Rapid, Long-distance Signal Transmission in Higher Plants

Rapid. Long-distance Signal Transmission in Higher Plants

M . MALONE

Horticulture Research International. Wellesbourne. Warwicks CV35 9EF. UK

I.

Introduction ............................................................................. 163 A . Shoot Responses to Change in the Root Environment ............... 164 B . Rapid Movements Induced by Localized Mechanical Stimuli ....... 164 C . Remote Responses to Localized Wounding .............................. 165 D . Miscellaneous Related Phenomena ......................................... 166

I1.

Mechanisms of Long-distance Communication Within Plants ............ A . Airborne Signals ................................................................. B . Phloem Translocation .......................................................... C . Hydraulic Pressure Signals .................................................... D . Hydraulic Dispersal Signals ................................................... E . Electrical Signals .................................................................

167 167 170 171 177 186

I11. Rapid, Long-distance Signalling in Plants: Case Studies ................... 188 A . Systemic Induction of P I by Localized Wounding in Tomato ..... 188 B . Signal Transmission in Mimosa ............................................. 196

IV . Implications and Directions for Further Research ............................ 200 A . Re-assessment of Electrical Signals in the Higher Plant ............. 201 B . Signalling of Non-wound Stimuli - an Hydraulic Mechanism? ... 209 V.

Conclusion ............................................................................... Acknowledgements .................................................................... References ................................................................................

216 216 217

I . INTRODUCTION Irritability. the capacity to sense and respond to the environment. is one of the characteristics of the living organism . It is particularly important in land Advances in Botanical Research Vol . 22 incorporating Advances in Plan1 Pathology

ISBN 0-12-005922-3

Copyright 0 1996 Academic Press Limited All rights of reproduction in any form reserved

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plants because they must cope in situ with environmental stresses, which may be long- or short-term, predictable or unpredictable. The entire sequence of events that link stimulus perception to response could be described as “signal transduction”. However, this term is usually restricted to the intracellular cascade of events which generate a response (reviews in Poovaiah and Reddy, 1993; Verhey and Lomax, 1993; Gilroy and Trewavas, 1994). Intracellular transduction alone will suffice for some local responses and for simple or unicellular organisms. Co-ordination of activity in the higher organism, however, will require long-distance signal transmission as well as intracellular signal transduction. For example, environmental stresses may not impinge uniformly on the higher plant, and appropriate responses will often involve tissues at considerable distance from those immediately affected by a stimulus. In this review, “long distance” refers to stretches that cannot be spanned by a couple of hours of diffusion in the aqueous phase: several millimetres or more, and sometimes up to metres. “Rapid” refers to rates of perhaps 0.2 mm s-’ and over. A broad view is taken of the concept of signalling, and almost any event that could connect a remote stimulus with a physiological response is considered. Discussion on the fundamental nature of signalling and signal transmission can be found in Bentrup (1979), Canny (1985), Firn (1985), Due (1989) and Wayne (1994). In higher animals, very rapid (40 m s-’) co-ordination within the organism is enabled by a specialized nervous system. Three hundred years of histology has not revealed nerves in plants. Nevertheless, plants can exhibit long-distance communication at rapid rates. According to some reports, rates of up to 300mm s-’ are possible (Sibaoka, 1966; Oda and Linstead, 1975; Umrath and Kasterberger, 1983). The major examples of rapid long-distance signalling within plants can be grouped into the following categories: A. SHOOT RESPONSES TO CHANGE IN THE ROOT ENVIRONMENT

Treatments applied to the root may cause dramatic changes in leaf growth rate beginning within a couple of minutes. This has been demonstrated with root cooling (Dale et af., 1990; Malone, 1993a; Pardossi etal., 1994), osmotica (Chazen and Neumann, 1994), salt (Cramer, 1992; Alarcon and Malone, 1995), and anaerobiosis (Nakahori etal., 1991). Over both short and longer time courses, stomata1 aperture in leaves can also respond sensitively to changes in conditions in the root environment (Davies and Zhang, 1991). B. RAPID MOVEMENTS INDUCED BY LOCALIZED MECHANICAL STIMULI

Rapid movements can be induced by touch in leaves of most species of the insectivorous genera Drosera, Dionaea, Aldrovanda and Utricularia (Darwin,

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1875; Williams, 1976) as well as in sensitive plants exemplified by Mimosa spp. Similar responses are found in a range of organs in Stylidium (Findlay, 1978), Berberis, Sparmania, Mahonia, Mimulus, Bignonia (Hill and Findlay, 198 l), Zncarvilka (Sinyukhin and Britikov, 1967), Oxalis, Carambofa (BurdonSanderson, 1882) and others (see Darwin, 1881). For centuries, these rapid visible responses have fascinated botanists (Jones, 1923, on Dionaea: “The most wonderful plant in the world”) and laymen (e.g. King Charles I1 of England, 1661, cited in Houwink, 1935) and they have attracted close study. By contrast, relatively few invisible responses to localized mechanical stimuli have been characterized (Bose, 1928; Jaffe, 1980; Braam and Davis, 1990; Galaud et al., 1993) and it seems likely that many more such responses, both local and remote, await discovery.

C.

REMOTE RESPONSES TO LOCALIZED WOUNDING

These include “defence reactions” in which damage at one leaf leads to accumulation of toxic compounds throughout the shoot. These responses have attracted particular interest because of their potential in crop protection (Ryan, 1989, 1992). Systemic accumulation of inhibitors of insect digestive enzymes is found after localized wounding in cultivated tomato (Green and Ryan, 1972), wild tomato (Wingate and Ryan, 1991), potato (Peiia-Cortes et a/., 1988), tobacco (Pearce‘ et al., 1993), maize (Eckelkamp et a/., 1993; Corder0 et al., 1994), poplar (Parsons er al,, 1989), alfalfa and Datura strumonium (Shukle and Murdock, 1983). Endogenous elicitors from tomato leaves can also induce proteinase inhibitors in leaves of squash, cucumber, strawberry, grape and clover (Walker-Simmons and Ryan, 1977). In tomato and potato, induction of proteinase inhibitors (PI) is detectable in remote leaves, within 20 min to 2 h from wounding (Peiia-Cortes et af., 1988; Graham et al., 1986; Malone er al., 1994b). The mechanism of wound signalling in this system is considered in more detail in section IIIA. Another defence reaction induced throughout the shoot by localized treatment is known as “systemic acquired resistance“ (SAR or ISR). It occurs after limited pathogen infection in a restricted (De Wit, 1985), or wide (Lawton et al., 1994) range of plants. When one leaf becomes infected by any of a diverse array of pathogens, uninfected leaves on the same plant may develop increased resistance to a similarly diverse range of pathogens (KuC, 1987). Normally, development of SAR requires many days, but after treatment of one leaf with certain chemicals, such as oxalic acid, SAR may develop within 20 h (Doubrava el al., 1988) and the signalling system could be much faster than this. Systemic emission of volatile pheromones and other semiochemicals or alIelochemicaIs may occur soon after localized insect feeding in certain plants. These compounds may attract predators or parasitoids to the attacking

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herbivore (Turlings and Tumlinson, 1992; Dicke et a / . , 1993; Dicke, 1994) and they are thus thought to be involved in defence. A variety of other remote or systemic responses, mostly with no known defence function, are reportedly induced soon after localized wounding. These include stomata1 closure (Van Sambeek and Pickard, 1976; Wildon etal., 1992), reduction in the rate of isoprene emission (Loreto and Sharkey, 1993), increased polysome formation (Davies and Schuster, 1981), changes in the pattern of bud break in Bidens (Desbiez et al., 1991), surface electrical phenomena (Houwink, 1935; Van Sambeek and Pickard, 1976; Malone and StankoviC, 1991; Stahlberg and Cosgrove, 1995), and changes in the activity of certain ions in the xylem (Ries etal., 1994). In many species, localized wounding also triggers a general increase in cell turgor pressure (Malone and StankoviC, 1991) and a swelling of the shoot (see Figs 3, 4 and 7; Boari and Malone, 1993). These effects are described in section IIC and IID. They are systemic except that tissues very close to the wound site may show the opposite effect: a contraction (Malone, 1993b, 1994; see section IID and Figs 10, 13, and the leaf marked * in Fig. 7). Swelling and/or contraction of the stem was also reported by Bose (1928) after application of small electric and mechanical stimuli to stems of Antirrhinum, Tradescantia, tomato, and “woody rose”. D. MISCELLANEOUS RELATED PHENOMENA

Many further examples of long-distance signalling in plants can be identified but the transmission rates are often relatively slow. Selected examples are considered briefly: a classic paradigm of signal transmission in plants was formerly provided by the Cholodny-Went theory of phototropism and geotropism. However, that theory has proved to be mostly incorrect (Firn and Digby, 1980). Even so, there is clear evidence that mobile signals from the root cap play some role in facilitating geotropism (Moore and Maimon, 1993). The polar transport of auxin (Goldsmith, 1977) provides a potential system for long-distance (basipetal) signalling at perhaps 10 mm h-’, for example, in apical dominance (Sachs and Thimann, 1967). Unidentified signals of daylength are generated in leaves and travel to the apical meristem to control the transition to flowering in various species (Penel et a/., 1985). Signals probably involving cytokinins, and which are generated mainly in the seed pod, trigger active senescence of soybean leaves (Nooden and Murray, 1982; Nooden and Letham, 1993). Plants may also transmit chemical or physical signals to neighbouring organisms. These include attractants (colour, scent, heat) produced by insectpollinated flowers, “warning” signals of heavy chemical defences (Augner, 1994), and phytotoxic root exudates to reduce competition from neighbours (Woods, 1960; Krebs, 1972, p.41). Some signals are released that have no obvious benefit to the transmitter and may even be deleterious. Many of these are probably unavoidable by-products of the plant’s biochemical or biophysical

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presence; they are hardly signals in a physiological sense but they can be ecologically important. Examples include the far-red light reflected from photosynthetic pigments of leaves in sunlight. This may induce competitive reactions in neighbouring plants (Ballare et a/., 1994). Also, volatile substances released from plants can be used by herbivores to locate a food source (Finch, 1986; Could, 1994). Water vapour is the major volatile compound released from land plants and, depending on the degree of physical coupling between canopy and atmosphere, water vapour released from one plant may have a significant influence on the microenvironment of neighbouring plants (Jones, 1992). Some reports discuss changes induced in one plant following wounding of a different plant (Baldwin and Schultz, 1984; Fowler and Lawton, 1985). Such interaction wouId seem to have little benefit to the transmitter unless the neighbouring plants are its close relatives. There are also occasional reports of more mysterious communication between plants. These tend to be on the metaphysical fringe (Backster, 1968; Wagner, 1989; and reinvestigation by Horowitz et al., 1975). In the following section, various mechanisms by which rapid, long-distance signalling within the plant might occur are considered.

11.

MECHANISMS OF LONG-DISTANCE COMMUNICATION WITHIN PLANTS

At least five potential mechanisms for rapid long-distance signalling within the plant can be identified: 1. airborne flow of volatile chemical messengers; 2. phloem transmission of chemicals;

3. self-propagating changes in hydraulic pressure; 4. xylem transmission of chemicals (=“hydraulic dispersal”); 5 . self-propagating changes in electrical potential.

In some cases the stimulus itself can propagate through the plant from a site of application: in Mimosa pudica, for example, vigorous shaking of one leaf can cause distant leaves to react, but the mechanical vibrations of shaking are themselves transmitted through the plant. The response is merely local (Sibaoka, 1991). Light may also be “piped” for considerable distances through the fabric of the plant (Mandoli and Briggs, 1984). This is probably best considered as stimulus transmission rather than true signalling. A.

AIRBORNE SIGNALS

Volatile messengers can move rapidly by diffusion through the plant’s air-space network. In some plants such movement may be facilitated by “internal winds”, which are mass flows of air within the plant (Armstrong and

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Armstrong, 1990). The flow velocity can reach 2 mm s-' in petioles of Nuphar lutea (Dacey, 1981). Few endogenous plant growth regulators are significantly volatile under biological conditions. Exceptions include ethylene (boiling point -103°C) and methyl jasmonate (MeJ). Ethylene is released by most plant tissues. It has a wide spectrum of effects and has been recognized as an important plant hormone for a century (Neljubow, 1911; Osborne, 1975). MeJ is also released by many plant tissues, especially after damage (Albrecht et al., 1993). It may arise during membrane breakdown (Farmer and Ryan, 1992; Peiia-Cortes etal., 1993). MeJ has recently been shown to be effective at extremely low levels in some systems (Farmer and Ryan, 1990; Falkenstein etal., 1991) and it should probably be considered as a true plant hormone. Under most circumstances, volatile compounds will quickly disperse from their sites of production into the surrounding atmosphere. They will therefore play little role in long-distance signalling within the plant. However, if plants are closely sheltered or enclosed in some way, volatiles released in one tissue may accumulate to significant levels and may reach and affect distant tissues on the same or neighbouring plants. This can be demonstrated by experimental enclosure of plants (Farmer and Ryan, 1990). In the natural environment, effective enclosure of plants occurs during submergence. This enables adjustment to changing water depth in a number of semi-aquatic plants (Ku et al., 1970). Nymphoides peltata, for example, has leaves that float on the surface of slow-moving water, but which are anchored by long petioles to roots in the substratum. The leaves produce ethylene continuously. Normally, the gas diffuses quickly away into the surrounding atmosphere, and there is no significant accumulation within the tissue. However, if the leaves become submerged by a rise in the water level, ethylene can no longer escape because its diffusion rate in water is slower by a factor of lo4 than that in air (Burg and Burg, 1965). Ethylene therefore accumulates rapidly in air spaces within the flooded plant; it may increase 20-fold to over 1 pI I-' within an hour of submergence (Fig. 1; Malone, 1983). At such elevated levels ethylene stimulates rapid petiole elongation in Nymphoides beginning within about 30 min (Fig. 2; Malone, 1983), as in many other water plants. This elongation returns the leaf lamina to the surface, whereupon the ethylene dissipates to the atmosphere, and petiole elongation ceases. In this system, therefore, elevation of ethylene levels within the petiole is both a signal of leaf submergence and a trigger of the depth-accommodation response. In the sundews (Drosera spp.) insects are captured by globules of viscous fluid secreted at the apex of glandular leaf hairs. Once an insect becomes entangled at one hair or group of hairs, these hairs quickly bend towards the leaf centre. Later, more distant hairs on the same leaf may bend towards the insect to enclose it further and to aid with its digestion (Darwin, 1875). In some species, the leaf lamina also bends markedly to enclose the insect (Darwin, 1875, p. 12). The signals that initiate bending in neighbouring leaf hairs and in the leaf lamina could be hormonal (Williams and Pickard, 1980) but there

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time from submergence (h) Fig. 1 . Kinetics of ethylene accumulation in leaves of a water plant after submergence. Air was vacuum-extracted from batches of nine submerged, excised leaves of Regnellidium diphyllum (the petiole of each leaf was first trimmed to 30mm). Ethylene content was determined by gas chromatograph (G C). From Malone (1983).

is anecdotal evidence that airborne factors released by the insect might also play a part. For example, the following observations on Drosera filiyormis, by one “Mrs Mary Trent” are recounted in Knight and Step (1905, p. 64): “At ten o’clock I pinned some living flies half an inch from the leaves, near the apex. In forty minutes the leaves had bent perceptibly toward the flies. At twelve o’clock the leaves had reached the flies and their legs were entangled among the bristles and held fast”. A “Mrs Treat” is mentioned and cited several times in connection with D. filiyormis in Darwin’s Insectivorous Plants (1875, pp. 278, 281). I presume that “Mrs Trent” is identical with “Mrs Treat”, and that she was a considerable authority on Drosera. Her observations here may seem unlikely, but airborne signalling is not inconceivable in Drosera. For example, the tentacles of D. rotundifolia are incredibly sensitive to ammonium ions, and can react to quantities “. . . comparable to those in rain water” (Darwin, 1875, p. 167); the level of ammonium in Darwin’s rainwater was probably considerably less than in present-day Kentish rainwater). Wounded or decaying insects might emit sufficient ammonia to trigger tropic reactions in nearby leaf tissue.

