Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply

Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply

A . J . S. McDONALD' 2nd W . J . DAVIES'

'Department of Plant and Soil Science. Aberdeen University. Cruick Shank Building. St . Machar Drive. Aberdeen. AB9 2UD. UK. 'Division of Biological Sciences. I.E.B.S., Lancaster University. Bailrigg. Lancaster. LA1 4YQ. UK

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230

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231

I.

Introduction

11.

Manipulating Water and N Supply

I11.

Acclimation of C and N Uptake ................................................ A . Framework for Analysing Limitations to CO, Uptake ............. B . Stomata1 Responses ........................................................... C . Changes in the Mesophyll .................................................. D . Acclimation of NO; Uptake ..............................................

235 235 238 240 243

IV .

Acclimation of Extension Growth .............................................. A . Framework for the Analysis of Extension Growth .................. B . Growth of Roots and Shoots when Water Supply is Restricted . C . Growth of Roots and Shoots when N Supply is Restricted .......

246 246 252 258

v.

Implications ........................................................................... A . Sink Strength ................................................................... B . Regulation of N Balance .................................................... C . Regulation of Water Use Efficiency .....................................

263 263 264 266

VI *

Information Transfer ............................................................... A . Responses t o Soil Drying ................................................... B . Responses t o N Limitation .................................................

267 267 273

VII

What is in the Xylem Sap and How Can Changes in Water and N Availability Change the Xylem Sap Contents? .............................. A . Collection of Xylem Sap ....................................................

275 275

Advances in Botanical Research Vol . 22 incorporating Advances in Plant Pathology

ISBN 0-12-005922-3

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

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A. J. S. McDONALD and W. J . DAVIES

Soil Drying and N Deprivation and the Effects on Xylem Contents ......................................................................... 276 C. Interaction and the Concept of Sensitivity Variation ............... 280 B.

VIII. Conclusions: An Integrated Stress Response System for the Plant? References .............................................................................

I.

..

286 289

INTRODUCTION

At the crop level, the framework of analysis provided by Montieth’s light interception model shows that when water or nutrient supply is limited, dry matter accumulation can be restricted by two broad classes of effects (Fig. 1) (Jarvis, 1985). These are direct effects of environmental perturbation on the processes that lead to the interception of solar radiation by plant parts and also possible effects on the efficiency with which intercepted light energy is converted into chemical energy. Although this model provides a very useful summary statement of how total biomass production may be influenced by restricted water or nitrogen (N) supply, it does not (and was not intended to) tell us anything about the underlying growth phenomena and physiology. For a more mechanistic appreciation of whole-plant response to perturbations in water and N supply, the extension growth of leaves and roots, in conjunction with the acclimation of specific uptake capacities for carbon (C) and N in leaves and roots, respectively, must be studied. Regulation at the level of single plant organs is, of course, only part of the response picture and it is important to note that effects of stress on root and leaf demography can assume progressively greater importance with prolonged drought and N deprivation. However, here we emphasize the more rapid responses of limitations to C and N uptake and the physiological mechanisms underlying the differential extension of roots and leaves at low water potentials and low N supply. First, we consider how water and N supplies can be manipulated around plant roots. These are important considerations, because the techniques employed can have a very great bearing on the results and conclusions reached. We then consider the acclimation of C and N uptake systems to deficits in water and N supply. We consider a framework for analysing extension growth and review the information on single leaf and root responses. We comment upon possible implications of response to whole plant functioning at limited availability of water and N. Finally, we review the literature on information transfer in response to water and N deficits and emphasize the importance of considering how the sensitivity of stomata1 and growth responses to chemical signals in the transpiration stream may be modulated by other variables.

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

23 1

/

c /

/

/

/

/

0

lo00 Photosynthetically active radiation intercepted (MJ m-2season")

Fig. 1. Dry matter production as a function of photosynthetically active radiation for C4 and C3 crops with adequate water, nutrients and temperature. Changes in leaf area decrease light interception and move production from A B. Impaired metabolism caused by stress decreases production at a given light interception (A --* D or B C). Stresses which decrease both area and efficiency decrease production via A + B C. From Jarvis (1985). -+

+

-+

11.

MANIPULATING WATER AND N SUPPLY

Our main consideration is the regulation of growth and uptake systems under conditions where the supply of water and N are growth-limiting. This can be termed supply-limitation and implies that uptake is insufficient to accommodate maximum growth in a given environment. Before we consider growth responses, it is appropriate to comment on the approaches used in manipulating water and N supply. In general, a useful approach to understanding the nature of any system, is t o perturb a relevant input variable and follow the time-series of response in output variables. Where soil drying and N deprivation are input variables, there are at least two aspects of perturbation that should be considered in designing any experiment, namely the magnitude of change and the rapidity with which it is effected. Because both of these aspects may affect conclusions with regard to growth response and regulatory

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mechanisms, it is important that the time-course of soil drying or N input should be very clearly documented. Much early literature shows that if plants are dehydrated rapidly, then stomata1 and other responses can be very different from those shown by plants that experience rather more gradual soil drying. In what follows, we cite many examples of what can result when the rate of soil drying is varied. One of the reasons for these differences can be the limited extent of solute regulation that can occur when rapid dehydration occurs. We argue later that plant growth regulators can mediate the responses of many plants to drought but Hartung and Slovik (1991) pointed out that synthesis of extra abscisic acid (ABA) is a relatively slow process and that rapid stress responses must involve redistribution of the hormone. Both of the above points must be borne in mind when, for example, investigating the molecular biology of drought stress. Here, detached shoot tissue is sometimes rapidly dehydrated on the laboratory bench, which is just about the most unrealistic droughting treatment imaginable. One common technique for studying the effects of limited water supply on plant growth has been to manipulate the water potential of plants by growing them in hydroponics and adding osmotica of different strengths. This technique has been used successfully on many occasions and has the merit that a relatively consistent degree of drought stress can be imposed. When soil is allowed to dry, the availability of water is restricted more severely as more and more water is lost and the analysis of the response can be complicated. One problem with osmotic treatments to simulate soil drying is that plant growth regulators generated in the roots may be leached from the roots, significantly altering the response of the whole plant to the drought stress treatment. There may also be other effects of solution culture on roots, one of which can be the structure of roots themselves. The production of root hairs is often inhibited in plants grown in solution culture. We will see later that low water potential treatments can have very different effects on cell wall properties and on turgor maintenance of maize primary roots, depending on whether they are imposed in vermiculite or in solution culture. One clever manipulation to overcome problems that may arise as a result of hydroponic treatments is to grow plants in soil inside large-diameter dialysis tubing. The tubes are sealed at the bottom and placed in large containers with osmotic solutions of the desired strength. In this way, plant roots are in contact with soil but the water potential around the roots is kept relatively constant. One important consideration in drought stress experiments is that, as a soil dries, both water and N supply may be restricted (Turner, 1986). If we are to distinguish between the effects of these two variables, we may have to use bathing solutions of different osmotic strength, rather than withholding water from a solid substrate. The literature contains a great many descriptions of techniques devised to restrict water supply to whole root systems or to parts of the root. Most recently it has become clear that to understand how plants respond to soil

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233

drying it is necessary to break the link between the soil drying process and the supply of water to the shoots. Davies and coworkers (Blackman and Davies, 1985; Neales e t a / . , 1989) used a split root system to try to ensure an unrestricted supply of water to the shoots of plants with some roots in contact with dry soil. These manipulations suggest that plants are sensitive to soil drying around the roots, even when water supply is not limited. Passioura and Munns (1984) describe a whole-plant pressure chamber that can be used to keep plants effectively fully turgid even when roots are in contact with very dry soil (Fig. 2). As the soil dries, pressure on the root soil system is increased so that the shoot water relations of unwatered plants are comparable to those of plants that have been watered regularly. Gollan eta/. (1986) showed that where this was achieved, stomata1 conductance declined, and both pressurized and non-pressurized plants showed essentially the same relationship between conductance and soil moisture status, indicating that much of the plant’s response to soil drying was not necessarily related to restricted water supply. The nature of the solid medium around the roots can also have an important effect on the response of plants in a soil-drying experiment. As soil dries, mechanical impedance of the soil (soil strength) can increase significantly and it is well known that this change will limit plant growth (Passioura, 1988). The morphology of the plant can also be changed. For example, roots growing in soil with a high mechanical impedance often show increases in thickness (see Scott Russell, 1977). Part of the response of roots to soil drying can involve a response to increased soil strength. We shall see later that roots grown in vermiculite (where soil strength is negligible) show restricted radial growth at low water potentials. Such techniques may be necessary to disentangle the separate effects of increased soil strength and restricted water supply as the soil dries. Passioura (1988) also used the whole-plant pressure chamber to address this question. In a great many studies of N uptake where supply limitation has been assumed, it would appear that insufficient thought has been given to the choice of N variable. Two emphases in manipulating N supply can be identified. In the vast majority of cases, the concentration of N in solution about the root has been varied. However, it has been argued that, where this is practised, interpretation of the growth response can be difficult (cf. Ingestad, 1982; Ingestad and Lund, 1986; Ingestad and h r e n , 1992). This is because plant growth can be maximized at very low concentrations of N in the bathing solution (cf. Olsen, 1950). Therefore, assuming that the external concentration is actually maintained, no effect of concentration on plant growth is to be expected over the wide range of N concentrations often investigated. In practice, treatment differences are often found but we assume these may be attributed to a lack of control over the N concentration in solution. If N concentrations are not closely monitored and maintained, then, as the plants become larger, N deficiency occurs. Typically, this will first be observed in the low-N treatments. With this approach, the plant will experience a whole range of N availability

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A. J . S. McDONALD and W. J. DAVIES

\

light

1

p .C r

U

1

I

Fig. 2. A Passioura-type pressure chamber to control the hydrostatic pressure of the xylem sap to atmospheric pressure. Plants are grown in soil in pots that can be enclosed in a pressure chamber, and xylem water potential is increased by applying pneumatic pressure. The pressure in the chamber is controlled by an electronic device that includes a light sensor, which is connected to the xylem of the plant via a waterfilled tube. From Gollan et al. (1986).

from excess consumption to extreme deprivation as it grows larger. It becomes extremely difficult to relate the growth response at any one time to a defined level of N deprivation. More recently, the flux of N to the root has been varied to control the growth of the plant (e.g. Ingestad and Lund, 1979). With this approach, the amount of N added to solution over a given period of time is manipulated. The N-flux approach can result in precise control of plant growth and is therefore most suitable for studies of growth regulation with respect t o supply limitation. Most studies with this approach have involved the addition of

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

235

variable, exponentially increasing amounts of N to plants grown in solution. This results in a range of plant relative growth rate (RGR) and plant N concentration which is maintained throughout the exponential phase of growth, and the plants are said to be at steady-state nutrition. We believe that the potential of this approach in preparing plant material for physiological studies in a growth context has yet to be fully realized. We are not saying that external concentration per se is unimportant to N uptake. However, for the most part, the range of external N concentration investigated has been much too high for supply limitation to take place. Recently, Macduff etal. (1993) varied the flux of nitrate such that a range of steady-state nutrition was achieved. These workers found that, for each value of flux and its associated plant growth, there was a unique, equilibrium value of external nitrate concentration. The crucial piece of information is that, in a separate experiment, when this range of equilibrium values of nitrate concentration was chosen as the experimental variable, plant growth and the values of all physiological and morphological variables investigated were identical to those where nitrate flux had been manipulated. In fact, in maintaining different external concentrations over the range investigated, Macduff et al. (1993) would necessarily have manipulated nitrate flux in the growth-limiting range. The point is that these were extremely low nitrate concentrations much lower than have generally been used in studies where supply-limitation has often been assumed.

111.

ACCLIMATION OF C AND N UPTAKE

A. FRAMEWORK FOR ANALYSING LIMITATIONS TO C02 UPTAKE

It has been standard practice to assess the stomatal and non-stomata1 contributions to the overall control of photosynthesis and consider these as quite separate influences. There is, however, considerable evidence that shows that there are significant interactions between the two levels of control. Historically, stomatal and non-stornatal limitations to photosynthesis have been assessed using electrical analogues to describe the resistances to carbon dioxide (CO,) uptake provided by the gas phase and the liquid phase of the transport pathway between the ambient air and the sites of fixation, Such calculations have several uncertainties (cf. Jones, 1992). A useful graphical device for investigating the relative contribution of gas and liquid phase resistances was first introduced by Jones (1973) and further developed by Farquhar and Sharkey (1982) and is illustrated in Fig. 3. By varying ambient CO, concentration and plotting the photosynthesis rate obtained against the calculated intercellular C 0 2 concentration (ci), a demand function for photosynthesis is obtained. As the photosynthetic rate increases, ci will fall below c, (the atmospheric CO, concentration) and the extent to which this occurs will

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A. J. S. McDONALD and W. J. DAVIES

t

A

cc

r

~ , - r

--

-

c, c, +-

Normal atmospheric concentration (c,)

cO, concentration (c,)

Fig. 3. (a) Response curve or demand function relation A (assimilation) to ci (intercellular CO, concentration). (b) Two supply functions relating A to ci for different gas phase resistances with the slopes of the lines equal to leaf conductance. (c) Calculation of photosynthetic limitation (see text for details). The response curve represents the demand function for a hypothetical leaf. The CO, concentration drop and that across the gas phase by across the mesophyll is represented by (ci (c, - ci). Farquhar and Sharkey’s definition of the gas phase limitation is given by a/ ( a b). The recommended definition is ( rg/ r g + r* ) . From Jones (1992).

r)

+

depend upon the resistance provided by the gas phase (essentially the stomata). High resistances can result in substantial limitation in ci. Figure 3 shows the relationship between the photosynthetic rate and ci for two different values of gas phase resistance; the straight lines represent the photosynthetic supply functions. The actual values of ci and the photosynthetic rate under any particular set of conditions is obtained from the intersection of the supply and demand functions (Fig. 3).

