Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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Anatomy of tomato fruit and fruit pedicel during fruit development
D. Rančić, S. Pekić Quarrie and I. Pećinar
Faculty of Agriculture University of Belgrade, Nemanjina 6 11080 Zemun, Serbia
Paper reviews on the anatomy of berry fruit development by comparative analysis of fruit and fruit pedicel in tomato
combining our results with other authors. We have presented data on developmental analysis of fruit: measurements of
different anatomical paramenters in fruit and fruit pedicel together with parameters of the fruit size. Anatomical
background of the water and solute transport during fruit development is elaborated by discussing all possible factors
affecting fruit development. The genotypes used in our research were tomato deficient mutant and wild type to try to
elucidate role of ABA in these proceses. Fruits of the ABA deficient mutant are smaller as consequence of smaller-sized
cells in pericarp since functional xylem area in fruit pedicels and diameter of xylem elements in both genotypes are
similar. Wild type has more nonfunctional xylem that could serve as a mechanical support for heavier fruits. Lower fruit
dry weight and phloem efficiency in pedicels of ABA deficient mutant compared to the wild type suggests important role
of ABA in the fruit sink activity.
Keywords xylem; phloem, ABA, tomato
1. Introduction
In tomato as well as in other crops size of the fruit is the key factor determining yield. This is why mechanisms of
regulating fruit growth and development were the research topic for many authors [1, 2]. Crop yield is the result of
many morfo-physiological and biochemical processes depending on environmental factors and genetical background
[3]. As the growth of fleshy fruits is to the great extent result of water accumulation, for the understanding of the fruit
developmental processes it is important to understand water transport as well as coordination between long distance
transport of water and solutes and short distance transports such as water absorption of the individual fruit cells [4].
Understanding of the plant and fruit anatomy could be, therefore, the key factor in understanding transport of water,
assimilates and signalling molecules within the plant [5] and, consequently its effects on yield. There are only few
papers reporting tomato fruit anatomy [6-8], while detailed anatomical and histochemical analysis during tomato fruit
development was done by Gillaspy et al. [9]. Cytological analysis of tomato fruit has shown that in the early phase of
fruit development cell division is the main limiting factor for fruit growth [10], and also that cytological changes at the
this phase are strongly dependent on transport of water and assimilates into the fruit [11]. For understanding factors
affecting fruit growth and development it is important to analyse comparatively anatomy of both, fruit and fruit pedicel.
Here we present detailed anatomical analysis of the tomato fruit and fruit pedicel anatomy during fruit development to
be able to show to which extent anatomy of the fruit pedicel determines/restricts water transport into the fruit and,
consequently, affects fruit size and fruit anatomy. Material used in this investigation: tomato ABA deficient mutant and
wild type represent good objects for examining of the role of ABA on plant morphology and physiology [12]. For
example, fruits of the ABA deficient mutant are smaller [13] and it is not known is it due to the smaller number of cells
in the pericarp or their smaller size due to reduced turgor. Comparative analysis of fruit anatomy in both genotypes
could provide explanation of these differences in fruit anatomy as well as to the possible role of ABA.
2. Materials and methods
Two tomato genotypes (Lycopersicon esculentum Mill.): wild type (Ailsa Craig) and flacca (CM Rick Tomato Genetics
Resource Center, USA) were investigated. Leaves of wild type in optimal water conditions have about 2.9 ng ABA per
mg dry weight [14], while flacca leaves only 26% amount of ABA compared with wild type [15]. The plants were
grown from seeds in commercial substrate (Potground H, Klasmann-Deilmann, Germany) in chamber operating with a
14h photoperiod with light intensity at plant level 300 µmolm-2s-1, temperature 25/18˚C and relative humidity 70%.
The root system was irrigated daily to a soil water content represents 80% of maximum soil water capacity.
Fruits and fruit pedicels were collected in three developmental phases: 12, 20 and 39 days after anthesis. Fruit
diameter, fresh and dry weight, thickness and number of cell layers in fruit pericarp, egzo and mezocarp, as well as
phloem area, diameter of xylem elements, xylem area and functional xylem area of the pedicel at the transversal
sections 5mm apart from the fruit were measured. Phloem efficiency was calculated by dividing fruit dry weight by
phloem pedicel area at the final stage of fruit development.
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2.1
Light microscopy
Slides for light microscopy were made according to standard procedure. Samples were fixed in FAA for 24h, postfixed
in 70% ethanol and dehydrated in a graded ethanol series. After tissue impregnation in Histowax (56-58°C) samples
were embedded. After cooling the blocks on a cold plate and solidifying paraffin, histological sections of about 5–7 µm
were cut using a microtome (Leica SM 2000 R). Before staining, the paraffin is removed from the sections by xylene,
followed by rehidratation in graded series of ethanol, and tissue was stained by safranine and alcian blue.
2.2
Xylem tracing
Water transport through fresh fruit pedicel was investigated using eosin, water soluble xylem mobile dye which easily
flows through vessels and pits and could be used as xylem tracer [16]. Pedicels were cut from plants, trimmed under
water and the end near cut stem was immediately immersed in 1 ml 1% water solution of eosin for 15 minutes. After
short rinsing, pedicels were cut transversally or longitudinally. Sections were investigated using Leica DMLS
epifluorescent microscope (filter A 340-380nm). Xylem, thanks to presence of lignin, in UV light shows
autofluorescence and emits blue light, but after eosin staining, fluorescence and emitted light is yellow. Since eosin
flows only through functional xylem elements, comparing blue areas with yellow ones it is possible to get information
about number of functional and non-functional xylem elements.
3. Fruit anatomy-developmental analysis
Tomato fruit consists of pericarp and seeds. Pericarp is composed from: egzocarp, mezocarp and endocarp. Outer layer
of cells in the egzocarp is epidermis and below there are two to three layers of hypodermal cells with thick cell walls.
Epidermis hasn’t stomata and has relatively thin cuticle, and the thickness of cuticle increases with the fruit growth.