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Fig. 2. Kinetics of ethylene-promoted growth in the petiole of Nymphoidespeltata. The height of a vertical column of 4 x 10-mm apical petiole segments was monitored using a displacement transducer. Air containing a saturating level of ethylene (0.1% v/v) was applied directly to the segments at time zero. From Malone (1983).

B. PHLOEM TRANSLOCATION

The phloem is the major pathway for long-distance transport of solutes in the plant (Baker and Milburn, 1989). Various chemical messengers are believed to move through the phloem, including abscisic acid (ABA) (Wolf et a/., 1990; Jackson, 1993), MeJ (Anderson, 1985), the tomato proteinase-inhibitor elicitor "systemin" (Pearce eta/., 1991), salicylic acid (Rasmussen eta/., 1991) and others (Hall and Baker, 1972). The phloem is the most obvious potential route for long-distance transmission of chemical signals in plants (Ishiwatari etal., 1995). In addition, the movement of many signals displays an association with the vascular system (Ryan, 1974; Van Sambeek and Pickard, 1976; Roblin and Bonnemain, 1985; Keil etal., 1989; Stanford eta/., 1990; Davis etal., 1991), and extends in both basipetal and acropetal directions. This again suggests movement in the phloem (Peiia-Cortes eta/., 1988; Davis eta/., 1991; Weiss and Bevan, 1991). Some workers perceive the phloem as being the only system capable of conveying solutes rapidly in both basipetal and acropetal directions in plants (e.g. Beraud eta/., 1992; Rigby etal., 1994); a view which is certainly incorrect (see section 1I.D).

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Estimates of the flow velocity of phloem sap range around 0.1-1 mm s - ' (MacRobbie, 1971; Nelson etal., 1983; Nobel, 1991, p. 515) with values up to 4 m m s-' (Baker and Milburn, cited in Wildon etal., 1992). Many longdistance signals in plants are slower than this, but a significant number are substantially faster. It is difficult to measure accurately the movement of signal molecules in the phloem. The most common method of extraction involves incubating the base of a cut petiole in a solution containing ethylenediaminetetraacetic acid (EDTA) as chelating agent (Anderson, 1985; Pearce etal., 1991). It is questionable whether extracts obtained in this way represent pure phloem sap (Jackson, 1993). Techniques based on severed aphid stylets (Ishiwatari et al., 1995) or on histochemical approaches (Narvaez-Vasquesz et al., 1995) should facilitate more precise analysis of the identity and flow rate of signal molecules in the phloem. Even here, however, caution must be excercised because various substances can pass between the xylem and the phloem (Van Bel, 1984; Minchin and McNaughton, 1987). Thus the presence of a molecule at any point in the phloem does not necessarily mean that it has arrived there entirely via the phloem. Some putative signal molecules, such as oligosaccharides, appear to have very limited phloem mobility (Baydoun and Fry, 1985). C. HYDRAULIC PRESSURE SIGNALS

Hydraulic pressure signals are propagating changes in water pressure. They will pass throughout the hydraulic continuum of the plant (Boyer, 1969, 1989; Malone, 1993b) and they offer "a potentially ubiquitous system of integration within plants" (Palta et al., 1987). Hydraulic theories of communication in plants date back to 1661 (Clarke, cited in Houwink, 1935); the fact that vessels were not then known was an inconvenience. Hydraulic theories thus predate electrical (Burdon-Sanderson, 1873) and hormonal (Neljubow, 1911) theories by a couple of centuries. A minority of tissues do not have good hydraulic connections to the main stem of the plant. These may include cortical regions of the mature root (Sanderson, 1983), and some unusual structures (Morse, 1990), as well as material destined shortly to part company with the mother plant, such as maturing fruit and seed (Darlington and Dixon, 1991; Welbaum etal., 1992), abscising leaves, etc. Most tissues, however, have plentiful hydraulic connections furnished by the xylem. This is particularly important in leaves because, except in some crassulacean acid metabolism (CAM) systems and plants of aquatic or exceptionally humid environments, the process of harvesting C 0 2 inevitably involves major loss of water (Cowan, 1977). Extensive xylem connections ramifying to all parts of the leaf (Canny, 1988) are required to replenish this water efficiently. These connections also provide a route for long-distance transmission of hydraulic signals.

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The front of a pressure wave will travel through the plant at up to the speed of sound (1500m s-’ in water). However, to be physiologically relevant, an hydraulic signal must cause a significant change in turgor pressure in the live cells receiving it. Most plant tissues are appreciably elastic; their turgor will change only if net influx (or efflux) of water occurs, that is significant relative to the hydraulic capacity of the tissue. Thus, all physiologically important hydraulic signals must involve significant mass flow of water. As a rough example, net water influx equivalent to 1-5% of the total volume of water in a leaf might be required to raise the turgor pressure of its cells through I bar (Malone, 1993a). The kinetics of pressure change in tissue receiving a hydraulic signal will depend on the magnitude and distribution of hydraulic resistance along the flow path, and of hydraulic capacitance of the receiver tissue. Despite several advances (Zimmermann, 1983; Canny, 1990; Tyree and Ewers, 1991 ; Peterson et al., 1993), the detailed information on plant hydraulic architecture required for prediction of the systemic pattern of pressure propagation is not available. The mass flow associated with hydraulic signals between organs will usually involve a relatively long axial pathway, through the xylem, and a short radial pathway through cells (or cell walls) of the tissue at each end. The volumetric rate of fluid flow through the open tubes of the xylem can be approximated from the Hagen-Poiseuille “law”:

where J, is volumetric flow rate through the tube, r is tube radius, AP is pressure gradient, q is kinematic viscosity of fluid, and I is tube length. This equation states that the flow rate (per tube) will depend on the prevailing gradient of hydrostatic pressure, on the 4th power of the tube radius, and on the viscosity of the fluid (Nobel, 1991; Niklas, 1992). The xylem usually contains only dilute solutes. These have negligible effect on flow rate. More concentrated solutes, such as sugars, can increase the viscosity of water considerably (Zimmermann, 1983). The viscosity of water can also change significantly with temperature over the biological range (Boyer, 1993). The Hagen-Poiseuille law often yields good approximations for water flow through xylem (Zimmermann and Brown, 1971, p. 199; Frensch and Steudle, 1989). Discrepancies can be ascribed to the various features by which xylem conduits differ from ideal tubes. These include occasional end walls, uneven internal walls (see Fig. 6 ) , non-circular members, and non-straight vascular bundles (Zimmermann, 1983; Tyree and Ewers, 1991). The radial pathway from the xylem into the tissue is comparatively short, but it appears to present more hydraulic resistance than the axial pathway, at least in herbaceous plants (Boyer, 1969; Malone, 1992). For example, Fig. 3 shows the kinetics of swelling at various points along an individual wheat leaf, in response to a remote wound. The half-time of swelling can be seen to vary

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Time from wound (min) Fig. 3. Kinetics of change in wheat-leaf thickness after wounding a neighbouring leaf. Five displacement transducers were distributed along a single wheat leaf, at the positions indicated in the diagram (inset). This leaf was about 120 mm in length. At time zero the neighbouring leaf was scorched with a flame for 3 s , at the position indicated by the vertical arrow. The curves show changing thickness a t each position, normalized so that the total change by 16min from wounding is the same for all. Symbols alternate between filled and open for successive curves. From Malone (1992).

inversely with the distance from the treatment site - that is, swelling appears faster further from the wound site. In fact, the kinetics depend on leaf thickness at the point of measurement, rather than on distance from the treatment site; the leaf becomes thinner towards its distal end, so the kinetics are faster there. This will occur only if the radial component of the flow path offers a larger resistance than the axial. In a range of herbaceous plants, change in xylem pressure at one location causes leaves over the entire shoot to swell with a half-time of 2-4 min (Fig. 4; Boyer, 1969; Boari and Malone, 1993). An abrupt change in xylem pressure can be imposed by cutting one leaf under water, or by damaging cells at one location and releasing their cell sap to the xylem (see Fig.9, section IIDl). Each of these treatments make water available to the xylem at the treatment

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Fig. 4. Remote wound-induced swelling in a range of plants. Each curve shows the mean change in leaf thickness from four similar plants treated simultaneously. A displacement transducer was placed on one leaf of each plant then, at the time indicated by the vertical line, one remote leaf on each plant was scorched for 3 s with a flame. Most of the plants were seedlings of about 15 cm in shoot height and having at least four expanded leaves. Time and vertical scales are indicated on the figure, as are the generic names. Wound-induced swelling of remote leaves was found in all plants tested. From Boari and Malone (1993).

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site. In young wheat leaves, a radial file of only three to four cells separates the xylem from the epidermis. Since the half-time for water-exchange across individual wheat-leaf cells is only 1 s or so (Malone and Tomos, 1990), it is surprising that the whole leaf displays a half-time of several minutes (Fig. 3). Even allowing for the concentric arrangement of cell layers around the xylem, the half-time for the whole leaf should be faster. This suggests the presence of an additional barrier to radial water efflux from xylem. The approximately exponential kinetics observed for leaf swelling (Fig. 3) also suggest the presence of a single major barrier to radial water flux, rather than a distributed series of resistances. This barrier may be similar to that identified by Canny (1990) in the bundle sheath that surrounds the vascular tissue in leaves of many monocotyledonous plants. Analogous barriers are present in the root endodermis and are postulated in some other tissues (Welbaum etal., 1992; Zimmermann et al., 1993). In a transpiring plant, the water status of all tissues will approach a dynamic equilibrium with their local xylem and thus with the entire plant. A change in the flux of water at any one site will alter local xylem pressure and will thus be transmitted throughout the xylem and throughout the plant. This means that a change in flux anywhere in the plant will be buffered by the hydraulic capacity of the entire plant, and it will affect the turgor pressure of live cells throughout the entire plant. Local changes in flux will occur, for example, when some roots encounter drying soil, or on a change in lighting or microenvironment at one leaf, even with a gust of wind or passing cloud. These events must cause small pressure transients to pass through the entire plant. Plants growing in the natural environment probably experience such pressure ripples throughout most of the day, but they may not be very large. 1. Transduction of pressure signals There is evidence that plant cells can be extremely sensitive to pressure (Staves et al., 1992). Various ways in which pressure changes might be transduced into physiological effects have been considered (Zimmermann, 1977). Two that deserve particular attention, from the whole-plant perspective, are:

Hydropassive effects on stomata. Raschke (1970) showed that small changes in the xylem pressure of maize leaves have rapid effects on stomatal aperture throughout the leaf. A decrease in xylem pressure causes opening of stomata, beginning almost immediately, and vice versa. The half-time here (a few seconds) is markedly faster than that for leaf swelling. This may indicate amplification of stomatal movement by mechanical interactions between guard cells and their neighbours, or the existence of an hydraulic by-pass direct from xylem to stomatal complex (Cowan, 1977; Boyer, 1985). In any case, the hydropassive opening of stomata by decreasing leaf water status produces a positive feedback loop: under transpiring conditions this could cause any

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small local decrease in water status to be amplified by acceleration of transpiration over the entire plant. Compensation for such effects occurs partly through change in leaf temperature, which automatically follows change in transpiration rate (Cowan, 1977), but it may also require active regulation of stomatal aperture. This suggests that ion-pumps and channels at the guard cell membrane could be very sensitive to change in leaf water status or guard cell turgor pressure. Many (but by no means all) transient or oscillating stomatal responses t o environmental change can be explained in terms of interactions between hydropassive and hydroactive stomatal movement (Cowan, 1977). Effects of turgor on cell growth rate. The commonly used model of Lockhart envisages a simple relationship between the extension rate of a cell and its turgor pressure (Lockhart, 1965; Cosgrove, 1986, 1993): dV Vdt

-=

0 ( P - Y)

where dV/Vdt is relative growth rate, 0 is cell wall extensibility, P is turgor pressure, and Y is wall yield threshold. Some recent models postulate lesser dependence on turgor (Zhu and Boyer, 1992), but all incorporate at least a threshold requirement. A decrease in the availability of water in the soil would therefore be expected rapidly to inhibit shoot growth rate, by reducing xylem pressure and cell turgor throughout the plant. Such effects are observed; treatments that cause a decline in root (or xylem) water status usually inhibit growth rate. The kinetics of growth inhibition in these cases are often very fast (Serpe and Matthews, 1992; Malone, 1993a; Chazen and Neumann, 1994; Stahlberg and Cosgrove, 1995); much faster than those that occur for transverse leaf shrinkage under the same treatment (Malone, 1993a). The faster effect on growth rate, compared to leaf swelling, may arise because: 1 . Cells that determine elongation rate (in grass leaves at least) may be immediately adjacent to the xylem and should thus respond more quickly to changing xylem pressure than the bulk of cells in the leaf (which determine transverse shrinkage). 2. Elastic changes in cell length will occur during change in turgor pressure, causing large but transient changes in apparent growth rate. The turgor change will act on the inherent elasticity in each cell (Cosgrove, 1981) and it may also be amplified through elastic interactions between the different tissues of the growing organ. Similar elastic interactions are manifested as “tissue tensions” in growing organs (Kutschera, 1989). 3. Only a small decline in turgor may be necessary to lower turgor to the yield threshold, and thus to stop growth (growth rate will be zero when P 5 Y , according to Lockhart’s equation, above; Boyer, 1985).

