Journal of Experimental Botany, Vol. 53, No. 366, pp. 83–88, January 2002
Mobilization of calcium in glasshouse tomato plants
by localized scorching
M. Malone1,2, P. White and M. Angela Morales1
HRI Wellesbourne, Warwickshire CV35 9EF, UK
Received 16 April 2001; Accepted 16 August 2001
Abstract
It is postulated here that significant amounts of
calcium will be mobilized into the plant by the scorching of one old leaf. This postulate was tested using
large (6 m) tomato plants in the glasshouse. Brief
scorching with a blowlamp was shown to release
some 35% of the leaf’s water into the plant. A range
of measurements was used to estimate the kinetics
and magnitude of this flow. The flow was found to
carry a pulse of up to 50% of the leaf’s total calcium
into the plant, probably via the xylem, and was estimated to increase xylem calcium levels transiently by a
factor of about 80. The potential value of scorching treatments in combating calcium-deficiency
disorders is discussed.
Key words: Calcium deficiency, Lycopersicon esculentum,
wound responses.
Introduction
The correct balance of mineral nutrients is essential for
healthy growth and high productivity in plants, especially
in glasshouse crops that are ‘forced’ by optimal growing
conditions.
Mineral nutrients such as potassium are transported
readily in the phloem. Localized deficiencies of these
nutrients are prevented by redistribution within the
shoot. By contrast, calcium (and perhaps boron) appears
to be virtually immobile in the phloem. It is transported through the plant almost entirely via the xylem
(Raven, 1977; Adams and Ho, 1993). This means that
organs of rapid transpiration, such as mature leaves, will
accumulate high levels of calcium while organs of low
transpiration will receive little. The latter includes the
1
2
fruit of tomato, pepper, and apple, the heart leaves of
lettuce, the tuber of potato, and many others (Bangerth,
1979). Xylem flow carries the transpiration stream and
is normally entirely acropetal. Thus, once deposited in
a transpiring tissue, calcium will be trapped there and
unable to exit to other parts of the shoot.
Because of these factors, low-transpiring fruit tissues
will have relatively low calcium levels and will be prone
to deficiency disorders such as blossom-end rot (BER),
a necrosis of the distal region of the fruit in tomato and
pepper (Adams and Ho, 1993). The situation can arise
in which older leaves contain very high levels of calcium,
whilst young developing fruit nearby on the same plant
are severely deficient.
Calcium distribution can be influenced by altering
patterns of transpiration, for example, by selective bagging of leaves. This can affect the incidence of BER in
tomato (Wiersum, 1966; Ehret and Ho, 1986) and
other plants (Palzkill et al., 1976). The flow of xylem
sap can also be altered by wounding (Malone et al.,
1994; Malone, 1996). For example, when leaf cells in a
transpiring plant are killed by heating, their vacuolar sap
will be released to the apoplast. From there it becomes
available to the xylem vessels that permeate the entire
leaf. Throughout most of the day, when the plant is
transpiring, these vessels will contain water under substantial hydraulic tension. Thus, any fluids released from
killed cells will be sucked into nearby xylem vessels. They
will flow down the petiole, in the basipetal (reversed)
direction, to the stem. From there they will flow to
other parts of the shoot in both basipetal and acropetal
directions (Malone, 1994). These flows are instrumental in
co-ordinating wound responses at the whole-plant level
(Malone, 1996).
These wound-induced flows are comprized primarily
of vacuolar sap, and will include most of the solutes
that were present in the vacuole in vivo, including the
Present address: Department of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
To whom correspondence should be addressed. E-mail: [email protected]
ß Society for Experimental Biology 2002
84
Malone et al.
accumulated calcium. It is therefore possible that localized wounding could mobilize significant quantities of
calcium from the wounded region to other parts of
the plant. Here, a quantitative analysis of this effect is
made, and its potential use in the management of calcium
deficiency in crop plants is considered.