Mezocarp is made from large thin wall cells and vascular tissue. Fruit vascular tissue is connected to pedicel vascular
tissue [17]. One vascular branch pass trough central and radial mezocarp to the seeds, while other vascular branches
radialy pass through outer layer of mezocarp [9] parallel to fruit surface [18], with week branching on proximal side,
but more on distal side with simultaneously decreasing ratio of xylem and increased ratio of phloem [19]. Endocarp is
unicellular layer boundaring locular cavity. Carpelar septe divide ovarium into two or more loculi. Elongated central
placenta, with attached seeds, is made of parenchima tissue and represents primary tissue which later fills the locular
cavities.
According to Gillaspy et al. [9] growth and development of tomato fruit could be divided into four phases. First
phase is development of ovary and fertilisation. Immediately after fertilization, starts the second phase - in ovary starts
cell divisions lasting 7-10 days, followed by tissue differentiation, seed development and early growth of embryo.
Number of cell divisions and duration of this phase could differ among fruits and both factors determine final fruit size.
After cell division phase, during 6-7 weeks fruit growth takes place due to increasing cell volume, until fruit reaches its
final size. Although fruit growth depends on cell division as well as on cell growth, in most plants, increasing cell size
has major role in determining final fruit size. In tomato, the size of placenta, locular tissue and mezocarp could be
increased more than 10-fold [9] due to the cell elongation. At the phase when fruits reach their final size, fruits are
green and have most of its final weight. The last phase starts after reaching final fruit size, and fruit development
continues with ripening when inflow of carbohydrates in fruits stops [20] and fruit change colour from green to orange
and finally red [21]. First change in colour is consequence of transformation of chloroplasts in chromoplasts, and
decreasing chlorophyll concentration, followed with increasing in beta-carotene concentration which gives fruit orange
colour. Final red colour is due to high concentration of lycopene [19]. Simultaneously with colour changes, metabolic
changes occur. At this phase, disintegration of inner and central layer of carpel walls [18] occurs, and placental tissue is
softening as a consequence of enzimatous degradation of cell walls and becomes gelatinous. Major biochemical events
at this stage are: increase in ethylene synthesis, increase in respiration, fruit acidity rapidly increases and decreases after
that, content of starch decreases and sugar content increases. Some other authors, contrary to Gillaspy et al. [9]
distinguish three major periods in the growth of tomato fruit: cell division, growth phase and ripening [22].
Illustration of the anatomy of developing tomato fruit in both genotypes is presented at Figs. 1 and 2.
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Fig. 1 Transversal sections of pericarp of wild type tomato fruit at 12, 20, 39 days after anthesis (from left to right, respectively)
(bar = 500 µm). (end – endocarp; mes – mesocarp; egz– egzocarp).
Fig. 2 Transversal sections of pericarp of ABA deficient tomato fruit at 12, 20, 39 days after anthesis (from left to right,
respectively) (bar = 500 µm). (end – endocarp; mes – mezocarp; egz– egzocarp).
Our results show that between 12 and 39 days after antesis (in Table 1) fruit diameter increases almost 4 times in
wild type as well as in flacca mutant, and fruit weight 40-50 times. Fruit diameter of flacca mutant is in all
developmental phases 1.3-1.6 times smaller, and fruit weight 2-3 times smaller than in wild type. On micrographs of the
fruit sections (in Fig. 1 and 2) it is possible to see that egzocarp is in both genotypes 3-4 layer of cells thick. Egzocarp
thickness does not change significantly during fruit development in any of the genotypes, but is about 14-24% thinner in
flacca mutant compared with wild type. Between 12 and 39 days after anthesis, the number of mezocarp cells is
constant and in both genotypes are 15-16 cells. As a result of intensive cell growth during this period, the thickness of
the mezocarp in both genotypes increased about 3 folds. Although in the early stages of development there are no
differences in the thickness of mezocarp between wild type and flacca mutant, in the final stage of fruit growth
mezocarp thickness in the wild type is about 20% greater than in the the flacca mutant. During this developmental phase
endocarp is presented as a single layer of cells, endocarp thickness is similar for the wild type and flacca, increasing
during the fruits development by about 2 fold. The number of cells in pericarp in both genotypes is 20-21 cells and does
not change from 12 to 39 days after anthesis. Although egzocarp thickness during this period remains the same, due to
increasing the size of the mezocarp cells and to less extent to increasing size of endocarp cells, pericarp thickness
increases about 3 times. In early phases of fruit development there are no major differences in the thickness of pericarp
between flacca and wild-type, but in the phase of final fruit size the thickness of pericarp is about 80% thicker in wild
type compared to the flacca mutant. Since there is no difference in the number of pericarp cells between flacca mutant
and wild type, the difference in the size of the fruit is, therefore, only a consequence of smaller-sized cells in ABA
deficient genotype. Smaller cells in ABA deficient mutants are probably consequence of water stress, because even in
optimal water conditions, due to permanently open stamata, ABA deficient mutant is loosing much more water than the
wild type. As the plants or tissues with smaller cells are generally more tolerant of lower water potential [23], drought
affects cell elongation more than cell division [24], so that tissue exposed to environmental conditions with less
available water is generally characterized by smaller size cells, and more prominent lignifications [25].