An increasing number of workers now argue that root-shoot hydraulic

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signals are not sufficient to account for shoot responses to soil conditions (Cowan, 1977; Passioura, 1988; Davies and Zhang, 1991; Munns, 1993). Many of these workers favour additional control by root-sourced chemical signals (section IID). However, while various additional factors will undoubtedly play a role in the longer term, the limited number of high-resolution measurements currently available often indicate a close correlation between purely hydraulic effects, and shoot growth responses in the short and medium term (Malone, 1993a; Pardossi etal., 1994; Alarcon and Malone, 1995). This indicates that chemical signals may not be necessary for such communication. Maturing fruit often show relatively long half-times for equilibration of water status with the rest of the plant. In these, hydraulic capacity may be large and xylem connections limited, and the majority of water influx may arrive via the phloem rather than the xylem (Ho etal., 1987). Since phloem translocation is thought to involve pressurized flow, it is possible that phloemborne hydraulic signals might become significant in large fruits. However, because of the higher sap viscosity, lower tube diameters, and internal membranes and constrictions, phloem-borne hydraulic signals are likely to be much weaker than those in xylem. Some plants have extensive tubular systems in addition to the phloem and xylem. Species such as Hevea have a systemic laticifer network. Mimosa pudica has a near-systemic network of large tube cells ("Schlauchzellen", Haberlandt, 1914) also termed "secretory cells" (Esau, 1970). Similar structures are present in many non-sensitive Leguminoseae. In Mimosa pudica there is also a large perivascular sheath of sclerenchyma that appears to consist of open tubes. In principle, any of these open tubular systems could also conduct rapid hydraulic signals. D.

HYDRAULIC DISPERSAL SIGNALS

Hydraulic dispersal refers to transmission of chemical signals by mass flow in the xylem. The xylem seems largely free of transverse membranes, and it offers potentially rapid distribution of solutes over long distances. Many inorganic nutrients, notably calcium, are believed to be delivered to leaves primarily in this way (Canny, 1993). However, for rapid signalling, chemical transmission in the xylem has a number of limitations: 1. Mass flow in xylem is generally unidirectional. Unlike hydraulic pressure signals, therefore, hydraulic disperal signals can usually contribute only to signalling from root to shoot. 2 . If hydrophobic barriers surround the xylem in the leaf (see section IIC), solutes may be trapped within the xylem by ultrafiltration (Canny, 1990) and may not reach the living tissue. Unless these peripheral ultrafiltering membranes are patchy or leaky, specific receptors may be necessary for transmission of chemical signals beyond the xylem.

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3. Xylem flow rate is unpredictable and will vary enormously with weather

conditions. Signal chemicals synthesized at a particular rate by some basal transmitting tissue will thus arrive in the xylem at a concentration that varies with the prevailing flow rate. The response of receiving tissues in the shoot could not, therefore, be based on the immediate concentration of a signal in the incoming stream. Receiving tissues would have to interpret the signal further, perhaps by integrating it over an extended period, or by somehow distinguishing its true delivery rate from its concentration (Gowing et al., 1993; Jackson, 1993). In principle, the system could operate if the transmitting tissue produced a chemically similar, but nonmodulated, reference signal in addition to the modulated signal. The ratio of signal: reference would be conserved regardless of the flow rate, and could be read off in the receiver tissue. A related problem is the “residence time” of solutes in the xylem. During periods of reduced transpiration, such as at night, chemical signals produced in the roots might not be conveyed to the leaves for many hours. Conversely, at other times the velocity of flow in the xylem might greatly exceed 1 mm s-I (Zimmermann and Brown, 1971; Nobel, 1991). Values of up to 200 mm s-’ are reported for some herbaceous plants (Zimmermann, 1983, p. 65) and, in young wheat plants, Passioura (1972) estimated a mean xylem-flow velocity of 250 mm s-I during the daytime in the basal part of the root. Excision of two of the three seminal roots from these wheat plants increased this velocity to some 800mm s-I in the remaining root. Peak flow rates would presumably have been even greater. A further consideration concerning solute movement is that during laminar flow, the pattern of velocities across the tube forms a parabola: the flow rate tends to zero at the periphery of the tube, while at the centre of the tube it tends to a peak velocity of twice the mean (Zimmermann, 1983). Thus, a compact pulse of solute added to one end of a tube will emerge from the other as an extended pulse, with a long tail. Interchange of fluid between the various vessels in a plant stem, which have a range of radii and correspondingly larger (squared) range of flow velocities, will further spread the solute elution profile, as could chemical interactions between the solute and the walls of the vessels. In view of such incoherent and unpredictable transport kinetics, it is difficult to envisage a root-shoot signalling system for close control (e.g. of whole-shoot stomatal aperture) which could operate efficiently using xylem-borne chemicals. Evidence from xylem anatomy and phyllotaxis, and from observation on the movement of tracers (Zimmermann and Brown, 1971; Jones and Lamboll, 1980; Zimmermann, 1983) suggests that any one region of the root (or shoot) may be preferentially coupled, by the xylem, to particular regions of the shoot. This could be problematic for root-shoot signalling by hydraulic dispersal. For example, signals for stomatal closure released from root tips in a partially dry soil (Davies and Zhang, 1991) might reach only certain leaves or parts of leaves, or they might reach some leaves long before others. The extent of this

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problem is unknown. In a number of herbaceous plants, localized treatments can induce systemic swelling of leaves (Fig. 4; section IIC) and the kinetics of this swelling are similar in most leaves throughout the shoot (see Fig. 7). This suggests that, if the xylem is separated into discrete sectors, there can be no significant barrier to pressure-driven flow between the sectors. During normal transpiration, however, the various parallel sectors could all be at similar pressure and there might then be virtually no interchange of water or solutes between them. Sectorial patterns might also develop or change with the age of the plant. This area merits closer study. Movement of chemicals with the transpiration stream will almost always be solely acropetal. Under some conditions, however, flow directions in the xylem can be disturbed and even transiently reversed. For example, reversal occurs if water is supplied artificially to the xylem at some point on the shoot (Bennett et al., 1984; Van de Pol and Marcelis, 1988). Transient reversal may also occur naturally, where the normal diurnal fluctuation in xylem water pressure meets tissues of large hydraulic capacitance and low transpiration rate, such as some fruit, or the heart leaves of lettuce (Malone, 1993b). Reversal of xylem flow also occurs on wounding (see below).

I.

Wound-induced hydraulic dispersal

A special situation develops around sites of localized wounding: hydraulic dispersal will occur rapidly and in all directions from such sites. This was first suggested by Ricca (1916) in connection with his experiments on Mimosa spegazzinii (Malone, 1994). This phenomenon occurs because water is released into the apoplast from damaged cells at the wound site, and it then becomes available at atmospheric pressure to the xylem. If the xylem is under tension, it will draw this water in and local xylem tension will be relieved. Local relief of xylem tension will spread rapidly, as an hydraulic pressure wave, throughout the xylem. Tissues over the entire plant tend toward a dynamic hydraulic equilibrium with the xylem (section IIC). Therefore, when xylem pressure suddenly rises systemically, after wounding, all the tissues of the shoot will begin to absorb water more rapidly from their nearest xylem. In “upstream” root tissues (those involved in water uptake from the soil), the cells will begin to lose water less rapidly to the xylem or, if the change in xylem pressure is sufficiently large, they may begin to absorb water from the xylem. Turgor pressure in all tissues will increase (Malone and StankoviC, 1991) and they will begin to swell (see Fig. 4; Malone, 1992; Boari and Malone, 1993). Swelling of the unwounded leaves requires a significant mass flow of water from the xylem (section IIC). This will tend to depress xylem pressure again, and cause more water to be drawn into the xylem at the wound site. Thus, mass flow will be pulled away from the wound site, through the xylem, and it will radiate towards all other parts of the plant. This wound-induced mass flow will continue until all the water released at the wound site is exhausted, or until tissues throughout the plant become saturated with water.

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The fluid released from damaged leaf cells is not pure water. It is mostly vacuolar sap, and contains considerable amounts of solutes including ions (chiefly K + but also Ca2+ and others; Malone and Tomos, 1992) as well as various organics. Mass flow from the wound site will convey all these solutes through the xylem. Given this situation, wound signalling requires merely that some soluble elicitor is present in the sap released from damaged cells, or is generated at the wound site from damaged cell constituents, or from interactions with the cell wall. Any such elicitor will be automatically conveyed, by the wound-induced mass flow (=hydraulic dispersal), from a wound site. Various such elicitors have been identified in tomato, including ‘‘systemin” (section IIIA). Systemic swelling can also occur by mechanisms other than wound-induced mass flow; for example, if transpiration were to decrease suddenly. However, this cannot explain the swelling that results from wounding because: 1 . Wound-induced swelling is faster than can be explained by water uptake across the root (Fig. 5 ) . This is because flows induced by wounding within the shoot do not have to traverse the large hydraulic resistance of the root endodermis. Thus, even if transpiration were to cease immediately and completely, the resultant swelling would not be as fast as that observed after wounding. 2. In shoots enclosed in polythene bags, and with their roots in mannitol solution, systemic wound-induced swelling can be induced as normal, even though transpiration rate remains negligible throughout (Malone and Alarcon, 1995). 3. Wound-induced swelling can occur even in excised shoots with no access to external water (Malone, 1992). Decreased transpiration could not account for actual swelling in this material; it could only slow the rate of shrinkage.

Excised plant organs placed in air normally contract rapidly as water is lost. However, internal redistribution of water within such organs can lead to transient localized expansion. This occurs because of internal pressure gradients generated by localized cell-wall relaxation and growth (Matyssek et al., 1991) or because of osmotic gradients generated by active redistribution of solutes, followed by passive redistribution of water (Weisz et al., 1989). In both these cases, the localized swelling is much slower (hours) than wound-induced swelling. The occurrence of rapid wound-induced swelling therefore provides a convenient diagnostic test for wound-induced hydraulic dispersal. One point needs to be emphasized: as described above, localized wounding destroys cell membranes and releases vacuolar water; it thus reverses local pressure gradients between tissue and xylem, and then within the xylem. It follows automatically that, basipetal to the wound site, the direction of xylem flow will be transiently reversed. Some workers have been reluctant to accept this point (e.g. Wildon etal., 1992) but it is supported by overwhelming evidence:

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seedling 1

Fig. 5 . Kinetics of wheat-leaf swelling after remote wounding and after rehydration of the root. Results from two seedlings run simultaneously are shown. A displacement transducer was placed on one leaf of each seedling. The steady-state leaf thickness obtained after substitution of the hydroponic root medium by 5-bar mannitol solution was first determined for each seedling (not shown). This serves as a reference point. The root medium was then changed back to water, and leaf thickness allowed to recover (not shown). Root rehydration (thinner lines): the roots were lifted clear of their hydroponic medium, and leaf thickness then began to decline because of water loss by transpiration. Severe dehydration of the roots was prevented by enclosing them in polythene bags. When leaf thickness had declined to the same level as previously reached in 5-bar mannitol, the roots were replaced in water (at the time indicated by the vertical line). The curves show recovery in leaf thickness with time after root rehydration. Wounding (thicker lines): the roots were returned to 5-bar mannitol solution (not shown). When leaf thickness had once again reached a steady level, a neighbouring leaf was wounded by scorching for 3 s with a flame (at the time indicated by the vertical line). These curves show the wound-induced swelling of leaves, with time. The thicker lines were taken subsequently from the same seedlings as the thinner lines. The transducers were not moved throughout, and the curves are therefore directly comparable. The absolute changes in leaf thickness were similar for both treatments, but the curves are normalized (to same total change over 0-7 min of treatment) to facilitate comparison of kinetics. The manipulations with mannitol ensured that both the wound-induced, and rehydration-induced swellings begin from approximately the same leaf water potential (thickness). It is evident that leaf swelling in response to wounding is faster than that in response t o rehydration of the root. This indicates that wound-induced leaf swelling is faster than can be accounted for by water influx across the root.

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1. Various tracers can be added to wound-induced mass flows, or to similar mass flows induced by submerged excision (see Fig. 9). These tracers are found to travel rapidly (c. 10mm s-I) through the plant, including in the direction basipetal to the wound site. Tracers that have been used in this context include heat (Houwink, 1939, and 14C-labelledcompounds such as Rhamnogalacturonan I (a pectic polysaccharide with PI-elicitor activity in tomato), adenine, sucrose and ABA (Malone et al., 1994a). In several cases the movement of these tracers has been shown to be unaffected by steam-girdling (Malone, 1993b). This demonstrates that they are xylemborne rather than phloem-borne. Further confirmation that basipetal xylem flow can occur comes from the classic experiment in which a pin is pushed into a transpiring herbaceous plant, through a droplet of dye on the surface of the stem: some of the dye is "sucked" almost instantaneously into the stem and it moves axially in both acropetal and basipetal directions (Simon etal., 1975, p. 189). The situation here involves local application of water at atmospheric pressure, to the xylem, and it is very much like that which occurs on wounding. 2. The rapid systemic increase in turgor pressure (Malone and StankoviC, 1991) and leaf thickness (Malone, 1992; Boari and Malone, 1993) that follows localized wounding, even in shoots with no access to water, can be explained only by mass flow of water from the wound site. Since the first stage of the flow is down the petiole of the wounded leaf, the initial mass flow, at least, must be mainly basipetal. 2. The kinetics of wound-induced h y d r d i c dispersal Velocity of transmission. The xylem itself presents a relatively low resistance to axial flow, and physiological pressure gradients can therefore induce very rapid flow in xylem. For example, if a pressure difference of 6 bar is applied to xylem vessels 25 pm diameter in a 40-mm petiole, the Hagen-Poiseuille equation predicts a mean flow velocity of about 300 mm s-' (Malone, 1994). Such rates will not normally be sustained after wounding in planta because radial flux of water from the xylem will quickly become limiting (but see section IID for examples of high flow rates sustained by transpiration). In the absence of data on xylem hydraulic capacity, it is difficult to model the kinetics of wound-induced flow. However, the velocity of this mass flow can be estimated experimentally. This was done in the petiole of wounded tomato leaves using three different methods (Malone, 1993b). For all three methods, the average flow velocity over the first 1 min after wounding was found to be about 8 m m s-I. The initial velocity could be much faster. Recently, the initial velocity of basipetal mass flow (close to a wound site) was measured directly using displacement transducers. In both tomato (see Fig. 10, section IIID) and Mimosa (see Fig. 13, section IIIB) this initial velocity exceeded the measurement resolution of 15-30 mm s-'.

