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Plant, Cell and Environment (1999) 22, 1377–1388

Novel approaches for examining the effects of differential soil compaction on xylem sap abscisic acid concentration, stomatal conductance and growth in barley (Hordeum vulgare L.) A. HUSSAIN,1 C. R. BLACK,1 I. B. TAYLOR,1 B. J. MULHOLLAND1,2 & J. A. ROBERTS1 1

School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK Current address: Horticultural Research International, Wellesbourne Research Station, Wellesbourne, Warwick, CV35 9EF, UK

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ABSTRACT Novel techniques were devised to explore the mechanisms mediating the adverse effects of compacted soil on plants. These included growing plants in: (i) profiles containing horizons differing in their degree of compaction and; (ii) split-pots in which the roots were divided between compartments containing moderately (1·4 g cm-3) and severely compacted (1·7 g cm-3) soil. Wild-type and ABA-deficient genotypes of barley were used to examine the role of abscisic acid (ABA) as a root-to-shoot signal. Shoot dry weight and leaf area were reduced and root : shoot ratio was increased relative to 1·4 g cm-3 control plants whenever plants of both genotypes encountered severely compacted horizons. In bartey cultivar Steptoe, stomatal conductance decreased within 4 d of the first roots encountering 1·7 g cm-3 soil and increased over a similar period when roots penetrated from 1·7 g cm-3 into 1·4 g cm-3 soil. Conductance was again reduced by a second 1·7 g cm-3 horizon. These responses were inversely correlated with xylem sap ABA concentration. No equivalent stomatal responses occurred in Az34 (ABA deficient genotype), in which the changes in xylem sap ABA were much smaller. When plants were grown in 1·7 : 1·4 g cm-3 split-pots, shoot growth was unaffected relative to 1·4 g cm-3 control plants in Steptoe, but was significantly reduced in Az34. Excision of the roots in compacted soil restored growth to the 1·4 g cm-3 control level in Az34. Stomatal conductance was reduced in the split-pot treatment of Steptoe, but returned to the 1·4 g cm-3 control level when the roots in compacted soil were excised. Xylem sap ABA concentration was initially higher than in 1·4 g cm-3 control plants but subsequently returned to the control level; no recovery occurred if the roots in compacted soil were left intact. Xylem sap ABA concentration in the split-pot treatment of Az34 was initially similar to plants grown in uniform 1·7 g cm-3 soil, but returned to the 1·4 g cm-3 control level when the roots Correspondence: A Hussain. E-mail: [email protected] © 1999 Blackwell Science Ltd

in the compacted compartment were excised. These results clearly demonstrate the involvement of a root-sourced signal in mediating responses to compacted soil; the role of ABA in providing this signal and future applications of the compaction procedures reported here are discussed. Key-words: Hordeum vulgare (barley); compacted soil; abscisic acid (ABA); root to shoot communication.

INTRODUCTION Soil compaction adversely affects crop growth and yield in many parts of the world (Voorhees 1991). External compression increases the bulk density and shear strength of the soil, restricting the growth of roots and thereby limiting their ability to exploit available water and nutrients. Moreover, the associated decrease in pore space volume reduces permeability and the diffusivity of gases and may result in the formation of anaerobic conditions (Greenland 1977). The stress imposed by soil compaction may therefore involve two key elements, impeded rooting conditions and an anaerobic rooting environment. Various methods have been used to examine the responses induced when roots encounter compacted soil. Goss (1977) used ballotini of varying size supplied with aerated nutrient solution, while others have employed wire mesh with differing grid sizes (Scholefield & Hall 1985), waxy substrates of varying strength (Taylor & Gardener 1960) or, more recently, pressurized columns of sand (Young et al. 1997). In contrast, Castillo et al. (1982) and Andrade, Wolfe & Ferres (1993) compacted soil under field conditions to create a range of compaction levels. Bengough, Mackenzie & Diggle (1992) and Mulholland et al. (1996a) used soil columns which differed in bulk density but were uniform in moisture content, whilst Masle (1998) controlled resistance to root penetration by altering the water content of compacted soil. These and other studies show that root and shoot growth are generally reduced whenever compacted soil is encountered. Wilson, Robards & Goss (1977) demonstrated that roots 1377

