CSG 15
MINISTRY OF AGRICULTURE, FISHERIES AND FOOD
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
MAFF, Area 6/01
1A Page Street, London SW1P 4PQ
An electronic version should be e-mailed to [email protected]
Project title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF project code
HH1323
Contractor organisation
and location
Horticulture Research International
Wellesbourne
Warwick, CV35 9EF
Total MAFF project costs
Project start date
£ 220,028
01/01/98
Project end date
31/12/00
Executive summary (maximum 2 sides A4)
This project aimed to develop our mechanistic understanding of the biophysical and biochemical factors
determining the fruit growth rate of tomato. Fruit size is an important determinant of quality in the tomato
industry and clearly, it depends on the rate and extent of fruit growth.
In a multicellular organ such as a 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. In the tomato fruit it
seems that most of the resistance to expansion is located within the outer skin (exocarp), which comprises a
single layer of epidermal cells plus about three layers of small compact thick-walled cells. Stress in the thin
walls of the large mesocarp parenchyma cells would become extreme unless much of it is exported to the skin.
An assay of tissue pressure showed that it was consistently about 75% of total turgor pressure. Furthermore, a
gradual reduction in turgor pressure, tissue pressure and epidermal stress was observed until ripening, when
there was a sharp fall in turgor pressure. Although the reason for this decrease cannot be stated with certainty,
accumulation of apoplastic solute appears probable. When the temperature was increased the turgor pressure
was slightly reduced although the proportion of turgor pressure exerted as tissue pressure remained constant.
Fruit growth was increased by reducing the number of fruits per truss, however, a slightly lower turgor pressure
was recorded. These data indicate that a change in turgor pressure was not responsible for the enhanced
growth rate observed with these treatments.
The mechanical properties of epidermal strips were tested with a constant load extensiometer which was able
to examine the extension (or “creep”) over a wide range of time scales. While material from mature and
growing fruits behaved in a similar way in the short term, after an hour the growing fruits had extended far
more. The long-term extensibility of epidermal strips was found to decline with fruit age. Furthermore,
increasing the temperature increased the extensibility as did reducing fruit numbers, which may explain why
these treatments promoted fruit growth.
CSG 15 (Rev. 12/99)
1
Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
The long-term extensibility increased when strips were incubated in a low pH buffer, this effect corresponds to
“acid growth” in other tissues. This effect was abolished by boiling and is consistent with the effect of
expansins. It may be that while xyloglucan-endotransglycosylase (XET) and peroxidase regulate the growth in
the long-term, creep mediating enzymes such as expansins facilitate short-term regulation of growth, probably
by active control of the pH of the epidermal apoplast. When calcium concentrations were increased from 1 to
10 mM extensibilities were considerably reduced. Furthermore, calcium chelators such as EDTA and EGTA
increased the long term extensibility, although the addition of sufficient calcium to saturate the chelator
reduced extensibility in proportion to the free calcium concentration, demonstrating that the effect was not
caused by the chelators themselves. Boiling tissues to inactivate cell wall enzymes did not reduce the effect of
EDTA and EGTA, nor were the effects of reduced pH and EDTA treatment additive. This suggests that
expansins and calcium chelators affect wall rheological properties in a similar way.
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. With respect to the termination of fruit growth, the enzyme most
often linked to decreases in wall extensibility is peroxidase. The fruit skin was found to contain more
peroxidase activity than the mesocarp and the wall-bound activity in the mesocarp was negligible. The skin of
mature fruits contained three additional peroxidase isoforms (Mr values of 44, 48, and 53 kD) which were
absent in the immature fruits. To assess whether the peroxidase isozymes were linked to ripening or growth, a
range of non-ripening mutants (nor, rin, Nr, Cnr, and Gr) were compared with those of the wild type (cv Ailsa
Craig) and the commercial variety (cv Counter). The fruit of these mutants grew in an apparently normal
manner and to a near-normal size, but they show greatly delayed or reduced ripening. In each of the mutants,
the additional isoforms of wall-bound peroxidase were apparent in the skin by the time of fruit maturity, as in
the wild type and commercial varieties, indicating that the new wall-bound peroxidase isozymes are unlikely to
be associated with softening or ripening. This was confirmed when immature fruits were prematurely ripened
with exogenous ethylene. Neither the ethylene treated fruit (which were showing colour) or the control fruit
detached from the vine developed the same peroxidase isozyme pattern as observed in mature fruit exocarp
tissue.