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Table 1 Developmental analysis of tomato fruit (*, ** and *** indicated differences between wild type and flacca
genotype significant at p≤0.05, p≤0.01and p≤0.001, respectively).
fresh fruit weight
fruit diameter
number of cell layers in
mezocarp
number of cell layers in
pericarp
number of cell layers in
egzocarp
thickness of mezocarp
thickness of egzocarp
thickness of endokarp
thickness of pericarp
genotype
12 days after
anthesis
20 days after
anthesis
39 days after
anthesis
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
0.81±0.26
0.29±0.42 **
12.3±12.1
7.9±9.6***
16.1±1.4
15.9±1.4
20.1±1.4
19.9±1.4
3±0
3±0
480.8±89.1
443.6±87.3
35.3±2.2
27.5±3.9***
15.4±5.3
14.5±3.2
524.1±100.8
487.1±90.4
7.41±0.70
2.80±1.00***
26.0±1.6
18.7±2.7***
16.2±1.1
16.2±1.6
20.2±1.6
21.0±1.7
4±0
3±0
784.5±116.0
730.2±105.4
36.5±3.9
27.7±1.3***
23.0±3.9
19.0±3.1**
846.5±99.6
774.4±110.6***
38.29±3.62
11.69±1.51***
47.9±2.3
29.8±1.7***
16.5±0.6
16.5±1.1
20.8±0.7
20.4±1.0
4±0
3±0
1670.9±209.7
1296.1±104.3***
36.7±5.1
31.7±3.3***
31.5±7.6
26.7±6.2
1740.6±211.3
1355.1±107.4***
4. Anatomy of the fruit pedicel during fruit development
On the petiole surface is epidermis with long narrow cells covered with cuticule and relatively small number of stomata.
In the epidermis are two types of hair: long multicellular nonglandular hairs and glandular trichome. Below the
epidermis there are several layers of chlorenchima cells. In the primary growth phase of the pedicel, characteristic only
for the flower stage, vascular system consists of bicolateral bundles separated with pith rays. As a result of secondary
growth, simultaneously with the fruit growth, there is significant increase of the cross sectional area of the pedicels.
Secondary growth begins with initiation of the vascular cambium. Secondary xylem is already well developed in fruit
size 6-10 mm (in Fig. 3 and 4) and at the cross sections of pedicels is evident ring of xylem tissue with narrow one-cell
secondary pith rays. At the outer and inner side of xylem is phloem tissue forming discontinued ring, separated from
each other and from xylem with parenchyma. New formed elements of secondary xylem and phloem pushed primary
xylem towards the centre and primary phloem toward the outer part of pedicel. As a result of forming secondary
phloem, in the later stages of pedicel development outer phloem becomes more or less continuous. In the outer region of
pericicle and somewhat less frequently at the inner side of inner phloem are located groups of mechanical fibres. A
central part of the pedicel is composed of large parenchimatous cells.
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Fig. 3 Functional analysis of xylem in wild type fruit pedicel 12, 20, 39 days after anthesis (from left to right, respectively): light
microscopy (above) and fluorescence microscopy (below) (bar = 500 µm). Note intensive fluorescence of functional xylem stained
with eosin
Fig. 4 Functional analysis of xylem in flacca fruit pedicel 12, 20, 39 days after anthesis (from left to right, respectively): light
microscopy (above) and fluorescence microscopy (below) (bar = 500 µm). Note intensive fluorescence of functional xylem stained
with eosin
Our results show (in Table 2) that during fruit development from 12 to 39 days after flowering, fruit pedicel cross
sectional area increases about 2 fold and there are no significant differences in cross sectional area between the flacca
and wild-type. Phloem area increases similarly with pedicel thickening and in this period of fruit development also
increases 2-3 times. Larger phloem area can provide greater assimilate inflow into fruits and faster growth of fruit, but
transport actually depends more on phloem efficiency than of phloem area on the stem cross section [26], [27]. There
are no differences in phloem area on the cross sections between genotypes in any growth phase, but the phloem
efficiency in the final stage of fruit growth in flacca mutant is about 30% less than in the wild type: in flacca mutant it is
10.19 ± 4 17 g / mm2, while in wild-type 14.40 ± 7.25 g dry mass per mm2 phloem area. More efficient phloem
transport in wild type could be the reason for almost twice higher fruit dry mass (2.63 ± 0.40 g) compared to the dry
mass of fruits in ABA deficient genotype (1.44 ± 0.14 g).
Xylem area in pedicel during fruit development from 12 to 39 days after anthesis increased about 6 fold, both in the
wild type and in the flacca mutant. During the early stage of fruit development xylem represents only 8-11% of the
pedicel cross section area, but until the 20th day after anthesis xylem takes about 25% in flacca and about 30% in wild
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type and this percentage does not change much until the final stages of fruit development. According to Garcia-Luis et
al [27] xylem of citrus pedicel is also the most developed tissue and takes a large part of the pedicel (about 42-46% of
total cross section area). Similar to our data, Bustan et al [28] have shown a rapid development of vascular tissue in
citrus pedicel occurring in a relatively short period of time, and, also, that pedicel thickening slows down at the
beginning of the linear growth phase of fruits. Development of pedicel preceeded the phase of the intensive fruit
growth, which means that the most intensive development of the pedicel vascular system occurs in the early stages of
fruit development, at a time when the demand for assimilates is still relatively low [27]. It is interesting that in tomato
fruit pedicels much of the xylem is not functional for water transport [29], with only 16-20% functional xylem in wild
type and 23-24% in flacca mutant. Although the xylem area on the cross section of the fruit pedicel of wild type in the
later stages of fruit development is larger by about 25% compared to flacca mutant, there is no difference in the
functional xylem area between these two genotypes. In addition, diameter of functional xylem elements in both
genotypes is in average about 8-10µm and does not change significantly during pedicel development. A similar area of
functional xylem and similar diameter of xylem elements indicate that the hydraulic conductivity of fruit pedicel in both
genotypes is similar. However, data reviewed by Holbrook et al. [30] showed that the ABA-deficient mutants have
reduced root hydraulic conductivity probably as a consequence of changes in the architecture of xylem [31]. Larger area
of non-functional xylem in wild type fruit pedicels compared to ABA deficient genotype may have an important
mechanical role to support much larger and heavier fruits [26]. It is not yet clarified what is the trigger or the
mechanism for formation of new vascular tissue in the pedicel. It is considered that probably growth regulators (for ex.
auxin) synthesized in young fruits are transported basipetaly and promote cambial activity [32]. In this way,
development of the pedicel vascular tissue is adapted for the future transport and mechanical requirements of the fruit.