1378 A. Hussain et al. may expand radially when confronted with pores smaller than their own diameter, thereby exerting pressure on the surrounding soil particles and weakening the soil ahead of the root tip (Hettiaratchi 1990). This response has been suggested to be under hormonal control (Russell & Goss 1974), possibly by ethylene (Kays, Nicklow & Simons 1974; Sarquis, Jordan & Morgan 1991, Sarquis, Morgan & Jordan 1992), although later studies suggested the involvement of abscisic acid (ABA) (Hartung & Davies 1991; Tardieu et al. 1991, 1992). It is well established that ABA accumulates in roots exposed to compacted soil (Tardieu et al. 1992) and exogenous applications have been shown to promote the growth of short thick roots similar to those produced under compacted soil conditions (Hartung & Davies 1991). As well as decreases in root growth, significant reductions in shoot growth and stomatal conductance may occur in plants growing on compacted soil (Masle & Passioura 1987; Mulholland et al. 1996a; Masle 1998) in the absence of discernible effects on the water or nutrient status of the shoot (Andrade et al. 1993). Chemical signals, such as ABA, accumulated in roots and transported to the shoot via the transpiration stream have been suggested to be responsible for inducing similar responses in water stressed plants (Zhang & Davies 1990a,b; Masle 1998). Several reports suggest that increases in xylem sap ABA concentration in plants growing in compacted soil are closely correlated with concurrent reductions in stomatal conductance (Mulholland et al. 1996a), and possibly also with the observed decreases in the rates of leaf emergence (Cook et al. 1996) and expansion (Tardieu et al. 1991, 1992; Hartung & Davies 1991). In contrast, Mulholland et al. (1996a,b) demonstrated that the increased xylem sap ABA concentrations observed in barley plants experiencing subcritical levels of soil compaction were necessary for the maintenance of shoot growth, while Munns (1992) argued that ABA is not the only growth inhibitor present in the xylem sap of water-stressed maize plants. The approaches employed to investigate the role of ABA in mediating responses to compacted soil have often attempted to simulate naturally compacted soils (Castillo et al. 1982; Seymour-Berg & Hsiao 1986; Tardieu, Katerji & Bethonod 1990; Andrade et al. 1993). Mulholland et al. (1996a) described a straightforward method for producing soil columns of known bulk density and moisture content. However, uniformly compacted soil columns may not be fully representative of naturally compacted soils, which are often highly heterogeneous in terms of bulk density and oxygen availability; thus roots commonly experience transitions between horizons which differ in the intensity of compaction. In the work reported here, the compaction technique of Mulholland et al. (1996a) was modified to produce soil columns containing horizons of differing bulk density, thereby enabling the timing of the physiological responses induced when roots encounter such transitions to be established. A novel split-pot approach was devised to allow plants to be grown with their root systems divided vertically between soil differing in its degree of compaction, in a manner analogous to previous root signalling studies

of the impact of soil drying (Zhang & Davies 1987; Gowing, Davies & Jones 1990). Wild-type (cv. Steptoe) and ABAdeficient mutant genotypes (Az34) of barley were used to elucidate the role of ABA in mediating plant responses to local variation in soil compaction.

MATERIALS AND METHODS Plant material Isogenic wild-type and ABA-deficient mutant genotypes of barley (Hordeum vulgare L. cv. Steptoe and Az34, respectively) were used. Az34 is a molybdenum cofactor (MoCo)deficient mutant (Sommers et al. 1983), in which enzymes involved in ABA biosynthesis, particularly ABA aldehyde oxidase, compete with others for the limited pool of available MoCo (Walker-Simmons, Kudrna & Warner 1989).

Seedling establishment and growth Seeds were germinated for 48 h on moist blotting paper in foil-covered Petri dishes under controlled conditions (18/12 °C day/night temperature, 14 h photo- and thermoperiod, 220 mmol m-2 s-1 PAR, 60% relative humidity) until the radicle was 2–5 mm long and the coleoptile was beginning to emerge. The germinated seeds were transplanted into the prepared soil columns at a density of three plants per pot or, in the split-pot experiment (see below), one plant per pot, and covered with a thin layer of loose soil to assist establishment. The plants were grown under the conditions stated above and harvests made at intervals of 5 d. When the coleoptile reached 60 mm in length, approximately 4 d after emergence (DAE), the soil surface was covered with 4 mm diameter polystyrene beads to minimize evaporation. The columns were weighed and rewatered twice daily until 10 DAE and three times daily thereafter as transpiration increased to maintain the soil close to its initial water content. This procedure has been shown to prevent significant soil drying (Mulholland et al. 1996a).