Localisation studies showed the wall-bound peroxidase activity was associated with the outer fruit exocarp,
vascular tissue towards the calyx end and the developing seed coats in young fruit; the latter two being sites of
lignification. There was negligible activity in the cuticle itself, although an involvement of peroxidase in the
formation of the cuticle cannot be disregarded. Localisation studies were also conducted on a sub-cellular
level. In young fruits (which only had the 58kD isozyme) the activity was restricted to the vacuolar tonoplast,
and may be associated with vacuole formation. In mature fruits the peroxidase activity was associated with the
cell walls, plasma membrane, large vesicles and vacuolar ‘debris’. To test whether the three new peroxidase
isozymes may be involved in stiffening of the exocarp cell walls, the rheological properties of a developmental
range of fruit exocarp strips were examined following incubation in the dialysed wall-bound fraction eluted from
mature fruit exocarp. When exocarp strips were subjected to freeze / thawing to rupture the cell matrix of the
strips and to allow penetration of larger molecules such as peroxidase both hydrogen peroxide and the
dialysed mature fruit exocarp wall bound fraction increased the stiffness of the tissue. It is concluded that the
newly identified peroxidase isozymes that appear in cell walls of the skin towards fruit maturity 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.
CSG 15 (1/00)
2
Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Scientific report (maximum 20 sides A4)
SCIENTIFIC OBJECTIVES
1. To quantify the relationship between pericarp cell turgor and pericarp “tissue tension” for various stages of
fruit development, and show how this is influenced by factors which alter fruit growth rate, such as light,
temperature, and water supply.
2. To determine the mechanical properties of the epidermis at various stages of fruit development, and to
identify the influence thereon of factors which alter fruit growth rate.
3. To characterise patterns of activity of the wall enzymes XET, expansin, and peroxidase, and determine how
these alter with changes in fruit growth rate and epidermal mechanical properties. This will clarify which
enzymes are responsible for promotion and cessation of growth, and it will indicate their potential as
biochemical markers of fruit expansion rate.
4. To identify aspects of the cell-wall environment, such as pH and Ca concentrations, which could influence
mechanical properties of the cell wall, and so affect fruit growth rate.
5. To determine the biophysical and biochemical properties of fruit tissue that lead to seasonal variation in fruit
size and fruit splitting in glasshouse tomato production.
All of these objectives were met in full and on time. An overview of the work conducted to fulfil these objectives
follows.
INTRODUCTION
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 determine
fruit size, which is a major determinant of quality in tomato. In addition, they are also important factors in major
disorders such as cracking and blossom-end rot.
In round-tomato cultivars grown commercially in the UK, fruit development takes about seven to eight weeks,
depending on temperature. During this time, the fruit grows from virtually zero to some 50-70 mm in diameter,
(depending on variety). Most of this growth takes place during the period 15-35 days 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 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, all the stress arising from a cell’s turgor pressure is borne by that cell’s wall. However, in a
multicellular organ such as a 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. 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. 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, and they have significant turgor pressure throughout most of the period of fruit
growth. 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. This research
project has aimed to develop a better understanding of the regulation of tomato fruit growth.
CSG 15 (1/00)
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
OBJECTIVE 1 - To quantify the relationship between pericarp cell turgor and pericarp “tissue tension”
for various stages of fruit development, and show how this is influenced by factors which alter fruit
growth rate, such as light, temperature, and water supply.
Assay of tissue pressure
Previously the turgor pressure transmitted from the mesocarp to the exocarp (known as tissue pressure) was
estimated by determining fruit water potential in vivo using a pressure probe and nanolitre osmometer and
comparing this with the water potential of pieces of excised mesocarp incubated in polyethylene-glycol
solutions of a range of osmotic pressures.
During the course of this project, an improved method for determination of tissue pressure has been
developed. It was predicted that turgor pressure of mesocarp cells should fall by approximately the tissue
pressure after excision from the fruit. Turgor pressures were determined in intact fruit using the single cell
pressure probe and then the fruit was rapidly dissected. A piece of the fruit was covered with a thin layer of
petroleum jelly and transferred to a transparent cuvette maintained at 100% RH using a constant flow of
humidified air. Further pressure probe measurements of the turgor pressures of cells in the excised piece of
tissue were obtained by inserting the probe through a small window in the side of the cuvette. From these
measurements it was found that the above prediction was correct. This provided a more accurate method for
determination of tissue pressure which was used in further experiments.