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

237

Farquhar and Sharkey (1982) suggested that the analysis described above can be used to calculate the gas-phase limitation to photosynthesis (I,). From the relationship shown in Fig. 3 we can see that a comparison of the photosynthetic rate at the normal operating point and the photosynthetic rate at a ci equal to c, (assumed to be an infinite gas phase conductance) can be used t o calculate lg ( a / ( a b ) in Fig. 3). Jones (1992) suggested that it would be more realistic to define the gas phase limitation as the relative sensitivity of photosynthesis to a small change in the gas phase resistance (I, = rg/(rg I-*), where r* is the slope dci/dP at the operating point (see Fig. 3). A critical assumption in analysis of this kind is that the stomata are equally open over the whole area of the leaf whose gas exchange is being measured. If this assumption does not hold, as for example where the stomata close in patches, the photosynthetic rate, gas phase resistance and the calculated value of ci only represent average values. This can give very misleading indications of the photosynthetic response to the environment. The relationship between photosynthetic rate and ci is non-linear, with changes in ci having little effect on photosynthesis when stomata are nearly fully open (Fig. 3). We can compare what happens if (i) a healthy leaf closes all its stomata by 50%, or (ii) if half the stomata in discrete patches close completely. In the first case, stomatal conductance (g) falls to g/2, assimilation falls according to the response curve (>A/2) and the calculated ci will decrease significantly, implying increased stomatal control of photosynthesis. In the second case, conductance will fall to g/2 but photosynthesis will now decrease to A/2, as only half the area is now photosynthesizing. Because conductance and photosynthetic rate have changed to a similar extent ci is apparently unchanged. Calculations of this kind made on an area basis where homogeneity of stomatal response is assumed can lead to the conclusion that photosynthetic capacity is directly reduced by the treatment (as shown by the apparent shift in the photosynthetic demand function in Fig. 3), even under circumstances where there may be no real shift in mesophyll properties. There is much analysis of this kind in the literature (e.g. Lange, 1988) but in the absence of a demonstration of a homogeneous stomatal response, interpretation of the results must be open to question. As early as 1983, Laisk proposed that inhomogeneities in stomatal response could cause problems for the analysis of gas exchange responses and since then he and others (e.g. Downton etal., 1988; Terashima etal., 1988) have shown a patchy stomatal response to a number of stomatal closing agents, particularly the application of the plant hormone ABA. Others have reported a patchy photosynthetic response which may be due to a collapse of parts of the mesophyll due to loss of turgor, with a resulting reduction in lateral C 0 2 diffusion capacity (Daley etal., 1989). Wise et al. (1992) note that patchy photosynthetic behaviour in severely stressed leaves cannot always be reversed by high C 0 2 concentrations, which would be the case if it was attributable to a patchy response of the stomata.

+

+

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A. J. S. McDONALD and W. J. DAVIES

More recent reports have shown that patchy photosynthetic responses do not always occur (Gunasekera and Berkowitz, 1992; Ort etal. 1994) but more detailed analysis of patchiness is probably required before we can unequivocally quantify the effect of soil drying or N deprivation on rnesophyll properties. It may be that patchy responses occur mainly when perturbation in the input variable is rapid. This is often the case, for example, when dehydration is rapid in experiments with drying soils and potted plants (Comic, 1994). B. STOMATAL RESPONSES

1. Soil drying Stomata are sensitive to changes in the availability of soil water but the mechanism of this response is currently the subject of some debate (see e.g. Davies and Zhang, 1991). Stomata1 movements depend on changes in turgor pressure inside the guard cells and in adjacent epidermal cells, and anything that causes an increase in the relative turgor of the guard cells will promote opening of the stomatal pores. If soil drying restricts the supply of water to the leaves, stomatal apertures are generally reduced. This observation tells us that stomatal responses to soil drying result from active, energy-requiring processes, rather than simple hydraulic responses. This must be the case because epidermal cells have a mechanical advantage over guard cells, such that equal increases in pressure in both, which might result from increased water availability, will cause some stomatal closure. Most stomatal movements, including those in response to soil drying, involve active changes in the osmotic status of the guard cells. A range of opening stimuli will increase the potassium content of guard cells, with uptake from surrounding cells being driven by ATP-powered primary proton extrusion at the guard cell plasmalemma. The proton gradient between the outside and the inside of the guard cell will drive cation entry. This may be accompanied by chloride uptake and/or by stimulation of malate synthesis from storage carbohydrate in the cytoplasm. There are many published relationships between stomatal conductance and leaf water relationships. If soil drying occurs relatively slowIy, closure of stomata will occur over a wide range of leaf water potentials. Rapid drying can result in a threshold response. Relationships can also be modified by previous exposure of plants to stress and hardening can greatly decrease the sensitivity of guard cells to decreasing leaf water potential. Although it is clear that stomata can respond to local changes in leaf water status, there is accumulating evidence that stomatal response to soil drying may often be controlled by additional factors. Turner et al. (1985) describe some experiments with sunflower and Nerium, where leaf water relations are manipulated by varying leaf-air vapour pressure difference. The relationship between stomatal conductance and leaf water potential depended on the leaf air vapour pressure

239

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

-2.0

-1.5

-1.0

-0.5

Leaf water potential (MPa)

0 0

20

40

60

80

Extractable soil water

100 (%)

Fig. 4. Relationship between leaf conductance and (left) leaf water potential and (right) extractable soil water in a single leaf in a temperature- and humidity-controlled gas exchange cuvette at A wi= 10 Pa kPa-', while the remainder of the plant in a growth cabinet was at A w , = 10Pa kPa-' (low transpiration, 0 ) or at Aw, = 30 Pa kPa-' (high transpiration, 0).A w, leaf to air vapour pressure difference. From Turner e t a f . (1985).

deficit (Fig. 4). There was a single relationship, however, when all of these data were plotted as a function of soil water status, suggesting that the stomata were actually responding to some function of soil water availability. Some data of Jones (1985) collected with Bramley apple trees growing in the field, suggested that leaf water status can often be controlled by stomatal behaviour rather than the converse. Here, stomatal closure in unwatered trees resulted in an increase in shoot water potential. The fact that stomata did not respond to this rehydration suggests that some interaction between roots and drying soil generated a non-hydraulic influence that kept the stomata closed. This possibility is supported by the manipulations of soil drying and water supply that are described in section 11.

2. N deprivation Stomata can also be sensitive to changes in N supply. Where N is withheld from roots of plants grown in solution, stomatal conductances are often reduced compared with values shown by plants supplied with more N (e.g. Chapin et al., 1988a,b). In other instances, stomatal response to N deprivation seems to be lacking. For example Palmer eta/. (1996) grew Helianthus plants according to the N-flux approach described above. When the N supply to these plants was abruptly reduced from 25% per day to 12% per day, no stomatal reaction was detected. Where it does occur, partial stomatal closure following

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N deprivation is not necessarily associated with a change in leaf water potential (Chapin et al., 1988a,b). Other studies have shown that decreases in leaf water potential can occur following N deprivation (Radin and Boyer, 1982). There is little information on how the rate of decrease in N supply or how previous exposure to fluctuation in N affect the reponse. However, there is evidence that the extent of the decrease in leaf water potential and stomatal conductance following N deprivation can be influenced by root temperature (Radin, 1990). C. CHANGES IN THE MESOPHYLL

1. Soil drying

In the estimation of photosynthetic limitation, problems of stomatal heterogeneity can be overcome with the use of several non-destructive techniques developed in the last few years. For example, the leaf disc oxygen electrode can be used to assess photosynthetic capacity even when the stomata are completely closed (see e.g. Kaiser, 1987). Figure 5 from Cornic (1994) shows variation in photosynthetic capacity, measured in an oxygen electrode, as a function of leaf water deficit. Leaf material was dehydrated either rapidly by excising leaves or slowly by withholding water from the plants over a period of 3 weeks. Measurements were made at CO, concentrations up to 17%, since Kaiser (1987) has shown that even at 5 % CO,, diffusion resistance of some plants is not completely overcome. For all of the plants measured, it was not until the leaf water deficit reached around 30%, that the leaf biochemistry began to be affected. Net photosynthetic rate is negligible at this water deficit and a comparison of the data suggests that stomatal limitation explains most of the observed decrease in photosynthesis in this range of water deficit. Rather surprisingly, the sensitivity of photosynthetic mechanisms is very similar across a wide range of plant material. We are also forced to conclude that ABA has little significant effect on the biochemistry of photosynthesis, since dehydration to this extent would be expected to result in massive accumulation of this plant hormone. Several reports seem to support this view (e.g. Downton etal., 1988). Maximum apparent quantum yield of photosynthesis measured at high CO, on three different plants did not vary much over a 40% range of leaf water deficit, suggesting that whole chain electron transport and related processes are also very resistant to leaf water deficit (Cornic etal., 1992). There are suggestions that the high CO, concentrations necessary for these measurements can themselves inhibit mesophyll activity but Cornic (1 994) argues that this is not a significant effect, at least in C3 plants. Several workers have now shown that maximum photosystem I1 (PSII) photochemical efficiency, estimated as the ratio of variable chlorophyll fluorescence to maximum fluorescence, is not significantly affected in the range of water deficit up to 30-40% (e.g. Bjorkman and Powles, 1984; Ben etal.,

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

1

24 1

\ 2

0

20 40 60 w ) 1 0 0 Water deficit (%)

Fig. 5 . (a) Relationship between photosynthetic capacity (C02 evolution, expressed 070 of maximum value) and leaf water deficit (LWD) for six different plant species: 1, Elatostema repens (understorey plant of the rain forest); 2,3 and 6, French bean, spinach and sunflower (cultivated mesophytes); 4, Arbutus unedo (xerophyte); 5 , Impatiens valeriana (hygrophyte). The relationship between leaf net C 0 2 uptake at 340ppm C 0 2 (9’0 maximum value, V) and LWD is also shown in the case of the French bean. (b) Relationship between apparent quantum yield of O2 evolution measured at high C 0 2 concentration and LWD. The numbers refer to the same plant species as specified in (a). From Cornic (1994). as

1987). The measurement of chlorophyll fluorescence from intact leaves using non-actinic modulated light means that, with an appropriate model, we can conveniently investigate photosynthetic whole chain electron transport and the regulation of light utilization by PSII (Weis and Berry, 1987; Genty etal., 1989). Again the conclusions from this work are that the regulation of PSII activity is the same whether or not the leaf is modified by changing leaf water status. These conclusions conflict with early work, largely involving isolation of organelles from wilted leaves, which seemed to suggest that photosynthetic mechanisms of higher plants were very sensitive to desiccation. We can now

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suggest that these responses described in early reports were either artefacts due to isolation procedures or the result of photoinhibition. Although the data described above do seem t o suggest that the photosynthetic metabolism of most plants is relatively robust as long as the water deficit is not too severe, there are data in the literature that suggest a down-regulation of photosynthetic processes as water potentials fall (see review by Ort eta/., 1994). One intriguing observation is that measured COz concentrations within leaves can increase as water deficit increases (Lauer and Boyer, 1992). It may be that this observation is consistent with the observations of Cornic, Genty and coworkers, if water deficit increases the resistance to diffusion of COz into the chloroplast more than the diffusion of COz into the leaf. Under these circumstances, ci could increase even under circumstances where the fixation of C 0 2 is not limited by photosynthetic biochemistry. One implication of the relative insensitivity of the photosynthetic apparatus to partial dehydration of the leaf is that the C 0 2 concentration in the chloroplasts of an illuminated, dehydrated leaf will be very low. This means that there will be light energy that cannot be utilized through CO, reduction. If photoinhibition is not to occur, this energy must be dissipated in other ways. Possibilities include leaf movements, non-radiative dissipation of energy and enhanced photosynthetic reduction of oxygen (see e.g. Cornic, 1994). If leaf water deficit is severe enough, the photosynthetic apparatus will eventually be damaged such that after rehydration photosynthetic activity will not be fully resumed. The nature of this damage is not well understood. 2. N deprivation Leaf N accounts for a large portion of the total-plant N, particularly in early vegetative growth. The majority of this N is in the enzymes of the photosynthetic carbon reduction (PCR) cycle and in the thylakoid proteins (Evans and Seeman, 1989). About haIf of this protein is involved in the light reactions of photosynthesis, including thylakoid membrane-bound proteins associated with light harvesting, electron transport and photophosphorylation. The other half is in soluble proteins involved in the dark reaction of the PCR cycle. These latter proteins include those involved in COz assimilation, photorespiration, ribulose bisphosphate (RuBP) regeneration, as well as sucrose and starch synthesis (Evans and Seeman, 1989). Where N supply and retranslocation are insufficient to support the growth rate of the plant, leaf N amounts decrease below values that are associated with more optimal supplies of N. In the short term, this may affect both the carboxylation efficiency and the C0,-saturated value of photosynthesis. With decreasing leaf N (per unit leaf area), the proportion of the total leaf N in the thylakoids remains about the same, the proportion in soluble proteins (such as Rubisco) decreases and the proportion in other nitrogenous compounds tends to increase (Evans, 1989). N-deficient plants may therefore tend to be more limited by Rubisco (decreased slope of an A/ci curve, Fig. 3) than by RuBP regeneration (i.e. the plateau). However, the Rubisco activity and

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

243

the electron transport capacity seem generally to be closely coupled (Evans, 1989). Rubisco and RuBP regeneration therefore often continue to co-limit photosynthesis even under N deprivation and a linear dependence of several photosynthetic variables upon leaf-N concentration has been reported (Fig. 6; Sage et al., 1990; Harley et al., 1992). Lawlor et al. (1989) reported similar proportions of chloroplast components (less than 20% variation) over a wide range (10-fold) of leaf N content in wheat. There are reports that indicate that longer-term acclimation to supplylimitation in N can be associated with a constancy in photosynthetic rates (Waring et al., 1985; McDonald et al., 1986b, 1992; Pettersson et al., 1993) and in the variables obtained from A/ci analysis (Pettersson and McDonald, 1994). This would indicate that any down-regulation or inhibition of photosynthesis in response t o variability in N supply can be of a transient nature. It was only at more extreme N deprivation that A/ci analysis revealed changes in photosynthetic characteristics (cf. Fig. 7). In addition to assimilation of COz, electrons are used to reduce nitrate and sulphate, resulting in the formation of amino acids (Oakes, 1986; cf. Lawlor, 1994). Nitrate (NO;) is reduced in the cytosol by nitrate reductase and, in the chloroplast stroma, nitrite is further reduced by nitrite reductase to ammonia. The reduction of CO, and NO; d o not occur in strict stoichiometry. Under conditions of N deprivation (a larger ratio of CO, to NO; supply), larger fractions of the products of the light reactions and electron transport are consumed in CO, assimilation, and more carbohydrates tend to accumulate than at higher availability of NO;. Where nitrogen supply is less limiting to growth (a smaller ratio of COz to NO; supply), amino acids tend to accumulate.