Table 2 Anatomical analysis of tomato fruit pedicel during fruit development (*, ** and *** indicated differences between
wild type and flacca genotype significant at p≤0.05, p≤0.01and p≤0.001, respectively).
diameter of xylem
elements (µm)
pedicel cross section
area (mm2)
xylem area (mm2)
functional xylem
area (mm2)
ratio of functional
xylem
phloem area (mm2)
genotype
12 days after
anthesis
20 days after
anthesis
39 days after
anthesis
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
wild type
flacca
8.8±3.2
8.2±2.5
1.64±0.59
1.62±0.34
0.18±0.15
0.13±0.14
0.03±0.02
0.02±0.01
20±10%
23±10%
0.05±0.02
0.06±0.02
9.3±2.8
9.3±3.0
2.58±0.61
2.34±0.79
0.77±0.24
0.57±0.27*
0.12±0.05
0.12±0.07
16±7%
23±10%**
0.08±0.03
0.10±0.06
8.9±3.4
9.7±3.4
3.37±0.33
3.01±0.62
1.02±0.24
0.77±0.21**
0.23±0.09
0.17±0.08
19±9%
24±9%**
0.13±0.04
0.15±0.07
5. Water and solute transport into developing fruit-anatomical background
Although photosynthesis of immature tomato fruits is not negligible, it represents only a small part of the total fruit dry
weight (less than 10%), and tomato fruit growth is mostly dependent on the input of water, minerals and assimilates
from other parts of plants [33]. Most of the material on which fruit growth depends on, is transported from the stem in
the fruit through the fruit pedicel, by xylem and phloem [4]. According to Guichard et al. [22] water transport in tomato
fruit pedicels by xylem and phloem occurs in the same direction, so tomato fruit continually accumulates water and
transpires [34]. The fruit can loose water in two ways: by transpiration and by xylem in the direction from the fruit to
the stem like in beans or apples [4, 35]. Tomato fruit has a low transpiration [11] because it has a thick cuticule [2] and
has no stomata [34], so significant impact on the xylem inflow into tomato fruit has only calyx transpiration [36-38]. In
berry fruits, xylem and phloem participate in the supply of fruit with water, but with a different amount depending on
the stage of fruit development. In young fruits of some species such as some cacti [39] phloem is the dominant source,
while in other fruits such as tomato [40], apple [35], kiwi [41] and grape [4, 42, 43] during fruit growth the transition
from xylem to predominantly phloem transport occurs. In grapes, for example, xylem water is the main source of water
for green berries and phloem contributing less than 10% [42], while after the beginning of ripening phloem input
represents more than 80% of the total amount of water [43, 44].
Transfer from xylem to phloem water transport for the ripening fruit has, also, been observed during tomato fruit
development [45]. It is estimated that tomato fruit is supplied mainly by phloem increasing from about 85% to about
95% of the total water input during the development of the fruit, while water input via xylem is almost completely
stopped approximately 25 days after flowering [33, 36, 37, 40]. It is assumed that the reduced xylem transport occurs
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because somewhere along the way between the stem and the fruit there is a place with a high hydraulic resistance [33] the abscission zone, often considered as the cause of the high hydraulic resistance for xylem water flow.
Results of several anatomical studies of xylem in fruit pedicels have shown that there is reduced xylem area at, both,
cross and longitudinal sections in the abscission zone in tomato pedicels [29, 46-48]. Near the abscission zone of tomato
fruit ended more than 90% of vessels, and in addition there are a significantly reducing their diameter [33, 47].
According to Rancic et al [48] hydraulic resistance of the abscission zone, calculated based on anatomical sections,
increases at least two orders of magnitude compared to the pedicel zone near the stem. Direct measurement of hydraulic
conductivity [33] showed that in total xylem hydraulic resistance in tomato fruit pedicel, dominant role has hydraulic
resistance in the abscission zone. In the studies of xylem in grape berry pedicels in experiments with dye infusion
different authors have shown that dye inflow in mature grape berries is much less than in green ones. Before the stage
of maturity, if the cut end of the grape pedicel were immersed in dye, the dye inflows in berries for about 30 minutes,
crossing at the distance of 5-10cm, but after the beginning of ripening, the dye flows through the pedicel xylem to the
fruit, but does not enter into the fruit, which led the general conclusion that the xylem in berries at the stage of
maturation is non-functional. This is most often explained as a result of rapid growth and consequential breaking,
stretching or damaging vessels [35, 42, 44, 49, 50, 51]. Phenomenon that after stopping transport water by xylem into
fruit, transport by phloem still continues could be explained by the fact that phloem remains functional due to its
structure. Phloem is, namely, composed of living cells [52] with elastic cell walls which could follow the growth
without being damaged. Even in the case of damage, such damage may be easily exceeded by transport through
simplast of parenhimatous cells [35].
However, experiments with visualization of the xylem transport demonstrated that vascular elements of xylem
remain intact and apparently functional during the development of grape berries because apoplast dye may enter the
pedicel through the central and peripheral vessels by the end of xylem ripening grape berries using the applied
hydrostatic gradient, applying pressure or using a absorbing material on the part of the fruit contrary to the pedicel [53].
