Journal of Experimental Botany, Vol. 53, No. 366, pp. 83-88,
January 2002
© 2002 Oxford University Press
Original Papers |
Mobilization of calcium in glasshouse tomato plants by localized scorching
HRI Wellesbourne, Warwickshire CV35 9EF, UK
Received 16 April 2001; Accepted 16 August 2001
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Abstract |
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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.
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Introduction |
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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 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 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.
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Materials and methods |
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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 screw-capped vials. They were weighed before and after drying for 48 h at 80 °C.
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 µmol m-2 s-1 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 °C and 78% RH, and the light regime was 12/12 h dark/light.
Scorching of leaflets was done with a small blowlamp powered by a butane/propane 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 °C, 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.
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Results |
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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 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.
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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=15). Similar leaflets had a total calcium content of 49.4±6 mg g-1 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 wound-induced 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 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 wound-induced 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 weight/fresh weight; D/F) of these undamaged control leaflets was found to be relatively constant at 0.13±0.014 (x±SD, n=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, D/F, 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 D/F from 2 min after burning must therefore reflect mainly a loss of water by reversed flow into the plant.
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The fraction of leaf water which has exited from these leaflets by time ‘t’ after scorching is given by:
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 | (2) |
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. 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.
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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 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 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.
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Effect of scorching on calcium levels in other leaves on the plant
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=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 µm h-1. 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).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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, 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 µl 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 µlx80 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 mMx700 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 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 g-1 (dry weight) of calcium (Adams and Ho, 1993) compared to 50 mg g-1 (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.
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Acknowledgments |
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Dr M Angela Morales thanks the MEC, Spain, for financial support. Part of this work was funded by MAFF (UK).
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Notes |
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1 Present address: Department of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.
2 To whom correspondence should be addressed. E-mail: m.malone{at}sussex.ac.uk
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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.[Web of Science]
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. H+ and Ca2+ 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.[Web of Science]
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.
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