Materials and methods
Tomato plants (Lycopersicon esculentum L. Mill) cv. Espero were
grown in rockwool in a commercial-type system (Van de Vooren
et al., 1986). At the time of the experiments described here
the main stems were about 6 m in length. Following standard
practice, the distal 3 m of stem were tied into a vertical orientation. Leaflets were selected at random from the east side of a
10 m row, about midway up the vertical part of the plant, unless
otherwise stated. Occasionally, leaves from the apex and base of
the plant were also sampled. For fresh and dry weight the leaflets were excised and immediately sealed in preweighed screwcapped vials. They were weighed before and after drying for
48 h at 80 8C.
Measurements of leaf thickness were carried out using displacement transducers as previously described (Turquois and
Malone, 1996). These thickness measurements were conducted
in the laboratory, using pot-grown tomato plants of about
4-weeks-old.
Fruit growth rate was measured using laser-displacement
sensors (Kitano et al., 1996; Malone and Andrews, 2001). These
experiments were conducted in a walk-in well-lit controlled
environment room (400 mmol m2 s1 PPFD), on large (5 m)
tomato plants (cv. Espero) growing in 20 l pots of peat-based
compost (Levingtons M2). The cabinets were held at a constant 22 8C and 78% RH, and the light regime was 12u12 h
darkulight.
Scorching of leaflets was done with a small blowlamp
powered by a butaneupropane canister. The flame was turned
low and directed at the underside of the leaf, with the tip of the
flame held about 10 cm from each leaflet for 1–2 s. Scorching
was quickly apparent as a change in leaf refractive properties
as the cells’ water was released to the apoplast.
For the determination of total leaf calcium, the dried leaflets
were ashed for 24 h at 550 8C, taken up in 1 ml concentrated
HCl then dissolved with 9 ml water. 1 ml of this solution was
diluted with 3 ml water and injected into an Ion-Coupled
Plasma spectrometer.
Leaf sap was collected by centrifugation from frozen-thawed
leaflets (Alarcon and Malone, 1995). Calcium content of leaf
sap was determined after ashing, as above.
All experiments were repeated at least three times.
blowlamp did evaporate a significant amount of water
(see Fig. 4). However, the remaining water was sufficient
to convey solutes into the plant. This is evident because
leaves of tomato plants showed a marked and prolonged decrease in thickness after brief scorching of a
neighbouring leaflet with the blowlamp (Fig. 1). This dip
in thickness is an osmotic consequence of the arrival in the
measured leaves of solute-rich sap from the wound site.
As they pass the position of the thickness transducer, the
solutes draw water out of the underlying cells causing
the dip in thickness (Boari and Malone, 1993). The larger
the dip, the more solutes have arrived from the wound
site. Compared to other flames, the blowlamp typically
induced a smaller initial (rising) phase, but a much larger
and faster dip in thickness (Fig. 1). This confirms that the
blowlamp was very effective at mobilizing solutes from
the scorched leaf.
Leaf and sap calcium levels
Undamaged, distal lateral leaflets were selected at random
from midway up mature tomato plants in the glasshouse.
Sap released from these leaflets by freeze–thawing was
found to contain calcium at 77.8"8 mM (mean"SE,
n ¼ 15). Similar leaflets had a total calcium content of
49.4"6 mg g1 dry weight which, if all the calcium was
in solution, would correspond to about 170 mM. This
indicates that about half the total calcium in the leaf
will potentially be available for mobilization with woundinduced flows of sap. Most of the remaining calcium is
presumably locked up in insoluble and wall-bound forms.
The oldest leaves, at the bottom of the plant, were found
Results
Effects of severe scorching
Work on wounding has usually used small wounds
(Malone, 1996). However, for effective mobilization of
leaf sap the entire leaflet should be damaged thoroughly
and rapidly. A blowlamp was therefore used. This could
scorch an entire leaflet in about 1 s. It seemed possible
that the blowlamp might drive water off as steam
rather than release it for reversed flow into the plant.
