is sufficient to permit the radicle to emerge and the consensus appears to be that, whilst this enzyme is required for endosperm weakening, it is not, by itself, sufficient to allow germination to be completed (Bewley, 1997; Hilhorst et al., 1998). In recent years, endo-ß-mannanase has been purified from tomato seeds and an antibody raised against it (Nonogaki et al., 1995). Also, cDNA clones for tomato endo-ß-mannanase have been reported, LeMan1 being for the mRNA in the lateral endosperm region, which is only expressed following germination (Bewley et al., 1997) and LeMan2 which is expressed in the micropylar endosperm prior to the completion of germination (Nonogaki et al., 2000).
Other enzymes associated with the hydrolysis of galactomannans have also been studied. 
-Galactosidase is required if the mannan backbone contains galactose side-chains. A cDNA (LeaGal1) clone has also been obtained for this enzyme (Feurtado et al., 2001), which is synthesized during seed development and is sequestered in protein storage vacuoles in the mature seed (Bassel et al., 2001). This enzyme exhibits high activity in the dry seed, during germination, and during early seedling growth. Most recently, ß-mannosidase has been purified from tomato seeds, and a cDNA (LeMside1) clone obtained (Mo and Bewley, 2002). This has allowed for comparisons to be made between the activities of the three galactomannan-degrading enzymes, and the expression of their genes. It is evident that -galactosidase is different from endo-ß-mannanase and ß-mannosidase with respect to the timing of its synthesis and expression, but the latter two enzymes exhibit similarities. Endo-ß-mannanase is a low-salt soluble enzyme, but ß-mannosidase requires high-salt buffers for extraction, indicative of its association with cell walls (Mo and Bewley, 2002), as it is in lettuce (Ouellette and Bewley, 1986). However, both enzymes increase in activity prior to the completion of germination, particularly in the micropylar endosperm, and both increase in the lateral endosperm following germination (Mo and Bewley, 2002). This change in activity parallels increased expression of transcripts for both enzymes in the micropylar endosperm, and the increase in both ß-mannosidase activity and transcripts in the lateral endosperm precedes those of endo-ß-mannanase by about 12 h. Hence these two enzymes which are intimately involved in the hydrolysis of mannan-rich polymers, endo-ß-mannanase to cleave the backbone to mannobiose and mannotriose oligomers and ß-mannosidase to hydrolyse these to mannose, appear to be regulated in a similar, but not identical, temporal manner.
To determine how linked the activities of these enzymes are, seeds and seed parts were used that were subjected to different physiological treatments, such as isolation and treatment with plant growth regulators. What is demonstrated is that while both enzyme activities appear to be related in their responses in populations of seeds, this is not the case in individual seeds within these populations.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material and germination conditions
Tomato (Lycopersicon esculentum Mill. cv. Glamour) seeds, purchased from Stokes Seeds, St. Catherines, Ontario, Canada were used for most experiments. The gib-1 mutant seeds of the cv. Moneymaker were generously provided by Dr Henk Hilhorst, Wageningen University, The Netherlands. Triplicate lots of 10–20 seeds of cv. Glamour were imbibed on 1.5 ml water or 100 µM abscisic acid (ABA) (Sigma), on Whatman No. 1 filter paper in 5 cm diameter Petri dishes in darkness at 25 °C and removed for dissection into the micropylar and lateral endosperm, and embryo at the required intervals for enzyme extraction and assay. Lots of the gib-1 seeds were treated similarly, but incubated on water or 100 µM GA4 (Sigma) prior to dissection. Triplicate lots of ten seeds were also incubated for up to 12 h in water before their endosperms were dissected, and placed in 1 ml water or 10 µM GA4 for up to 72 h at 25 °C before enzyme extraction and assay.
Assays for endo-ß-mannanase (EC 3.2.1.78) and ß-mannosidase (EC 3.2.1.25)
Ten seeds or 20 seed parts (micropylar endosperm, lateral endosperm and embryo) were ground in an ice cold mortar in 1.5 ml 0.1 M HEPES-KOH buffer pH 8 and a small amount of washed sea sand. The extract was centrifuged at 21 000 g for 10 min at 4 °C and the supernatant was adjusted to 1.5 ml and used to assay for endo-ß-mannanase activity. The pellet was resuspended in 1.5 ml buffer plus 0.5 M NaCl and mixed in a ground-glass tissue grinder. The extract was allowed to stand at room temperature for 15 min (McCleary, 1982), centrifuged as before, the volume adjusted to 1.5 ml, and used to assay for ß-mannosidase.