D. ACCLIMATION OF NO;

UPTAKE

There are many reports that V,,, for NO; uptake in roots, which have been pregrown with a plentiful supply of NO;, increases when NO; is withheld (Clarkson, 1986; Jackson et al., 1986). This is apparent from significantly higher uptake rates in these roots when subsequently exposed to higher concentrations of nitrate, than in roots that have been maintained at the higher concentration for longer periods of time. Evidence that this can relate to the N demand of the whole plant is provided by results from split-root experiments where it has been reported that NO; uptake by roots fed with full nutrient solution may be stimulated by NO; starvation of other parts of the root system (cf. Touraine et al., 1994). Where NO; is freely available at the root surface and potentially nonlimiting to growth, uptake of NO; is considered to be demand-limited and it is apparent that there is a feedback of NO; usage in plant growth upon subsequent rates of NO; uptake (cf. Ismande and Touraine, 1994; Touraine et al., 1994). The evidence indicates that signals of N-demand probably d o not

244

A. J. S. McDONALD and W. J. DAVIES 150

V,

-

(35)= -9.6 + 60.0 N (P = 0.90)

125 100 75

50 25

/4

0

V,

-

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Fig. 6. The relationship between key model parameters describing the A/ci response and total leaf N in cotton grown in ambient and elevated COz. Estimates of V,,,, (maximum rate of carboxylation), J,,, (maximum rate of electron transport) and TPU (triose phosphate utilization), obtained by non-linear least squares regression, plotted as a function of leaf N. Estimates are obtained from recently fully-expanded and from leaves up to 18 d after full expansion (0).For V,,,,. indepenleaves (0) dent linear regressions were obtained for leaves grown in ambient and elevated COz . Filled symbols denote elevated C 0 2 grown plants. Regressions of J,,, and TPU data on leaf N content are based on combined ambient and elevated COz grown plants. From Harley etal. (1992).

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7

arise in (or are not confined to) roots themselves but are emitted in the shoot and transported to the root (Edwards and Barber, 1976; Simpson et al., 1982; Burns, 1991). Larsson (1994) recently reviewed studies of N deprivation at supply limitation where NO; flux has been varied. Two types of response of specificuptake capacity (V,,,,,) to nitrate supply have been reported. Oscarson et al. (1989) found proportionately higher values of V,, at higher supply rates of NO;. In other studies, V,, was found to increase with increasing supply over the lower range of supply rates (phase I) but was found to decrease at higher rates of supply (phase 11) (Fig. 8; Mattsson et al., 1991). An apparent discrepancy exists between the acclimated response of V,,, in the phase I studies reported by Larsson and the findings of other workers. The findings of Larsson and his colleagues show that V,,, can be lower in NO;-deficient roots. Presumably the discrepancy between the dependency of V,,, on NO; supply in phase I and that more generally reported in depletion studies, may be accounted for by differences in the acclimation period and the sustained nature of N deprivation at extremely low fluxes and concentrations of NO; in the studies of Larsson and his colleagues.

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A . J. S. McDONALD and W. J . DAVIES

>I

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Fig. 8. Relative growth rate (RGR) and V,,, for net nitrate uptake, in vegetative barley maintained at different relative addition rates of nitrate (RA). Data from Mattsson etuf. (1991). Phases of uptake responses (see text) indicated by Roman numerals. From Larsson (1994).

IV. ACCLIMATION OF EXTENSION GROWTH A.

FRAMEWORK FOR THE ANALYSIS OF EXTENSION GROWTH

An understanding of plant response to environmental perturbations, assumes the measurement of relevant growth and physiology. However, recent advances indicate that accepted dogma on growth phenomena and traditional methods of measurement may be inadequate. In this section we provide support for this statement and advocate the use of a more mechanistic model of growth and more focused methods of measurement. During the past 30 years, it has been common practice to consider the expansive growth of plant organs in terms of a model originally formulated to describe the expansion of single cells (Lockhart, 1965). A driving force for growth, the cell turgor, is assumed to stretch the cell wall irreversibly at a rate determined by its yielding properties. In this model, the relative growth rate of the cell (r) is related to turgor ( P ) by two yielding properties of cell walls, the extensibility (m)and the yield threshold (Y). We can write the equation r = m(P - Y), where the amount of turgor that drives growth is the difference between P and Y . Lockhart's model tells us nothing about the nature of wall yielding or how wall properties can change in response to a change in the environment. For many years, Y and m were viewed as parameters that

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

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were properties of cell-wall structure and which only varied over an extended time-scale. More recently, however, it has become clear that wall-yielding properties can be highly variable over very short time-scales (e.g. Frensch and Hsiao, 1994) and that this variation can act to maintain a relatively constant elongation rate. While there have been many reservations over the use of this equation to describe the growth of whole organs and even over its validity as a model for single cells (Ray etal. 1972; Zhu and Boyer, 1992), it continues to be a commonly used framework for analysing growth (Cosgrove, 1993). Is it really relevant? One of the main criticisms of Lockhart’s model has been the lack of evidence in support of turgor as a regulatory variable. In later sections, we discuss with respect to water and nitrogen supply, instances where differences in growth rate within the extension zone of plant organs were apparently associated with constancy in turgor. In these cases, it may be assumed that metabolism associated with cell-wall growth was regulating the rate of expansion in single cells. In some cases, turgor has been manipulated without any apparent effect on growth. For example, from their study of irreversible extension as a function of manipulated turgor in single giant algal cells of Chara coralha, Zhu and Boyer (1992) concluded that the crucial requirement with regard to turgor was that it should exceed the yield threshold value. Above this value (and assuming turgor did not attain damagingly high values) these authors found no regulatory role for turgor in cell extension. Their data indicate that the turgor-driven component of cell expansion ( P- Y ) in the Lockhart equation may be conceptually inadequate and that cell expansion may respond to a turgor switch: either ON when P - Y > 0, or OFF when P - Y I0. The regulatory mechanisms would then relate to growth processes in the cell wall. We consider this to be a definitive observation where turgor did not regulate growth. However, its relevance to cell growth in the tissues of higher plants has yet to be ascertained. For many years, it was only possible t o estimate turgor in tissues (not single cells) of higher plants by measuring water potential and osmotic potential and calculating the difference. This was commonly done on large plant parts using the pressure chamber and an osmometer. Water relations of small samples of plant tissue can be measured using the psychrometer but this is problematical with growing tissue because cells continue to grow in the psychrometer and, with a restricted water supply, cell turgors decrease with time to the yield threshold (Cosgrove, 1985). This can be avoided by using particular precautions (Michelena and Boyer, 1982) and other techniques (Matsuda and Riazi, 1981). Interpretation of earlier results from studies with dicot. leaves, where turgor has been varied by manipulating the transpiration rate and calculated from measurements on tissue samples, indicates that growth rate in cells of higher plant organs may be dependent upon P - Y . For example, Taylor and Davies (1986) made stepwise decreases in calculated turgor and measured corresponding decreases in the expansion of whole dicot. leaves (Fig. 9). However, interpretation of such data is problematical for two reasons. First, extension is measured for the whole organ and, second, turgor is calculated on tissue

248

A. J. S. McDONALD and W.J . DAVIES

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Fig. 9. Relationship between growth and turgor for illuminated (0)and darkened ( 0 ) leaves of Betulu pendula. Regression lines: illuminated, Y = 0.46~-0.031, r = 0.726; darkened Y = 0.13~-0.009.r = 0.750. From Taylor and Davies (1986). samples without taking into account any spatial variation in developmental stage of different cell populations. One intepretation of the Taylor and Davies data is that cell expansion was proportional to P - Y . This would be consistent with a turgor-driven plastic deformation of the cell wall, relating to the mechanical properties of the wall as measured, for example, by an Instron apparatus (Van Volkenburgh et al., 1983). The mechanical properties presumably cannot persist indefinitely in the absence of further metabolic loosening and incorporation of new wall material. Moreover, the extent to which irreversible deformation can occur without damaging wall structure and reducing its capacity for further growth is uncertain. Zhu and Boyer (1992) found evidence of damage to the extension process after turgor had been artificially increased to a higher value. For the several-hour period in which they investigated the response, these authors found no recovery of extension growth and it may well be that the wall was irreversibly damaged. It is uncertain whether a longer delay would have shown an eventual recovery to the metabolically driven rate. It has been common practice to measure the plastic extensibility of tissues and discuss the findings in terms of m in the Lockhart equation (cf. Pritchard and Tomos, 1993; Taylor etal., 1993, 1994). However, apart from the problems of measurement (Cosgrove, 1993) and the possible irrelevance of the forces applied (potentially excessive) to those normally associated with living cell walls, the approach can be criticized on other grounds. For example, increase in cell-wall size is dependent upon loosening and synthetic processes

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

249

of which wall plasticity is a result. The crucial point here is that the rates of these processes may change, affecting the rate at which the wall grows, without necessarily affecting the plasticity of the end product. It is even conceivable that, where P - Y I0, wall growth processes may continue, without the cell expanding. It is to be expected that Y might decrease (cf. Frensch and Hsiao, 1994), but, until this results in a resumption of cell expansion, it is possible that wall plasticity might increase. We are proposing a possible negative feedback of cell expansion on rn which might therefore be inversely related to current expansion. On recovery of P - Y > 0, we might expect an expansive spurt (a type of stored growth) until the metabolically limited rate is resumed. We do not dispute that plastic extensibility is less in older, more slowly growing tissues (e.g. Taylor et al., 1993) but we doubt the relevance of this property to the regulation of cell expansion. Moreover, reliable measurements of turgor in conjunction with relevant growth data are sufficient to assess the likely importance of wall phenomena to the regulation of growth. We have argued that plasticity measurements on cell walls are potentially incorrect and misleading. They are also probably unnecessary since biochemical studies have now provided us with fundamental models of wallloosening phenomena (see below). For all of these reasons, we now find it hard to motivate the measurement of plasticity in studies of the regulation of plant growth. The observed dependence of leaf extension upon manipulated turgor in Fig. 9 may not necessarily have been attributable to turgor-driven plastic deformation. If there were populations of cells within the leaf at different developmental stages, with predictably higher yield thresholds (Y) in older cells than in younger cells, an alternative explanation may be proposed. With decreasing turgor, the pressure in an increasingly large number of cells would fall below the yield threshold value. If a turgor switch was operating (similar to that apparent in the Zhu and Boyer study) there would have been zero growth where P - Y 5 0. Cell expansion would occur at a rate limited by cellwall metabolism where P - Y > 0 without any further regulatory significance being necessarily attached to turgor. If this latter interpretation is valid, then a decrease in leaf growth, such as that in Fig. 9 caused by increasing transpiration rate, would be attributable to a decrease in the number of cells rnomentarily contributing to leaf expansion. We can postulate that the expansion of an organ might be manipulated by varying the pressure in its constituent cells even though turgor pressure may have no regulatory role beyond a discrete ON/OFF switching. This argument illustrates the possible inadequacy of discussing growth regulation at the cell level from measurements of extension growth on whole organs and from turgor pressure calculated from tissue samples (cf. Spollen etal., 1993; section IVB). Such a n approach does not provide information for estimating parameters and variables in models of regulation at the single-cell level. We have discussed two possible ways in which a step-decrease in humidity

250

A. J . S . McDONALD and W. J . DAVIES

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Time (h) Fig. 10. Time-series of dependence of leaf expansion rate upon humidity. Note that the rate (slope) is less at lower humidity. A direct measure of turgor would confirm that this is because of a reduction in cell turgor. Wall loosening has been unaffected by the humidity (turgor) change and leaf size at the end of the run is the same as would have been attained had the humidity been maintained at 68% throughout. Unpublished data of the authors for Betula pendula.

about a dicot. shoot might cause a temporary reduction in the extension growth of leaves. If such a reduction is followed by a step-increasein humidity to its original value, a recovery in extension growth is observed (Fig. 10). Initially, the rate of extension is in excess of the original value but, after a delay, the original rate of extension is resumed. Apparently, total extension over the period investigated is unaffected by perturbations in turgor. (Although not discussed by Zhu and Boyer, this phenomenon is also apparent from their data.) Irrespective of the manner in which turgor affects growth (ON/OFF switching or plastic deformation), it is apparent that the longer-term rate of extension growth in Fig. 10 was dependent upon something other than turgor. Presumably, this related to cell-wall metabolism. What then of Lockhart? In our view, the most convincing data currently available to help us understand the regulation of cell growth are those reported by Zhu and Boyer (1992) where turgor did not have a regulatory role beyond a switching function. We know of no data where cell turgor and growth measurements are of sufficiently high spatial resolution that we can argue for a turgor-dependent regulation of cell growth in organs of higher plants. Thus we find no reason to pursue the Lockhart equation as a framework for analysing growth response to environmental perturbations. However, it is possible

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

25 1

that a degree of turgor-dependent plastic deformation can occur in the short term but not predictably in the longer term. A possible scenario is thus one where the long-term metabolically controlled rate of irreversible expansion may be punctuated by irreversible deformations associated with short-term variation in turgor pressure. It remains one of the more immediate challenges in plant science to demonstrate whether or not turgor pressure has a regulatory role in the expansion of cells in higher plant organs. What are the alternatives to Lockhart? It has become clear that to make progress in understanding the factors regulating plant growth requires increased understanding of the biochemistry of the cell wall, coupled with the development of a model of yielding properties at the molecular level (Fry, 1989; Passioura and Fry, 1992; Passioura, 1994). The main postulates of Passioura’s latest model are (i) that there are two functionally disparate populations of hemicellulose molecules in the wall that tie cellulose microfibrils together: those that are taut and load-bearing and those that are slack and not load-bearing; and (ii) that there are enzymes that cleave or loosen the loadbearing molecules. Passioura suggests that as the wall stretches during growth, slack molecules become taut, are recruited to load bearing and thereby stiffen the wall. Stiffening can be undone by enzymes that cut or loosen load-bearing tethers. The rate of expansion of the cell wall can depend on the frequency with which load-bearing molecules are loosened, a function of the activity of wall-loosening enzymes which may be a highly dynamic variable, and presumably also a function of tension which can also unzip hydrogen bonds between molecules. Expansion rate will also depend on the number of loadbearing molecules that are recruited per unit increase in wall length. This latter property may depend on the composition of the wall (e.g. the ratio of cellulose to hemicellulose) and could be modulated on a time-scale pertaining to synthesis of new wall material. Our current understanding of the biochemical basis of wall stiffening and loosening during growth is described below. Data are now accumulating t o allow us to analyse the effects of different environmental variables on the growth of plant parts in terms of variation in the properties described above. In some cases we have information on specific wall biochemistries in conjunction with improved measurements of turgor in cells of the growing zone. A great deal of progress has been made in recent years with the development of the cell pressure microprobe (see e.g. Steudle, 1993). Using this instrument, it is possible to measure directly the turgors of individual growing cells and even to investigate the spatial distribution of turgor within the elongation zone of an organ. Indeed, in view of the advances being made in single cell measurements, it is unlikely that measurements at the tissue level comprising cells of different developmental stages can any longer be motivated in growth studies where the regulating model pertains to cell turgor and the wall phenomena of single cells. It remains a challenge to adapt the use of the pressure probe to a wider variety of plant cells and measurement environments than have so far been investigated.