Developmental changes in tissue pressure
This procedure was used to examine changes in tissue pressure throughout fruit development. It was
observed that tissue pressure was consistently about 75% of total turgor pressure and therefore followed the
same developmental pattern as turgor pressure (i.e. a good empirical linear relationship with fruit age for the
majority of development). These results are shown in figure 1a. The stress created by tissue pressure is also
affected by the architecture of the fruit; especially the changing relationship between pericarp area and fruit
circumference. Allowing for these changes did not substantially alter the developmental pattern of epidermal
stress (the relationship between fruit age and epidermal stress is shown in figure 1b). A gradual reduction in
turgor pressure, tissue pressure and epidermal stress was observed until ripening, at which point all three
values fell to approximately zero. This late fall in turgor pressure is likely to be an important part of fruit
softening (it will for example considerably affect penetrometer results). The fall in turgor pressure occurs
despite a gradual increase in cell sap solute concentration (measured using a nanolitre osmometer) and
therefore must correspond to a decrease in apoplastic water potential. Although the reason for this decrease
cannot be stated with certainty, accumulation of apoplastic solute appears probable.
Figure 1a illustrates turgor pressure measured in vivo ( ), the turgor pressure after excision ( ) and the
calculated tissue pressure ( ) for a series of measurements throughout fruit development. Figure 1b shows
the resultant epidermal stress ( ).
4
Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Effect of environment on tissue pressure
The effect of temperature upon tissue pressure was also examined. Although turgor pressure was slightly
reduced at increased temperatures, the proportion of turgor pressure exerted as tissue pressure was not
affected, so that a slight reduction in tissue pressure was observed. “Gape” tests using hoops of tomato fruit
incubated in solutions of different water potentials also show that tissue pressure was only eliminated when
turgor pressure fell to zero, again indicating that as fruit water potential is reduced, tissue pressure remains
proportional to turgor pressure.
Fruit from plants in which trusses were reduced to a single fruit early in development were found to have
slightly lower turgor pressures than control plants, even though growth rates of these fruit were increased. This
observation was slightly surprising, but corresponded well with increased epidermal extensibility in fruit from
reduced trusses and an increase in extensibility in mid-summer, when assimilate supply was expected to be
greatest.
OBJECTIVE 2 - To determine the mechanical properties of the epidermis at various stages of fruit
development, and to identify the influence thereon of factors which alter fruit growth rate.
Analysis of rheological data
Epidermal mechanical properties were characterised in a number of experiments employing a custom built
constant load extensiometer constructed in Lancaster. This type of instrument can be used to examine the rate
of extension (or “creep”) of plant material over a wide range of time scales. Quantitative interpretation of this
data has not been straightforward and has required extensive modelling of the rheological behaviour of plant
cell walls. The model breaks the total extension of a strip of tomato epidermis subjected to a constant stress
into a number of mathematical functions which together account for the observed creep with a high degree of
precision (r2 > 0.999 in most cases). As is common in such rheological models, each component of the model
primarily affects creep over particular periods in the overall duration of extension. Comparison with analogous
artificial polymer systems has suggested that the longer term components are of greatest importance in
growth. This can be illustrated by examination figure 2, which is a plot of the logarithm of length of epidermal
strips from growing and mature fruit relative to its initial length against the logarithm of time elapsed since
stress was applied. As can be seen, the mature and growing material did not differ much in the early part of
the time course, but the strip from the growing fruit exhibited a distinctive upward turn in this type of plot after
about an hour. This behaviour corresponds to the point at which flow of polymers becomes the primary
process in creep of artificial polymer systems and is a consistent signature of growing cell walls. This is
perhaps intuitive, as growth also occurs over prolonged time scales. An additional point of some interest is that
creep of stem and leaf tissue has been found to be best described by the same model. It therefore appears to
be of general relevance despite being developed from analysis of a rather unusual tissue. It should also be
noted that pH has a profound effect upon epidermal properties, especially long term behaviour.
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Figure 2 is a plot of natural log length against natural log time for epidermal strips from growing ( )and mature
fruit ( ) incubated at pH 5.0 and from growing fruit incubated at pH 6.0 ( ).