A gel diffusion assay was used to assay for endo-ß-mannanase activity (Bewley et al., 2000), with locust bean gum (Sigma) as substrate. Two mm diameter wells were made in the gels and duplicate samples of 2 µl buffer-soluble extracts were introduced by micropipette. After incubation of the gels for 14–16 h at 25 °C, the gels were stained with Congo Red (Sigma) and the clearing zone, indicative of enzyme activity, around each well was measured. Enzyme activity was expressed in relation to that of a serial dilution of Aspergillus niger endo-ß-mannanase (Megazyme, Bray, Eire).
To assay for ß-mannosidase activity, duplicate 60 µl aliquots of the high-salt soluble extract were incubated with 90 µl 2 mM p-nitrophenyl ß-D mannopyranoside (Sigma) in McIlvaine buffer (0.1 M citric acid, 0.2 M Na2HPO4, pH 5) for 2 h at 37 °C. The reaction was terminated and colour developed by the addition of 75 µl 0.2 M aqueous sodium carbonate. The extinction coefficient for p-nitrophenyl was taken as 18 400 (Reid and Meier, 1973) to calculate the amount released in pmol min–1 g–1 fresh weight.
Single seed part assay for endo-ß-mannanase and ß-mannosidase
Seeds of the cv. Glamour were dissected to isolate the micropylar endosperm region after imbibition in water for 36 h (before radicle emergence) or on ABA (50 µM) for 48 h (no seeds completed germination). Gib-1 seeds were also imbibed on GA4 (100 µM) for 48 h and seeds taken prior to radicle emergence. Each individual micropylar endosperm was kept on ice in a 1.5 ml Eppendorf tube after dissection until being ground to a fine powder in liquid N2 and 80 µl high-salt buffer (0.1 M HEPES-KOH pH 8, 0.5 M NaCl) was added to extract both enzymes. Samples were vortexed and allowed to stand for 15 min before centrifuging at 13 000 g for 10 min at 4 °C and the supernatants used for enzyme assays as above.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Previously it was shown that endo-ß-mannanase and ß-mannosidase increase in activity during and, particularly, following germination of the tomato seed. The initial increase in activity of both enzymes is in the micropylar endosperm, followed by an increase in the lateral endosperm, with only relatively small changes occurring in the embryo (Mo and Bewley, 2002). The changes in enzyme activity are mirrored by changes in the expression of the mRNAs for both enzymes, which led to the suggestion that the expression of these enzymes is quite closely regulated.
This possibility is explored further here by following the activities of the enzymes induced in seeds subject to different physiological conditions. The activities of endo-ß-mannanase and ß-mannosidase were measured in the micropylar endosperm, since it is this area where the galacotmannan-rich cell walls have to be weakened to allow penetration of the radicle, and in some experiments in the lateral endosperm, the region in which the cell walls are mobilized as a carbon source to support early seedling growth.
Abscisic acid and enzyme activities
Germination of intact tomato seeds cv. Glamour was completely inhibited by 50 µM ABA. The effect of this on ß-mannosidase activity in the micropylar endosperm was relatively small, even after 72 h from the start of imbibition (Fig. 1A), and was almost equal to that in seeds which had completed germination on water (25% germinated at 48 h, and 90% at 72 h). Endo-ß-mannanase activity in the micropylar endosperm was not sensitive to ABA either, at least up to 48 h from the start of imbibition, but by 72 h was approximately 75% lower compared with the seeds germinated on water (Fig. 1B). These observations on endo-ß-mannanase are consistent with those made previously, that the production of this enzyme in the micropylar endosperm is not sensitive initially to ABA during germination, and led to the view that the presence of this enzyme alone is not in itself sufficient to allow germination to occur (Toorop et al., 1996; Still and Bradford 1997). The decline in endo-ß-mannanase activity after longer times of imbibition has also been noted previously and has been related to the failure of a second phase of endosperm weakening necessary for germination to be completed (Toorop et al., 2000). Now it is apparent that the initial presence of both mannan-degrading enzymes, endo-ß-mannanase and ß-mannosidase, is insufficient to permit germination to occur. It is also apparent that there is a less substantial decline in the activity of ß-mannosidase in the prolonged presence of ABA and that sustained activity of this enzyme, which may also hydrolyse mannan chains as an exo-enzyme, is not sufficient to allow for the completion of germination.