Measurements on detached leaflets showed that the
Fig. 1. Effect on leaf thickness of scorching with a blowlamp. Leaf
thickness was monitored while applying a scorch wound to one
neighbouring leaflet on the same plant. Brief scorching with the
blowlamp (lower) is compared with mild and severe scorching with
Ca mobilization
85
to contain about 50% more total calcium than those from
the middle.
Sap extracted from heat-killed leaves contained consistently higher levels of calcium than did sap from
frozen–thawed leaves. This elevation was in proportion
to the amount of water lost as steam during heating
(see Fig. 4) and is probably a simple concentration effect.
Thus, the figure of 70 mM calcium is probably an underestimate of that which would occur in flows from
scorched leaves.
Partitioning of water flows
When a leaflet is wounded by scorching, its water will
be lost by a combination of the following processes:
(1) xylem-borne reversed flow into the plant; (2) evaporation as steam during and immediately after the scorch
wound; and (3) evaporation direct to the atmosphere over
the first few hours after the wound. The measurements
described below were conducted to partition the woundinduced flow quantitatively into these three components.
It should then be possible to estimate the amount of
calcium that will be mobilized to the plant with any
wound-induced flow.
Reversed flow into the plant: Evaporation from tomato
leaflets was virtually eliminated by wrapping them in
plastic film and aluminium foil. This was confirmed by
periodic weighing of scorched leaves that had been
wrapped, detached, and hung in the canopy so as to
mimic the position, orientation, and exposure of attached
leaves. Such leaflets lost weight only very slowly.
Undamaged leaflets were chosen from positions near
those to be scorched, and excised into preweighed bottles.
This was done shortly before scorching of the damaged
leaflets, so that the leaflets would be comparable in
terms of water content. The dry weight fraction (dry
weightufresh weight; DuF ) of these undamaged control
leaflets was found to be relatively constant at 0.13"0.014
(x"SD, n ¼ 20). This value was taken as representative
of the dry weight fraction of non-damaged leaflets, and
was termed fn.
Intact leaflets were scorched then wrapped (as above)
whilst still attached to the plant. At various times thereafter, these leaflets were excised, unwrapped, and sealed
into preweighed bottles. The dry weight fraction, DuF, of
these burnt leaves was found to increase with time after
scorching (Fig. 2). Evaporation from these leaves was
prevented by wrapping, and the observed increase in DuF
from 2 min after burning must therefore reflect mainly
a loss of water by reversed flow into the plant.
The fraction of leaf water which has exited from these
leaflets by time ‘t’ after scorching is given by:
1
Mt
F0 Ft
¼
M0
F0 D
(1)
Fig. 2. Dry weight fraction of wrapped leaflets with time after scorching.
Scorched and wrapped leaflets were excised from the plant at various
times after scorching. Their fresh (F ) and dry weights (D) were
measured and plotted as DuF with time after scorching (mean"SE,
n ¼ 10). The curve is a fitted second-degree polynomial.
where Mt, the mass of water remaining in the leaflet at
time t; M0, the initial mass of water in the leaf; F0, the
initial fresh weight of the leaf; Ft, the fresh weight at
time t; and D, the dry weight of the leaflet. F0, the initial
fresh weight of these leaflets cannot be measured directly
because they are attached. However, it can be estimated
using fn:
D
F0 ¼
(2)
fn
Substituting this into equation 1 yields:
fn
1 Ft
Mt
D
¼
1
1 fn
M0
(3)
This transformation can be applied to the data in Fig. 2.
It shows that about 50% of the leaflet’s water exits to
the plant during the period from 2 min–2 h after scorching (Fig. 3). In a typical leaflet, this amounts to about
0.7 cm3.