Figure 3 illustrates changes in the long term “extensibility” of strips of tomato fruit epidermis during fruit
development. The additional effect of pH is of considerable importance and will be further discussed under
objective 4. These results are consistent with an effect of XET upon overall extensibility, but potentially
extensively modulated by a pH effect.
Figure 3 shows the long term extensibility of fruit epidermal strips throughout development. The effect of
incubating the strips in buffers of pH 4.5 ( ), pH 5.0 ( ), pH 5.5 ( ) and pH 6.0 ( ) are shown.
Environmental effects upon extensibility
Increasing the temperature at which strip extensibility was assayed by 10°C had a considerable effect upon
the long term extensibility, causing an increase by a factor of between 2.5-3.5. This does not correspond with
the observed effect of increased temperature on fruit growth rate where a 10°C temperature increase caused
fruit growth rate to increase by a maximum of 1.6 and by rather less at higher temperatures. This confirms that
the observed effect of temperature on fruit turgor pressure and tissue pressure is of considerable importance
in determining final fruit size.
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
An additional seasonal effect upon epidermal long term extensibility has also been observed. It was found that
extensibilities in mid-summer were almost twice those in spring and autumn (even though the extensibilities
were assayed at a constant temperature). It was not possible to ascertain whether this effect was caused by
increased light levels or temperature, but an effect of light intensity would be consistent with an observation
that fruit of plants transferred from a greenhouse to a constant temperature growth chamber exhibit a gradual
reduction in growth rate. It was also found that after pruning adjacent trusses and reducing the number of fruit
on a truss increased extensibility was observed within 24 hours, further suggesting that wall extensibility is
regulated in response to assimilate availability.
Cherry tomatoes
Cherry tomatoes have also provided an interesting system for examination of the roles of tissue pressure and
epidermal extensibility. Turgor pressures (Fig. 4a), expansion rates (Fig. 4b) and epidermal extensibilities (Fig.
4c) of cherry tomatoes were measured throughout development and found to be comparable to those of round
fruit of a similar age. The cherry tomatoes ripened slightly earlier and the associated fall in turgor pressure can
be seen in Figure 4a.It appears that the final difference in fruit size is largely caused by events very early in
development.
Figure 4a is a time course showing developmental changes in turgor pressure in cherry and round tomatoes.
Figure 4b illustrates fruit relative expansion rates of cherry and round tomatoes throughout development and
Figure 4c long term extensibilities (cherry tomato results are filled circles and round tomatoes unfilled circles
in all three figures).
7
Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Atomic force microscopy
We have begun to develop a new method for analysis of cell wall rheological properties in collaboration with
the research group of Dr Hubert Pollock at the Physics Department at Lancaster. This method involves use of
an atomic force microscope to trace the surface of sections of plant material, providing images of extremely
high resolution under conditions comparable with those in vivo. Figure 5a is a transverse section of a tomato
leaf rachis cell wall at the surface of tissue bathed in solution (in this case a low calcium buffer) clearly showing
the fibrils of the wall structure. This image is striking but Figure 5b is potentially even more interesting. Here
the effect of the hardness of the surface upon the tapping frequency of the atomic force microscope tip has
been mapped to create the image, therefore demonstrating a method for analysis of wall biomechanical
properties on individual walls. It is expected that it will soon be possible to direct this method at specific cell
walls within tissues and to examine the effect of changing composition of the bathing solution.
A
B
Figure 5a is an image of a tomato stem obtained using one of the atomic force microscopes in the Lancaster
University Physics Department. Near the centre of the image a cell wall has been cut across revealing the
cellulose fibrils in the wall structure. In Figure 5b the effect of the surface mechanical properties upon the
frequency of the microscope tip has been used to indicate the hardness of the surface for the same area.
OBJECTIVE 3 - To characterise patterns of activity of the wall enzymes XET, expansin, and peroxidase,
and determine how these alter with changes in fruit growth rate and epidermal mechanical properties.
This will clarify which enzymes are responsible for promotion and cessation of growth, and it will
indicate their potential as biochemical markers of fruit expansion rate.