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Extent of transmission. Leaf lamina are often only a few hundred micrometres thick. In absolute terms, therefore, the quantity of fluid released at wound sites on leaves is small (up to 0 . 3 ~ 1mm-2 wound area, for a leaf 300pm thick). However, the cross-sectional area of the xylem is also very small, and small volumes of water can thus displace xylem contents over substantial distances (see below). For example, consider a long, linear leaf such as that of maize. If we assume that the xylem in this leaf occupies 1% of the cross-sectional area of the leaf (excluding air space) then, assuming uniform flow across all the xylem, it follows that a heat-wound that kills a 1-mm wide strip across the leaf will release enough fluid to displace the xylem contents through a length of 100 mm of similar leaf tissue. This is not a particularly impressive distance, considering that a wound which killed an entire 1-mm strip of leaf was used. In the wounded plant, however, two additional factors operate that amplify the extent of this mass flow. First, the total area of the xylem in transverse section (TS) is virtually irrelevant for flow patterns: the r4 term in Hagen-Poiseuille dictates that the few largest vessels will carry the vast majority of any flow (Zimmermann, 1983). Thus, because most of the flow is confined to the few larger vessels, it must displace xylem contents through a much greater maximum distance for the same volume flux. Taking the above model leaf again, the few largest vessels might constitute only 20% of the total TS area of the xylem, giving a mass flow distance of up to 500 mm, rather than 100mm, for the same wound. The distribution of vessel radii in tomato petiole was measured using scanning electron microscopy (Fig. 6). In the example petiole described in Malone (1993b) the single largest vessel (of 26 in total), had a radius of 14pm, and carried over 13% of any flow through the petiole. This vessel occupied only about 0.06% of the total TS area of the petiole, and 8% of the total TS area of the xylem. A wound that damaged 1 mm2 on a subtending leaflet would therefore release sufficient fluid (0.3~1, above) to displace the contents of this vessel through 63mm ( = 63312pm = 13% x 0.3 x 109pm3/(?rx 142pm2)). The second source of amplification of wound-induced mass flow arises from the osmotic pressure of vacuolar sap. As discussed above, sap released from damaged cells enters the xylem and begins to flow away from the wound site. Healthy cells surrounding the xylem immediately basipetal to the wound site are therefore suddenly exposed to xylem sap of high osmotic pressure. These healthy cells therefore begin to lose water to the xylem, down the new osmotic gradient (despite the fact that xylem tension is reduced). This increases the volume of water available for mass flow and thus increases the extent of that mass flow. The extra water flowing from these healthy cells progressively dilutes the sap originating from the wound site, and so this effect diminishes with distance. This effect can be followed experimentally because it causes tissue close to the wound site to decrease in thickness (see Figs 10, 13), while more distant tissues increase. At a certain distance from the wound site the tissue initially increases in thickness, as xylem tension is released, but it then

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Fig. 6. Xylem vessel in a tomato petiole seen in transverse section under the scanning electron microscope. The section was from a position subtending the terminal leaflet of leaf three of a plant with four expanded leaves. The white vertical lines are part of a gauging tool, from which vessel diameter can be estimated. The distance between these lines in the example shown is 24.3 pm. The specimen was excised using a fresh razor blade, rinsed in water, then freeze-dried, shadowed with gold, and mounted.

changes sharply and begins to decrease in thickness (leaflet nearest the wound site in Fig. 7, marked *; Malone, 1993b). The onset of the decrease indicates the time at which solutes from the wound site arrive in that tissue and begin to exert their osmotic effect. It can thus be used to estimate the rate of solute flow from a wound site (Boari and Malone, 1993; see section IIIA). Because of these t w o amplifying mechanisms, even small wounds can generate sufficient fluid for hydraulic dispersal through considerable distances. In tomato leaves, for example, wounds imposed by the feeding of an individual Spodoptera larva can produce sufficient mass flow for hydraulic dispersal over a distance of 270 to 2700mm (Alarcon and Malone, 1994). As described above, hydraulic dispersal requires xylem tension. Its kinetics will therefore vary with the water status of the plant. This provides a useful diagnostic test for the involvement of hydraulic dispersal in physiological signalling: in the extreme case, when the plant is near-saturated with water there will be negligible xylem tension and no hydraulic dispersal (Malone and Alarcon, 1995). This situation obtains in a well-watered plant enclosed at high humidity, in which transpiration is minimal. Under these conditions, wound

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If4 If5

If3

Fig. 7. Systemic wound-induced increases in leaf thickness in tomato. The lines show simultaneous transducer recordings of leaf thickness, with time, in various leaves of an individual tomato plant. Leaf numbers are shown (right; the oldest leaf would be number 1). Two blank transducers (B), which did not contain leaves, were also run. At the time indicated by the long vertical line, one leaflet of leaf five was scorched for 3 s using a cigarette lighter. The transducer nearest to the wound site is marked with an asterisk. From Malone (1993b).

signalling in tomato can be eliminated (example in section IIIA). If signalling is not eliminated under these circumstances, then hydraulic mechanisms can normally be ruled out (but see section IIIB). Wound-induced mass flow is transient. It ceases when there is no further water available at the wound site. However, once soluble elicitors have been delivered into the xylem, they will continue to be carried by the transpiration stream long after the wound-induced mass flow has stopped. There will therefore be a second more prolonged and slower phase of hydraulic dispersal corresponding to flow with the transpiration stream. This will be in the acropetal direction only, and it will continue until all the solutes released from the wound site have been swept from the xylem into the leaves. Completion of this second phase may take up to an hour in tomato (see Fig. 10) and Mimosa (see Fig. 14; Malone, 1994). Most work on wound-induced hydraulic dispersal has involved localized

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wounding by heat. This procedure is very convenient for experimental use, and it releases large amounts of water at the treatment site, but it probably bears little relation to wounding in the natural environment. More realistic wounding can be achieved by mechanical damage or by insect feeding. These treatments also induce mass flows which, although small relative to those caused by scorching, are sufficient for long-distance hydraulic dispersal (Alarcon and Malone, 1994; Malone etal., 1994b). It is also conceivable that some nonwound stimuli generate limited basipetal hydraulic dispersal. This possibility is discussed in section IVB. E. ELECTRICAL SIGNALS

It has been known for a century that brief electrical transients, usually termed “action potentials” (AP), precede or accompany rapid movement in the motor tissues of certain plants. Burdon-Sanderson, with encouragement from Charles Darwin (see Williams and Pickard, 1980), recorded electrical transients from the surface of the trap lobes of Dionaea muscipula (Venus flytrap) during rapid trap closure (Burdon-Sanderson, 1873; Burdon-Sanderson and Page, 1876). Such electrical events reflect transmembrane ion fluxes, and they can be monitored using either intracellular or extracellular electrodes. A close correspondence is usually found between the patterns with these two types of electrode (Frachisse-Stoilskovic and Julien, 1993; Due, 1993). Extracellular electrodes cost very little. For example, the six-electrode recording system described by Malone and StankoviC (1991) cost about f 150 to build, including power supplies, “Faraday” cage, and amplifiers, but excluding the PC and A-D convertor card. Extracellular electrodes are simple to use, but they are typically quite large and will measure a net response from many individual cells. Detailed studies of electrical events, including some intracellular measurements, have been carried out on several mechanically-stimulated sensitive plants, including Dionaea (Stuhlman and Darden, 1950; Benolken and Jacobsen, 1970; Sibaoka, 1980; Iijima and Hagiwara, 1987; Hodick and Severs, 1988), Aldrovanda (Iijima and Sibaoka, 1981, 1982), Drosera (Williams and Pickard, 1972a,b, 1980; Williams and Spanswick, 1976), and Mimosa (Sibaoka, 1962, 1980; Abe and Oda, 1976; Samejima and Sibaoka, 1983; Stoeckel and Takeda, 1993). It is widely believed that some electrical transients in sensitive plants are self-propagating action potentials akin to those of animal nervous systems, which permit rapid signalling and co-ordination over substantial distances (Sibaoka, 1966, 1969, 1991 ; Pickard, 1973; Roblin, 1979; Davies, 1987; Thain and Wildon, 1992). Transients recorded from sensitive plants often resemble A P from animal nerves, except that the plant A P is slower by a factor of about l o 3 (Burdon-Sanderson and Page, 1876; Sibaoka, 1991).

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Electrical activity is also found to spread rapidly from sites of localized wounding in most non-sensitive herbaceous plants, including laboratory favourites such as Phaseolus, Helianthus, Vicia, Pisum and Lycopersicon (Pickard, 1973; Van Sambeek and Pickard, 1976; Roblin and Bonnemain, 1985; Boari and Malone, 1993). Systemic electrical activity is especially pronounced following localized wounding by heat. However, the pattern of wound-induced electrical events in these herbaceous plants is more variable and prolonged than the single spikes or spike trains seen in mechanicallytriggered sensitive plants. An example is shown in Fig. 15. These patterns are considered to incorporate at least two distinct electrical phenomena (Pickard, 1973): (i) AP, as described above, and (ii) “variation potential” (VP, sometimes termed the “slow wave”), which is usually more prolonged than the AP . In herbaceous plants, A P are usually said to move with velocity of 1-20mms-’, while in sensitive plants, they move at up to 200mms-’ (Sibaoka, 1969). The pathway of AP is uncertain but intracellular recordings tend to locate most activity in the phloem parenchyma (Samejima and Sibaoka, 1983) or in the phloem sieve tubes (Sinyukhin, cited in Pickard, 1972; Eschrich et al., 1988; Wildon et al. 1995). The phloem cannot be the only location of AP because A P are also reported from the glandular tentacles of Drosera, which have no phloem (Williams, 1976). Many workers envisage a long-distance signalling role for A P in plants (Sibaoka, 1966, 1969, 1991; Pickard, 1973; Roblin, 1979; Davies, 1987; Thain and Wildon, 1992). By contrast, there is widespread agreement that the VP is not a self-propagating signal; rather, it is a local electrical response to the underlying passage of chemical substances (Houwink, 1935; Sibaoka, 1966, 1969; Pickard, 1973). These substances are released from the wound site and they move through the plant in the xylem (by hydraulic dispersal), eliciting electrical changes in any living cells which they contact, especially in those cells adjacent to the xylem. The nature of the chemical substance(s) that elicits the V P is not known. It may be a specific (hormonal) depolarizing substance, and various workers note parallels with Ricca’s factor in Mimosa (Houwink, 1935; Pickard, 1973; Sibaoka, 1991; Malone, 1994) and perhaps with “traumatin” (Zimmermann and Coudron, 1979) or “turgorins” (Schildknecht, 1983; Kallas et al., 1990). Alternatively, the VP may be a purely electrochemical reaction to the sudden appearance in the xylem and surrounding apoplast, of the cocktail of ions released from the wound site. For example, vacuolar sap from cells of the wheat-leaf epidermis contains about 200 mM K’, as well as some 3 0 m ~each of Ca2+ and C1- (Malone and Tomos, 1992). A solution containing these ions, even in diluted form, could well disturb the Nerstian component of the membrane potential in any cells that it contacts. It is often stated that the stimulating chemical(s) responsible for the variation potential moves in the xylem “with the transpiration stream” (Sibaoka, 1969; Hill and Findlay, 1981; Davies, 1987) and some workers have

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commented that the VP do not exhibit the slow unidirectional (acropetal) movement that this should imply (Hill and Findlay, 1981). In fact, of course, when the stimulating substance enters the xylem at a wound site, it will move by a process of hydraulic dispersal. As discussed above (section IIDl), woundinduced hydraulic dispersal can transport material in both acropetal and basipetal directions, and at rates considerably faster than the normal transpiration stream. The onset of the VP, at successive distances from the wound site, will therefore provide a further indication of the movement of solutes by wound-induced hydraulic dispersal. The topic of plant electrical signals is examined in more detail in section IV, and an alternative interpretation of some plant electrical phenomena is there discussed.