Water driven off during scorching: A large decrease
occurs in the leaflet’s water content during the first
2–5 min after scorching (Fig. 3). There will be a small
rapid exit of water to the plant at this time (as in Fig. 1)
but most of this loss represents water driven off to the
atmosphere as steam. This component can be quantified
by periodic weighing during scorching of detached leaflets (from which there can be no flux of water to the
plant). Examples in Fig. 4 show that 15–30% of leaf
fresh weight (about 20–40% of leaf water content) is
86
Malone et al.
Fig. 5. Fate of leaf water after scorching. After brief scorching with the
blowlamp, leaf water falls into each of four competing pools (burned
off as steam; evaporated to the atmosphere; reversed flow into the plant;
remaining in the leaf ). The relative magnitudes of the four pools, with
time, is shown.
Fig. 3. Kinetics of water exit by reversed flow from a scorched leaf.
Proportion (left axis) and estimated volume (right axis) which has exited
evaporation from such leaflets but no reversed flow to
the plant. The rate of evaporation was found to be
about 0.7% of leaf water content per minute.
This component completes the balance sheet of water
loss after scorching of intact leaves. The various components are compiled in Fig. 5. No correction was made
for the dry matter content of leaf sap, which is about
4%, but the error thus admitted will be small.
Effect of scorching on calcium levels in other
leaves on the plant
Fig. 4. Water driven off as steam during scorching. Four detached
leaflets were weighed periodically. At the times indicated by the arrows
they were scorched briefly with the blowlamp. In two of the leaflets,
a patch was also scorched briefly with the flame from a cigarette lighter
(at around 300 min).
driven off as steam when leaflets are scorched with the
blowlamp.
Water lost by evaporation after scorching: The data in
Figs 2 and 3 was generated from leaflets covered to
minimize evaporation. In leaflets without such covering,
a further portion of the leaflet’s water will be lost by
evaporation during the hours after scorching. This component was estimated by measuring weight loss from
detached, non-wrapped, scorched leaves. There will be
Young, undamaged leaflets were taken from plants upon
which a lower leaf had been scorched on the previous
day. These were found to contain about 30% more
total calcium than similar leaflets from neighbouring
undamaged plants (dry weight basis, P ¼ 0.01). In extreme
cases, undamaged leaves from scorched plants had up
to 80% more calcium than those from non-scorched
plants. These higher levels were observed in undamaged
leaflets neighbouring scorched ones (i.e. from 10–30 cm
distant on the same leaf ), and in upper leaflets on plants
of which one lower leaf had been scorched each day for
four consecutive days.
Patterns of fruit growth
Fruit growth was monitored continuously in a controlled
environment (CE) room. Younger fruit grew in diameter
at rates often exceeding 100 mm h1. During the day,
these fruit sometimes showed bursts of growth that
coincided with irrigation events (Fig. 6). Older fruit grew
much more slowly and showed little evidence of such
bursts (Fig. 6).
Discussion
Water was released rapidly from leaflets of glasshouse
plants by severe scorching (Figs 1–3). The various fates of
this water were estimated as described in the Results,
Ca mobilization
Fig. 6. Patterns of fruit growth rate. These experiments were done on
large (2.5 m tall) pot-grown plants in a brightly-lit controlled environment. The rate of change in fruit diameter is plotted against time for
each of two fruits and one blank. Values on the figure are approximate
fruit diameter. For each of the three growth-rate traces, the position of
zero growth rate is indicated by a horizontal line. The middle trace is
from a rapidly growing young fruit; the lower trace is from a mature
(breaker) fruit. The timing of irrigation events is indicated by the arrows.
Light and dark periods are indicated on the bar (lower). Time and scale
markers are included.
and are summarized in Fig. 5. This shows that about 35%
of the leaflet’s water will be mobilized to the plant by
brief scorching with a blowlamp. Typically, this will
amount to about 500 ml from each leaflet. Estimates
(below) indicate that this water flux will deliver a
significant pulse of calcium into the plant.