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, xyloglucanendotransglycosylase (XET) activity shows a general correlation with growth rate in tomato fruit (Thompson et
al. 1998) and expansins may also be involved in wall loosening. 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. In the tomato fruit,
growth slows and ceases as fruits approach maturity and this is correlated with a marked increase in the
activity of wall-bound peroxidase in the fruit skin (Thompson et al., 1998).
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Appearance of peroxidase isozymes
Those peroxidases that are thought to be responsible for cell wall stiffening via their cross-linking activity are
ionically associated with the cell wall. The ionic elution of these peroxidases using high ionic strength solutions
(e.g. 1M NaCl) represents the wall-bound fraction. PAGE on native gels showed that one or more peroxidase
isozymes were present in most fractions from the mesocarp and skin (exocarp) of tomato fruit throughout their
development (Fig 6). On a fresh-weight basis (Fig 6), 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.
Figure 6. 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).
Tentative molecular weights are assigned to the peroxidase isozymes in Fig 6. 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 localisation 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 localisation on gels. In our gels, the molecular-weight marker
proteins stained with coomassie, 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 kD, while the additional bands appearing
towards fruit maturity had apparent Mr values of 44, 48, and 53 kD. Trials on iso-electric focusing gels
revealed that the wall-bound peroxidase from mature fruit comprised only a single band at pI = 4.6. This
indicates that the various isoforms all have similar charge properties.
Extraction procedures used here probably measure 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 mM CaCl2). 1 M NaCl was used
routinely for consistency with previous work.
Is the peroxidase linked to ripening or growth ?
9
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Elevated temperatures were found to increase fruit growth rates, although a corresponding decrease in the
duration of growth was observed. Native PAGE revealed that the new more mobile isoforms also appear in the
fruit skin earlier when fruit are exposed to elevated temperatures. However, 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. To determine whether the late-appearing peroxidase isoforms are
associated with ripening, peroxidase patterns in the skin of mature fruit of several non-ripening mutants (nor,
rin, Nr, Cnr, and Gr) were compared with those of the wild type (cv Ailsa Craig) and the commercial variety (cv
Counter). 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 7), as in the wild type and commercial varieties. In addition, wall-bound peroxidase in the skin of
the rin mutant showed a progressive development of isozymes towards fruit maturity (Fig 7, lower) similar to
that in the commercial material (Fig 6). 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.
Figure 7. Wall-bound peroxidase isozymes in fruit of non-ripening mutants. Upper: wall-bound isozymes from
the skin of mature fruit of various mutants. Lower: wall-bound isozymes from the skin of rin fruit at various
stages of development from immature (left) to mature (right).
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. 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.
To further justify this hypothesis immature fruit (20dpa), which do not contain the three new peroxidase
isozymes in their outer exocarp were prematurely ripened with exogenous ethylene (500ppm). Fruit were
stored after harvest at 22°C in two sealed containers with (treatment) and without (control) exogenous
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
ethylene (500ppm). The atmosphere in each was replenished daily. After 13 days from the start of the
treatment a colour change was observed in those fruit subjected to exogenous ethylene. No colour change
was observed in the control fruit. Although the 53kD peroxidase isozyme appeared (Fig. 8), neither the
ethylene treated fruit or the control fruit detached from the vine developed the same peroxidase isozyme
pattern as observed in mature fruit exocarp tissue (Fig. 6). The results support the hypothesis that the
induction of the three new peroxidase isozymes is not ripening related.
Figure 8. Native gels stained for wall-bound peroxidase activity in the exocarp from detached fruits (20dpa)
prematurely ripened with 500 ppm exogenous ethylene or left untreated.
Tissue based peroxidase localisation.
Biochemical studies showed both a localisation of peroxidase activity in the outer fruit exocarp and an
associated developmental increase in peroxidase activity within this tissue layer. It was also demonstrated that
this developmental increase within the fruit exocarp is associated with the appearance of three new
isoperoxidases appearing as the rate of fruit expansion declines at the onset of fruit maturation. Using nitrocellulose tissue printing these results can be visualised on a tissue basis. The relatively high activity of wallbound peroxidase activity in the skin, especially in mature fruit, is clear from ‘tissue prints’ of salt-extractable
peroxidase activity (Fig 9). 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 figure 9 such
vascular bundles are cut mostly transversely. In young fruit much of the activity is associated with the
developing seeds.
Figure 9. Tissue prints of peroxidase. These were made by pressing the washed, cut surface of a halved fruit
onto nitrocellulose paper impregnated with 1M NaCl.