Soil preparation and compaction A brown earth of the Arrow series (Thomasson 1971), which is susceptible to compaction (O’Sullivan & Ball 1982), was used. Its physical properties were: sand (60–2000 mm) 78%, silt (2–60 mm) 13·3% and clay (< 2 mm) 8·7% by weight; organic matter content was 14% (Brereton 1986). Topsoil was air-dried for 1–3 d, passed through a 5 mm sieve, and oven-dried at 105 °C for 48 h. As its initial pH was 5·9, 2 g kg-1 of lime was added to raise this to the optimum for barley of 6·5–7·0. Nitrogen, phosphorus and potassium were added to the dry soil at rates of 31·3, 15 and 35 mg kg-1, respectively, following chemical analysis to identify the requirements. The soil was compacted in rigid 75 mm internal diameter transparent perspex tubes to allow root extension to be observed directly at daily intervals; the tubes were wrapped

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1377–1388

Novel approaches for soil compaction studies 1379 in black polythene to exclude light at all other times. The tubes were divided vertically into two halves, which were taped together during experiments, but could be separated to facilitate extraction of the roots. Soil was mixed in 15 kg batches with ice at a rate of 0·145 g g-1 and allowed to equilibrate in polythene bags for 48 h to facilitate compaction; the quantity of ice required was determined using the Proctor Compactibility Test (Proctor 1933). Soil was packed into the tubes using a metal stage (75 mm diameter) attached to an Instron Model 1140 (Instron, High Wycombe, UK) fitted with a 5000 N load cell. The plate was lowered vertically within the tubes to a pre-determined depth at a rate of 200 mm min-1. Predetermined quantities of soil were compacted in 20 mm deep layers; the surface of each layer was scarified to a depth of 5 mm before adding the next to maximize uniformity. Soil moisture content was then brought to 0·192 g g-1 by melting ice on the surface of the columns and allowing them to equilibrate for 48 h in sealed polythene bags prior to sowing. Initial air-filled porosity values for the 1·4 and 1·7 g cm-3 bulk density treatments examined in the present study were, respectively, 19·3 and 2·0% (Mulholland 1994; Mulholland et al. 1996a). No significant variation in soil moisture content between horizons was found for any of the treatments examined.

Compaction treatments Previous work (Mulholland et al. 1996a) has shown that shoot growth in barley cv. Steptoe is greatest in moderately compacted soil (1·4 g cm-3; penetrometer resistance 1·43 MPa at a soil moisture content of 0·192 g g-1) but is greatly reduced in severely compacted soil (1·7 g cm-3; penetrometer resistance 2·40 MPa). Both were used in the present study to examine the physiological responses induced when roots encounter transitions between soil horizons differing in the severity of compaction.

drawing a length of dental floss between the two halves of the surrounding perspex tube. Half columns of moderately compacted and severely compacted soil were taped together to form vertically divided columns. Heavy duty polythene membranes securely taped to the walls of the perspex tubes provided a rigid barrier against lateral movement of the adjacent half columns of soil and consequent changes in bulk density, and also prevented any penetration of roots or exchange of water, nutrients and gases between the two compartments. Uniform 1·4 and 1·7 g cm-3 columns were prepared by taping together half columns of the same bulk density, again separated by rigid polythene membranes. Germinated seeds were transplanted into a 30-mm layer of loose soil spread over the surface of the columns; as the seedlings grew, their roots became divided between the two halves of the underlying split columns. At 10 DAE, the roots growing in the severely compacted side of the 1·7 : 1·4 g cm-3 columns were severed for 10 replicate plants of both genotypes to allow their physiological responses to be compared with intact plants in which part of the root system remained in compacted soil.

Seedling growth Seedlings of both genotypes were grown for 30 d or, in the split-pot experiment, 20 d. The columns were randomly arranged in the growth room and randomly selected for harvest; 10 replicates of each genotype and treatment were harvested at 5 d intervals, or five replicates in the split-pot experiment. Leaf area (Li-Cor Model 3100 Meter, Lincoln, NB, USA) and shoot dry weight (85 °C for 48 h) were determined immediately. After collecting xylem exudate for ABA analysis, the soil columns were divided into 30 mm horizons, the roots carefully washed from each horizon, and their dry weights determined.

Infra-red gas analysis and leaf water relations Horizontally stratified compaction layers Three treatments were used to examine the timing, nature and sequence of responses induced when roots encounter differentially compacted soil profiles. In the first, 90 mm of moderately compacted soil overlay 90 mm of severely compacted soil; the reciprocal treatment comprised 90 mm of 1·7 g cm-3 soil overlying 90 mm of 1·4 g cm-3 soil. In the third treatment, three 60 mm horizons were stacked vertically to provide 180 mm deep profiles containing two 60 mm horizons of 1·7 g cm-3 soil separated by a 1·4 g cm-3 horizon. This design permitted responses to be examined as roots emerged from severely compacted into moderately compacted soil and then encountered a second severely compacted horizon. The control treatments comprised uniform 1·4 or 1·7 g cm-3 soil columns.