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An increase in activity of ß-mannosidase occurred in the lateral endosperm in seeds imbibed in water, but this was sensitive to ABA in that at 72 h it was reduced by about 50% in the presence of the inhibitor (Fig. 1C). The reduction in activity of endo-ß-mannanase was much greater, by comparison, being as much as 90% less at 72 h (Fig. 1D). Again this reduction in endo-ß-mannanase is consistent with the observation that, in the absence of the completion of germination, there is little or no production of this enzyme (Toorop et al., 1996). The activity of ß-mannosidase is similarly reduced by ABA, but to a relatively lesser extent.
Gibberellin induction of enzyme activities in gib-1 mutants
Isolated endosperms of the gib-1 mutant of tomato seed, a line that does not synthesize gibberellins, respond to applied gibberellins by synthesizing both endo-ß-mannanase and -galactosidase (Groot et al., 1988). An increase in mannosidase activity was also noted; the substrate used for enzyme assays was incorrectly stated as being p-nitrophenyl--D-mannopyranosidase, but p-nitrophenyl-ß-D-pyrannoside was in fact used (Steven Groot, personal communication). To achieve full germination of a population of intact seeds of the gib-1 mutant, GA4 at a concentration of 100 µM was required; there was 10–15% visible germination at 48 h, about 50% at 72 h, and germination was completed for all seeds at 96 h. In the absence of GA there was a small amount of ß-mannosidase produced over 96 h in all seed parts (Fig. 2C), although no seeds imbibed on water completed germination, and there was negligible endo-ß-mannanase activity (Fig. 2D). In the presence of GA, activities of both enzymes were induced in all regions (Fig. 2A, B), particularly in the micropylar and lateral endosperm regions following the completion of germination. Thus, in the intact gib-1 seed, the activities of both enzymes respond similarly, being low in the absence of a germination stimulus, but both increasing in a similar temporal manner in the presence of GA. The behaviour of the enzymes in the GA-induced gib-1 mutant seeds is also similar to that of the wild-type seeds, although they are of a different cv. (Fig. 1A, C).
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A stimulus from the embryo induces enzyme activities
On the basis of these and previous data, it is assumed that the activities of the galactomannan-degrading enzymes are under the control of gibberellins and that this phytohormone diffuses out from the embryo to effect the induction (Bewley, 1997). Thus an experiment was conducted to determine for how long the endosperm has to be in contact with the embryo for endo-ß-mannanase and ß-mannosidase synthesis to be induced and if both enzymes increase at the same time. Intact seeds of the cv. Glamour were imbibed on water for up to 12 h and at 2 h intervals the endosperms were dissected from the seed and placed in water or GA4. At 72 h from the time of initial imbibition the activities of the two enzymes were determined. Seeds from which the endosperm was separated from the endosperm prior to imbibition did not exhibit any endo-ß-mannanase activity after 72 h, and about 20% of maximum ß-mannosidase activity (Fig. 3A, B). By the time the endosperms had been in contact with the embryo within the intact seed for as short a time as 6 h, however, maximum activities of both enzymes were obtained following endosperm isolation and placement on water. This period of contact with the embryo of 6 h is very brief, considering that neither enzyme activity increases appreciably until about 48 h and maximum activities are not achieved in the endosperm regions until 60–72 h from the start of imbibition.
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GA4 (10 µM, or higher concentrations) was not completely able to replace the requirement by the endosperm for contact with the embryo (Fig. 3A, B). Imbibing endosperms isolated from the dry seed on GA resulted in an increase in activity of both enzymes, and in the presence of GA 4-h-isolated endosperms exhibited the same amount of activity of endo-ß-mannanase and ß-mannosidase as those in contact with the embryo for 6 h before isolation and being placed in water. GA also increased the activity of endo-ß-mannanase in endosperms dissected at all times following the start of imbibition, raising the maximum amount produced. This occurred to a lesser extent for ß-mannosidase.