In many organs, peroxidase is associated with sites of lignification. It may be involved in polymerisation of
lignin components (Lagrimini, Bradford & Rothstein 1990). This may account for the concentrations of
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
peroxidase found over the vascular tissue in older fruit, and over the developing seed coats in young fruit (Fig
9). 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.
Cellular peroxidase localisation in the fruit exocarp
In some organs, such as the hypocotyl of Lupins, 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 10). However, the cuticle
represents a major component of the mature tomato fruit exocarp (Fig 11), and the possible involvement of
peroxidase in the formation of the cuticle cannot be disregarded.
Figure 10. Localisation 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 a. was boiled briefly with dithiothreitol to inactivate peroxidases prior to addition of the chloronaphthol
substrate. Staining is apparent as dark regions over the cell-wall region in b. but not the cuticle. No staining is
apparent in a.
Figure 11. A spurr’s embedded section of mature (45 dpa) tomato fruit stained with auramine O showing the
ingress of the cuticle into the exocarp cells.
Sub-cellular localisation in tomato fruit exocarp.
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
In common with many previous workers, 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 homogenisation. However, this possibility seems
unlikely given the extensive washing procedures employed to remove the symplastic component. Also, a
number of consistent differences were observed between the pattern of isozymes in the wall-bound and
soluble fractions: for example, the 58 kD band diminishes substantially in the wall-bound, but not in the
soluble, fraction of the skin towards full fruit maturity. Likewise, the cell walls of the mesophyll show negligible
salt-extractable peroxidase even when the soluble activity in that tissue is high. Furthermore, preliminary
studies in our laboratory failed to find any significant rebinding of soluble peroxidase onto salt-extracted cell
walls from skin or mesocarp of mature fruit. These various lines of evidence indicate that the salt-extractable
peroxidases are present in the wall in vivo.
However, rebinding of soluble peroxidase does not allow for possible changes in the binding capacity of the
walls following ionic elution. Therefore, it was necessary to examine the localisation of peroxidase at a subcellular level, to establish whether peroxidase is present in the cell walls in-vivo. Two developmental age
groups of tomato fruit exocarp were examined. 14dpa, which had only one peroxidase isozyme at 58kD and
45dpa which had four peroxidase isozymes 58, 53, 48 and 43kD. The results from fruit at 14dpa (Fig. 12)
showed that peroxidase activity was restricted to the vacuolar tonoplast; there appeared to be no wall bound
activity in the cell walls. It is speculated that peroxidase activity on the tonoplast membrane may be associated
with vacuole formation from vacuolar vesicles (Fig. 12b). From these sub-cellular observations under
transmission electron microscopy (TEM), we deduce that ‘wall-bound’ activity ionically eluted from immature
tomato fruit exocarp is more likely to be peroxidase activity associated with the tonoplast membrane.
A
B
Figure 12. A TEM image of immature fruit (14 dpa) exocarp cells stained with 3,3’-diaminobenzidine for
peroxidase activity.
In mature fruits (45dpa), peroxidase activity is associated with the cell wall, plasma membrane, large vesicles
(8 m) and vacuolar ‘debris’ (Fig. 13). We speculate that ‘wall-bound’ peroxidase activity will include the
activity associated with the wall together with a component associated in-vivo with the plasma membrane and
vesicles, whereas the soluble component will include activity associated with vacuolar ‘debris’ and a
component from the plasma membrane and vesicles.
The TEM images suggest that vesicles are formed from the plasma membrane and move into the vacuole
where they burst and fragment. If this is the case the peroxidase activity associated with the vesicle membrane
may be a remnant of that associated with the plasma membrane. The function of these vesicles is unclear,
although they may function as a way of re-mobilising excess plasma membrane laid down by exocytosis.
However, the energetics for movement of such large vesicles by endocytosis is questionable.
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Figure 13. A TEM image of mature green fruit (45 dpa) exocarp cells stained with 3,3’-diaminobenzidine for
peroxidase activity.