Measurements on frozen–thawed material showed
that about half the leaf’s calcium could be mobilized
with wound-induced flows of sap, and that such flows
will contain about 78 mM calcium. This seems surprisingly high if, as some authors suggest, most of the leaf’s
calcium is bound to cell walls (Bagshaw and Cleland,
1993). It seems more likely that a large proportion of leaf
calcium is present in solution in the vacuolar sap. This
view is supported by direct measurements of vacuolar
calcium concentrations, at least in wheat leaf (Malone
et al., 1991).
Recordings with xylem-feeding insects show that
xylem sap in the glasshouse tomato plants used here
contains about 1 mM calcium, and that this is remarkably constant throughout the diurnal cycle (M Malone,
unpublished results). Sap flow from the scorched leaflet
would thus boost xylem calcium levels transiently by a
factor of about 80. The calcium delivered to the plant
by scorching a single leaflet from the middle of the plant
would amount to about 500 ml 3 80 mM, or about 1.5 mg.
Scorching of one old leaf comprizing 7 leaflets would
thus deliver about 14 mg of calcium into the plant. This
compares with the normal total daily calcium uptake for
these large plants of about 28 mg (1 mM 3 700 ml).
Clearly then, scorching will rapidly mobilize a significant pulse of calcium into the shoot. Scorching of
an entire leaf may seem rather traumatic, but the
oldest leaves contribute little to photosynthesis. Current
87
horticultural practice is to remove these older leaves
periodically to reduce the risk of infection. Their loss by
scorching would thus impose no yield penalty. There
could also be additional benefits of scorching in terms of
systemic wound-induced increases in resistance to insects
and pathogens (Vigers et al., 1992; Ryan, 1990).
Calcium deficiency can be a problem in growing leaves
of tomato crops under certain conditions, but it is in the
fruit that calcium deficiency symptoms are most severe
and costly. Fruit typically have much lower calcium
levels than other parts of the shoot. The berry part of
a typical mature fruit might contain only 0.3–1 mg g1
(dry weight) of calcium (Adams and Ho, 1993) compared
to 50 mg g1 (dry weight) in leaves. Thus, scorching of
one leaflet would release as much calcium as is contained
in the berries of an entire truss of six large fruit. Clearly,
to be of benefit in reducing BER, a significant proportion
of the pulse delivered by scorching would have to enter
the fruit. Otherwise, the calcium mobilized would simply
be transpired away back into the leaves. Fruit normally
obtain most of their water and solutes from the phloem
(Ho et al., 1987). However, continuous xylem connections
exist from the stem all the way into the fruit in tomato
(Malone and Andrews, 2001) and these offer a pathway
for the entry of calcium-rich fluid.
Figure 6 demonstrates surges in fruit growth rate.
These surges were most noticeable during the day, and
in younger fruit. They coincided with episodes of watering (Fig. 6). This, together with their rapid kinetics,
suggests that they involve xylem rather than phloem
water. A scorch-induced calcium pulse timed to coincide
with one of these surges might greatly increase fruit calcium status. Alternatively, pulses of fruit growth can be
induced by small temperature spikes (Pearce et al., 1993)
which can be programmed into the glasshouse environment. These also probably involve a significant contribution from xylem water, and they might offer a further
opportunity for promoting delivery of wound-induced
calcium spikes into the fruit.
New varieties and improvements in nutritional regimes
have reduced the economic importance of BER. However, it is still a sporadic nuisance especially in some niche
varieties (Ho et al., 1999). It could become more serious
with the trend towards recirculating media with associated higher salinity and increased incidence of BER
(Spurr, 1959; Adams and Ho, 1993). The results reported
here indicate that scorching of older leaves could offer
a useful tool against calcium deficiency in tomato and
related crops.
Acknowledgements
Dr M Angela Morales thanks the MEC, Spain, for financial
support. Part of this work was funded by MAFF (UK).
88
Malone et al.