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A. J. S. McDONALD and W. J. DAVIES

With the combined awareness of how wall chemistry may relate to loosening and yielding phenomena, and with an improved resolution of turgor measurement, it has become possible in recent years to make progress in understanding the nature of growth response to perturbations in variables such as water and nitrogen supply and make more definitive statements about regulatory mechanisms than was previously possible. These mechanisms are now discussed in the following sections. B. GROWTH OF ROOTS AND SHOOTS WHEN WATER SUPPLY IS RESTRICTED

When plants experience a reduction in water availability from the soil, shoot growth is often more inhibited than root growth (Sharp et al., 1988). The total growth rate of the plant might be expected to decrease but, in some cases, the absolute biomass of roots has been shown to increase relative to the biomass of well-watered controls (Sharp and Davies, 1979; Malik etal., 1979; Blum etal., 1983). Absolute increases in root growth probably only occur under rather specialized circumstances where carbohydrate supply would otherwise be limiting root growth. Where shoot growth is restricted but photosynthesis is able to continue, increased carbohydrate supply from the shoot to the root might be expected. It is generally accepted that this situation can arise during soil drying because the effects on stomatal behaviour and photosynthesis are not manifest until shoot growth is very significantly restricted (Hsiao, 1973). It is important to understand why shoot growth is generally more sensitive to drought than stomatal behaviour and also ask why root cells are able to continue to expand under circumstances where shoot-cell growth is restricted. There is no dispute that, under many circumstances, reduced water availability in the soil will result in reduced leaf turgor and a reduction of growth. However, it is difficult to know the extent to which the limitation in growth can be attributed to reduction in turgor. Indeed, from our previous discussion (section IVA), it is apparent that a causal dependence of growth on turgor is not necessarily to be expected. It is interesting to note that, in a range of grass species, restricted growth has been found in low water potential treatments, despite the complete maintenance of turgor in the growing cells as a result of osmotic adjustment (e.g. Michelena and Boyer, 1982; Nonami and Boyer, 1989). This result indicates that cell walls in the leaves become less yielding under reduced water availability. Results of Nonami and Boyer (1990a,b) support this view. Appreciation of this point has considerable significance for those interested in developing genotypes for drought-prone environments. It has been suggested that selection of plants showing effective osmotic adjustment in aerial plant parts can result in yield advantage because turgor maintenance can be equated with growth maintenance at low water potential. However, if growth at low water potentials is actually restricted by wall yielding, then we might expect to find solutes accumulating in shoots that have stopped growing or where growth rates have been slowed (see Munns, 1988).

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

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In fact, this is the case, and growth sensitivity to water deficit can commonly be related to the degree of osmotic adjustment in a genotype, with those genotypes showing the greatest solute accumulation being those in which growth is most significantly reduced by water deficit (Fig. 11; Kuang etal., 1990). This is not to say that selection for osmotic adjustment in aerial plant parts cannot result in yield advantage for other reasons, such as the postponement of cell death or the avoidance of desiccation damage to reproductive plant parts. The results described above indicate that, if we are t o understand how low water potential treatments restrict growth of above-ground plant parts, we must understand how the yielding properties of cell walls are regulated. The same is also true for roots. Here, several workers have shown that, following immersion of roots in solutions of low water potential, root elongation recovered more rapidly than root-tip turgor, indicating that the low water potential treatment actually increased the yielding properties of cell walls, maintaining growth at low water potential (Hsiao and Jing, 1987; Itoh etal., 1987; Kuzmanoff and Evans, 1981). It seems extremely important to understand why the wall properties of leaf and root cells respond so differently to a given treatment.

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A . J. S. McDONALD and W. J . DAVIES

A series of well-controlled studies has now confirmed early suggestions (Weaver, 1926) and shown that root growth is intrinsically less sensitive to low water potential than is the growth of the shoot (Westgate and Boyer, 1985; Spollen et af., 1993). In these experiments, small seedlings were grown in moist vermiculite under non-transpiring conditions so that water potentials of roots and shoots were in equilibrium with the water potential of the vermiculite. Under these conditions, growth and water relations of roots and shoots can be monitored with precision. In the maize primary root (the best-studied root system in investigations of the effects of low water potential on growth), longitudinal expansion rates are maintained preferentially towards the apex of the root, even when the water potential is reduced to - 1.6 MPa (Sharp et af., 1988). This results in a shorter elongation zone than at high water potential (Fig. 12). Pressure probe measurements show that cell turgor is essentially constant throughout the elongation zone, suggesting spatial variation in wall-yielding properties over only a few millimetres. Turgor at -1.6 MPa is 50% less than at -0.02 MPa, showing that, while osmotic adjustment was not complete, it was sufficient to maintain growth close to the apex (Fig. 12). Sharp et al. (1990) and Voetberg and Sharp (1991) have investigated osmotic adjustment in the growing regions of maize primary roots at low water potential, They found that concentrations of the amino acid proline were particularly high towards the apex of the root where elongation rates have been shown t o be completely maintained over a wide range of water potentials. Proline accumulation accounted for almost half of the osmotic adjustment in the apical millimetre of roots growing at -1.6 MPa, indicating that proline deposition plays an important role in the maintenance of P - Y > 0 and root growth at low water potential. Ober and Sharp (1994) have recently shown that application of the carotenoid synthesis inhibitor fluridone to root tips held at low water potential will decrease proline accumulation throughout the root. This suggests that ABA may play a regulatory role in maintaining the osmotic status of roots at low water potential. This is particularly interesting because Saab et af. (1990, 1992) have shown that ABA plays important roles in both the maintenance of root growth and the inhibition of shoot growth in maize seedlings at low water potential. Again, fluridone has been used to reduce the accumulation of endogenous ABA at low water potential and the effect of this treatment is to reduce the growth rate of roots and increase the growth rate of shoots at a water potential - 1.6 MPa. These authors have described spatial variation in growth and ABA distribution through the root and mesocotyl. In both cases, low water potential treatment reduces the length of the growing zone. In roots, fluridone treatment reduces this further. In the mesocotyl, fluridone treatment restores the growth rate almost to that of the well-watered control. Comparison of the ABA distribution and the ABA profile suggests a gradient in sensitivity of the growing cells to the ABA treatment. Young cells are relatively insensitive, compared to more mature cells. It should also be noted

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

255

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that Saab et al. (1990) report a relative insentivity to ABA of growth processes of plants maintained at high water potential. Later in the chapter, we discuss the importance of sensitivity variation in the hormonal response as a more general phenomenon. In section IVA, we discussed the necessity of making relevant measurements of growth and physiology in understanding growth regulation in plant organs. The work of Sharp and colleagues on the regulation of root growth at low

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A. J. S. McDONALD and W. J. DAVIES

water potential shows the importance of being able to assess spatial variation of cell properties in this kind of study. Spollen eta/. (1993) noted that one of the results of treating a growing zone as a homogeneous mass and plotting overall root elongation rate versus root-tip turgor can be the conclusion that elongation rate is reduced as a function of a reduction in turgor when in fact the reduced elongation rate of the whole root is simply a result of a reduction in the size of the growing zone. If we are to explain the maintenance of cell growth rate close to the tip of the maize primary root, even when the turgor is reduced by around 50%. it is necessary to argue that wall yielding is increased in this region. Passioura’s molecular model of cell-wall properties allows us to think of such changes in terms of an enzyme cleaving tethers between adjacent cellulose molecules. One enzyme that has recently received much attention in this context is xyloglucan endotransglycosylase (XET) (Fry et al., 1992). This enzyme has the property of both cutting and rejoining xyloglucan polymers which bind strongly to cellulose and are usually long enough to link adjacent microfibrils (Fry, 1989). Spollen et al. (1993) and Wu eta /. (1994) report on the spatial distribution of XET activity in the apical few millimetres of maize primary roots growing at two water potentials (Fig. 13). At low water potential, activity was significantly enhanced immediately behind the apex, where wall yielding is thought to increase, In contrast to this result. Pritchard and Tomos (1993) report that maize roots growing in mannitol at -1.OMPa showed no change in XET activity compared to roots growing at higher water potential. In their study, however, root-tip turgor was completely maintained and therefore this result may not be entirely inconsistent with the study discussed above. Wu etal. (1994) show that treatment of maize roots at low water potential with fluridone delayed the increase in XET activity at low water potential. This effect was largely overridden when internal ABA concentrations were restored by external application. The loss of activity associated with decreased ABA was associated with inhibition of the deposition of activity. This calculation allows the determination of the deposition rate profile that must occur to maintain the density of XET in the face of tissue expansion and displacement from the root apex. The calculation is necessary to show that the effects of ABA are due to variation in XET synthesis and/or activity and not to accompanying changes in root morphology. To demonstrate that the reported variation in XET activity is important for the maintenance of root growth at low water potential it will be necessary to alter XET activity in vivo. From other systems, there is some evidence against XET as a wall-loosening enzyme supporting wall extension (McQueen-Mason et al., 1993). Long-term extension activity of the isolated cucumber cell wall was not promoted by this enzyme but was dependent on a novel class of proteins named expansins (McQueen-Mason etal. , 1992). Wu et at. (1994) report that preliminary experiments have also shown that expansin-like activity is also increased in the maize root apical region by low water potential treatment.

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d 2

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6

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Fig. 13. Spatial distribution of xyloglucan endotransglycosylase (XET) activity in the apical 10 mm of maize primary roots at high \k, ( , 0; -0.03 MPa) or low \k, (A; - 1.6 MPa, 48 h after transplanting). High 'Ir, measurements were made either 20 h ( ; development control) or 48 h (0; temporal control) after transplanting. XET activities are expressed o n the basis of fresh weight (FW; a), total soluble protein (b), and cell wall dry weight (DW; c). Data are means f SE (n = 3 [high *,I, n = 4 [low +,I. From Wu etal. (1994).

25 8

A. J. S. McDONALD and W. J . DAVIES

Passioura (1994) has suggested that the effects of changing water status on expansion rate may be mediated by changes in the hydration of the cell wall. Shrinkage at low water potential may inactivate enzyme molecules by restricting their freedom of movement. Some changes in cell-wall components have also been reported in roots held at low water potential. Net synthesis of cellulose and other cell-wall polysaccharides may be inhibited in both shoots and roots (e.g. Zhong and Lauchli, 1988) as water potential falls but the amount of particular proteins in the cell wall can increase (Bozarth et al., 1987; Surowy and Boyer, 1991). In soybean hypocotyls, genes for both a 28- and a 31-kDa protein were expressed at high water potential, while the production of the 28-kDa protein was up-regulated by water deficit. In the roots, the mRNA for the 31-kDa protein accumulated as water potential fell. The cDNAs for these messages were homologous to those of vegetative storage proteins. The authors suggest that they may also have a dual function as growth proteins. Surowy and Boyer also report on increased expression of an H+-ATPase mRNA in roots of soybeans at low water potential. Such an enzyme could play a role in the acidification of the apoplast that may be necessary for the maintenance of growth. Creelmann and Mullet (1991) reported differential expression of a proline-rich cell-wall protein in shoot and root elongation zones of soybean. In roots, low water potential reduces the expression of this gene, while the converse is true in the elongation zone of the hypocotyl. C. GROWTH OF ROOTS AND SHOOTS WHEN N SUPPLY IS RESTRICTED

When plants experience a reduction in N supply, the growth of shoots is reduced more than that of roots and, in terms of dry matter increment, the growth rate of the whole plant decreases (e.g. Ingestad and Lund, 1979). This is not a general phenomenon with regard to all nutrient stresses. Deprivation of other mineral nutrients (e.g. K, Mg) can be associated with little or no shift in favour of root growth and can even result in higher shoot to root ratios than are found in control plants (Ericsson and Kahr, 1993; Cakmak etal., 1994). However, with respect to N deprivation, the observation is similar to that previously made with respect to reduction in soil-water availability (section IVB). Apparently, for a given degree of N deprivation, the extent to which a shift in dry matter partitioning occurs can depend upon the type of plant (cf. Aerts, 1994). This may possibly relate to inherent differences in modes of phloem loading (Van Be1 and Visser, 1994). In studies where plant growth has been manipulated by varying the flux of Mg, the allocation of dry matter between roots and shoots has been affected by the amount of N absorbed (T. Ericsson, pers. comm.). Where excess uptake of N was decreased, allocation of dry matter to the root increased while plant growth rate was still being limited by Mg supply. This may indicate a central