Split root system The 90 mm deep columns of moderately or severely compacted (1·4 and 1·7 g cm-3) soil were divided vertically by

Stomatal conductance and net photosynthesis were measured using a CIRAS 1 combined infra-red gas analyser and a Parkinson leaf cuvette with a 2·5 cm2 chamber (PP Systems, Hitchin, Herts, UK). Measurements began when the first leaf reached full size at 4 DAE, and were repeated on alternate days commencing 4 h after the start of the photoperiod. On each sampling date, 10–20 measurements were made for the youngest expanded leaf for each genotype and treatment within a 60–90-min period under ambient light (220 mmol m-2 s-1 PAR). Leaf area within the cuvette was calculated as the product of length and mean width to enable stomatal conductance and net photosynthesis to be expressed per unit area. Water (yw) and osmotic potentials (yS) were measured for the youngest expanded leaf using a pressure chamber (PMS Instrument Co, Corvalis, OR, USA) and freezing point osmometer (Hermann Roebling, Berlin, Germany). Turgor potential (yp) was calculated as yp = yw – ys. Five replicate measurements were made for each variable and treatment at each harvest.


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Xylem sap collection and ABA determination Xylem sap was collected and analysed for ABA content at each harvest as described by Mulholland et al. (1996a). After excising the shoots 20 mm above the soil, silicone rubber tubing (3–8 mm internal diameter) was attached to the detopped root system to collect the first 200 mL of xylem sap exuded under root pressure. The sap was transferred to pre-weighed 0·5 mL vials (Sarstedt, Leicester, UK) and stored at –20 °C prior to determination of its ABA concentration by radio-immunoassay (RIA) using AFRC MAC252, a monoclonal antibody specific to ABA (Quarrie et al. 1988). The accuracy of RIA measurements for barley was verified by parallel GC-MS analysis (Mulholland 1994), as recommended for each new tissue for which the RIA approach is used (Walker-Simmons & Abrams 1991).

sible to establish the nature and timing of effects on the shoots. Shoot dry weight (Fig. 1a) and leaf area (Fig. 1c) at 30 DAE were significantly reduced (P < 0·001) in both genotypes relative to 1·4 g cm-3 control plants when the roots encountered a transition between 1·4 g cm-3 and 1·7 g cm-3 soil. Root : shoot ratio was increased (P < 0·001; Fig. 1d), although total root dry weight was not significantly affected (Fig. 1b). Root distribution was altered in both genotypes, with root growth being reduced in the 120–180 cm horizons relative to the 1·4 g cm-3 control (Fig. 2; P < 0·001). However, the impact of the compacted horizon on growth was smaller than in plants grown in uniform 1·7 g cm-3 columns. The wild-type and ABAdeficient genotypes exhibited generally similar responses. In Steptoe, stomatal conductance decreased (P < 0·001) within 4 d of the first roots encountering the 1·7 g cm-3 horizon (Fig. 3b), declining from 210 to 120 mmol m-2 s-1,

Statistical analysis The data were analysed by analysis of variance using Genstat 5 (Lawes Agricultural Trust, IACR Rothamsted, UK).

RESULTS Control treatments Leaf area and shoot dry weight were greatly reduced (P < 0·001) in the severely compacted treatment (1·7 g cm-3) relative to the 1·4 g cm-3 control plants (Figs 1 a & c). The reductions in root dry weight in severely compacted soil were smaller than those for shoot growth, with the result that root : shoot ratio was approximately doubled in both genotypes (Figs 1a & d). Roots penetrated to the 150–180 mm layer in the 1·4 g cm-3 treatment, but failed to penetrate below the 60–90 mm horizon in the 1·7 g cm-3 treatment of both genotypes (Fig. 2). Stomatal conductance was significantly reduced (P < 0·001) in the 1·7 g cm-3 treatment of Steptoe throughout the experimental period (Fig. 3a), but did not differ significantly between the uniform 1·4 and 1·7 g cm-3 treatments of Az34, in which conductances were consistently greater except during the final 4-d period. Xylem sap ABA concentration was greatly increased (P < 0·001) in the 1·7 g cm-3 treatment of Steptoe, particularly between 5 and 15 DAE (Fig. 3a), reflecting the substantial reduction in stomatal conductance at this time (Fig. 3a). Xylem sap ABA concentrations in the 1·7 g cm-3 treatment of Az34 were double those for 1·4 g cm-3 control plants during this period, although the values were significantly lower than in Steptoe (P < 0·001); these results clearly demonstrate the ability of Az34 to accumulate limited quantities of ABA when its roots encounter severely compacted soil. Figure 1. Influence of transitions between horizons differing in