References
Adams P, Ho LC. 1993. Effects of environment on the uptake
and distribution of calcium in tomato and on the incidence
of blossom-end rot. Plant and Soil 154, 127–132.
Alarcon J-J, Malone M. 1995. The influence of plant age
on wound induction of proteinase inhibitors in tomato.
Physiologia Plantarum 95, 423–427.
Bagshaw SL, Cleland RE. 1993. The effects of enhanced levels
of calcium on the gravireaction of sunflower hypocotyls.
Plant, Cell and Environment 16, 1091–1097.
Bangerth F. 1979. Calcium-related physiological disorders of
plants. Annual Review of Phytopathology 17, 97–122.
Boari F, Malone M. 1993. Wound-induced hydraulic signals:
survey of occurrence in a range of species. Journal of
Experimental Botany 44, 741–746.
Ehret DL, Ho LC. 1986. Translocation of Ca in relation to
tomato fruit growth. Annals of Botany 58, 679–688.
Ho LC, Grange RI, Picken AJ. 1987. An analysis of the
accumulation of water and dry matter in tomato fruit.
Plant, Cell and Environment 10, 157–162.
Ho LC, Hand DJ, Fussell M. 1999. Improvement of tomato
fruit quality by calcium nutrition. In: Papadopoulos AP, ed.
Proceedings of an International Symposium on Growing
Media and Hydroponics. Acta Horticulturae 481, 463–468.
Kitano M, Hamakoga M, Yokomakura F, Eguchi H. 1996.
Interactive dynamics of fruit and stem growth in tomato
plants as affected by root water condition. 1. Expansion and
contraction of fruit and stem. Biotronics 25, 67–75.
Malone M. 1994. Wound-induced hydraulic signals and
stimulus transmission in Mimosa pudica L. New Phytologist
128, 49–56.
Malone M. 1996. Rapid, long-distance signal transmission in
higher plants. Advances in Botanical Research 22, 163–228.
Malone M, Alarcon J-J, Palumbo L. 1994. An hydraulic
interpretation of rapid, long-distance wound signalling in
the tomato. Planta 193, 181–185.
Malone M, Andrews J. 2001. The distribution of xylem
hydraulic resistance in the fruiting truss of tomato. Plant,
Cell and Environment (in press).
Malone M, Leigh RA, Tomos AD. 1991. Concentrations
of vacuolar inorganic ions in individual cells of intact
wheat leaf epidermis. Journal of Experimental Botany 42,
305–309.
Palzkill DA, Tibbits TW, Williams H. 1976. Enhancement of
calcium transport to inner leaves of cabbage for prevention
of tipburn. Journal of the American Society for Horticultural
Science 101, 645–648.
Pearce BD, Grange RI, 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.
Raven JA. 1977. Hq and Ca2q in phloem and symplast: relation
of relative immobility of the ions to the cytoplasmic nature of
the transport paths. New Phytologist 79, 465–480.
Ryan CA. 1990. Proteinase inhibitors in plants: genes for
improving defences against insects and pathogens. Annual
Review of Phytopathology 28, 425–429.
Spurr AR. 1959. Anatomical aspects of blossom-end rot in
the tomato with special reference to Ca nutrition. Hilgardia
28, 269–295.
Turquois N, Malone M. 1996. Non-destructive assessment
of developing hydraulic connections in the graft union of
tomato. Journal of Experimental Botany 47, 701–707.
Vigers AJ, Wiedemann S, Roberts WK, Legrand M,
Selitrennikoff CP, Fritig B. 1992. Thaumatin-like pathogenesis-related proteins are antifungal. Plant Science 83,
155–161.
van de Vooren J, Welles GWH, Hayman G. 1986. Glasshouse
crop production. In: Atherton JG, Rudich J, eds. The tomato
crop. Chapman and Hall, 581–602.
Wiersum LK. 1966. The calcium supply of fruits and storage
tissues in relation to water transport. Acta Horticulturae
4, 33–38.