In addition, the switch in grape from phloem to xylem transport is fast, and occurs in 2 days [54], which is a relatively
short period of time for major changes in the anatomical level, especially if they are a consequence of cell growth, so
therefore, a simple anatomical explanation (such as xylem interruption) for change from xylem to phloem transport
which appears in the phase of berry ripening is unlikely [4]. In our experiment with tomato, the dye is transported by
xylem and passed through a few cm long pedicel relatively fastly reaching the opposite end of the pedicel (near the
fruit) in 15-30 minutes. Similar results to ours in experiment with tomato were obtained by van Ieperen et al. [33]. In
their experiment of functional vessel staining, the most intense colour was near the place where the dye is added to the
petiole (part of the pedicel near the stem), while the pedicel near the fruit was less coloured, but there are still was a lot
of vessels [33]. The experiment with dye infusion in tomato pedicel performed by Malone and Andrews [56] also
showed that there is a continuous functional xylem along the pedicel and the pericarp and according to these authors,
over 90% of hydraulic resistance between stem and fruit is in the fruit pericarp and not in the abscission zone. The
results from our experiments also showed that in any stage of pedicel development there are no disruption of xylem
transport since the data presented on table 2 show that functional xylem increased during fruit development in both
genotypes. It should, also, be taken into account that the experiment was carried out with the pedicels that were cut off
from the parent plants and all fruits were bigger than 0.5 cm when removed before immersion pedicel in staining
solution. In these circumstances the only driving force for the xylem flow was transpiration of calyx as in the case of the
removal of sepals transport was negligible. In the case when the fruit is on the pedicel, an additional driving force for
the xylem flow from stem into the fruit is, besides sepal transpiration cuticular transpiration of the fruit. However
because of thick tomato fruit cuticle [2] and the lack of stomata [34] tomato fruit has weak transpiration [11]. On the
other hand, the presence of the fruit could possibly have a negative impact on the flow of water by xylem into fruit. In
direct measurements of hydraulic conductivity of tomato pedicels Van Ieperen et al. [33] concluded that in the zones
before and after abscission zone there are increased conductivity during fruit development which is in line with the
formation of the new xylem elements during secondary growth. However, thanks to the high resistance in the abscission
zone, due to the small xylem area and absence of secondary growth in this area [33, 46, 47], the total hydraulic
resistance of the fruit pedicels between 11 and 31 days after anthesis is not changed and does not increase. It is known
that in the pericarp of tomato fruit 13-14 days after flowering accumulates starch, and the rapid accumulation of hexose
begins 23-25 days after flowering [45]. It is interesting that at the same time with the accumulation of hexose,
approximately 25 days after anthesis fruit water intake by xylem almost completely stops [33, 36, 37, 40]. The facts that
the hydraulic conductivity of pedicel does not change during the period from 11 to 35 days after anthesis [33], and that
the stop of xylem transport occurs 25 days after anthesis [36, 37, 40], indicates that although the hydraulic resistance in
the abscission zone is much less than in other parts of the pedicel, it is constant during fruit development and apparently
did not cause interruption of water xylem transport. Rapid transition from significant to minimal contribution of xylem
to the fruit water balance at the initial stages of fruit ripening phase in grape also coincides with an increased intake of
sugar in the fruit [53, 43], while phloem input increases about 10 times [42]. Since our results have shown that in
tomato fruit petiole from 12 to 39 days after anthesis there is no interruption in the xylem functionality, in a situation
where the fruits are intact on the plant, the cessation of water xylem input into the fruit can be explained by the fact that
there are flow through xylem but that it occurs in the reverse direction. Considering that the fruit is a plant organ with a
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great sink activity, it is possible that the excess of water which enters in fruit by phloem is forwarded by xylem into the
stem. In this way the water that comes out from the fruit by xylem, prevents xylem transport in the direction toward the
fruit. In line with this hypothesis are the data Ehret & Ho [36, 37] and Ho et al. [40] that significant xylem transport in
tomato fruit occurs only at night when the phloem transport is on minimum [34].
Tomato fruit growth is also limited by the elasticity of cell walls of egzocarp [57]. Parenchymatous mezocarp cells
which grow rapidly make pressure on egzocarp, and this is the reason why the mechanical properties of egzocarp are
important in controlling growth of fruits. It is thought that enzymes such as peroxidase, which reduce the elasticity of
cell walls, may play an important role in controlling growth of the fruit, since it has been observed that the cessation of
growth of the fruit is associated with an increase in peroxidase activity in egzocarp [2, 58]. Increased phloem water
intake may increase the pressure inside the fruit due to the limited extensibility egzocarp of fruits [57], which leads to
the return of water by xylem especially if the excess of entered water by phloem exceeds the needs of berries for growth
and transpiration [43]. Restoring water by xylem makes fruit less susceptible to plant water status [33] and can reduce
their vulnerability to cracking as it serves as a overflow mechanism [59]. A similar conclusion gave Keller et al. [43]
based on grape experiment. While in the grapevine there are apparently intact and functional xylem vessels, at the
beginning of ripening grape berries there are significant reduction of water transported to the fruit through xylem
comparing to the phloem [42, 53, 60]. Keller et al. [43] have explained this phenomenon by excising phloem water by
xylem recirculation. It is known that the apoplast pressure in grapes fruits is higher than in plants [61], so the entry of
water by xylem in the berries is probably inhibited by the positive pressure within the mature berries [43]. In
accordance with this theory are data by Choat et al. [59] who made direct measurements of hydraulic conductivity of
grapevine berry stems from 20-100 days after flowering. They found that indeed there is a significant increase in
resistance to pedicel in the later stages of ripening (80-100 days after flowering), but this increase was not strong
enough and occurs too late, that could explain the interruption of xylem transport that happens between 60-75 days after
flowering. Malone and Andrews [56] have experimentally demonstrated the flow of fruit to the leaves, which also
indicates the presence of functional xylem connection between stem and fruit. Although the use of xylem transport as a
way for transport water into the fruit can be mush reduced, it does not necessarily mean that it is consequence of the
physical blockade or other direct reduction of hydraulic conductivity of xylem, and it is more probably a consequence
of the loss of the driving force (hydrostatic gradient) in berry apoplast. There are examples that, during development of
the fruit, the relative importance of flow by xylem and phloem can be reversed and is probably more dependent on the
sink/source relationships, and not on the physical loss of continuity or xylem conductivity [4]. In plum fruit, xylem
entry is for example, dominant during phase II (mid-June), phloem transport becomes dominant at the start of phase III
(early July), while later in the phase III (mid-July) intensity of both flows is similar, which again indicates the presence
of functional xylem [4]. Temporary reduction in size of plant organs is a common occurrence during the drought and it
reflects the water loss by transpiration directly or water divert from the plant organ to the rest of the plant by xylem
[42]. During the day, conditions which enhance leaf transpiration led to decreased size of the immature fruit of grapes,
apples and other species [35, 4]. In organs with low transpiration rate, like many fleshy fruits, fruit shrinkening during
the day indicates that transport of water from the organ by xylem, exceeds water intake by phloem. Shrinkening of
grape berries during the soil drying occurs in green immature fruits, but fruits that are in maturation phase are much less
sensitive to plant water status [42]. Although this was interpreted as a break in functional xylem as a consequence of
rapid growth [4, 35, 44, 50, 51] we believe that the insensitivity of ripening berries on the plant water deficit is actually
evidence of strong phloem component. Tomato fruit does not exhibit or has very little variation in fruit size due to
temporary lack of water in the stem [34, 36, 37]. Fruit shrinking is low even when the water potential gradient between
fruit and stem apoplasts clearly favours the transport of water in the direction to the stem during the drought period [34].