These experiments are consistent with the hypothesis that a diffusible factor from the embryo is required for an increase to occur in the galactomannan-degrading enzymes in the endosperm. This factor enters the endosperm within the first 6 h from the start of imbibition, although the initial increases in endo-ß-mannanase and ß-mannosidase activities do not occur until many hours later. The requirement for the embryo is not completely satiated by the addition of GA to the endosperms isolated during the first 0–4 h, suggesting that factors in addition to this growth regulator are involved. Nevertheless, GA is able to elicit an increase in activities of both enzymes, as in the gib-1 mutant line (Fig. 2). With respect to the response of the endosperms to the presence of the embryo, both behave in a similar manner, with ß-mannosidase being somewhat less sensitive to its absence over the first 6 h (Fig. 3).
Enzyme production by individual micropylar endosperms
The previous results generally indicate that the activities of both endo-ß-mannanase and ß-mannosidase behave in a similar manner in the presence of ABA, GA or isolation of the endosperm, although not necessarily quantitatively. Still and Bradford (1997) showed that individual tomato seeds in a population exhibit different amounts of endo-ß-mannanase activity in their micropylar endosperm regions. To determine if endo-ß-mannanase and ß-mannosidase are relatively equal in their activities in individual seeds, i.e high activity of the former accompanies high activity of the latter, individual micropylar endosperms were dissected at a selected time point prior to the completion of germination (36 h) and their enzyme activities assayed. The results are expressed for a sample of 62 individual micropylar endosperms, based on increasing ß-mannosidase activity (Fig. 4A) and endo-ß-mannanase activity (Fig. 4B). It is apparent that, within an individual endosperm region, there is no consistent correlation between the activities of the two enzymes. Whilst some of the highest endo-ß-mannanase activity is present in micropylar endosperms exhibiting higher ß-mannosidase activity, this is by no means consistent and, in some endosperms, endo-ß-mannanase activity is very low while ß-mannosidase activity is high (Fig. 4A). Conversely, the highest ß-mannosidase activity is in endosperms containing the lowest endo-ß-mannanase activity.
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These studies were then extended to determine if these differences in activities persisted in the micropylar endosperm of seeds treated with ABA, which do not germinate, and gib-1 mutant seeds treated with GA to stimulate germination. In the ABA-treated cv. Glamour seeds, as in the water-imbibed seeds, there was a large disparity in endo-ß-mannanase activity between individual micropylar endosperms isolated after 48 h. Also, the same variance existed between ß-mannosidase activity and endo-ß-mannanase activities within individual micropylar endosperms (Fig. 5). Therefore, preventing germination and, in effect, regulating the seeds so that none will germinate, does not effect a synchronization of activities of the two enzymes. Likewise, in gib-1 seeds promoted by GA to complete germination, there was still a large fluctuation in the activities of endo-ß-mannanase and ß-mannosidase in individual micropylar endosperms at a time just prior to the completion of germination of the greater part of the population (only micropylar endosperms of ungerminated seeds were assayed) (Fig. 6).
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In conclusion, it is apparent that the enzyme activity data obtained using populations of seeds do not necessarily reflect the behaviour of individual seeds within that population, in agreement with the contention of Still and Bradford (1997). In the present study on germinating and germinated tomato seeds, their observations have been extended by conducting a comparison of the activities of two enzymes, endo-ß-mannanase and ß-mannosidase, in individual micropylar endosperms. By the nature of their substrates, these enzymes might be expected to be regulated in a similar manner to effect the mobilization of the galactomannan-containing cell walls of this region. Cell wall weakening would aid in achieving the release of the embryo from its surrounding constraints to permit the completion of germination and also in the subsequent mobilization of the cell walls as a carbon source for the growing embryo. Studies of the regulation of these enzymes by ABA and GA in a seed population indicate that the increases or decreases in enzyme activity are regulated in a similar manner, particularly in the micropylar endosperm. Furthermore, both increase in response to a signal from the embryo with which the endosperm must be in contact for at least 6 h. But what is also apparent from the single micropylar endosperm assays, in the presence or absence of germination regulators, is that the changes in the activities in two enzymes, while apparently similar at the seed population level, are not the same in every individual within that population. This brings into question whether it is prudent to search for common elements in the induction of the genes for ß-mannosidase and endo-ß-mannanase.
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Acknowledgements |
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These studies were completed by BM in partial fulfillment of the requirements for the PhD degree, during which she was supported by an Ontario Graduate Scholarship. The work is supported by NSERC grant A2210 to JDB.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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