Effect of peroxidase on the mechanical properties of the Exocarp
To test whether the three new peroxidase isozymes may be involved in stiffening of the exocarp cell walls, the
rheological properties of a developmental range of fruit exocarp strips were examined (using an instron)
following incubation in the dialysed wall-bound fraction eluted from mature fruit exocarp. When exocarp strips
excised from 30dpa fruit were incubated in 10mM mes buffer at pH 6.0, adding 18mM hydrogen peroxide
resulted in a significant increase in stress, strain and stiffness, although adding the dialysed mature fruit
exocarp wall bound fraction had no effect. However, when exocarp strips were subjected to freeze / thawing to
rupture the cell matrix of the strips and to allow penetration of larger molecules such as peroxidase both
hydrogen peroxide and the dialysed mature fruit exocarp wall bound fraction increased the stiffness of the
tissue. These results support our hypothesis, however further studies are required to verify these results.
Results presented here demonstrate that new peroxidase isozymes of apparent Mr 44, 48, & 53 kD, appear in
cell walls of the fruit skin coincident with the period of cessation of fruit growth. Given that fruit growth depends
mainly on the mechanical properties of the skin, and that peroxidases appear to stiffen cell walls, these
isozymes could be causally involved in the cessation of fruit growth.
OBJECTIVE 4 - To identify aspects of the cell-wall environment, such as pH and Ca concentrations,
which could influence mechanical properties of the cell wall, and so affect fruit growth rate.
pH effects
In section 2 it was noted that pH exerted a considerable effect upon long term extensibility. This was not
surprising, and clearly corresponds with the “acid growth” observed in many other tissues. This effect was
abolished by boiling. These properties are entirely consistent with the effects of expansins. From Figure 3 it is
apparent that although extensibility is considerably affected by pH, there is a developmental trend of
decreasing extensibility even in pH 6.0 buffers. These observations suggest that extensibility is regulated in
the long term by enzymes regulating wall composition such as XET and peroxidase, but that creep mediating
enzymes such as expansins facilitate short term regulation of growth, probably by active control of the pH of
the epidermal apoplast. It was hoped that we would be able to assay the pH of the epidermal apoplast,
unfortunately, this proved to require more time than could be accommodated within the current project.
Calcium effects
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Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Because of the known requirement of tomato plants for high concentrations of calcium for normal fruit
development, the effect of calcium upon the extensibility of epidermal strips and leaf mid-rachis tissue was
also examined. The results from these experiments have proved to be very interesting and the effect of
calcium concentration and a calcium chelator (EDTA) upon the rheology of strips from a growing tomato fruit
are illustrated in Figure 14. It was found that extensibilities were considerably reduced as calcium
concentrations were increased from 1 to 10 mM. Furthermore, calcium chelators such as EDTA and EGTA
affected extensibilities in a similar manner to reduced pH. Addition of sufficient calcium to saturate the chelator
reduced extensibility in proportion to the free calcium concentration, demonstrating that the effect was not
caused by the chelators themselves. Boiling tissues to inactivate cell wall enzymes did not reduce the effect of
EDTA and EGTA, nor were the effects of reduced pH and EDTA treatment additive. This suggests that
expansins and calcium chelators affect wall rheological properties in a similar way. This may have
considerable implications for tissues such as tomato fruit which are prone to calcium deficiency. If tissue
growth rate is normally modulated by alteration of expansin activity by regulation of expansin gene expression
and apoplastic pH, then extremely low concentrations of calcium in the cell may lead to uncontrolled rapid
growth, in turn diluting cell wall calcium still further. Cell wall rigidity is required for plant cells to survive their
high internal turgor pressures, and it is possible that excessively flexible cell walls could lead to a loss of
cellular integrity. It could be argued that this might plausibly contribute to blossom end rot. Likewise the
elevated calcium levels to be expected in the vegetative tissues of plants treated with the high calcium
concentrations required to prevent calcium deficiency in the fruit might lead to some inhibition of vegetative
growth. This may be advantageous provided the inhibition is not extreme enough to significantly reduce
assimilate supply. It appears that under many circumstances growth of reproductive tissue is selectively
maintained under conditions leading to reduction of vegetative growth.
Figure 14 is a plot of natural log length against natural log time for epidermal strips from growing fruit
incubated in buffers containing 10 mM CaCl2 ( ), 1 mM CaCl2 ( ) and 5 mM EDTA ( ).
OBJECTIVE 5 - To determine the biophysical and biochemical properties of fruit tissue that lead to
seasonal variation in fruit size and fruit splitting in glasshouse tomato production.