Transition between moderately and severely compacted soil Examination of the responses induced as roots penetrated from moderately into severely compacted soil made it pos-

the degree of soil compaction on (a) shoot and (b) root dry weight (c) leaf area and (d) root : shoot ratio (R : S) in Steptoe and Az34. C1 and C2 denote the uniform 1·4 and 1·7 g m-3 treatments; 1, 2 and 3, respectively, represent the 1·4 : 1·7 g m-3, 1·7 : 1·4 g m-3 and the 1·7 : 1·4 : 1·7 g m-3 treatments. Bars show pooled values for the standard error of the difference (SED).


Novel approaches for soil compaction studies 1381

Figure 2. Influence of transitions between horizons differing in the degree of soil compaction on root dry weight profiles in Steptoe and Az34. Bars show single standard errors of the mean (SEM).

before recovering to 170 mmol m-2 s-1 during the ensuing 16 d period. In contrast, Az34 showed no significant change in conductance as its roots penetrated from 1·4 to 1·7 g cm-3 soil, suggesting an inability to regulate stomatal aperture effectively in response to local differences in the severity of compaction (Fig. 3b). The reduction in stomatal conductance when the roots of Steptoe penetrated into the 1·7 g cm-3 horizon was reflected by a corresponding increase in xylem sap ABA concentration (Fig. 3b). Xylem sap ABA concentration increased four-fold, from 45 to 180 mmol m-3, within 5 d of the first roots entering the 1·7 g cm-3 horizon, before decreasing again during the ensuing 15 d period to 90 mmol m-3 at 30 DAE. ABA concentrations were consistently lower in Az34 than in Steptoe, with a maximum of 55 mmol m-3 being recorded 5 d after the roots encountered the transition between horizons; the 25% increase in xylem sap ABA in Az34 at this time was much smaller than in Steptoe. Despite the observed effects on stomatal conductance, net photosynthesis and the components of leaf water potential showed no detectable response to any of the bulk density transitions in the severity of soil compaction examined (Hussain 1998).

Transition between severely compacted and moderately compacted soil This experiment aimed to determine the extent to which the symptoms induced by severe compaction are alleviated when roots emerge into less compacted soil. Shoot growth

was significantly reduced in the 1·7 : 1·4 g cm-3 treatment of both genotypes relative to 1·4 g cm-3 control plants (P < 0·001; Figs 1a & c), but was not significantly different from the uniform 1·7 g cm-3 treatment. Root distribution contrasted with the reciprocal 1·4 : 1·7 g cm-3 treatment (Fig. 2), as a greater proportion of the roots of both genotypes were found in the 90–180 mm horizons. Stomatal responses in the 1·7 : 1·4 g cm-3 treatment (Fig. 3c) contrasted with the reciprocal 1·4 : 1·7 g cm-3 treatment (Fig. 3b). The wild type, Steptoe, initially showed a substantial decrease in conductance from 200 mmol m-2 s-1 at 4 DAE to 120 mmol m-2 s-1 at 8 DAE, a value which was maintained over the next four days. Following the penetration of roots into the 1·4 g cm-3 horizon at 14 DAE, conductance increased significantly (P < 0·001) to 160 mmol m-2 s-1 during the ensuing 2 d period and continued to increase to 200 mmol m-2 s-1 at 30 DAE. No significant treatment-specific effects on conductance were detected in Az34, although the values decreased with time from 300 to 220 mmol m-2 s-1. Wild-type plants whose roots established in severely compacted soil before extending into a 1·4 g cm-3 horizon showed a close correlation between stomatal conductance and xylem sap ABA concentration (Fig. 3c). In Steptoe, a 50% reduction in xylem sap ABA, from 160 to 80 mmol m-3, was apparent 5 d after the roots penetrated the 1·7 : 1·4 g cm-3 interface; Az34 exhibited a smaller 20% decrease from 50 to 40 mmol m-3. Net photosynthesis and foliar water relations were again not significantly affected (Hussain 1998).