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role for N in determining the allocation of dry matter between shoots and roots, even where other nutrients are limiting the growth rate of the plant. Total dry matter increment of the plant seems to be a function of the total N availability to the roots and it may be unimportant that N deprivation occurs about one part of the root if N supply is increased by the same amount around another part. In split-root studies, Samuelson et al. (1992) showed that the growth rate of the plant (Hordeurn vulgare) was proportional to the total amount of N absorbed, irrespective of how it was supplied t o two parts of the root. The two parts of the root system, however, grew at quite different rates, in response to the amount of N that they were absorbing. With time, this resulted in a stable root to shoot ratio at the whole-plant level (Fig. 14). It was also noted that the frequency of lateral roots and their ultimate length was unaffected by reduced supply of N. However, laterals took much longer to develop and attain final length at lower N supply than at higher supply. Following N deprivation, an inhibition of expansion growth can proceed more rapidly than the shift in dry matter partitioning (Fig. 15, McDonald et al., 1986a). Because it was concurrent with accumulation of starch in leaves, this rapid inhibition of shoot expansion in Betula pendula was not attributed to a carbohydrate limitation of expansive growth. However, it has yet to be definitively demonstrated that, following N deprivation, carbohydrate supply is in fact non-limiting to the growth of cells in individual, expanding leaves. Presumably, carbohydrate metabolism and transport phenomena associated with, for example, the availability of xyloglucan in the growing cell wall could be crucial. It has been observed that N deprivation leads to rapid reduction in the expansion of single leaves of Helianthus annuus (Fig. 16, Palmer et a f . , 1996). Here, reduction in leaf expansion was attributed to reduced cell growth in older expanding leaves but may also have partly been attributable to a reduced rate of cell production in younger leaves. Reduction in final leaf size was accounted for by a decrease in the size of epidermal cells in Salix viminalis (McDonald, 1989). However, reduced cell numbers were more important in accounting for decreased blade extension in N-limited Festuca sp. (MacAdam etal., 1992). We conclude that cell division and cell expansion, can both be reduced following N deprivation and that reduced numbers and activity of meristems may assume increasing importance with time in inhibiting the development of shoot area (cf. Dale and Milthorpe, 1983). Here, we confine our thoughts to the regulation of cell expansion following N deprivation. At the outset, it can be stated that the quality of physiological and growth information available in the context of N deprivation is lacking compared with that for soil drying. We do have a good understanding of morphological development following N deprivation (cf. Clarkson and Touraine, 1994; Rooney, 1994) but it is apparent that critical studies of growth, physiology and biochemical gradients within the growth zones of leaves and roots have

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--

J. DAVIES

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s

0.15

c

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0.10

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Total plant

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E

0, .a) 5

50 I;

c

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P

z

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20 Yo

30 0

5

10

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20

Days of N addition

Fig. 14. (a) Relative growth rate (RGR) of whole plant, root and shoot as a function of relative addition rate of nitrate (RA) in standard cultures. Dashed line: RGR = RA. (b) Weight proportions of subroots of split-root cultured plants growing at RA 0.09d- , with the nitrate addition divided at a ratio of 20:80 between the subroots, as a function of time. Also shown are data from a culture with perturbed nitrate additions. From Samuelson et al. (1992).

to be carried out before definitive statements on the regulation of cell expansion can be made. Radin and Boyer (1982) addressed the question of possible mechanisms of growth reduction in leaves of H. annuus when plants were grown in solutions of lower-N concentration compared with leaves on plants at higher-N

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

26 1

0.4

0.3 0.2 0.1 h

a,

0.0

cn

a .-K

0.3

'c

0

a,

c

2

p

.c

0.2 0.1

a

5 0.0

a

0.3

0.2 0.1

0.0 -10

0

10 20 Time (d)

30

40

Fig. 15. Time-series of relative rates of increase (d-') in (a) leaf nitrogen ( 0 ) . root nitrogen (0)and total plant nitrogen (+); (b) leaf starch (a), root starch (0) and plant starch (+); (c) in root dry weight (O), plant dry weight (+) and in leaf area ( ). On Day 0, the relative rate of increase in nutrient supply decreased from 0.20 d - ' to 0.05 d-I. The dashed vertical lines denote four stages of the experiment. From McDonald et al. (1986a).

concentration. (The problems with this approach to studying the effects of N deprivation are dicussed in section 11.) They found that the hydraulic conductivity of the root was decreased at lower N concentration and that, during periods of high transpiration, the water potential of growing leaves was reduced in plants at lower N compared to leaves of plants at higher N. They concluded that the reduction in calculated turgor may have accounted for the observed reduction in leaf expansion at lower N. In the light of the discussion

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A. J. S . McDONALD and W. J. DAVIES

A

B

m

E

a

ij

F

c'

0

3

6

9

12

1 5 0

3

6 ; 9 12

15

t step

Time (Days in unit) Fig. 16. Time-series of leaf area and turgor in leaf epidermal cells of sunflower plants grown at (A) 26g N N-' d'-' or grown at (B) 26g N gN-' d'-' and then transferred to 0.04 g N g N-'d-'. The dashed vertical line shows the occasion, tstepr of decrease in nitrate supply. Data are for the first leaf pair (0)and for the second leaf pair (V). From Palmer et al. (1996).

in section IVA, we might conclude that this may have been attributable to either a reduced turgor-driven rate of plastic deformation or to expansion ceasing in a number of cells where P - Y I0. More recently, Palmer etal. (1996) measured turgor with a pressure probe in epidermal cells of growing leaves of H . annuus following a rapid depletion of N about the roots. Palmer did not find a significant difference in turgor between cells measured either before or after N deprivation or between cells in similar leaves of control and N-deprived plants (Fig. 16). Here, it would seem likely that the rapid reduction in leaf expansion was attributable to changes in cell-wall properties. At present, we do not know the reason for the discrepancy between the data of Radin and Boyer and those of Palmer but suppose that it may relate either to the

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263

measurement methods, the nature of N deprivation or to some unidentified interaction. Other attempts have been made to identify mechanisms regulating leaf expansion following N deprivation (Taylor et al., 1993; Stadenberg et al., 1994) but interpretation of the data is difficult. This is because growth has been measured at the organ level (without targeting specific growth zones) and mechanisms a t the cell level have been inferred from measurements of water relations and mechanical properties made at the tissue and organ level. We conclude that priority must be given to direct measurements of turgor and relevant wall biochemistry in well-defined regions of growth in plants where N availability has been varied. This is necessary if definitive statements about the regulation of cell expansion, consistent with current models of cell-wall growth, are t o be made with respect to N deprivation. It is possible that effects of N supply on cell-wall growth relate directly to N substrate limitations to the synthesis of wall enzymes such as XET and other wall proteins in the expansin class (see section IVB). It is also possible that N deprivation may be more indirect, affecting the activity of XET and expansins in the cell wall at the time of N decrease. Hormonal balance (ABA and cytokinins) in the plant can be affected by perturbation in N supply (cf. Clarkson and Touraine, 1994) and increased ABA concentrations in plants with decreased availability of N or all nutrients have been reported (Goldbach etal., 1975; Mizrahi and Richmond, 1972; Radin e t a f . , 1982; Chapin etal., 1988a,b; Thorsteinsson etal., 1990; Palmer et al., 1996). The causal significance, however, of ABA involvement in regulating growth processes in the cell wall following N deprivation, has yet to be established. An interesting possibility is that N deficiency may accelerate the process of cross-linking wall components, thus making the wall less yielding. MacAdam et al. (1992) provided evidence of a link between growth deceleration and a dramatic increase in the activity of peroxidase enzymes in the meristems of tall fescue leaves. Chaloupkova and Smart (1994) recently reported the ABA stimulated induction of a novel peroxidase in Spirodela. Apparently, induction is antagonized by high cytokinin concentrations.

V.

IMPLICATIONS A. SINK STRENGTH

Because the initial events in cell expansion are associated with loosening in the cell wall and, because there is no apparent mechanistic basis for assuming a carbohydrate limitation to the activities of wall-loosening enzymes and proteins discussed above, it may be more logical to think of shifts in carbohydrate partitioning between shoot and root growth as a consequence of differential wall-loosening activities rather than a cause of such. Following a decrease in

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A. J. S. McDONALD and W . J. DAVIES

nitrate supply, the proportional decrease in average-cell length was found to be less in the root cortex than in epidermal cells of the leaf in sunflower (unpublished data of the authors). This was associated with a decrease in plant-growth rate and a shift in dry-matter partitioning in favour of root growth. If the wall-loosening events produce important components of sink strength by, for example, creating sites for the subsequent incorporation of xyloglucan, then differentials in loosening could create differentials in sink strength, ultimately affecting the directional fluxes of sucrose. With time, other components of sink strength such as secondary wall thickening and the differential activity of root and shoot meristems will presumably contribute to the fate of sucrose in the phloem. However, we propose that part of the shift in dry matter partitioning in favour of root growth may be a direct result of differences in sink strength induced by differences in the extent to which the expansion of root and leaf cells is inhibited by N deprivation or a restriction in water supply. B. REGULATION OF N BALANCE

It is apparent from longer-term studies of growth response to N availability that acclimation tends to confer a stability of C : N ratio in the whole plant. Under conditions of demand limitation, Touraine eta/. (1994) cite case studies with root temperature (Clarkson et a/., 1986), sodium-chloride salinity (Helal and Mengel, 1979; Luque and Bingham, 1981; Touraine and Ammar, 1985), aluminium toxicity (Tan and Keltjens, 1990) and soil pH (Schubert e t a / . , 1990). The common finding to all of these studies is that growth rate was limited by a variable in the root environment but N concentration in the plant was unaffected. Under conditions of demand-control, enhancement of N uptake by some roots can be obtained by withdrawing N from the solution surrounding other roots (cf. Touraine e t a / . , 1994). The general conclusion is that some internal regulation ensures that N0;uptake matches the N requirements of the plant in growth. A similar conclusion might be drawn from the data of Ingestad and McDonald (1989) where birch plants were grown over a wide range of photon flux density. Here, different growth rates were achieved but very similar plant N concentrations were found where N was made freely available in solution to plants which had different C assimilation rates (cf. McDonald etal., 1992). However, it should be noted that there are examples where growth limitations caused by one mineral nutrient do not always result in conservation of N concentration. For example, Goransson (1994a,b) reported significantly higher N concentrations in birch at growthlimiting supplies of Mn or Fe, where N was made freely available in solution. Stability of plant N concentration has also been reported as an acclimated response to sustained N deprivation (e.g. Ingestad and Lund, 1979; Ingestad and McDonald, 1989). The split-root studies of Samuelson e t a / . (1992)

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indicate that, at supply limitation, it is the total N absorbed, irrespective of how it is made available to different parts of the root system, that confers stability to the prevailing C : N ratio of the whole plant. The actual value and stability of plant-N concentration may best be considered as a consequence of regulatory activity in the C and N uptake systems, without necessarily conferring any importance to the maintenance of plant N concentration as a set point. Lawlor (1994) makes this point with regard to relating mechanisms of CO, and N0;assimilation to C :N ratios in the whole plant. Rooney (1994) comes to a similar conclusion with regard t o plant development, Although there is much evidence that indicates that the external C and N supply influences development, there is little to support the importance of C : N ratio per se in the regulation of morphological changes and stages through which a plant progresses. Presumably, there are concurrent feedbacks of the end products of C and N metabolism in shoots on both the subsequent uptake of C in leaves and N0;in roots but there may be no mechanistic reason for assuming a complete matching of these fluxes. Larsson (1 994) discussed the combined significance of acclimation of V,,, for N0;uptake and changes in root extension to the further acquisition of N at supply limitation. In the type of hydroponic culture system used by Larsson and his colleagues, the shift in favour of root extension following N deprivation may be of little consequence to the further uptake of N. However, if what is being observed reflects an adaptation to reduced NO; availability, then the findings are presumably of significance to the uptake of nitrate from a soil, where root extension allows new soil volumes t o be exploited. An interesting calculation has been made by Larsson (1994) of relative V,,,, in which he relates the uptake capacity to the amount of N in the plant. This allows a discussion of uptake capacity with respect to the instantaneous rate of NO; uptake which is required to maintain a given plant RGR (Fig. 17). The conclusion from the data of Mattsson etal. (1991) is that relative V,,, is far in excess of the uptake requirement at low supply rates associated with low plant RGR but approaches the actual uptake requirement at highest supply rates associated with highest plant RGR. Because these are steady-state responses of the uptake system and root extension, it may be concluded that, on NO;-deficient sites, the plant can maintain a high scavenging capacity for available NO;. Interestingly, the relative V,,, at the lowest supply rate is sufficient to accommodate an NO; uptake which would shift the plant from a low RGR to a maximum RGR, should a flush of sufficient NO; occur (McDonald and Stadenberg, 1994: calculated from the data of Oscarson et al., 1989). Down-regulation of uptake capacity at higher supply rates (Mattsson eial., 1991) resulting in a relative V,,, which approximates the uptake requirement for steady-state growth might be interpreted as an economy of energy investment in the uptake system. The data of Oscarson etal. (1989), where V,, was maintained at a high value, indicate that down-regulation at high supply rates may not always be the case. The extent to which there may

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2.5

-k c

2.0

increased carrier density _ increased R.T increased tissue N conc

-

increased metab reg

[I

U

1.5

-*

I

-

0

I

0.10

II

>

)n

0.20

RA (d.') Fig. 17. The relative V,,, for net nitrate uptake in vegetative barley cultures grown at different relative addition rates of nitrate (RA). Relative growth rate (RGR) values included for comparison. Directions of physiological responses (observed and putative) of significance for the relative V,,, are shown in the top of the figure. From Mattson etal. (1991) and Larsson (1994).

be a species difference in the trade-off between regulation of V,,, and root extension in the maintenance of high uptake capacity for the whole plant, is predictably an interesting phenomenon contributing to the competitive fitness of individual plants and species on sites of variable N supply. Over a very wide range of N supply, the acclimated response can be such that plant growth rate, in terms of biomass increase per plant N and unit time (plantN productivity), tends to be conserved (e.g. Ingestad, 1979). This is in the same range of N supplies for which a tendency towards the conservation of photosynthetic rate (leaf area basis) has also been found and means that the regulation of leaf extension by N supply can be associated with a conservation of photosynthetic rate and plant-N productivity. Apparently, this acclimation can coincide with that for NO; uptake such that a capacity sufficient to accommodate the N requirement for sudden, rapid growth can be maintained. C.