Lack of fruits shrinkening during periods of drought does not necessarily imply lack of diverting water by xylem [33],
but water intake by phloem may run contrary to gradient of water potential and could easily exceed the amount of water
that is returned from the fruit in stem by xylem, resulting in a net import of water.
6. Role of ABA in fruit development
It is believed that ABA stimulates assimilate intake into fruit and accelerates the fruit maturation [62, 63]. Important
role of ABA in the fruit sink activity is confirmed by our data showing the lower efficiency of phloem and almost twice
lower dry weight of fruit of ABA deficient genotype in comparison with the wild type. It is possible that ABA acts by
increasing sugar intake by phloem or releases glucose from stored carbohydrates [64]. Investigations based on ABA
deficient mutants have indicated that ABA has little effect on long distance transport of photoassimilates, but has a role
in regulating distribution of photoassimilates in the place of their accumulation [65]. Some authors believe that the
ABA has a role in redirecting assimilates from phloem to the fruit by enhancing the sink activity of pericarp and locular
tissue during the phase of the most rapid growth [65, 66]. Experiments with injection of egzogenous ABA in citrus
fruits showed that ABA increased concentrations of glucose and fructose, but did not affect concentration of organic
acids [64]. It is possible that ABA increases the accumulation of sugars in sink organs by activation of enzyme (acid
invertase) which catalyzes the irreversible hydrolysis of sucrose into glucose and fructose [67]. Similarly, results of
Cheikh and Brenner [68] indicate that the ABA can affect the accumulation of starch through the action on the activity
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of fructoso-1,6-biphosphatase [65]. The concentration of ABA in the pericarp of tomato in the later stages of fruit
development in comparing with the initial value first decreases [9], and then rapidly increases [63] reaching the
maximum five weeks after flowering, which coincides with the phase of final fruit growth [69]. Fruit maturation in
apples and cherries correspond with increases concentration of endogenous ABA in fruits [70]. Since the greatest
concentration of free ABA coincides with the phase of final fruit size and changing colour of fruits [62] implies that
ABA is a regulator in ripening tomato fruits [63] and it is considered that ABA enables the initiation of maturation by
increasing the fruit sensitivity to ethylene [71].
Acknowledgements We wish to thank the CM Rick Tomato Genetics Resource Center, USA for seeds used in this study and to
Project CROPWAT for financial support. We also acknowledge invaluable help and technical support of Maja Terzic and Radenko
Radosevic.
References
[1] Bussieres P. Potential Dry Matter and Water Import Rates in the Tomato Fruit in Relationship to Fruit Size. Annals of Botany
1993;72:63-72.
[2] Andrews J, Adams SR, Burton KS and Evered CE. Subcellular localization of peroxidase in tomato fruit skin and the possible
implications for the regulation of fruit growth. Journal of Experimental Botany, 2002;53:2185-2191.
[3] Kulkarni M, Deshpande U. Comparative Studies in Stem Anatomy and Morphology in Relation to Drought Resistance in
Tomato (Lycopersicon esculentum). Am. J. Plant Physiol. 2006;1:82-88.
[4] Matthews MA, KA Shackel. Growth and Water Transport in Fleshy Fruit. In, Vascular Transport in Plants, N.M. Holbrook and
M.A. Zwieniecki (Eds.) 2005 pp 181-197.
[5] Luković J, Maksimović I, Zorić L, Nagl N, Perčić M, Polić D, Putnik-Delić M. Histological characteristics of sugar beet leaves
potentially linked to drought tolerance. Industrial Crops and Products. 2009; 30:281–286.
[6] Stertz SC, Espirito Santo AP, Bona C, Freitas RJS. Comparative morphological analysis of cherry tomato fruits from three
cropping systems. Scientia Agricola 2005; 62: 296-298.
[7] Lemaire-Chamley M, Petit J, Garcia V, Just D, Baldet P, Germain V, Fagard M, Mouassite M, Cheniclet C, and Rothan C.
Changes in Transcriptional Profiles Are Associated with Early Fruit Tissue Specialization in Tomato, Plant Physiology.
2005;139:750–769.
[8] Xiao H, Radovich C, Welty N, Hsu J, Li D, Meulia T, van der Knaap E. Integration of tomato reproductive developmental
landmarks and expression profiles, and the effect of SUN on fruit shape. BMC Plant Biology. 2009;9:49 doi:10.1186/1471-22299-49
[9] Gillaspy G, Ben-David H and Gruissem W. Fruits: a developmental perspective. Plant Cell 1993;5: 1439–1451.
[10] Bertin N, Gautier H, Roche C. Number of cells in tomato fruit depending on fruit position and source–sink balance during
plant development. Plant Growth Regulation 36, 2002. 105–112.
[11] Guichard S, Gary C, Leonardi C, and Bertin N. Analysis of Growth and Water Relations of Tomato Fruits in Relation to Air
Vapor Pressure Deficit and Plant Fruit Load. J Plant Growth Regul. 2005;24:201–213.