1382 A. Hussain et al.

Figure 3. Influence of transitions between horizons differing in the degree of soil compaction on stomatal conductance and xylem sap ABA concentration in Steptoe and Az34. (a), uniform 1·4 and 1·7 g m-3 treatments; (b) (c) and (d), 1·4 : 1·7 g m-3, 1·7 : 1·4 g m-3 and 1·7 : 1·4 : 1·7 g m-3 treatments. Arrows indicate when the first roots encountered transitions between horizons of differing bulk density. Bars show pooled values for the standard error of the difference (SED).

Sequential transitions between severely/moderately/severely compacted soil By exposing roots to sequential transitions between horizons differing in the severity of compaction, it was possible to determine the ability of the signalling system to respond

to repeated contact with severely compacted soil. Shoot dry weight and leaf area were significantly reduced (P < 0·001) relative to the 1·4 g cm-3 control treatment when plants of both genotypes were grown in columns containing sequential 1·7, 1·4 and 1·7 g cm-3 horizons. The inhibition of growth was similar to the uniform 1·7 g cm-3 treatment (Figs 1a &


Novel approaches for soil compaction studies 1383 c), although root penetration to depth was greater in the sequential treatment of both genotypes (Fig. 2). Stomatal conductance in Steptoe initially declined to 100 mmol m-2 s-1 at 8 DAE, when the roots emerged into the 1·4 g cm-3 horizon (Fig. 3d), but increased (P < 0·001) during the ensuing 4 d period to a maximum of 175 mmol m-2 s-1 at 14 DAE. The roots then encountered the second severely compacted horizon, after which conductance decreased within 4 d to a minimum of 100 mmol m-2 s-1 at 20 DAE, before recovering to 140 mmol m-2 s-1 at 30 DAE. Az34 exhibited no significant change in conductance during root penetration of the various horizons, although the values declined from 285 to 200 mmol m-2 s-1 during the 30 d experimental period. Wild-type plants in the sequential compaction treatment initially exhibited relatively high xylem sap ABA concentrations (200 mmol m-3; Fig. 3d), which decreased to 75 mmol m-3 during the 10 d period following root penetration into the 1·4 g cm-3 horizon at 8 DAE, before increasing sharply to 175 mmol m-3 at 20 DAE following root extension into the second 1·7 g cm-3 horizon at 14 DAE. Xylem sap ABA concentration in Az34 declined from 75 mmol m-3 at 5 DAE to 40 mmol m-3 at 30 DAE.

Split-pot experiments Although highly effective in identifying the nature of root : shoot signalling systems, split-pot approaches have not previously been applied to soil compaction studies. When Steptoe was grown in the 1·7 : 1·4 g cm-3 split-pot system, there was no significant effect on shoot growth relative to 1·4 g cm-3 control plants at 20 DAE (Fig. 4a), whereas leaf area and shoot dry weight were reduced by 33% in Az34 (Fig. 4a & c; P < 0·05). Excision of the roots of Az34 in the compartment containing 1·7 g cm-3 soil at 10 DAE restored shoot dry weight to the 1·4 g cm-3 control level by 20 DAE (Fig. 4a); leaf area was also partially restored (Fig. 4c). During the first 10 DAE, stomatal conductance in the 1·7 : 1·4 g cm-3 split-pot treatment of Steptoe was similar (100–150 mmol m-2 s-1) to the uniform 1·7 g cm-3 treatment (Fig. 5), and significantly lower (P < 0·01) than in 1·4 g cm-3 control plants. However, conductance in Steptoe increased significantly (P < 0·05) within 4 d of excising the roots in the 1·7 g cm-3 compartment and was comparable to 1·4 g cm-3 control plants by 20 DAE. Conductance was consistently much greater in all treatments of Az34 (300–250 mmol m-2 s-1) than in Steptoe (200–100 mmol m-2 s-1); the values for Az34 did not vary significantly between treatments and declined steadily during the experiment (Fig. 5b). The relatively low stomatal conductances exhibited by Steptoe in the 1·4 : 1·7 g cm-3 treatment were reflected by significantly higher xylem sap ABA concentrations (P < 0·001) during the first 10 DAE relative to 1·4 g cm-3 control plants; the values were even higher in the uniform 1·7 g cm-3 treatment. Wild-type plants exhibited a significant decline (P < 0·05) in xylem sap ABA within 5 d of sev-

ering the roots in the 1·7 g cm-3 compartment and the values returned to the 1·4 g cm-3 control level by 20 DAE (Fig. 5a). Plants in which the roots in compacted soil were not severed showed no detectable decline in ABA concentration between 10 and 15 DAE. Xylem sap ABA concentrations in the 1·4 : 1·7 g cm-3 treatment of Az34 at 10 DAE were comparable to the uniform 1·7 g cm-3 treatment, although significantly below those observed for Steptoe (P < 0·001). Severing the roots in the 1·7 g cm-3 compartment induced a significant decline (P < 0·05) in ABA concentration to a level similar to 1·4 g cm-3 control plants (Fig. 5b).