Our previous observation that fruit turgor pressure and tissue pressure follow an extremely stable
developmental progression, which is not altered even when the water relations of the rest of the plant are
altered have been reinforced by this work. Surprisingly, it appears that tissue pressure is only affected by
temperature and source-sink relations and under these circumstances tissue pressure falls even though the
growth rate increases. It is unclear whether a causal or regulatory relationship exists, but it appears certain the
large effects of temperature and source-sink relations upon epidermal extensibility would cause a much
greater increase in fruit growth rate if the observed decrease in tissue pressure did not occur.
15
Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
It also appears that epidermal extensibility is tightly controlled and that this regulation allows control of fruit
growth in the long, medium and short term. In the long term the structural properties of the wall are
developmentally regulated, probably primarily by XET and cell wall bound peroxidase. Our results show that
several new isoforms of peroxidase appear in the cell walls of the skin at around the time that fruit growth is
terminating. 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).
Extensibility is also altered in response to greenhouse conditions (probably primarily assimilate supply) and
fruit number. This alteration includes both pH dependent (i.e. expansin) and pH independent (i.e. structural
properties correlated with XET activity) components. Finally, extensibility is affected by temperature and the
ionic composition of the apoplast. Controlling the pH of the apoplast is likely to be an important mechanism of
short term growth regulation. Furthermore, calcium affects epidermal extensibility and if it’s concentration falls
to very low levels, growth rates may increase.
REFERENCES
Andrews J. (1995) The climacteric respiration rise in attached and detached tomato fruit. Postharvest Biology
& Technology 6, 287-292.
Bestwick C.S., Brown I.R. & Mansfield J.W. (1998) Localised changes in peroxidase activity accompany
hydrogen peroxide generation during the development of a non-host hypersensitive reaction in lettuce. Plant
Physiology 118, 1067-78.
Ferrer M.A., Munoz R. & Ros Barcelo A. (1991) A biochemical and cytochemical study of the cuticleassociated peroxidases in Lupinus. Annals of Botany 67, 561-568.
Grange R.I. (1995) Water relations and growth of tomato fruit pericarp tissue. Plant Cell & Environment 18,
1311-18.
Pearce B.D., Grange R.I. & Hardwick K. (1993) The growth of young tomato fruit I. Effects of temperature and
irradiance on fruit grown in controlled environments. Journal of Horticultural Science 68, 1-11.
Thompson D.S., Davies W.J. & Ho L.C. (1998) Regulation of tomato fruit growth by epidermal cell wall
enzymes. Plant Cell & Environment 21, 589-99.
PUBLICATIONS RESULTING FROM THIS WORK
D.S. Thompson, W.J. Davies & L.C. Ho. (1998) Tomato fruit growth regulation by epidermal cell wall enzymes.
Plant, Cell and Environment 21, 589-599.
W.J. Davies, D.S. Thompson and J.E. Taylor. (1998) Manipulation of growth of horticultural crops under
environmental stress. In: Genetic and Environmental Manipulation of Horticultural Crops, eds. K.E. Cockshull,
D. Gray, G.B. Seymour and B. Thomas. pp 157-174
Thompson D.S., Smith P.W., Davies W.J., Ho L.C. (1999). Interactions between environment, fruit water
relations and fruit growth. Acta Horticulturae 487, 65-70.
Andrews J., Malone M., Thompson D.S., Ho L.C., Burton K.S. (2000). Peroxidase isozyme patterns in the skin
of maturing tomato fruit. Plant, Cell & Environment 23, 415-422.
16
Project
title
Biophysical and biochemical regulation of tomato fruit growth.
MAFF
project code
HH1323
Davies, W.J., Bacon, M.A., Thompson, D.S., Sobeih, W. and Gonzales-Rodriguez, L.. (2000) Regulation of
leaf and fruit growth in plants growing in drying soil: exploitation of the plant's chemical signalling system and
hydraulic architecture to increase the efficiency of water use in agriculture. Journal of Experimental Botany 51,
1617-1626.
Presentations:
Thompson DS. Rheology of the epidermis of growing tomato fruit. At: Plant Protein Club Workshop ‘Plant
proteins and the mechanical properties of cell walls’. Alicante, Spain. 10-12 April 1999.
Davies WJ. Using plant stress to improve quality. At: HDC/TGA/HRA 1999 Tomato Conference. Coventry, UK.
7 October 1999.
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