REGULATION OF WATER USE EFFICIENCY

Implicit in our discussion of the regulation of growth and C gain in response to soil drying is the assumption that plant water status is maintained within

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a range favourable to growth. We understand that below some critical water status threshold, the plant may be damaged or at least growth and gas exchange will be limited. In a competitive situation, however, there is little to be gained by conserving water which will be used by competitors although, once captured, efficient use of water can convey some advantage. The concept of water-use efficiency is relevant here (see Davies and Pereira, 1992). Much has been written about the instantaneous water-use efficiency of different species and we know that closure of stomata can increase instantaneous water-use efficiency under certain conditions (see e.g. Jones, 1993). The early writings of Jones (1976) and Cowan and Farquhar (1977) show clearly that maximizing water-use efficiency on a day-to-day basis does not necessarily mean that the instantaneous ratio between transpiration and C gain must always be maximized. Further analysis suggests that over the longer term, plants may regulate growth and stomatal behaviour in an optimal fashion with respect to the amount of water available in the soil and, to some extent, the uncertainties in the future environment (Cowan, 1982). Regulation of this kind requires that the plant has some means of estimating the amount of water available in the soil. Such mechanisms are discussed in the following section. Jones and Sutherland (1991) and Jones (1993) suggested an alternative kind of regulation. This involves a trade-off between open stomata for high assimilation and closing stomata to prevent damage to the water conducting system (Tyree and Sperry, 1989). The model proposes that high productivity could be achieved by closure of stomata only when a proportion of xylem vessels are embolized. Beyond this point, catastrophic xylem failure is avoided by stomatal closure. Since such catastrophic failure rarely if ever occurs in natural environments, Jones (1993) suggests that a major function of stomata may be to avoid this phenomenon. It is interesting that the model proposed by Jones and Sutherland was able to explain many of the well-known features of stomatal response to soil drying, including stomatal regulation by soil water status and atmospheric humidity rather than by leaf water potential.

VI.

INFORMATION TRANSFER A.

RESPONSES TO SOIL DRYING

When the soil dries, water uptake by the roots declines and this reduction in water supply will eventually result in the development of a water deficit in the shoot. The extent of this water deficit will depend not only on the extent to which water supply is restricted but also on the rate at which water is lost from the shoot. This is determined by the evaporative demand of the atmosphere and the diffusion resistance provided by the Ieaf. We have described above some experiments where stomatal behaviour is linked to the water supply from the soil rather than to the water status of the shoot and in these plants, the

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A. J . S. McDONALD and W. J. DAVIES

water status of the shoot is often controlled by the stomata, such that shoot water potential does not vary as the soil dries. Plants that exhibit this type of behaviour can be described as isohydric, while those that show reduced water potential as soil dries are anisohydric. At least part of the stomatal control shown by anisohydric plants in drying soil can be a response to the changing water status of the shoot. Because of the influence of both edaphic and climatic variables on plant water status, it is difficult to see how the plant can assess the water supply from the soil from the shoot water status. During the day, this can vary significantly over a very short time-scale and cannot therefore be a suitable developmental regulator. Predawn shoot water status is much more stable and may therefore be a more suitable regulator but in very dry soil this variable may come into equilibrium for only a short time before the end of the dark period. Quite recently, several authors (see e.g. Davies and Zhang, 1991) have suggested that interaction between the plant and drying soil may generate a chemical signal which can move from the root to the shoot to provide a measure of soil water status and/or soil water availability. There are many possible candidates for such chemical signals (see section VIIB) but most attention has been given to the possibility that the plant growth regulator ABA can act as a signal in this context. We know that ABA is synthesized in increased quantities in root cells as they dehydrate (e.g. Cornish and Zeevaart, 1985) and that ABA will move in the transpiration stream to the shoot to regulate both stomatal behaviour and growth. For potted maize plants, Zhang and Davies (1989) have shown a relationship between soil water content and the ABA content of roots in contact with this soil, while Tardieu etal. (1992a) have shown a clear relationship between xylem ABA concentration and predawn leaf water potential for plants growing in the field. When xylem ABA concentrations are plotted against soil water availability for the field crop, there is a clear relationship for non-transpiring plants (sampled at night). For plants sampled during the day, the ABA signal seems to depend upon soil properties as well as the water status of the soil, as different relationships are obtained for plants in well-mixed and compacted soil (Fig. 18). This is presumably a function of different water fluxes through the roots of plants in the two types of soil. Clumped roots in compacted soil might be expected to dry the soil locally and generate stronger ABA signals as roots dehydrate further and water fluxes are reduced. These data indicate that the ABA signal cannot provide the shoot with an absolute measure of the amount of water available in the soil. Rather, the signal seems to indicate the access that plant roots have to soil water, a property that depends both on soil water status and on the distribution of roots in relation to the water. Figure 18 also indicates that the ABA signalling system is relatively insensitive to soil drying, which we would expect if stomata are not to be partially closed most of the time. It has been suggested (Passioura, 1988) that root signals may be generated as a result of an interaction between

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

500

.

400 '

300' 200 '

.

Predawn

1

0

269

Day time

40 80 120 Transpirable soil water (mm)

Fig. 18. Relationship between abscisic acid (ABA) concentration in the xylem and transpirable soil water beneath a crop of maize plants growing in the field in France. Data was collected before dawn o r during the day. and 0 represent plants grown in soil with low mechanical impedence and A and A represent plants growing in soil with a compacted plough layer. From Tardieu et al. (1992a).

the root and the changing physical properties of the soil. This is clearly not the case in the system described in Fig. 18, as the root signalling pattern during the day is different from that generated at night, suggesting a response to root dehydration. We would not necessarily expect the physicaI influence of the soil to vary between the day and the night. Although there are many papers that report relationships between xylem ABA concentration and stomatal behaviour of droughted plants (e.g. Loveys 1984; Zhang and Davies, 1990; Khalil and Grace, 1992, 1993), the many feedbacks in the control system mean that perturbations in the system do not always produce responses that are easily predicted. Tardieu and Davies (1993) modelled the control of stomatal behaviour by ABA signals and described a system that is comprised of five simple equations (Fig. 19). ABA is generated in the roots by dehydration of the root cells as the soil/root interface resistance

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A. J . S. McDONALD and W. J. DAVIES

- J, = (w, - VJ 1R, [ABA] = a y, (J, + b)

Fig. 19. Representation of variabIes and equations of control in an interactive model describing water flux through maize plants [model described by Tardieu and Davies (1993)l. Input variables: a,,, net radiation; T, and T,, air and dew point temperatures; *s, soil water potential. R, and R,, are the plant and the soil-plant resistances to water flux. Unknowns: g, , stomatal conductance; and \k,, root and water flux; [ABA], concentration of abscisic acid in the leaf water potentials; .Iw, xylem. Other symbols are constants (see Tardieu and Davies, 1993). Arrows symbolize transfers of water and/or ABA.

is increased. The concentration of ABA in the xylem is determined by the extent of root dehydration and the flux of water though the plant. This latter variable is important because we might expect that on hot dry days high rates of water loss will dilute the signal. Evidence for this is provided by our observation of a relatively stable xylem ABA concentration as the day progresses (Tardieu et al., 1992b). The effects of increasing root dehydration on ABA concentration in the xylem are apparently offset by increasing water flux as evaporative demand increases through the day. In the field, stomatal conductance is commonly restricted late in the day and it is not easy to reconcile this observation with the relative stability of

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

27 1

ABA concentration in the xylem. It appears that this can be done if the sensitivity of the stomata to the ABA signal is made to vary with time of day, as is apparently the case in the field (Tardieu and Davies, 1992). We have shown that this variation may be a function of varying shoot water potential (see section VIIC) and the model therefore calculates stomatal conductance as a function of ABA concentration in the xylem with a variable sensitivity depending on shoot water status. We can therefore explain limitation in conductance late in the afternoon as a function of increased stomatal sensitivity to an ABA signal as the leaf water potential falls. Output from the model (Fig. 20) of Tardieu and Davies (1993) shows control of stomata on a diurnal basis t o be largely a function of sensitivity variation, with increased restriction in conductance on a daily basis as the soil dries and the baseline concentration of ABA in the plant increases. Interestingly, shoot water potential is also controlled by the system. A crucial role for sensitivity variation is highlighted when the model is run with chemical control alone (no interaction with water status to increase sensitivity). This version of the model fails to control plant water potential and fails to restrict stomatal conductance to any appreciable extent (Tardieu, 1993). Our analysis seems to suggest that plants growing in the field can regulate stomatal behaviour as a function of the access that plant roots have to water in the soil. The extent to which the stomata respond to the root signal will depend upon a n interaction with leaf physiology and the microclimate around the leaf and may be viewed as a sensitivity modulation. The nature and extent of this type of interaction is discussed further in section VIIC. Control of stomata can be important in the regulation of gas exchange, particularly in rough, relatively uncoupled canopies. The system of regulation that we have described above will also be important in as much as the ABA concentrations in the plant and the water status of the shoot will also be regulated. This can have important consequences for development of plants growing in situations where water supply is restricted. There has been considerable interest in the possibility that chemical or genetic modification of the plant’s ABA balance in the field will modify its response to soil drying and possibly modify its drought resistance (e.g. Quarrie, 1991). Despite much early promise, it is not clear that even quite substantial genetic variation in the capacity for ABA accumulation has a significant influence on plant growth and development. This may be because of the many feedbacks between the water and the ABA relations of the plant (e.g. Tardieu, 1993) which may mean that a capacity to accumulate large amounts of ABA will not always be realized. Recent work on Mediterranean forages by Puliga etal. (1996) has highlighted one character that may be important for the control of leaf growth in the field. It seems that summer-active species - that is, those that continue to produce leaves even when soil drying is quite severe - may show a restricted capacity to produce ABA when expressed as a function of soil water status, even though the ABA production per unit of

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A. J. S. McDONALD and W. J. DAVIES

a

8

12 Time (h)

b

16

20

8

12

16

20

Time (h)

Fig. 20. Simulations of the daily pattern of stomata1 conductance (gs),water flux in the soil-plant atmosphere continuum (J,,,), leaf and root potentials (9, and qr), abscisic acid concentration in the xylem sap ([ABA]) and ABA flux to the leaf (JAB*). Soil water potential (9) is -0.3 MPa, meteorological conditions (net radiation, R,, and air vapour pressure deficit, VPD) are those measured on 26 July 1990. Soil characteristics: silty clay loam. (a) Calculations with values of soil-root resistance to water transfer (Rsp) simulating a root system in favourable conditions. (b) Calculations with a value of R,, multiplied by 20, simulating unfavourable characteristics of the root system for water uptake (such as root clumping). From Tardieu and Davies ( I 993).

plant water deficit is not unusual. These data seem to provide further evidence that plants may regulate their growth and development in the field as a function of a measure of soil water availability and that there may be some genetic variation here that may be of interest.

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B. RESPONSES TO N LIMITATION

1. Stomata1 conductance and leaf growth We consider two possible types of signal to N deprivation. The first is a possible role for ABA, either on its own or in conjunction with cytokinins, and the second relates to the possible importance of hydraulic signals in the plant. There is evidence that these may not be independent variables. Apparently, an external supply of ABA can increase the hydraulic conductivity (Lp) of root membranes (Karmoker and Van Steveninck, 1978; Pitman and Wellfare, 1978; Glinka, 1980). It might be supposed that a synthesis of ABA in response to N deficit might therefore oppose the apparent decrease in hydraulic conductivity but, as Clarkson and Touraine (1994) point out, this apparent paradox may presumably be resolved by distinguishing between hydraulic conductivity of root cell membranes at the site of ABA synthesis in the root tips and the hydraulic conductance of other (the vast majority) of root cells effectively isolated from this region. It is also possible that shifts in Lp of roots may cause changes in the water potential of the leaf, triggering synthesis of ABA in the leaf (cf. Clarkson and Touraine, 1994) or causing shifts in the compartmentation of leaf ABA. The combination of simultaneous increase in ABA and decrease in cytokinins might constitute a strong signal in response to N deprivation. Jackson (1993) recently reviewed the occurrence of decreased cytokinin synthesis in roots following mineral nutrient shortage. A number of studies were cited in support of the earlier findings of Kulaeva (1962) that cytokinins had a central role in delaying leaf senescence and that, following N deprivation, cytokinin synthesis in roots can be reduced. In two separate studies with Solanurn tuberosum, Sattelmacher and Marschner (1978) and Krauss (1978) showed that NO; deficiency induced decreases in cytokinin-like activity and increases in ABA in the xylem, respectively (cf. Clarkson and Touraine, 1994). Importantly, Radin etal. (1982) found that increased application of cytokinin decreased the sensitivity of stomata to ABA in response to water stress. Thus, there is evidence that the ABA :cytokinin ratio may constitute a strong signal but one that is not necessarily specific to N deprivation.