DISCUSSION The use of novel techniques to study plant responses to compacted soil has provided a substantial body of new data concerning the timing and nature of the effects induced, the ability of plants to recover following contact with localized areas of compacted soil, and the positive nature of the signals that are transmitted from the roots to the shoots. Stomatal conductance and leaf expansion are commonly reduced in plants that are growing in compacted soil (Tardieu et al. 1992; Andrade et al. 1993; Young et al. 1997), and it has been suggested that both responses may be mediated by increases in xylem sap ABA concentration (Tardieu et al. 1992). However, the results reported here, together with those from previous experiments with barley (Mulholland et al. 1996a,b), suggest that xylem sap ABA may have differing roles in the regulation of stomatal conductance and leaf expansion. An inverse correlation between stomatal conductance and xylem sap ABA concentration was consistently observed for the wild-type genotype, Steptoe, with conductance invariably being reduced when at least part of the root system encountered severely compacted soil. However, the ABA-deficient mutant, Az34, did not demonstrate similar stomatal responses, suggesting that the much smaller changes in its xylem sap ABA levels induced when its roots encountered transitions between differentially compacted horizons were insufficient to induce any alteration in stomatal conductance. Thus xylem sap ABA concentration exhibited characteristic increases when the roots of wild-type plants encountered transitions between moderately and severely compacted horizons, and corresponding decreases following the reverse transition. The increase in xylem sap ABA concentration when roots encountered a second severely compacted horizon was comparable to that induced by the first; there was therefore no evidence of habituation, as has been reported in response to wind stress (Teweski & Jaffe 1986), or that the second pulse of ABA was larger than the first. The split-pot system involved simultaneous exposure of the roots of individual plants to vertically divided soil columns in which the two halves differed in the degree of compaction. Even though the majority of the root system of wild-type plants was growing in moderately compacted soil, those roots which encountered severely compacted soil


1384 A. Hussain et al. raised xylem sap ABA concentration sufficiently to induce a substantial reduction in stomatal conductance, as has been reported for other species in response to soil drying (Blackman & Davies 1985; Zhang & Davies 1987; Gowing et al. 1990). In contrast, the much more limited accumula-

tion of ABA exhibited by Az34 was again insufficient to induce detectable effects on stomatal conductance. When the roots of wild-type plants growing in the severely compacted compartment were severed, xylem sap ABA concentration decreased and conductance returned to control

Figure 4. (a) Shoot and (b) root dry weight (c) leaf area and (d) root : shoot ratio (R : S) for Steptoe and Az34 plants grown in splitpots at 20 d after emergence (DAE). C1 and C2 denote the uniform 1·4 and 1·7 g m-3 treatments; 1, plants with root systems divided between moderately and severely compacted soil (1·4 : 1·7 g m-3); and 2, plants in which the roots growing in severely compacted soil were severed at 10 DAE. (b) shows total root dry weights and their distribution between the two compartments of the uniform 1·4 and 1·7 g cm-3 and 1·4 : 1·7 g cm-3 split-pot treatments 1 and 2; the stippled areas in the latter two treatments denote values for the 1·7 g cm-3 compartment. Bars show pooled values for the standard error of the difference (SED). © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1377–1388

Novel approaches for soil compaction studies 1385

Figure 5. Time courses of stomatal conductance and xylem sap ABA concentration for Steptoe and Az34 plants grown in split-pots. and , respectively, denote the uniform 1·4 and 1·7 g m-3 treatments; represents plants with root systems divided between moderately and severely compacted soil (1·4 : 1·7 g m-3), while ¥ denotes plants in which the roots growing in severely compacted soil were severed at 10 DAE (arrow). Bars show pooled values for the standard error of the difference (SED).