111. RAPID, LONG-DISTANCE SIGNALLING IN PLANTS: CASE STUDIES A. SYSTEMIC INDUCTION OF PI BY LOCALIZED WOUNDING IN TOMATO

During a biochemical study of endogenous PI in solanaceous plants, Ryan and coworkers noticed large and seemingly erratic variations in the levels of certain PI in tomato leaves. This led them to the discovery that damage to one leaf of a tomato plant can trigger accumulation of PI in other leaves (Green and Ryan, 1972, 1973). PI accumulation was later shown to involve woundinduced genes (Graham eta/., 1986). Because PI interfere with insects’ digestive enzymes, and are toxic to insects (Broadway eta/., 1986; Hilder eta/., 1987; Orozco-Cardenas eta/., 1993), wound-inducible PI is thought to be part of a systemic defence system against leaf-eating insects and possibly other pests (Bowles, 1992; Ryan, 1992). PI may also render the wounded tomato plant less attractive to insects (Edwards eta/., 1985). Similar systemic woundinducible PI occurs in potato shoots and in several other species (listed in section IC). The induction of PI in tomato and potato may not be truly systemic. For example, there is little or no induction in most root tissues (Stanford eta/., 1990; Narvaez-Vasquez eta/., 1993), nor within parts of the stem and petiole (Peiia-Cortes eta/., 1988; Keil eta/., 1989; Stanford et al., 1990). However, PI accumulation can occur throughout all the leaves (at least in young plants, see below) including those above and below the wound site (Peiia-Cortes eta/., 1988). Clearly, a signal must pass throughout the shoot from the wounded leaf. This signal is known as the proteinase-inhibitor inducing factor, or PIIF (Ryan, 1974). The velocity of this signal has been estimated by various means: Green and Ryan (1973), and Ryan (1974, 1977) excised the damaged leaf at various times after wounding, and found that the signal exited the wounded leaf with a half time of 40min or less. Wildon eta/. (1992) found that some

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of the wound signal exited from damaged cotyledons of tomato within 5 min. Increased PI activity can be detected in remote leaves within as little as 1 h after localized wounding in tomato (Malone et al., 1994b). Peiia-Cortes et al. (1988) showed that the mRNA for PI became detectable systemically within 20min of localized wounding in potato. The similar mRNA in tomato may become detectable in remote leaves within 15 min of wounding (StankoviC, pers. comm.) Even allowing zero time for signal generation, reception and transduction, these data indicate that the velocity of the wound signal can exceed 0.2 mm s-’. Each of the five mechanisms of signal transmission discussed in section I1 has been considered as a possible explanation for wound signalling in tomato: 1 . Volatile methyl jasmonate can be released from plant tissue, perhaps especially if wounded (Albrecht et al., 1993), and it can induce PI in tomato leaves (Farmer and Ryan, 1990, 1992; Bolter, 1993). Ethylene might also modulate PI induction (Weiss and Bevan, 1991). However, under most conditions involvement of these airborne signals in systemic wound induction can be discounted because wounding of a leaf on one plant does not induce PI in an adjacent plant, even if the two plants are held very close together (noted in Malone etal., 1994b); to be effective, the wound must be on the same plant. 2. Phloem transmission of elicitor chemicals is believed by many workers to be the mechanism of long-distance wound signalling in tomato (Ryan, 1992; Orozco-Cardenas etal., 1993). This is partly by default: many workers have imagined that the phloem is the only system to offer rapid bidirectional transport in plants (e.g. Weiss and Bevan, 1991; Rigby etal., 1994; but see section IIDl). However, transport of chemicals in the phloem would be consistent with the observed association of wound signals with the vascular system (Ryan, 1974; Keil etal., 1989; Stanford et al., 1990). Recent work has also demonstrated inhibition of systemic PI induction by p-chloromercuribenzene sulfonic acid (PCMBS), a known inhibitor of phloem translocation (Narvaez-Vasquez et al., 1994). Various endogenous chemicals are capable of inducing PI in unwounded tissue. These substances are known as PIIF (Ryan, 1992). There is evidence that the most active of these chemicals (“systemin”) can move in the phloem (Pearce et al., 1991; Narvaez-Vasquez et d.,1995). Some others are virtually immobile in phloem (Baydoun and Fry, 1985). Nelson et al. (1983) reported that steam girdling (which kills phloem) prevents passage of the wound signal in tomato. Conversely, other workers report that blockage of phloem transport by steam girdling (Fig. 8, Malone and Alarcon, 1995) or by chilling (Wildon etal., 1992) does not impair wound signalling in tomato. The phloem is notoriously sensitive to damage (Oparka, 1990) and, in the immediate vicinity of any wound site, it would presumably be disabled for a considerable period of time. The positive pressure of phloem

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sap would also tend to drive any flow towards the damaged sieve tubes at the wound site. This would not encourage rapid wound signalling through the phloem. 3. Hydraulic pressure signals are transmitted throughout the tomato plant from sites of localized wounding (evident as systemic leaf swelling in Fig. 7; Malone, 1993b). Systemic pressure signals can be mimicked without significant wounding, by excision through a petiole submerged in water (Fig. 9). However, this treatment does not induce PI in remote leaves and pressure changes have therefore been discounted as wound signals in tomato (Malone et al., 1994b). 4. Electrical events appear throughout the tomato plant soon after localized wounding and it has been proposed that they are travelling wound signals (Pickard, 1973; Wildon etal., 1989). Wildon e t a / . (1992) showed that neither electrical events nor the wound signal were stopped by a chilled region. However, there are two problems with the electrical theory. First, it is not clear whether the electrical events are self-perpetuating signals, or merely local responses to chemical signals (see section IVB). Second, there is no evidence that electrical events will induce PI; for example, electrical transients are also induced in tomato leaves by changes in lighting (Cheeseman and Pickard, 1977), but these do not induce PI. Much of the wound-induced electrical activity reported from wounded tomato plants looks more like variation potentials than action potentials (e.g. Fig. 1 in Wildon e t a / . , 1989; Fig. 15, and see section IVB). This is important because variation potentials are not self-propagating signals; they are local responses to chemicals travelling in the xylem (section HE). Variation potentials cannot therefore be long-distance wound signals in tomato. Wounded tomato plants often exhibit spike-like electrical events that resemble action potentials (Wildon et al., 1989; see Fig. 15) but again, it is questionable whether these events propagate through the plant (see section IVB). Finally, any involvement of self-propagating electrical signals in the tomato-PI system appears to be eliminated by the finding that wound signals readily traverse extensive heat-killed zones of the tomato petiole (Fig. 8; Malone and Alarcon, 1995). Electrical signals would not be able to traverse such zones because they require live membranes along which to propagate. 5 . The involvement of hydraulic dispersal in wound signalling in tomato was suggested by Malone and StankoviC (1991). Localized heat wounds cause rapid and systemic swelling of the tomato shoot (Fig. 7). This indicates that wounding triggers large and systemic mass flows from the damaged region in tomato. Quantitatively similar mass flows, induced by submerged excision, have been shown to be capable of conveying various solutes rapidly throughout the plant (Malone et al., 1994a). It follows that solutes present in the sap released from damaged cells will also be carried throughout the

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plant after localized wounding. Various chemicals with PIIF-activity, including “systemin” which is active at extremely low levels, are known to be among the solutes present in tomato cell sap (McGurl et al., 1992; Ryan, 1992). These will be carried rapidly away from wound sites along with the wound-induced mass flow. Various lines of evidence indicate that hydraulic dispersal occurs rapidly, and over long distances in the xylem of the tomato plant (section IID1). The fastest flow rates occur basipetal to the wound site, along the petiole of the wounded leaf. From the pattern of decreasing thickness in tissue close to the wound site (Fig. 10; see section IID2), solutes evidently traverse

n

a

.->

0.6

I

I

I

I

5

10

I

I

I

15

20

1

1

CI

0

5 0.5 L

v

.-E

> 0.4 -= 0

(Is

.Q.-=b

0.3

r

.-r g

0.2

Q

.-ra

0 0.1

CI

k

0.0 0

25

time from wounding (h) Fig. 8. Wound signals pass through the heat-killed petiole of tomato. One day prior to the experiment, a zone of the petiole of leaf two was heat-killed (closed symbols), or not (open symbols). At time zero, the terminal leaflet of leaf two was wounded (triangles), or not (circles) by scorching for 3 s with a flame. At various times thereafter, the terminal leaflets of leaf three were harvested for PI assay. Each symbol shows mean &SE ( n = 10). These data show that passage of the wound signal is not blocked at heat-killed petioles. From Malone and Alarcon (1995).

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subrnerged excision 1

2,

scorch wound

Fig.9. Systemic swelling induced by wounding, and by submerged excision in tomato. Plants had three fully-expanded leaves. A displacement transducer was placed on the terminal leaflet of leaf three. The curves show change in leaf thickness with time, and each is a mean for six plants run simultaneously. At the time indicated by the arrow (upper trace only) one leaflet of leaf two was excised in air. At the time indicated by the vertical line, one leaflet of leaf two was wounded by scorching for 3 s with a match flame (lower curve) or was excised by a clean cut through its submerged petiole (upper curve). The two curves were taken successively from the same plants, without moving the transducers, and they are therefore directly comparable. Several hours were allowed for recovery between wounding treatments (not shown) and the curves are offset arbitrarily for clarity. From Malone etal. (1994b).

this stage at rates of at least 15-30mms-' in tomato, as in Mimosa (Malone, 1994; section IIIB, see Fig. 13). Eventually, after 30-60min, the returning transpiration stream sweeps any remaining solutes from the xylem of the wounded leaflet (Fig. 10). Wounding of less than 1% of a single leaflet has been estimated to generate sufficient mass flow for quasi-systemic signalling in tomato (Malone, 1993b; section IID2). This calculation is based on the volume of sap required to displace the contents of the major xylem vessels in the petiole of the wounded leaf, as far as their junction with the stem. From that point, even without further wound-induced mass flow, xylem-borne dispersal will occur with the normal transpiration stream, at least in the acropetal direction. Even wounds imposed by individual leaf-eating insects release sufficient sap to generate long-distance hydraulic dispersal in tomato (Alarcon and Malone, 1994). Significant hydraulic dispersal will occur only if there is a prevailing tension in the xylem of the shoot. If the plant is well-watered and enclosed in a polythene bag to minimize transpiration, there will be negligible xylem

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193

Fig. 10. Kinetics of change in leaf thickness close to a wound site in tomato. Four displacement transducers were spaced along the terminal leaflet of leaf four of an intact plant, at positions shown in the diagram (left). The plant had five expanded leaves in total, the oldest and lowest is designated leaf one. An area near the tip of the leaflet bearing the transducers (as indicated on the diagram) was wounded by scorching for 2 s with a flame, at the time indicated by the vertical line. The curves (centre and right) indicate changing leaf thickness at each transducer position, normalized so that the total change by 7 min from wounding is the same at each position. The two sets of curves (centre, right) are presented at different temporal resolutions (note separate time-scale markers). The central set shows the early wound-induced change at high resolution, while the right-hand set continues the data at a slower resolution. From the central set of curves it is evident that the onset of the decrease in thickness occurs almost simultaneously at all positions along the wounded leaf. This indicates that hydraulic dispersal of solutes (in cell sap) from the wound site occurs at rates of at least 15-30mms-I. The subsequent recoveries in leaf thickness shown in the right hand set of curves indicate the pattern of displacement of cell sap from the xylem underlying each transducer position, by “clean” water in the returning transpiration stream. From the onset of the steep part of these recoveries, at successive positions (indicated by vertical dashed arrows) the rate of this returning transpirational flow can be estimated as: 3. I mm min-’ (T4-+ T3); 3.3 mm min-’ (T3 + T2); 2.6 mm min-’ (T2 Tl). +

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tension and no hydraulic dispersal. Under these conditions, therefore, localized wounding should induce neither systemic swelling nor systemic PI. Both these predictions have been shown to hold true (Malone and Alarcon, 1995). This is very strong evidence in favour of wound signalling by hydraulic dispersal, as is the finding that wound signals pass freely through steam-killed regions of petiole (Fig. 8). Only the xylem remains functional within such regions. Two experiments that are supposedly inconsistent with hydraulic dispersal in the wounded tomato have been mentioned in the literature. First, Wildon et al. (1992) noted that transpiration continued from the wounded leaf, and they argued that this indicated no reversal of flow in xylem in the petiole of the wounded leaf. However, this argument is based on a misconception: transpiration from the leaf lamina can occur simultaneously with basipetal flow in the petiole, if the water for both processes comes from an intermediate position - that is, the wound site. Second, Thain and Wildon (1992) note that wound signalling can still occur in tomato shoots that have been excised and placed with their cut ends in water. They state that the xylem flow should be “very different” in this situation compared to that in the intact plant. The assumption here is that shoots with their cut ends in water should have no xylem tension, and therefore no wound-induced mass flow. Our experiments have confirmed that where xylem tension is eliminated (by enclosure in polythene bags) then wound signalling is also eliminated (Malone and Alarcon, 1995). However, we tested the assumption that shoots placed in water have no xylem tension, and found it to be false: such shoots show systemic swelling in response to wounding, exactly as in the intact plant (Fig. 11). This indicates that excised shoots can exhibit wound-induced hydraulic dispersal in the normal way. Evidently, xylem blockage occurs near the cut end of the excised shoot. Blockage may be associated with damage, active wound healing, or embolism. The block can be removed by excising a few millimetres from the base of cut stem. This causes transient swelling of the excised shoot (Fig. 11) exactly as with submerged excision through the petiole of an intact plant (Fig. 9) but the block soon regenerates. The hydraulic resistance of the (blocked) stem base is thus sufficient to ensure a substantial xylem tension (and therefore wound-induced hydraulic dispersal) in the excised shoot, as long as it is transpiring. Hydraulic dispersal is thus the most likely mechanism of systemic wound signalling in the tomato. The involvement of airborne- and hydraulic pressure signals can be discounted, and it remains to be established whether electrical events are signals in themselves or merely local responses to other travelling signals. The fact that wound signalling is unimpeded by an intervening heatkilled zone strongly favours xylem-borne signals over those requiring live cells - that is, electrical and phloem-borne signals. Note that heat-killing the

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excised shoots

intact plants

blank

1 30 min

Fig. 11. Wound-induced hydraulic signals in intact tomato plants and in excised shoots. Change in leaf thickness with time was measured using displacement transducers placed on the terminal leaflet of leaf three of intact tomato plants (lower traces) or of excised shoots with the base of the stem placed in water (upper traces). All six plants were at the three-leaf stage, and were run simultaneously. At the time indicated by the first vertical lines the soil was watered generously (lower traces, intact plants) or 3 mm was sliced, by a submerged cut, from the stem bases (upper curves, excised shoots). At the second vertical line, one leaflet of leaf two of each plant was wounded by scorching for 3 s with a match flame. Both the intact and the excised shoots showed marked wound-induced swelling.

petiole does not hinder xylem-borne flow, as indicated by the continued occurrence of wound-induced swelling in tissues on either side of such dead petioles (Malone et a(., 1994b). Given that hydraulic dispersal is involved, a general interaction between tomato shoot water relations and wound signalling can be predicted. This may account for the observed effects on PI induction of light, temperature, and C 0 2 (Green and Ryan, 1973; Ryan, 1977); each of these variables will affect transpiration rate (and thus shoot water status), and their effects may therefore be via hydraulic dispersal. The evidence from cold blocks and from steam girdling emphasizes that the

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phloem cannot play a primary role in wound signalling in tomato. Similarly, when the phloem could potentially function in isolation (in wounded plants held at high humidity to eliminate xylem tension) no wound signalling is observed (Malone and Alarcon, 1995). However, there remains a possibility that phloem transport could play some secondary or auxiliary role. For example, some reports indicate that exit of PIIF from wounded leaves can continue for over an hour (Green and Ryan, 1973). This is longer than would be expected for a mechanism involving only hydraulic dispersal. It is conceivable that the phloem can become secondarily involved in wound-signal transmission, by xylem-to-phloem transfer after there has been an initial ingress of PIIF into the xylem. Interchange between the xylem and phloem is reported in several systems (Van Bel, 1984; Minchin and McNaughton, 1987) and it is not inconceivable that it occurs with PIIF chemicals in tomato. This scenario would be compatible with the findings of Narvaez-Vasquez et al. (1995): 15 min after application to a wound site, ‘‘C-labelled systemin was located primarily in the xylem, but by 30min and thereafter, the label was located primarily in the phloem, and it appeared to spread progressively through the phloem over a period of 3 h or more. There are some question marks regarding the ecological significance of the wound-inducible PI system in tomato. Most work on this system has used young plants ( c 6 weeks old) which clearly show systemic induction of PI. In older plants, however, systemic wound induction of PI may be absent (Wolfson and Murdock, 1990). This is not because older leaves cannot accumulate PI; they can, but perhaps only in response to a nearby wound (Fig. 12). It is possible that the larger hydraulic capacity or more complicated hydraulic architecture of older plants dictates that hydraulic dispersal signals will extend only over a relatively limited range from wound sites in such plants. Local wound-induction could still confer a significant defence benefit to the plant. B.