2. Nitrate uptake There is no evidence for effects of ABA on the NO;-uptake system. Chapin et al. (1988a,b) applied ABA externally and found no effect and, even where small changes in the ABA concentration of roots were measured after N deprivation, these were not in phase with changes in NO; transport. In mutants that are defective in ABA synthesis, the NO; transporter activity increased in ways similar to wild-type tomato. Thus, Clarkson and Touraine (1994) make the important point that the NO; transport responses appear to be controlled separately from the morphological responses. At demand limitation, the evidence is in support of a shoot-sourced signal producing feedback

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inhibition of N uptake. A consequence of NO; assimilation in the shoot is a stoichiometric synthesis of amino acids and organic acids. Both these components are among the main nitrogenous components of phloem sap (Hall etal., 1971; Richardson etal., 1982) and thus are potentially important as regulatory signals of NO; uptake (Touraine et al., 1994). Touraine et al. (1994) discuss the stimulation of NO; uptake in the root by carboxylates transported in the phloem from the shoot. Details of the mechanism by which carboxylate (mainly malate), imported into the roots, stimulates NO; uptake are still not clear. Anions are believed to enter root cells via transporters with protons or via antiports with hydroxyl ions or bicarbonate ions at the plasma membrane (Clarkson, 1986; Glass, 1988). The overall control loop in the whole plant relates to the models of Dijkshoorn et al. (1968) and Ben Zioni et al. (1971) in which a cycling loop of K ions supplies the accompanying cations for NO; in the xylem and carboxylate ions in the phioem. Decarboxylation of malate in the root leads to the formation of bicarbonate ions which are released into the external solution. The tight stoichiometry between NO; absorbed and hydroxyl released provides a regulatory possibility. There is good evidence that amino acids in the phloem can play a regulatory role in the uptake of NO;. Touraine etal. (1994) make the important point that the relative isolation of the cycling pool of amino-N might make its composition sufficiently specific to convey information. Where protein synthesis is reduced in the shoot, both qualitative and quantitative changes in the aminoacid composition of the phloem may be expected. The idea is that an increase in one or more amino acids transported from the shoot will cause a downregulation of NO; uptake. The mechanism of this down-regulation is uncertain but it is thought to relate to transcriptional events rather than any direct effects of amino acids on the NO; transporter (see review by Touraine et al., 1994). It may be concluded that, although some details of mechanism remain obscure, a good working model for NO; uptake and its regulation in the whole plant at demand limitation can be assumed, in which the key components are a stimulation of uptake by phloem-sourced carboxylate and an inhibition of uptake by phloem-sourced amino acids (Fig. 21). Larsson (1994) suggests that the mechanisms behind the change in V,,, in phase I1 (Fig. 8) may be the same as those involved during N starvation of plants pregrown with a plentiful supply of NO;. He considers the possibility of a central role for the cytosolic NO; pool in control of NO; uptake. This possibility exists because the cytosolic concentrations are extremely low in phase I but rise sharply in phase 11. It also seems feasible that regulation at demand limitation, based on the phloem content of amino acids and carboxylate, might be relevant to that at supply limitation. A sudden decrease in NO; supply might initially stimulate the uptake system as a result of a decrease in the amount of amino acids and

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Fig. 21. A model for the control of nitrate uptake by leaf-generated signals during (a) rapid vegetative growth and (b) rapid pod fill. During vegetative growth (a), nitrate ions are rapidly absorbed by the root and transported via the xylem to the leaf. In the leaf, nitrate reduction produces organic acids (OA) and amino acids (AA). Most of the newly formed OA are translocated to the root where a carboxyl group is released in exchange for a nitrate ion, whereas the newly assimilated N is incorporated primarily into leaf N compounds. During rapid pod fill (b), leaf proteolysis occurs and much of the amino N in the leaf is exported to the filling pods. Consequently, the phloem is enriched with amino compounds, which repress nitrate uptake and consequently diminish the rate of nitrate reduction. From Ismande and Touraine (1994). a n increase in the amount of carboxylate cycling via the phloem. This would be consistent with the commonly reported increase in V,,, following NO; deficiency. However, with time, a decrease in carbon assimilation by the shoot a n d changes in C and N usage in growth will occur which might result in changes in the composition of the amino acid pool. It is possible that longerterm changes in the composition of amino acids in the phloem, associated with retranslocation a n d cycling of N (cf. Mattsson etal., 1991), might then downregulate the uptake system, giving the type of acclimated response in phase I discussed by Larsson (1994).

VII. WHAT IS IN THE XYLEM SAP AND HOW CAN CHANGES IN WATER AND N AVAILABILITY CHANGE THE XYLEM SAP CONTENTS? A.

COLLECTION OF XYLEM SAP

One substantial difficulty in assessing the effects of any root perturbation o n the concentrations of the different substances in the xylem stream is to take

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samples for analysis without disrupting contents or concentration (see e.g. Jackson, 1993). Xylem sap is commonly expressed from cut leaves or cut stumps under pressure (e.g. Zhang and Davies, 1990) or under partial vacuum (Pate et al., 1994) and may also be collected from bleeding stumps exhibiting root pressure (e.g. Loveys, 1984). All of these techniques may be criticized on the grounds that plants are severed before sampling so that the transpiration stream is no longer moving through the plant part sampled. It is argued that this can result in the concentration of substances in the xylem and Else et al. (1994) demonstrated that this may be a problem when sap is sampled from relatively small roots of tomato. These authors also demonstrated that contamination of xylem sap samples with sap from damaged cells at the cut surface can also lead to a substantial overestimate of concentrations of hormones in the xylem stream. Tardieu and others sampled xylem sap from large maize leaves from plants grown in the field (e.g. Tardieu etal., 1992a). These experiments revealed large effects of transpiration flux on the concentration of ABA in the transpiration stream and these effects have been elucidated using a destructive sampling technique. These authors argued that xylem sap samples from such large leaves can be taken from sap which was present in the xylem before the leaf was sampled. Of course the amount of redistribution of xylem contents between xylem conduits and xylem parenchyma is unknown. It is generally accepted that problems of the concentration of xylem sap as a result of sampling non-transpiring plants can be avoided if the whole plant pressure chamber shown in Fig. 2. is used to sample sap (see e.g. Munns, 1989; Jeschke and Pate, 1991). Here, a small overpressure can be applied to roots to force sap from the cut tip of a transpiring leaf. Of course, the pressure chamber cannot be used with field-grown plants but it probably is a good idea to validate particular sap collection methods against the pressure chamber to check for overestimations of concentration. B. SOIL DRYING AND N DEPRIVATION AND EFFECTS ON XYLEM CONTENTS

I . Plant hormones As we have discussed above, considerable attention has been given to the possibility that plant hormones can move in the xylem stream to act as signals to the shoot of the availability of soil water. Many authors have reported that soil drying can increase xylem sap ABA concentrations by several orders of magnitude (see e.g. Wartinger et al., 1990; Davies and Zhang, 1991; Khalil and Grace, 1992, 1993; Jackson et al., 1995; Correira and Pereira, 1995). Munns (1989) suggested that the high concentrations of ABA detected by these authors may be artefactual due to the techniques used for sap collection but similar or even greater concentrations were detected by Schurr et al. (1992) in sunflower sap sampled with the whole plant pressure chamber. In all of the studies cited above and in many others, generally good correlations were

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reported between xylem sap ABA concentration and stomatal conductance. If we are to argue for a regulatory role for ABA, it is important to determine that this is something more than a correlation and that it is the ABA that is influencing the stomata and not the converse. In their model of chemical control of stomatal behaviour described above, Tardieu and Davies (1993) show that there can be a relationship between stomatal conductance and the concentration of chemical constituents in the xylem, even when these constituents have no effect on conductance. This relationship arises simply as a result of the concentration of constituents as stomata close and transpiration is restricted. The activity of xylem sap constituents can be tested by comparing concentration :response relationships produced by droughting the plants with those generated by applying the constituent of interest externally. Zhang and Davies (1990) have done this for ABA in maize plants and found an excellent correlation between relationships generated by the two methods. Further confirmation that the constituent in question is active and indeed is the only active component can be obtained by removing it from the xylem sap sample and testing the remaining sap for activity in an appropriate bioassay. Munns and King (1988) have done this with xylem sap extracted from wheat plants. ABA was removed from sap samples with a immunoaffinity column but when sap was tested in a transpiration bioassay, much antitranspirant activity still remained (Fig. 22). Similar results were obtained by Trejo (1994) working with Phaseolus. Subsequent experiments by Munns (1992) suggest that there is similar non-ABA growth-inhibiting activity in the xylem stream of wheat and Chandler etal. (1993) report on the enhanced expression of dehydrins as a result of treatment with sap extracted from droughted plants but with ABA removed. Although there has been some confusion over whether this non-ABA activity can be found in newly collected xylem sap (Munns et al., 1993), it has recently been suggested that it may be some kind of ABA-adduct that could release free ABA in the leaf under certain conditions (Munns and Sharp, 1994). Netting et al. (1992) reported on the existence of such a compound but unequivocal identification has not yet been achieved. Zhang and Davies (1 991) failed to find any non-ABA-like antitranspirant activity in the xylem sap of maize plants (Fig. 22) but they did filter their sap to remove large molecular-weight compounds which blocked up the xylem and caused wilting and it is therefore possible that they also removed any ABA-adduct by this process. Interestingly, Trejo (1994) used an identical filtering procedure in their experiments but still detected non-ABA antitranspirant activity. In the experiments reported above, xylem sap collected from well-watered and droughted plants of a variety of species contained ABA at concentrations which are between micromolar and nanomolar. In most experiments, micromolar ABA will close stomata but this is not always the case and Trejo et al. (1993) have shown that this is because of the activity of the mesophyll cells

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Fig. 22. Transpiration of detached wheat leaves as a function of concentration of abscisic acid (ABA) in assay solutions. The solutions were: synthetic ABA in artificial xylem sap (O), xylem sap from well-watered maize plants (m), xylem sap from unwatered maize plants (A),and xylem sap from unwatered maize plants but with ABA removed by immunoaffinity colum (A). The dotted line shows the dose response of detached wheat leaves to synthetic ABA solutions. The crossed point is a typical result for xylem sap extracted from unwatered wheat plants and fed to detached wheat leaves. Data are expressed as percentages of the rate of water loss from control leaves fed with artificial xylem sap only (no ABA) and are means of five observations. The bars are double standard deviations divided by the mean transpiration rate of the control leaves. From Davies and Zhang (1991).

in metabolizing and compartmentalizing ABA. If the mesophyll tissue is removed and ABA is applied to epidermal tissue alone, stomata will respond to ABA concentrations as low as lo-'' M (Trejo etal., 1995). These results suggest that there is enough ABA in xylem sap of well-watered plants to close stomata, assuming that the sap reaches the guard cells without modification. Clearly, since stomata are not permanently closed, this cannot be the case. Hartung and coworkers (see e.g. Hartung and Slovik, 1991) have worked hard to elucidate the factors influencing compartmentation of ABA in the leaf. We know comparatively little, however, about ABA metabolism. It seems likely that the regulation of these processes will have an important controlling influence on stomata1 behaviour by determining what proportion of ABA in the transpiration stream gets through to the guard cells. We need more information on how rates of metabolism and compartmentation might be

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influenced by the daily pattern of variation of climate and by soil drying. When one considers the massive flux of ABA into a leaf on a daily basis, it is clear that the removal of ABA from sites of action in the leaf must be extremely effective. Nevertheless, Gowing et af. (1993) show that stomatal behaviour of cherry leaves is influenced by the amount as well as the concentration of ABA arriving in the leaf. Trejo et af. (1995) report a similar phenomenon for Commefina epidermis with the mesophyll removed. When whole leaves received different fluxes of different ABA concentrations, stomatal responses could be interpreted mostly as a function of a concentration response. This indicates that we are justified in using concentration as a variable in the analysis of control of gas exchange. This is not to say that we should not estimate flux in an attempt to demonstrate unequivocally that signalling is taking place (see Jackson, 1993). Thus far, we have concentrated on the role for ABA, a growth inhibitor, in the process of signalling between roots and shoots. The possibility that root to shoot signalling may also involve the modification of the transport of promoters has also received considerable attention. Indeed, much of the early work in this field concerned the possibility that cytokinin transport from roots to shoots could be reduced by soil drying. Cytokinins are required for the normal functioning of shoots and may be required for the opening of stomata. Incoll and Jewer (1987) considered the possible importance of cytokinins in the drought responses of stomata and made a strong case for their involvement. The effects of augmenting the supply of cytokinins on stomatal behaviour can be seen particularly in older leaves, where cytokinin contents may be declining (Blackman and Davies, 1984) and there is some suggestion that modification in cytokinin supply may modify the responses of stomata to C 0 2 . Blackman and Davies (1983) showed that high cytokinin concentrations could reduce stomatal responses to COz and suggest that the observation that soil drying enhances the C 0 2 response of stomata may be explained by a soil dryinginduced reduction in the supply of ABA to leaves. We have noted above that relatively mild soil drying may enhance the concentration of ABA in the xylem stream by perhaps two orders of magnitude, making this molecule a powerful candidate for a signal molecule in this context. It seems unlikely that even quite severe soil drying can do more than reduce cytokinin transport by 50 or even 75%. We can perhaps argue that along with many other components of the xylem stream, this molecule has a role in providing the shoot with a general indication of root functioning. The potency of these compounds as growth regulators perhaps make them particularly important in this regard. There has been very little attempt to address the importance of cytokinins in the control of stomatal behaviour in field-grown plants. Fussader et af. (1992) suggest that these compounds may be important. 2. Ions, amino acids and pH Although many authors have addressed the possibility that part of the influence

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of soil drying on the physiology of droughted plants may be a function of reduced uptake of ions (e.g. Turner, 1986), it is only since the development of the whole plant pressure chamber that it has been possible to quantify reliably the concentrations and fluxes of different ionic components of the xylem sap as plants are exposed to increasing degrees of soil drought. Gollan et al. (1992) sampled sap from turgid, transpiring sunflower plants and found that concentrations of NO; and orthophosphate decreased with soil water content, whereas concentrations of the other anions remained unaltered (Fig. 23). Calcium concentrations also decreased, presumably as a function of the relative immobility of this ion in the soil as the soil water content is reduced. A similar argument can be made for orthophosphate (Nye and Tinker, 1977). K, Mn, Mg and Na concentrations in the xylem sap were not affected by soil drying. The pH, the buffering capacity at a pH below 5 and the catiodanion ratio increased after soil water content fell beyond a certain point. Amino acid concentration of the xylem sap increased as the soil dried, although the concentration of amino acids transported in the xylem sap of sunflower is negligible compared to the NO; concentration. Jeschke and Pate (1991) used the whole plant pressure chamber to collect xylem sap from transpiring castor bean seedlings. NO; concentrations and concentrations of reduced N were measured at various levels up the shoot in xylem exudates and in phloem sap. An experimentally based modelling technique allowed some quantification of N fluxes into different leaves and it was apparent that younger leaves were heavily dependent on xylem import for their N requirements. In the N-flux experiments performed by Palmer eta/. (1996) and described earlier, an abrupt decrease in N supply to roots caused a very abrupt decrease in NO; concentration in the xylem and therefore in NO; flux into leaves. It seems possible, therefore, that reduced NO; flux in the xylem could act as a chemical signal to the shoots of NO; deprivation around the roots.