[12] Jones HG, Sharp CS, Higgs KH. Growth and Water Relations of Wilty Mutants of Tomato (Lycopersicon esculentum Mill.)
Journal of Experimental Botany. 1987;38:1848-1856.
[13] Liu Y, Bino RJ, Karssen CM, Hilhorst HMW. Water relations of GA- and ABA-deficient tomato mutants during seed and fruit
development and their influence on germination. Physiologia plantarum 2006;96:425 – 432.
[14] Cornish K, Zeevaart JAD. Phenotypic Expression of Wild-Type Tomato and Three Wilty Mutants in Relation to Abscisic
Acid Accumulation in Roots and Leaflets of Reciprocal Grafts. Plant Physiology 1988; 87:190-194.
[15] Neill SJ, Horgan R. Abscisic Acid Production and Water Relations in Wilty Tomato Mutants Subjected to Water Deficiency.
Journal of experimental botany. 1985;36:1222-1231.
[16] Baum SF, PN Tran, WK Silk. Effects of salinity on xylem structure and water use in growing leaves of sorghum. New. Phytol.
2000;146:119-127.
[17] Bussieres P. Water Import Rate in Tomato Fruit: A Resistance Model. Annals of Botany. 1994;73:75-82.
[18] Roth I. Fruits of angiosperms 1977; Gebrüder Borntraeger, Berlin 675 pp.
[19] Hayward HE. The structure of economic plants, 1938 New York, Macmillan,
[20] Wang F, A Sanz, M Brenner, A Smith. Sucrose Synthase, Starch Accumulation, and Tomato Fruit Sink Strength. Plant
Physiology 1993;101:321-327.
[21] Carrari F, Fernie AR. Metabolic regulation underlying tomato fruit development. Journal of Experimental Botany. 2006;57:
1883–1897.
[22] Guichard S, Bertin N, Leonardi C, Gary C. Tomato fruit quality in relation to water and carbon fluxes Agronomie 2001;21:
385-392.
[23] Cutler JM, Rains DW, Loomis RS. The importance of cell size in the water relations of plants. Physiol. Plant. 1977;40:255–
260.
[24] Hsiao T. Plant responses to water stress. Annual Review of Plant Physiology 1973;24:519–80.
[25] Kulkarni M, Deshpande U. In Vitro screening of tomato genotypes for drought resistance using polyethylene glycol. African
Journal of Biotechnology, 2007;6:691-696.
[26] Zhang C, Tanabe K, Tamura F, Matsumoto K and Yoshida A. 13C-photosynthate accumulation in Japanese pear fruit during
the period of rapid fruit growth is limited by the sink strength of fruit rather than by the transport capacity of the pedicel.
Journal of Experimental Botany. 2005;56:2713–2719.
©FORMATEX 2010
859
Microscopy: Science, Technology, Applications and Education
A.
Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
[27] Garcia-Luis A, Oliveira MEM, Bordon Y, Siqueira DL, Tominaga S, Guardiola JL.Dry matter accumulation in citrus fruit is
not limited by transport capacity of the pedicel. Annals of Botany 2002;90:755–764.
[28] Bustan A, Erner Y, Goldschmidt EE. Interactions between developing Citrus fruit and their supportive vascular system. Annals
of Botany 1995;76:657–666.
[29] Rančić D, Pekić Quarrie S, Terzić M, Savić S, Stikić R Comparison of light and fluorescence microscopy for xylem analysis
in tomato pedicels during fruit development. Journal of Microscopy. 2008;232: 618–622.
[30] Holbrook NM, Shashidhar VR, James RA, Munns R. Stomatal control in tomato with ABA-deficient roots: response of
grafted plants to soil drying. Journal of Experimental Botany (2002) 53:1503–1514
[31] Hose E, Steudle E, Hartung W. Abscisic acid and hydraulic conductivity of maize root: a study using cell- and root-pressure
probes. Planta 2000;211: 874–882.
[32] Sachs T. The control of patterned differentiation of vascular tissues Advances in Botanical Research 1981;9: 151-262.
[33] van Ieperen W, Volkov VS and van Meeteren U. Distribution of xylem hydraulic resistance in fruiting truss of tomato
influenced by water stress. Journal of Experimental Botany. 2003;54:317-324 .
[34] Johnson RW, Dixon MA, Lee DR. Water relations of the tomato during fruit growth Plant, Cell and Environment 1992;15:
947-953.
[35] Lang A. Xylem, phloem and transpiration flows in developing apple fruits. Journal of Experimental Botany 1990;41:645–651
[36] Ehret DL, Ho LC. Effects of osmotic potential in nutrient solution on diurnal growth in tomato fruit. Journal of Experimental
Botany. 1986;37:1294-1302.
[37] Ehret DL, Ho LC. Translocation of Ca in relation to tomato fruit growth. Annals of Botany 1986;58: 679-688.
[38] Araki T, Kitano M, Eguchi H. Respiration, sap flux, water balance and expansive growth in tomato fruit. Biotronics 1997; 26:
95–102.
[39] Nobel PS, De la Barrera E. Carbon and water balances for young fruits of platyopunitas. Physiologia Plantarum 2000;109:
160–166.
[40] Ho LC, Grange RI, Picken AJ. An analysis of the accumulation of water and dry matter in tomato fruit. Plant, Cell and
Environment 1987;10:157–162.
[41] Dichio B, Remorini D, Lang S. Developmental changes in xylem functionality in kiwifruit: implications for fruit calcium
accumulation. Acta Horticulturae 2003;610:191–195.
[42] Greenspan MD, Shackel KA, Matthews MA. Developmental changes in the diurnal water budget of the grape berry exposed to
water deficits. Plant, Cell and Environment. 1994;17: 811–820.
[43] Keller M, Smith JP and Bondada BR. Ripening grape berries remain hydraulically connected to the shoot. Journal of
Experimental Botany, 2006;57:2577–2587.