SIGNAL TRANSMISSION IN MIMOSA

Signalling in the “sensitive plant” Mimosa, especially M. pudica and M . spegazzinii, has attracted attention for centuries. The subject is summarized

Fig. 12. Local, but not systemic induction of proteinase inhibitor (PI) by wounding in large, mature tomato plants. Tomato plants were grown under natural lighting in a large outdoor polythene tunnel. By 42 days these plants were already larger than those normally used in our laboratory experiments, and by the end of the experiment (84 days old) they had over 15 leaves. Leaflets were harvested from the top, middle or bottom of the plant, and their PI level was assayed. Leaflets from the middle and bottom of the plants were harvested after wounding o f an apical leaflet (A “remote wound”). Leaflets from the top, middle and bottom of the plant were also harvested after wounding of an adjacent leaflet ( 0 “local wound”). Controls ( 0 ) were

SIGNAL TRANSMISSION TRANSMISSION IN IN HIGHER HIGHER PLANTS PLANTS SIGNAL

197 197

-

0.50

-

0.25

-

0.00

-

0.50

-

0.25

-

0.00

-

unwounded. In each case, 24 h elapsed between wounding and harvest. Leaves from all regions of the plant showed PI-induction in response to a local wound, but a remote (apical) wound did not induce PI in leaves on the middle and bottom of the plant. Such remote wounds induce substantial PI in young plants (Malone etal., 1994b).

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here only briefly. Further consideration can be found in Houwink (1939, Weintraub (1952), Sibaoka (1966), Pickard (1973), Schildknecht (1983), and Malone (1994). Mimosa shows rapid and remote leaf movements in response to wounding. Its responses to non-wound stimuli, such as touch, tend to be limited to the stimulated leaf. Both hydraulic and electrical theories of wound-signalling in Mimosa have been debated for many years (Ricca, 1926; Ball, 1927). For example, Bose (1914) states, rather loftily, that “The question of whether the transmitted effect in Mimosa is due to a hydro-mechanical or excitatory impulse is held to be of much interest in Plant Physiology”. The work of Ricca (1916), and others, provided strong evidence that the wound signal in Mimosa is a chemical messenger which moves in the xylem that is, in the terms used in this review, that wound signalling involves hydraulic dispersal. Ricca argued that the signal must be chemical rather than electrical since it could be isolated and stored in a dry form. The putative chemical has since been referred to as “Ricca’s factor”. Ricca also reported that the signal would pass through sections of stem that were steam-killed, or that had been removed and replaced by a water-filled glass tube. Malone (1994) re-examined Ricca’s ideas and demonstrated that hydraulic dispersal from wound sites in Mimosa would pass in both acropetal and basipetal directions. The initial rate of this hydraulic dispersal was shown to be 15-30 mm s-’ or more (Fig. 13) which is sufficiently rapid to explain signal transmission. The pattern of lag times for responses of leaves progressively further from the wound site is entirely consistent with an initial, brief but rapid bidirectional hydraulic dispersal, succeeded by a prolonged period of solely acropetal movement with the transpiration stream (Fig. 14; these two phases of hydraulic dispersal are described in section IID2). Mimosa shoots contain at least one tubular laticifer-like system. The fluid in this system is under considerable pressure, 15-20 bar or more, even in transpiring shoots (unpublished data); a precise determination of the pressure is difficult because the fluid coagulates spontaneously soon after puncture, and so it blocks up the tip of a pressure probe. This pressurized system ejects copious fluid at wound sites, and so permits extensive hydraulic dispersal even from wound sites which would be hydraulically negligible in plants like the tomato (Malone, 1994). I can find very little foundation for the view of various modern writers that Mimosa represents a classic model of electrical signalling in plants (e,g. Roblin, 1979; Satter, 1989; Tinz-Fiichtmeier and Gradmann, 1990; Thain and Wildon, 1992). Remote electrical events do occur in the wounded Mimosa plant, but they appear to be incidental to wound signalling. They may represent local responses to the underlying passage of Ricca’s factor (Malone, 1994). In the case of the slower signalling that occurs with touch stimuli in Mimosa, Ricca (1926) considered a form of stepwise hydraulic dispersal: when one pair of leaflets move (in response to a direct mechanical perturbation), the motor

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Fig. 13. Kinetics of hydraulic dispersal close t o a wound site in Mimosa. Four transducers (TO-T3, plus a blank) were arranged along one pinna-rachis and petiole of Mimosa in a similar manner t o the arrangement shown for tomato in Fig. 10. At the time indicated by the vertical line, the tip of the same leaf was scorched for 1 s with a flame. The numbers in brackets are the distance, in mm, of each transducer from the wound site. The vertical arrows shown on the upper curve indicate times of application of “blank” wounds. These involved holding a flame near to, but not touching, the leaf tip (From Malone, 1994).

cells in their pulvini collapse. This collapse involves ejection of a small amount of fluid which may be sufficient for hydraulic dispersal through the 1 mm or so of rachis to the next pair of leaflets, thus triggering their collapse with further ejection of fluid, and so on (see section IVB). Note that this mechanism could work even without xylem tension, because positive apoplastic pressures

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0

200

400

600

800

time to leaf movement (s) Fig. 14. Response times for leaves above and below a wound site in Mimosa. One leaf in the centre of the plant was wounded by scorching for 3 s with a flame. The lag time to the motor reaction (leaf fall) in the main pulvini of successive leaves above and below the wound site is plotted against their distance from the wound site. There were leaves more than 15 cm below the wound site but they never responded. Data from three trials with the same plant are plotted, with a different symbol for each trial. The pattern indicates a brief initial phase of rapid flow in acropetal and (especially) basipetal directions, succeeded by a prolonged phase of purely acropetal movement (From Malone, 1994).

would be generated by the collapse of successive pulvini (Malone, 1994). Flow at positive pressure could travel through the intercellular space instead of, or as well as, through the xylem.

IV.

IMPLICATIONS AND DIRECTIONS FOR FURTHER RESEARCH

Various aspects of plant signalling that merit closer study have been identified in previous sections. These include the possible role of hydraulic dispersal in

20 1

SIGNAL TRANSMISSION IN HIGHER PLANTS

co-ordination of systemic acquired resistance (SAR, section lC), and the need for further information on sectorial patterns within the xylem (section IID). There is also a need for reappraisal of the nature of electrical signals in higher plants. In addition, recent information on hydraulic signalling may have wider implications, for example, for signalling of non-wound stimuli. These areas are discussed below.

A.

RE-ASSESSMENT OF ELECTRICAL SIGNALS IN THE HIGHER PLANT

The physiological role and even the existence of long-distance electrical signals in higher plants is a vexed question. The area is summarized briefly in section IIE. It is revisited here with some more speculative discussion on the nature of these electrical events. Physiologists have long been reluctant to accept the idea of long-distance electrical signalling in higher plants. Some interesting ideas on the origin of this reluctance have been advanced by advocates of plant electrical signals (Pickard, 1973; Simons, 1981; Davies, 1987; Thain and Wildon, 1992; Wayne, 1994). These ideas include: the desire of nineteenth-century botanists to emphasize differences between plants and animals; the emphasis on plant hormonal messengers that stems from influential work on auxins during the 1930s; doubts among some plant physiologists about whether a plasma membrane actually exists in plant cells and the unhelpful influence of contributions from the metaphysical fringe. Probably a more important reason is that the highly variable nature of some electrical phenomena in higher plants raises doubts about whether they could control anything. In addition, there is the long-standing suspicion that electrical events in plants represent local responses to the underlying passage of chemical signals (Ricca, 1916, 1926; Malone, 1994; Stahlberg and Cosgrove, 1995). Furthermore, the evidence for self-propagating electrical signals in most higher plants is slim and at best, correlative. As an example of the latter, consider the interesting and careful work of Williams and Pickard (1972a) on tentacles of Drosera: electrical events in the stalk were shown to follow stimulation of the head, and precede bending of the stalk. This is certainly a useful correlation. However, the conclusion made in the paper that . . the impulse has been identified with a train of AP" is premature because correlation alone will not necessarily indicate causal relationship; there remains the possibility that both the electrical activity and the bending are reflections of a local response induced by some other travelling signal. There seems no a priori reason why the tissues of higher plants should not be capable of generating, transmitting and responding to action potentials: the cells commonly maintain large transmembrane electric fields (Findlay and Hope, 1976) and the membrane permeability can change abruptly (as in Mimosa; Samejima and Sibaoka, 1982). The absence of nerves from plants

".

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is not a particular problem since various primitive animal tissues can propagate electrical events between ordinary cells (Thain and Wildon, 1982). Also, in many plant tissues, numerous plasmodesmata run between adjacent cells, and these could provide routes of low electrical resistance leading to strong electrical coupling between the cells (Spanswick, 1972; Cheeseman and Pickard, 1977; Iijima and Sibaoka, 1982; Lew, 1994). Moreover, nerve-like action potentials can be generated and transmitted in cells of Nitella and some other algae (Gradmann and Mummert, 1980; Sibaoka and Tabata, 1981; Beilby, 1989; Wayne, 1994). By analogy with the animal nervous system, the plant action potential should begin when a stimulus is perceived in particular receptor cells, it should propagate itself across the tissue without decrement, and it should initiate a response in distant cells (Thain and Wildon, 1992). The evidence for these stages is considered below; first in the sensitive carnivorous plants, then in non-sensitive plants.

I . Sensitive carnivorous plants The most convincing evidence for electrical signals in plants comes from the sensitive carnivores; Dionaea muscipula and Aldrovanda vesiculosa (Drosera to a lesser extent because its movements are rather slower). Utricularia might also fit into this category but the trigger cells are very close to the motor cells of the trapdoor, and there has been little study of its signal transmission (Sydenham and Findlay, 1975). The linkage in Utricularia might be purely mechanical (Lloyd, 1942). No further sensitive carnivores are known, although many other plants use static traps to catch insects (Darwin, 1875). There is evidence from Dionaeae and Aldrovanda of each of the three stages discussed above: Generation of A P. Membrane depolarization perhaps corresponding to a “receptor potential” has been observed during mechanical stimulation of cells in the base of the trigger hairs of Dionaea and Aldrovanda, and in the tentacle head of Drosera (Williams and Packard, 1980). The membranes can also be depolarized artificially by an injected current pulse. When this depolarization exceeds a critical value, a much larger depolarization is triggered: the AP. The AP is presumably caused by a massive transient increase in the permeability of the membrane toward particular ions: Na’ in the animal nerve, C1(and/or Ca2+) in the characeans (Gradmann and Mummert, 1980; Wayne, 1994). The electric currents associated with this strong depolarization may be sufficient to depolarize adjacent membranes beyond their critical threshold. If so, the AP will regenerate itself in neighbouring regions of membrane or in neighbouring cells, and thus become self-propagating at a rate independent of the initial depolarization. This is the basis of the all-or-nothing behaviour characteristic of AP in animal nerves. Propagation of AP. The propagation of AP can be assessed by comparing the

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203

timing of the electrical transient at successive distances from the site of stimulation. This approach has yielded propagation rates of 50-200 mm s-' across the trap lobes of Dionaea and Aldrovanda (Sibaoka, 1980; Iijima and Sibaoka, 1981), and about 20 mm s-' in the tentacle of Drosera (Williams and Pickard, 1972b).

Initiation of motor activity. There is a strong temporal correlation between occurrence of the A P and induction of movement in the motor tissue. The AP usually precedes movement, indicating that it may be the trigger for movement (Sibaoka, 1980, 1991). These sensitive carnivorous plants thus provide clear evidence of electrical signalling. Even in these, however, an alternative mechanism of signal transmission is conceivable (see section IVB). 2. Non-sensitive plants In non-sensitive plants, the situation is less clear. The most important classes of electrical events in these plants are the AP and the V P (introduced in section IIE). Other classes have been distinguished (Sibaoka, 1969; Bentrup, 1979) but these are probably unimportant in signalling. Obviously, when considering signalling of wound stimuli, it is critical to distinguish between the A P and the VP; the former is potentially a self-perpetuating signal, while the latter is merely a local response to passing solutes. In principal, the two phenomena are distinct (Pickard, 1973): AP should be brief, rapid, fullyreversible spiky events that conform to all-or-none characteristics. VP by contrast, will be (almost) any other electrical disturbance induced (usually) by wounding. In practice, however, it may be difficult to distinguish between these two phenomena. They often occur together and may be superimposed. They may also travel at roughly the same rate. Further entanglement may occur if the VP triggers new A P at sites distant from the original wound (Pickard, 1973). Moreover, V P are very variable and may incorporate rapid monophasic or biphasic changes in potential that resemble parts of an AP, and vice-versa (Van Sambeek and Pickard, 1976). Not surprisingly then, there are several published examples that are referred to as A P by the authors, but which could well be interpreted as VP by other workers (see e.g. Pickard (1973) discussion on Umrath (1959)). Pickard (1973) also notes problems of interpretation of potentials in the work of Kawano, and Lou; similarly Pickard (1972) queries some of the A P in the work of Sinyukhin. Further examples are mentioned in Findlay and Hope (1976). Unfortunately therefore, AP and VP can be far less distinct than some authors imply, and it becomes difficult to distinguish putative electrical signals (AP) from electrical responses

(W.