C.

INTERACTION AND THE CONCEPT OF SENSITIVITY VARIATION

In most studies of the effects of environmental perturbation on the physiology, growth and development of plants, the effects of individual environmental variabIes are considered in isolation. As we focus in on the mechanisms of response it is necessary to become more and more reductionist in our approach. It has, however, become increasingly clear that many of the plant responses to stress are whole-plant phenomena and that by focusing on cellular and even molecular responses in isolation, we risk being unable to elucidate the whole story. Work with whole plants presents problems but these are issues that we must address if we are to understand how plants respond to perturbations in the natural environment. In the context of the present chapter, a central issue is the extent to which responses of plants to soil drying can be

PLANT RESPONSES TO WATER AND NITROGEN SUPPLY

28 1

Fig. 23. Relationship between the concentrations of the cations: potassium, magnesium, calcium and sodium, and the anions: nitrate, phosphate, sulphate and chloride, and the soil water content in the xylem sap samples of eight plants. Values of significance P < 0.001 are indicated. From GolIan eta/. (1992).

understood simply in hydraulic terms and the extent to which we must consider the importance of the various chemical effects that are produced by soil drying. We have made a case here and in other writings for a central role for ABA in the regulation of functioning of droughted plants. Nevertheless, as we have discovered more and more about the operation of the hormonal control

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system, it has become necessary to introduce concepts such as sensitivity of response t o explain what we see. For example, the responses of root cells to ABA, reported by Saab el al. (1992) seem to show clear increases in sensitivity as cells age. It seems important to examine further the idea of sensitivity variation and determine the extent to which this idea will allow us to account for whole-plant responses to deprivation in water and N supply. In our discussions above, we have noted that increases in endogenous ABA concentration occur both in response to soil drying and in response to a reduction in N supply. We examine in turn interactions involving ABA which are important in the context of the regulating physiology, growth and development of plants deprived of water and/or N. We also examine the possibility that N may act in its own right as a plant hormone. I . Interactions between the effects of ABA and plant water relations In their work on the behaviour of maize stomata in the field, Tardieu and Davies (1992) noted that stomata were apparently more sensitive to endogenously produced ABA during the afternoon hours than during the morning. Burschka etaf. (1983) made a similar observation in their experiments where ABA was applied to field plants at different times throughout the day. Working in the laboratory, Tardieu and Davies (1992) were able to show that the basis of this response was an apparent interaction between ABA and the water status of the plant, such that stomata became more sensitive to ABA as leaf water potential declined. These experiments were performed with isolated epidermal strips held in polyethylene glycol solutions at different water potentials. Trejo and Davies (1994) confirmed these results with experiments performed with whole leaves. Here, water potentials were reduced by adding a fine capillary to the end of a petiole of detached Phaseolus leaves. A reduction in water potential of only a few tenths of a megapascal brought about a significant increase in sensitivity of the stomata to ABA. This appeared to be a relatively dynamic response, however, as closure of stomata caused rehydration of the leaves and the difference of stomatal sensitivity to the ABA dose decreased. There are many reports of variation in stomatal sensitivity to ABA following drought stress. A general response seems to be that drought stress or ABA treatment reduces stomatal sensitivity to ABA (see e.g. Dorffling et al., 1977) but this is not always the case (e.g. Peng and Weyers, 1994). Work by MacRobbie (1990) on ion fluxes through guard cell membranes shows that following an initial challenge with ABA, a repeat application after as long as 28 min has no effect on ion flux. This work and the work of Tardieu and Davies (1992) suggests a fundamental variation in sensitivity of guard cell membrane processes to ABA application. This may have to d o with binding of the hormone on the guard cell but we have little information on binding sites for this hormone and therefore, speculation in this area is unrewarding. One further complication is current uncertainty over the loca-

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tion of ABA-binding sites on guard cells. The work of Hartung (1983) seemed to show conclusively that ABA binding was restricted to the outside of the plasma membrane of the guard cell. Anderson et al. (1994) seemed to confirm this conclusion, but work by Allan et al. (1994) suggested that there may be some binding on the inside of the membrane. Much ABA can be sequestered in the guard cell and if this pool of the hormone is important for guard cell functioning, then we need to reassess our whole concept of stomatal functioning in stressed plants. Many reports of apparent variation in stomatal sensitivity to ABA dose may simply be a function of variation in the amount of ABA reaching the guard cells (see e.g. Trejo et al., 1993). We have discussed above how compartmentation and metabolism can influence apparent sensitivity and it is clearly important to distinguish between these two types of sensitivity variation. We know that soil drying will result in an increase in pH of the xylem sap (Hartung et al., 1988) and we would expect this to release ABA from sites of sequestration in the leaf (e.g. mesophyll cell chloroplasts) to increase the availability of the hormone t o the sites of action on the guard cell. It may be then that water deficit and other stresses can influence both fundamental and apparent sensitivity of guard cells to ABA. One other very important effect of plant water status on the plant’s sensitivity to ABA is shown by Saab etal. (1990) who reported that ABA will act to sustain root growth at low water potential but has little effect at high water potential. Saab and coworkers offer no explanation for this phenomenon but it seems likely that cellular water status has caused a fundamental change in cellular properties such that the binding of the hormone or the signal transduction chain have been influenced. In fact, the situation may be even more complicated than this.

2. Interactions between the effects of ABA and other chemical components of the xylem sap We have noted above that experiments with the whole plant pressure chamber have revealed substantial effects of soil drying on the ionic status of xylem sap. Osonubi etal. (1988) have shown similar changes for spruce trees growing in the field. It is well known that stomatal functioning depends upon perturbation of ion fluxes across guard cell membranes and that changes in the mineral nutrition of plants (e.g. K nutrition and Ca nutrition) can be shown to influence stomatal behaviour (Atkinson et af., 1990). Nevertheless, it seems likely that changes in ion composition seen as a result of soil drying may not be large enough to function on their own as modifiers of stomatal behaviour. The possibility of interaction with ABA effects have been considered, however, and Radin etal. (1982) have shown that both N and P deficiency can enhance stomatal sensitivity to an ABA signal. These possibilities have been addressed in greater detail by Schurr etal. (1992) who note that for their

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J. DAVIES

\

Fig. 24. Stornatal conductance of individual plants versus abscisic acid (ABA) concentration in their xylem sap during the first day with significant reduction of leaf conductance due to soil water depletion. From Schurr et al. (1992).

sunflower plants exposed to a soil drying treatment, a single relationship between ABA in the xylem sap and leaf conductance could not be shown. Rather, a series of relationships was found for individual plants (Fig. 24). These differences were explained by variation in the ionic status of xylem sap. Both the concentration of NO; and Ca in the xylem sap were positively correlated with sensitivity to an ABA signal and pH was negatively correlated with sensitivity. These changes are of a very substantial magnitude and whiIe we have some mechanistic understanding of the interaction between the effects on stomata of ABA and Ca (De Silva etal., 1985; McAinsh etal., 1990), we have little understanding of how ABA and NO; can interact to influence stomata unless this is via effects of N metabolism on changes in the pH relations of the leaf (Raven and Smith, 1976). If this is the case, this will be an apparent change in stomata1 sensitivity, while the Ca interaction may be a more fundamental effect on guard cell ion fluxes. Trewavas (1981) raised the possibility that NO; should be classified as a plant growth substance in the sense that this term is used to describe auxins, gibberellins and the other major classes of growth substances. Of these substances, we have discussed signalling by ABA and cytokinins. Implicit in our discussion of the communication of information around the plant is that the source of the substance conveying the information is localized in one part of the plant and transport of the substance is to an area which is relatively defi-

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cient in that substance. These criteria can apply to both ABA (see e.g. Davies and Zhang, 1991) and cytokinin (see e.g. Incoll and Jewer, 1987) although even this is a controversial area (Trewavas, 1981). The criteria may not necessarily apply to N, where there is considerable internal circulation (Pate et al., 1979) but there is evidence that variation in NO; supply to leaves as a result of soil drying can regulate NO; reductase activity in maize leaves (Shaner and Boyer, 1976). These authors differentiated between an effect of NO; already in the leaves of these plants and that arriving in the transpiration stream. In other words, the NO; is acting as a signal in this system, rather than a substrate. We have no evidence that this is the case in the regulation of leaf growth as a result of soil drying but this possibility should be considered. Leaves generally have a very high N content compared to stems and roots, probably because of a substantial protein requirement for construction of the photosynthetic apparatus. Despite this, the apical transport of N in a wellwatered plant may exceed N use by only 20% or so (Schulze, 1991). We have seen above that when the soil dries substantially, the N supply to leaves can be very substantially restricted by a combination of a reduced concentration in the xylem stream and a reduced transpiration flux. It may be then that a link between a soil-drying induced reduction in N supply and a reduction in leaf growth can be postulated, with N acting as both a signal and a substrate. This will depend to some extent on the amount of N that can be remobilized from stores to make up for the shortfall in supply from the roots. In plants with a relatively small vacuolar store of N, this can be drawn down rapidly in response to inadequate external supply (e.g. Chapin et al., 1988a,b).

3. Interactions between the effects of ABA and temperature We consider here the interaction between ABA and temperature because of the link between high temperature and soil drying. This link may be particularly important in the control of leaf growth of grasses where the meristem is commonly at or even below the surface of the soil. As high insolation drives evapotranspiration, the soil and the leaf meristem will warm. Dodd and Davies (1994) showed that ABA supplied to the leaf through the transpiration stream is more effective as leaf temperature increases. Patterns of leaf growth of plants in drying field soil can be interpreted in terms of this interaction (see e.g. Gallagher and Biscoe, 1979). These authors reported that soil drying has little effect on leaf extension rate early in the morning when temperatures are low, while the soil-drying induced reduction in leaf growth is increased later in the day as leaf temperature increases (Fig. 25). These differences in apparent sensitivity may be explained by differences in the accumulation of ABA in the meristem of the leaf and/or the result of a fundamental variation in sensitivity of the growing cells to the ABA signal. A similar interaction between ABA and temperature was reported in a study of stomata1 responses of maize plants

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2.1

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May 1975. The numbers on the lines refer to the time at the start of the 2 h period. The dashed line AB represents the expected response of extension rate of temperature in the absence of water stress. From Gallagher and Biscoe (1979).

(Rodriguez and Davies, 1982) and Allan el al. (1994) recently noted that changing the growth temperature for plants may alter the responses of the guard cells to the hormone by changing the signal transduction chain.

VIII. CONCLUSIONS: AN INTEGRATED STRESS RESPONSE SYSTEM FOR THE PLANT? In this chapter, we have argued for the regulation of stomata1 behaviour and leaf growth of plants in drying soil by root-derived chemical signals. In our view, ABA plays a central role in this signalling system. There is now good evidence for enhanced ABA synthesis in roots and transport to leaves following soil drying. Increasing evidence suggests that some of this extra ABA may

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result from reduced N availability. Clearly, ABA does not operate in isolation. There is evidence that synthesis and transport of other growth regulators, some as yet unidentified, are affected by soil drying but we need much more information in this area. We need to quantify both concentrations and fluxes of regulators in plants subjected to soil drying and N deprivation. It appears that altered fluxes of some other xylem components (e.g. cytokinin, N and Ca) will influence stornatal behaviour and leaf growth in their own right but changes in the concentrations and fluxes of these substances may not be large enough to explain the sensitive responses of leaf growth and stornatal behaviour that are sometimes seen. We propose here that soil-drying induced variation in the xylem transport of regulators such as cytokinin, N and Ca will interact with a modified supply of ABA to regulate leaf growth and functioning. Chapin (1989, 1991) proposed a similar hypothesis. We extend his ideas by arguing a modulating role for leaf water status and for several climatic variables as well as for N. Figure 26 shows an outline of the control system. The production of ABA and cytokinin will reflect the extent of soil drying but the supply of these regulators to leaves will be highly variable depending on the transpiration rate of the plant (see Tardieu and Davies, 1993) and the penetration of the transpiration stream to the sites of action within the leaf (see e.g. Trejo etal., 1993). The sites of action for stomata are either on the inside or the outside of the plasmalemma, while there is some uncertainty over sites of action for the regulation of leaf growth. The degree of penetration of the hormones to the sites of action will depend on metabolism and compartmentation of the hormones. Variation in the sensitivity of response to what hormone does reach the site of action is another crucial variable in our system but we have little information on the mechanistic basis of this variation. Our integrated stress response system also contains several feedback responses which are discussed in detail above. Hormonal accumulation in roots will influence root growth and development which will affect uptake of both water and N. We also show feedback effects of the modification of growth and development and gas exchange on water and N availability and balance. There may also be feedback effects of growth on N uptake capacity. The variation in the sensitivity of stomata to an ABA signal reported by Schurr et al. (1992) can be of a very significant magnitude and so it is clear that the processes determining this are at least as important in our control system as variation in the supply of the major substrates for growth and variation in the supply of our main hormonal regulator, ABA. In the extreme, even the ABA content of the xylem sap of well-watered plants will close stomata and may well reduce leaf growth. To understand why this does not occur and how sensitivity is nearly always damped down is a major challenge for the years ahead. In recent years, the subject of root to shoot signalling has received a considerable amount of attention. Some authors, in focusing on these

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between hydraulic and chemical control should be carefully considered. A shoot response to a modified chemical signal arriving from the roots and providing information on edaphic conditions is a simple but attractive concept. Nevertheless, this idea has never been able to explain the more dynamic minute by minute or even hour by hour responses of stomata, particularly where these are occurring in tall trees where a signal may take 10 days to arrive from the roots. The idea of sensitivity variation provides a more dynamic link between the root message and the climatic environment. In our revised formulation, the root signal in response to soil drying or N deprivation may provide a stable long-term regulation of development, while interaction with temperature, leaf water status etc. will determine the extent of short-term, dynamic response to the signal.

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