[44] Creasy GL, Price SF, Lombard PB. Evidence for xylem discontinuity in Pinot noir and Merlot grapes: dye uptake and mineral
composition during berry maturation. American Journal of Enology and Viticulture 1993;44:187–192.
[45] Ruan YL, Patrick JW. The cellular pathway of post phloem sugar transport in developing tomato fruit. Planta 1995;196: 434–
444.
[46] Lee DR. Vasculature of the abscission zone of tomato fruit: implications for transport. Canadian Journal of Botany.
1989;67:1898–1902.
[47] Andre´ JP, Catesson AM, Liberman M. Characters andorigin of vessels with heterogenous structure in leaf andflower
abscission zones. Canadian Journal of Botany 1999;77:253–261.
[48] Rančić D, Pekić Quarrie S, Radošević R, Terzić M, Pećinar I , Stikić R, Jansen S. The application of various anatomical
techniques for studying the hydraulic network in tomato fruit pedicels. Protoplasma 2010;DOI 10.1007/s00709-010-0115-y.
[49] Châtelet DS, Rost TL, Matthews MA and Shackel KA. The peripheral xylem of grapevine (Vitis vinifera) berries. 2. Anatomy
and development. Journal of Experimental Botany. 2008;59:1997–2007.
[50] Findlay N, Oliver KJ, Nii N, Coombe BG. Solute accumulation by grape pericarp cells. IV. Perfusion of pericarp apoplast via
the pedicel and evidence for xylem malfunction in ripening berries. Journal of Experimental Botany. 1987;38:668–679.
[51] Lang A, During H. Partitioning control by water potential gradient: evidence for compartmentation breakdown in grape
berries. Journal of Experimental Botany. 1991;42:1117–1122.
[52] Esau K. Anatomy of seed plants, New York, USA. John Wiley and Sons 1977
[53] Bondada BR, Matthews MA and Shackel KA. Functional xylem in the post-veraison grape berry. Journal of Experimental
Botany. 2005; 56:2949–2957.
[54] Düring, H, Lang A, Oggionni F. Patterns of water flow in Riesling berries in relation to developmental changes in their xylem
morphology. Vitis 1987;26:123-131.
[56] Malone M, Andrews J. The distribution of xylem hydraulic resistance in the fruiting truss of tomato. Plant, Cell and
Environment. 2001;24:565–570.
[57] Matthews MA, Cheng G, Weinbaum SA. Changes in water potential and dermal extensibility during grape berry development.
Journal of the American Society of Horticultural Science 1987;112:314–319.
[58] Savić S, Stikić R, B Vucelić Radović, B Bogičević, Z Jovanović and V Hadži-Tašković Šukalović. Comparative effects of
regulated deficit irrigation (RDI) and partial root-zone drying (PRD) on growth and cell wall peroxidase activity in tomato
fruits. Scientia Horticulturae 2008;117:15-20.
[59] Choat B, GA Gambetta, KA Shackel, MA Matthews. Vascular Function in Grape Berries across Development and Its
Relevance to Apparent Hydraulic Isolation. Plant Physiology 2009;151:1677-1687
[60] Rogiers SY, Smith JA, White R, Keller M, Holzapfel BP, Virgona JM. Vascular function in berries of Vitis vinifera (L) cv.
Shiraz. Australian Journal of Grape and Wine Research 2001;7: 46–51.
[61] Tyerman SD, Tilbrook J, Pardo C, Kotula L, Sullivan W, Steudle E. Direct measurements of hydraulic properties in
developing berries of Vitis vinifera L. cv. Shiraz and Chardonnay. Australian Journal of Grape and Wine Research
2004;10:170–181.
860
©FORMATEX 2010
Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
[62] Kojima K, S Kuraishi, N Sakurai, K Fusao. Distribution of abscisic acid in different parts of the reproductive organs of
tomato. Scientia Hortic. 1993;56: 23-30.
[63] Buta JG, Spaulding DW. Changes in indole-3-acetic acid and abscisic acid levels during tomato (Lycopersicon esculentum
Mill.) fruit development and ripening. Journal of Plant Growth Regulation. 1994;13:163-166.
[64] Kojima K, Yamada Y, Yamamoto M. Effectso f Abscisic Acid Injection on Sugar and Organic Acid Contents of Citrus Fruit.
J.Japan Soc.Hort. Sci. 1995;64:17-21.
[65] Smith GS, Klages KU, Green TGA, Walton EF. Changes in abscisic acid concentration, surface conductance, and water
content of developing kiwifruit. Scientia Horticulturae 1995;62:13-27.
[66] Kojima K. Simultaneous measurement of ABA, IAA and GAs in citrus: Role of ABA in relation to sink ability. Plant
physiology and biochemistry 1995;29:179-185.
[67] Pan Q-H, Li M-J, Peng C-C, Zhang N, Zou X, Zou K-Q, Wang X-L, Yu X-C, Wang X-F and Zhang D-P. Abscisic acid
activates acid invertases in developing grape berry. Physiologia Plantarum 2005;125:157–170.
[68] Cheikh N, Brenner ML. Regulation of Key Enzymes of Sucrose Biosynthesis in Soybean Leaves. Plant Physiol. 1992;
100:1230-1237.
[69] Abdel-Rahman M, Thomas TH, Dossand GJ, Howell L.Changes in endogenous plant hormones in cherry tomato fruit during
development and maturation. Physiologia plantarum 1975; 34:39-43.
[70] Setha S, Kondo S, Hirai N, Ohigashi H. Xanthoxin, abscisic acid and its metabolite levels associated with apple fruit
development. Plant Science 2004;166:493–499.
[71] Jiang Y, DC Joyce, AJ Macnish. Effect of Abscisic Acid on Banana Fruit Ripening in Relation to the Role of Ethylene.
Journal of Plant Growth Regulation
©FORMATEX 2010
861