One important distinguishing criterion should hinge on whether the electrical event can pass through dead regions of tissue (Houwink, 1935): V P can, but A P cannot because there are no live polarized membranes at which the

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AP can regenerate itself. This useful indicator has rarely been exploited. Even this may not be infallible because: (i) VP can sometimes peter out while crossing dead tissue (Pickard, 1973); (ii) in characeans at least, AP can sometimes jump across gaps of several millimeters if aided by salty media or by salt bridges (Ping etal., 1990; Osterhout and Hill, 1930); and (iii) an AP might appear to traverse a dead zone if it is retriggered in the living tissue at the other side by an accompanying VP (Pickard, 1973). If AP are to be viable as physiological signals they must propagate through the plant in a reasonably stable manner. There is evidence that they do so in the sensitive carnivores (above). In non-sensitive plants, the VP may spread in a logical fashion from wound sites (as expected since the mechanism involves hydraulic dispersal; section IIE and IID). However, the progressive spread of AP after wounding in such plants is much less clear. This is not only because of uncertainty about whether AP or VP are being measured, but also because in many published results too few, and sometimes only a single recording electrode (plus reference of course), have been employed. In some other cases, the recording and reference electrodes are placed rather close together on the plant, so that it may be difficult to distinguish whether the electrical event occurs at the position of the recording electrode, or at the reference electrode (e.g. Wildon etal., 1992; see legend to Fig. 17). A recording of surface electrical activity measured at multiple electrodes in a wounded tomato plant is shown in Fig. 15. Two major types of electrical feature are visible at all four electrodes along the stem below the wound site in this record: the first is the large early shoulder which recovers slowly over 3-4 min. From its asymmetry and long duration, this can be identified as the VP. It moves progressively from one electrode to the next at a mean rate of about 10 mm s-'. This reflects the underlying passage of xylem-borne solutes travelling by hydraulic-dispersal from the wound site. The second type of event in Fig. 15 is the spiky transient. These are symmetrical and brief, each lasting some 10s at '/2 peak height. Although these are considerably longer than AP in the carnivores, they can reasonably be identified as AP. However, it is evident from the figure that these spikes do not travel along the stem in any logical manner. Rather, the pattern at any one position appears to be virtually independent of those at preceding or succeeding positions on the same stem. Electrical recordings from wounded tomato plants are also shown by Van Sambeek and Pickard (1976) and by Wildon et al. (1989). The former show a pair of recordings very similar to those in Fig. 15, and suggesting a similar conclusion: the VP moves from one electrode to the next, but the AP do not. The results of Wildon etal. (1989) also show movement of the VP, but movement of AP cannot be judged from their data because spikes are shown only for a single electrode. Thus, although spiky electrical events occur in the wounded tomato plant, there is no convincing evidence that they propagate. A distinction must therefore be drawn between these events and the self-perpetuating AP of animal nerves.

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\

\

Fig. 15. Surface electrical patterns induced by wounding in tomato. Six electrodes

(EO-ES)were distributed along the stem and petiole of a tomato plant, at the positions

arrowed in the diagram (left). A common reference electrode was pushed into the soil. The curves show change in surface electrical potential with time, at each electrode. At the time indicated by the vertical line, the terminal leaflet of leaf five (indicated by * on the diagram) was wounded by scorching for 3 s with a flame.

Single spikes and spike trains are common in electrical recordings from the wounded tomato plant. In our experience, however, they are atypical of herbaceous plants in general. In a survey of over a dozen species, we usually found only quite smooth V P after wounding, with few or no spikes (Boari and Malone, 1993). It may be that this type of spiky activity results from some morphological peculiarity. For example, spikes are found in tomato and some cucurbits; all of these have large glandular hairs at their surface which might generate local electrical activity, or which might alter the appearance of underlying activity, perhaps by offering low-resistance windows to the interior of the tissue. Such windows could have a large effect on recordings made with surface electrodes (Williams and Spanswick, 1976). It was concluded above that the spiky so-called “AP” do not travel in the wounded tomato plant. Spikes that do not appear to travel have also been reported from several other tissues (e.g. Pickard, 1972). In several reports on higher plants, however, workers claim to show the movement of AP. The beautiful recordings of Zawadzki and Trebacz (1985) using two recording

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A

.

B.

A

I

A-

Ref

p 3

”

-

Fig. 16. Basipetal (part A) and acropetal (B) travelling electrical events in the sunflower stem. At the times indicated by the vertical arrows, electrical stimuli were applied by passing current between the wires marked “ + n and ”-”. The positions of recording electrodes (1-3) are shown on the diagrams, together with that of the reference “Ref“. The curves show change in surface potential, with time, for each recording electrode. From Zawadzki et al. (1991) with permission.

electrodes on Lupinus, and of Zawadzki etal. (1991) using three recording electrodes on Helianthus (Fig. 16) serve as good examples: after localized stimuli, electrical phenomena are evident that clearly pass along the stem, affecting successive electrodes sequentially and in a logical manner. These are termed “AP” by the authors, and are referred to as good examples of propagating AP by several other workers (e.g. Thain and Wildon, 1992). However, I argue that these events are not AP; they are VP. This is because the duration of the electrical peak is rather long for an AP (c. 20s or more at ?hpeak height), and because the rate of travel (c. 8mms-’) is typical for the hydraulic dispersal associated with VP. Note that, although electrical stimulation, rather than wounding, was used in these studies, the applied voltage (4 V applied for 3 s) is very likely to have caused cell damage, or at least caused a change in membrane permeability, leading to release of solutes and water, and therefore to hydraulic dispersal, exactly as for wounding (see section IVB). This view is supported by the statement of Paszewski and

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Zawadzki (1976) that small heat wounds, involving “approaching or touching the stem with a hot glass rod” produce effects similar to those with electrical stimulation. The recurring dilemma of plant electrical “signals” is evident here: are these events AP or VP? That is, are they propagating electrical signals or merely local responses to a chemical signal travelling through the underlying tissue? The mechanism of the VP is known, at least in outline: cell sap is released at the wound site and is drawn along, via the xylem, by the pre-existing demand for water in all the tissues of the plant. It involves hydraulic dispersal (section IID). A number of predictions can therefore be made about VP: 1. The VP should not be blocked at dead regions. 2. (a) The propagation rate of hydraulic dispersal, and therefore VP, will depend on the prevailing water status of the plant; lower water status should tend to drive faster VP movement for a given wound; and (b) in the extreme case, where shoot water status is saturated (as with wellwatered plants enclosed for a few hours in polythene bags) xylem tension becomes negligible and VP should not travel at all. 3. The VP, because it is associated with rapid flows of water and solutes through the xylem, should also be associated with systemic changes in thickness of the tissue.

Higher plant AP are rather ill-defined in both character and mechanism. For example, Hill and Findlay (1981) stated that ”. . . it is quite disappointing to realise that a century after their discovery, the nature of propagated action potentials in higher plants is still poorly understood and we have no clear idea of their ionic basis”. It is therefore difficult to predict precisely how AP should behave in higher plants. Presumably, however, any self-propagating AP should be blocked at dead regions, should not be associated with large flows of water, and should be little affected by plant water status. Thus, AP ought to behave differently from VP under all three of the test conditions postulated above. These tests have not been reported from the species used by Zawadzki and colleagues. However, we have recorded similar electrical phenomena from various plants, including wheat seedlings, and we have tested the above predictions on these seedlings. Figure 17 shows that the wound-induced electrical event is accelerated when plant water status is depressed. Figure 18 shows that the electrical activity can pass across heat-killed tissue. Both of these observations are consistent with a mechanism involving hydraulic dispersal and VP, rather than one involving AP. It is also known that strong and systemic leaf swelling occurs in wheat seedlings and in other plants after wounding (see Fig. 4), and that this swelling is associated closely with wound-induced electrical events (Malone, 1992; Boari and Malone, 1993). Concerning prediction 2(b), we have found in wheat (not shown), as in tomato (Fig. 19), that enclosure of well-watered plants at high humidity will completely eliminate woundinduced electrical phenomena. Thus, according to each of the four criteria

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~~

A. roots in water

B. roots in mannitol

wound

E4 electro E3 arranger E2 as part

El

1 min

(El isolated) -

Fig. 17. Acceleration of wound-induced electrical events by reduced water status in wheat. Four electrodes were distributed along a wheat seedling (two-leaf stage) as shown in the diagram (A, right); three were placed along the blade of leaf two (E2-E4) and one was placed on the sheath of leaf one (El). A common reference electrode was placed in the hydroponic root medium. The curves show change in surface electrical potential with time, for each electrode. A. Roots were held in water. At the time indicated by the vertical line a small region near the tip of leaf one (vertical arrow) was wounded by scorching for 2 s with a match flame. B. The root medium was replaced by 5-bar mannitol solution and the seedling allowed to equilibrate for 4 h (not shown). Then, at the time indicated by the vertical line, a small region near the tip of leaf one (adjacent to that wounded in part A) was scorched as above. It is evident that, in the presence of mannitol (which reduces plant water status) electrical events are accelerated. The value recorded from each electrode is the difference in potential

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above, we can identify these wound-induced electrical events as VP rather than AP. The same conclusion is likely to hold for the (similar) recordings of Zawadzki and coworkers. I argue that most, possibly all, of the apparentlytravelling wound-induced electrical phenomona in higher plants are VP, reflecting hydraulic dispersal of solutes, rather than self-propagating AP. The tests described above should be applied in various other species, to assess whether their electrical activity is VP or AP. Tests involving enclosure in polythene (test 2 (b)), and transmission across dead tissue (test l), are particularly simple to apply. The spiky electrical phenomena from tomato cannot be explained as local responses to xylem-borne solutes, because these spikes do not appear to travel. However, the observation that spiky events in tomato are abolished if the plants are wounded at high humidity (Fig. 19) suggests that these too, may occur in response to hydraulic events. These spiky events normally occur during or shortly after the major wound-induced swelling of the tissue, and they may reflect pulses of ions emitted by cells during turgor- and/or volume regulation, as in Acetabularia (Wendler et al., 1983) and many animal cells (Sardaki and Parker, 1991). See section IVB, below. B. SIGNALLING OF NON-WOUND STIMULI - AN HYDRAULIC MECHANISM?

In addition to the electrical responses induced by wounding, there are many examples of electrical activity induced by non-wound stimuli, such as touch, cold water and small depolarizing currents. In these cases, it may seem that a mechanism based on hydraulic dispersal would be impossible because nonwound stimuli do not release the sap necessary to fuel hydraulic dispersal. However, non-wound stimuli can cause transient changes in membrane permeability (Jaffe, 1980). These would result in efflux of ions, followed passively by water, and they might release sufficient fluid to generate limited hydraulic dispersal. Rapid decreases in the axial electrical resistance of various (non-sensitive) plant tissues were recorded by Bose (1928) after gentle mechanical distortion, and by Jaffe (1980) after mild rubbing of the stem. This

between that electrode and the reference electrode in the root medium. In both A and B the three electrodes along leaf two (E2-E4) show an almost identical electrical pattern. These synchronous events are probably reflections of the electrical event at El (a variation potential) passing onward to register on the reference electrode (which is effectively at the root surface). Thus, they appear synchronously at all three positions and are inverted compared to that at E l . It follows that electrical events at the reference electrode will also be registered in electrode E l . The pattern at the reference electrode can be isolated approximately, as the inverted mean of the pattern in E2-E4. This mean reference pattern can then be subtracted from the trace El to yield the true pattern at E l (“El isolated”; dotted line). Applying the same reasoning, there is only negligible electrical activity at any of the positions E2-E4.

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E4

E3 E2

El

Fig. 18. Passage of electrical events across heat-killed regions of the wheat leaf. Four electrodes (El-E4) were arranged along a wheat seedling as shown in Fig. 17A. The four traces show change in surface potential with time, for each electrode. Four hours previous to this recording, the central region of the neighbouring leaf one was killed by scorching for 5 s with a match flame. By the start of this recording, the central part of leaf one was brown and shrivelled. At the time indicated by the vertical line, a small region near the tip of leaf one (distal to the previously killed zone), was wounded by scorching for 2 s. Wound-induced electrical events are evident at positions proximal to the dead zone, and they must therefore have traversed it. As argued in the legend to Fig. 17, much of the apparently synchronous electrical activity seen here at electrodes E2-E4 actually represents events occurring at the position of the reference electrode, which is also proximal to the dead zone.

probably indicates significant ion efflux from cells in the stimulated region. The rapid loss of ions postulated here would almost certainly be associated with water loss, and it could also depolarize the membrane potential, generating an electrical spike rather like an AP. Depolarization, the first portion of the AP, involves efflux of C1- in Charu, whereas repolarization to the resting potential involves efflux of K + (Wayne, 1994). The rrhimum ion flux necessary to cause electrical changes would be negligible in osmotic terms (Tinz-Fuchtmeier and Gradmann, 1990) but it appears that far more than the electrical-minimum flux often occurs in these cases. Thus, AP are associated with measurable water loss in Chara (Barry, 1970; Oda and Linstead, 1975; Wayne, 1994) and Nitella (Sandlin etul., 1968), and Wendler etal. (1983) discuss the role of AP in osmoregulation in Acetabularia. The latter authors show that the burst of C1- efflux associated with each periodic action potential imposes a significant stepwise decrease in cell turgor pressure and volume. Thus, an electrical spike, even if non-propagating and restricted to one region of the cell, will be associated with ion efflux which could influence the

SIGNAL TRANSMISSION IN HIGHER PLANTS

a. before mannitol addition

21 1

b. after mannitol addition

T1

Fig. 19. Elimination of wound-induced electrical activity in tomato plants at saturating water status. The shoot of a well-watered tomato plant (four-leaf stage) was enclosed in a polythene bag. Electrodes were placed on the base of the petiole of leaf three (El) and on the stem between leaf two and leaf three (E2). A displacement transducer was placed on the terminal leaflet of leaf two. A. The plant was allowed to equilibrate for 4 h inside the polythene bag then, at the time indicated by the first vertical line, half of the terminal leaflet of leaf three was wounded, through the polythene bag, by placing a hot (c. 90°C) metal block on it. B. The soil was irrigated to run-off with mannitol solution (10 bar) and the plant allowed to equilibrate for 4 h (not shown). At the time indicated by the vertical line, the remaining half of the terminal leaflet of leaf three was wounded, through the bag, as in part A. Parts A and B were conducted on successive days on the same individual plant. The transducer and electrodes remained in the same positions throughout and the two parts are therefore directly comparable. It is evident that, at saturating water status, wounding induces neither remote hydraulic nor electrical responses. Both types of response are restored if the plant's water status is reduced (still inside the bag) by irrigation with mannitol. Note especially that the spiky electrical events, as well as the slower variation potential, are eliminated at saturating water status.

remainder of the cell by causing a transient depression of turgor. A local water current could also be established transiently across the membrane, with efflux at the depolarized patch of membrane, where apoplastic solutes are most concentrated, and influx across the remainder of the cell where the solute gradients are unchanged. In the higher plant, where turgid, elastic cells are packed together and surrounded by an apoplast of negative pressure and small volume, a modest release of water and ions (causing depolarization) at one cell could provide enough sap to fuel hydraulic dispersal to neighbouring cells. Therefore, even without any electrical propagation or plasmodesmatal connections, events associated with depolarization at one cell could be signalled to neighbouring cells. If the membranes of these neighbouring cells are sensitive to a sudden change in turgor or apoplastic ion composition, they might also depolarize in

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response to this signal, with further loss of fluid. Thus, an hydraulic-dispersal signal, induced without wounding, could propagate across plant tissue. Electrically, such a signal might appear very much like a propagating AP, because successive ranks of cells would depolarize as the signal passed. An extreme example of this process, involving loss of ions from stimulated but unwounded cells, may happen in the “motor tissue” of some sensitive plants (Findlay, 1978). In Mimosa, for example, rapid movement of the leaf is driven by collapse of turgor pressure and volume in cells of the pulvinar motor cortex (Hill and Findlay, 1981; Sibaoka, 1991). This collapse is caused by a sudden and massive loss of solutes from the cells. K + and C1- are the major components, but other ions are probably also involved (Samejima and Sibaoka, 1982; Kumon and Suda, 1984, 1985). Because the cell walls are elastic, when turgor pressure coIlapses, cell volume must also collapse and water will be ejected from the collapsing cell. The water efflux could follow as a response to the efflux of ions, by diffusional flow down the new osmotic/ hydrostatic gradient. Alternatively, some workers argue that rapid efflux of sap from the motor cells of Mimosa resembles bulk flow rather than diffusional flow (Hill and Findlay, 1981; Iijima and Sibaoka, 1983; Kumon and Suda, 1984; Sibaoka, 1991); in this case, cell sap, including all the solutes it contains, will be squirted into the surrounding apoplast. If rapid flow in the Mimosa pulvinus is indeed a bulk flow, the membranes of this tissue might contain some very unusual transmembrane proteins, perhaps giant versions of the “aquaporins” reported from other tissues (Chrispeels and Maurel, 1994). Regardless of the mechanism of efflux, the fluid released to the apoplast will flow away from the cell which ejected it, because of its positive pressure, and because of apoplastic water tension. Such fluid will quickly reach neighbouring cells. If one of the ions or other constituents expelled from a collapsing motor cell, such as Ca2+ or Ricca’s factor, can stimulate opening of membrane channels, it will trigger a sudden change in membrane permeability in neighbouring cells leading to their collapse with further release of sap containing this active substance. In this way a cascade would occur such that the entire motor cortex will react, with each cell triggered by hydraulic dispersal of active substance from its collapsing neighbours. Furthermore, the catastrophic ion efflux from collapsing cells will generate an electrical transient, and this will appear to propagate across the tissue, as successive ranks of cells collapse. The initial ion efflux (and thus the associated electrical transient) could appear significantly in advance of actual leaf movement. This is because leaf movement might not begin until a large proportion of cells in the motor cortex had completed their collapse, since the few remaining turgid cells might support the leaf for a time. The lag of about 0.5 s observed between electrical transient and leaf movement in Mimosa (Sibaoka, 1966; Samejima and Sibaoka, 1980), might therefore be consistent with an hydraulic-dispersal mechanism of signal propagation, as well as with an electrical theory. Thus, even where “AP” appear to propagate rapidly across non-wounded tissue immediately prior to

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a motor response, hydraulic-, rather than electrical-, transmission could be involved. This possibility should be a priority for further study. At the molecular level, the electrical and hydraulic-dispersal mechanisms might not be totally distinct since, if the signal carrier is an ion, it will move according to local gradients of both pressure and electrical potential. One fundamental difference is that the major signal molecule could be neutral according to the hydraulic mechanism, but it would have to be electrically charged to be involved in a propagating AP. Note that, where many cells are collapsing catastrophically, as in the pulvini of Mimosa, positive apoplastic pressures could be generated transiently. Bose (1928) demonstrated by clever use of dyes, that sap was forcibly ejected from the primary pulvinus of Mimosa during its motor reaction. This probably also explains why the tertiary pulvini of Mimosa darken visibly for a moment as they collapse - the forcible displacement of intercellular air by flooding will change the refractive index of the tissue. In this case, hydraulic dispersal could occur over limited distances without the normal requirement for xylem tension. From the foregoing, it is concluded that non-wound stimuli, as well as wound stimuli (section IIDl), may be signalled by hydraulic dispersal in both nonsensitive plants and in the sensitive non-carnivores like Mimosa. The question then arises as to whether the motor reactions in the sensitive carnivores (principally Dionaea and Aldrovanda) might also be co-ordinated hydraulically, with the AP following the front of the motor reaction, and as a reflection of it rather than a trigger of it. This view would overturn a century-old conception of signals in these plants, but it is difficult to find other than anecdotal evidence pertaining to it in the literature. Examples of such evidence include: 1 . The trap of Dionaea closes much faster in light than in dark (Jaffe, 1973). This is suggestive of an hydraulic mechanism because hydraulic dispersal should also be faster under light (transpiring) conditions, when xylem tension is greater. 2. Wounding of a small number of cells, particularly in the motor region of the trap, can induce rapid closure in both Dionaea and Aldrovanda (Darwin, 1875, p. 292; DiPalma et al., 1966; Sibaoka, 1966; Williams, 1976; Iijima and Sibaoka, 1982). This is consistent with a mechanism involving hydraulic dispersal because the wounded cells would release sap, including any putative active substance, to the apoplast. Thus the normal receptor process would be circumvented and the signal cascade would begin from the wound site. 3. The trigger hairs are the structures that normally perceive mechanical stimuli from insects in the trap lobes of Dionaea and Aldrovanda. One particular cell layer in Dionaea has indented cell walls and, since these indentations will focus any bending stresses in the hair, these cells are believed to be the major receptors (Bentrup, 1979). They have been identified as such

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from their electrical behaviour (Benolken and Jacobsen, 1970). These cells are markedly larger than their neighbours (Haberlandt, 1914; Bentrup, 1979; Williams and Mozingo, 1971; Mozingo etul., 1970). One might predict that receptor cells at the start of an hydraulic signalling chain would be of large volume so that they can release a strong pulse of fluid to get the signal underway. 4. The organization of the xylem in Dionueu appears compatible with hydraulic transmission across the lobes. There is a massive midrib with vascular bundles branching off at right angles to it into the trap lobes. Signal transmission is fastest parallel to these vascular bundles (Darwin, 1875; Sibaoka, 1966). In Droseru, each tentacle contains central xylem elements, which extend into the glandular head (Williams, 1976; Darwin, 1875, p. 5). These might provide a route for hydraulic dispersal. Histology of the trap lobes of Aldrovundu is discussed briefly by Iijima and Sibaoka (1982) but they make no mention of vasculature. A strongly suberized or cutinized “endodermoid layer” is noted by Williams (1 976) in the mechanoreceptor tissues of various sensitive carnivorous plants. Williams uses this layer to trace evolutionary relationships between the genera, but he assigns no function to it and presumes it is vestigial in some species. The hydraulic viewpoint would predict the presence of such a layer to permit accumulation of aqueous secretions at the trap surface (against the prevailing hydrostatic gradients), and to prevent their escape into the apoplast and xylem. Accumulation of such secretions is necessary to trap insects in Droseru and to facilitate their digestion in both Droseru and Dionueu (Darwin, 1875). One major obstacle for speculations concerning the nature of signal transmission in traps of the sensitive carnivores is that the cellular water relations of trap closure are not understood. Almost all rapid plant movements involve rapid, reversible turgor loss and cell collapse, as in the pulvinus of Mimosa. Contrary to popular belief (Williams, 1976), however, there are no hinge-type pulvinar zones in the traps of Dionueu or Aldrovandu (or in the tentacles of Droseru), and their closure involves a different mechanism. Several workers have concluded that trap closure in these plants involves rapid and irreversible expansion of the entire outer surface of the trap lobe (Brown, 1916; Williams and Pickard, 1980; Williams and Bennett, 1982), while other workers favour the involvement of elastic changes instead or as well (Darwin, 1875; Stuhlman, cited in Hill and Findlay, 1981; Ashida, cited in Iijima and Sibaoka, 1981; Hodick and Severs, 1989). Williams and Bennett (1982) concluded that during trap closure in Dionueu, the outer epidermis undergoes irreversible cell growth at rates of up to 10% per second (dV/V), and they proposed the involvement of an acid-growth mechanism. But cell-wall loosening and cell growth at these unprecedented rates seems unlikely. Also, there would have to be a net water influx into the entire outer epidermis to support this growth, and water flows into leaves usually have half-times of minutes rather than seconds (see Figs 3,

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4, 9). An alternative view is that the basic mechanism involves rapid decrease

in turgor of epidermal cells over the outer or inner trap lobes, and that preexisting tissue tensions within the lobe then cause it to distort into the closed position (Iijima and Sibaoka, 1983; Hodick and Severs, 1989). This view seems inconsistent with the careful observations of Brown (1916), which revealed that the entire trap became larger with each cycle of closure and opening. These fascinating trap systems warrant re-examination with modern techniques for cellular water relations, such as the pressure probe. If their closure really does involve cell growth at fantastic rates, then hydraulic transmission of the impulse can be ruled out: signalling by hydraulic dispersal would not be possible if motor cells absorb water, rather than release it, when triggered. In this case, signalling by a neuroid, self-propagating action potential would appear to be the only available mechanism capable of delivering the observed high transmission rate. It is not inconceivable that the sensitive carnivores use AP in a manner fundamentally different from all other plants. If cells of these traps can grow at 10% per second, their cell walls must have some unique properties. A biochemical analysis of cell-wall constituents, including proteins, in the trap lobes might be very interesting. Finally, some tests which might be used to distinguish between hydraulic and electrical signalling in the sensitive carnivorous plants can be considered: 1. If the signal is a hydraulic dispersal, then many cells of the motor zone

must contain the putative “active substance”. An aqueous extract of the motor zone should therefore be capable of inducing closure when applied to the trap via the xylem. The putative active factor might then be purified using trap closure as a bioassay. Aqueous leaf extract can trigger movement in Mimosa (Ricca, 1916) and Berberis (Sibaoka, 1969). Large scorch wounds applied t o one trap should also trigger long-distance hydraulic dispersal of the active substance, leading to closure in other traps on the same plant. 2. As discussed earlier, transmission across dead zones of tissue provides a method of distinguishing between hydraulic and electrical transmission. As far as I am aware, this has not been tried with the sensitive carnivores, although Darwin (1875) noted that transmission could pass around transverse cuts in the trap Iobe of Diunaea. One approach might be to use a hot metal cylinder to apply a circular heat wound to one trap lobe of Dionaea. The wound should encircle at least one trigger hair. Then, after the leaf had recovered and reopened, the trigger hair could be brushed or the enclosed leaf surface stimulated by wounding. If the entire trap can still be induced t o close under these circumstances, then the involvement of selfpropagating electrical signals can probably be ruled out. 3. Similarly, the enclosure of well-watered plants at saturating humidity should eliminate hydraulic signals and should inhibit or at least retard leaf

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closure, even if a positive-pressure hydraulic signal is involved (as suggested for the Mimosa leaf). Afdrovanda is aquatic and its traps capture aquatic insects, but I am not clear whether the plant normally grows in an entirely submerged position. Iijima and Sibaoka (1981) state that Aldrovanda “floats just below the water surface” whereas Le Maout and Decaisne (1876, p. 407) state that it “floats on stagnant waters in S. France, N. Italy, and Bengal”. Even if a part of the plant normally protrudes above the water surface (and thus generates transpiration and xylem tension) Iijima and Sibaoka (1981) state that isolated traps of Aldrovanda can be triggered when completely submerged. It would be interesting to determine whether the rate of closure is reduced under fully submerged cf. partially submerged conditions. Dionaea is indigenous to damp boggy habitats in N. Carolina, but it is not habitually submerged, neither are the Drosera spp. Investigations of these types should be carried out to resolve the mechanism of rapid signalling in the sensitive carnivores.

V.

CONCLUSION

Higher plants exhibit various forms of rapid communication. Hydraulic pressure signals, phloem-borne- and airborne chemical signals can travel long distances in plants. Each offers a mechanism for long-distance co-ordination within the plant. Electrical “signals” in plants are more enigmatic. Many electrical events could represent local responses t o the passage of other signals. The most convincing evidence of self-propagating electrical signals comes from the leaf traps of the sensitive carnivores (Dionaea and Afdrovanda).Further analysis of signalling in these trap structures will require fundamental information on the water relations of trap closure. Transmission of chemical signals by hydraulic dispersal in the xylem could be the major mechanism of wound signalling in plants, and it may be of widespread ecological importance in the co-ordination of plant defences against leaf-eating insects and some lesion-inducing pathogens. Sucking insects which precisely target the phloem (or xylem), such as aphids, cause little cell damage and probably trigger negligible hydraulic signals. Plants may not be able rapidly to mobilize defences against these pests. Hydraulic dispersal may also play a role in signalling of non-wound stimuli.

ACKNOWLEDGEMENTS The author is funded by the Biotechnology and Biological Sciences Research Council (BBSRC, UK). Helpful comments on this chapter were provided by Professor Lyn Jones and Dr David Gray of HRI Wellesbourne. Professor E. Davies (University of Nebraska) kindly granted permission to reproduce

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Fig. 16. Grateful t h a n k s are due t o Jim Justice of HRI Wellesbourne f o r help with electron microscopy, and t o m y colleagues, Drs Bratislav Stankovii-, Francesca Boari, G i n a P a l u m b o and Juan Alarcon.

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