levels within 5 d. Thus, stomatal behaviour was clearly affected by a root-sourced signal, and the evidence presented here strongly suggests that ABA was an essential component of this signal. The role of ABA in mediating the effects of severe soil compaction on leaf expansion appears more subtle. The treatments involving transitions between differentially compacted horizons produced effects similar to those obtained using columns of uniformly compacted soil (Mulholland et al. 1996a). Thus, when a sufficient proportion of the roots encountered severely compacted soil, leaf expansion was inhibited and shoot dry weight was reduced

in both genotypes. The observation that the ABA-deficient mutant, Az34, also exhibited reduced leaf expansion when its roots encountered severe soil compaction suggests either that increases in xylem sap ABA concentration are not required to induce this response or, alternatively, that the mutant, with its much lower background ABA levels, is sensitive to relatively small stress-induced increases in ABA concentration. Zhang & Davies (1989) have previously suggested that it is the relative increase in the ABA signal rather than the absolute concentration of ABA that is important in determining plant responses. However, the increases in xylem sap ABA concentrations exhibited by


1386 A. Hussain et al. Az34 were insufficient to induce stomatal closure in response to severe soil compaction. The leaf expansion response would therefore have to be triggered by much smaller changes in ABA concentration than was the case for stomatal conductance or, alternatively, a signal other than ABA may have been involved. The results from the split-pot experiment indicate that increased xylem sap ABA concentrations are important in maintaining shoot growth when part of the root system encounters compacted soil, rather than reducing growth as has been suggested for water-stressed plants (Saab & Sharpe 1989). These findings closely parallel those obtained by Mulholland et al. (1996b) for wild-type barley plants grown in uniform columns providing a subcritical level of soil compaction (1·6 g cm-3). In the present study, the leaf area at 20 DAE for wild-type plants whose root systems were divided between compartments containing moderately and severely compacted soil was only 15% lower than in 1·4 g cm-3 control plants, in agreement with the suggestion by Hartung, Zhang & Davies. (1994) that shoot characteristics may not be greatly affected when plants are grown in heterogeneously compacted soil. The ABAdeficient mutant, Az34, exhibited a much greater reduction (40%) in leaf area in the 1·7 : 1·4 g cm-3 split pot treatment, even though the values for both genotypes were similar when both compartments contained either moderately or severely compacted soil. The split-pot system may therefore be used to provide subcritical levels of soil compaction which promote similar responses to those observed previously using uniform 1·6 g cm-3 soil columns (Mulholland et al. 1996a). Both methods of exposing barley to subcritical levels of compaction suggest that increased xylem sap ABA concentrations are necessary to maintain leaf expansion under such conditions. Indeed, Mulholland et al. (1996b) demonstrated that restoration of wild-type xylem sap ABA concentrations by supplementing xylem sap from mutant plants with synthetic ABA produced a phenotypic reversion of the mutant to wild-type leaf expansion rates. Increased xylem sap ABA also appeared to act as a positive message in terms of maintaining leaf expansion when part of the root system encounters severely compacted soil, possibly by countering the influence of a second rootsourced signal with a ‘negative’ impact on leaf expansion (Munns 1992). When the roots in the severely compacted compartment of the split-pot system were excised, these signal(s) were abolished and leaf growth was restored to levels comparable to 1·4 g cm-3 control plants. The adverse effects of soil compaction may arise primarily from two interrelated factors, mechanical impedance and oxygen availability (Soehne 1958; Greenland 1977). Compaction inevitably increases resistance to root growth to an extent related to the severity of compaction and the consequent increases in bulk density and soil strength; oxygen supplies are also reduced by the associated decline in air-filled porosity. As oxygen availability was not determined in the present study, the relative importance of these factors in promoting the observed responses cannot be established with certainty. However, similar stomatal and

growth responses have been reported in several previous compaction studies in which oxygen supplies were shown not to be limiting (Masle & Passioura 1987; Masle 1990; Andrade et al. 1993); these workers therefore concluded that the observed responses were induced by mechanical impedance alone. As none of these studies attempted to establish the nature of the root-sourced signals involved in inducing the observed responses, the role of ABA was not established. The results obtained provide strong evidence that increased ABA export from roots encountering compacted soil is responsible for the concurrent reductions in stomatal conductance. However, the associated reductions in shoot growth cannot be attributed unequivocally to increased xylem sap ABA concentrations in view of the limited increases observed in Az34. A possible explanation is that leaf expansion in the mutant is more sensitive than stomatal conductance to small changes in endogenous ABA levels, although the phenotypic reversion of the mutant to wild-type leaf expansion rates induced when xylem sap ABA concentrations were increased to wild-type levels (Mulholland et al. 1996b) is inconsistent with this proposal. An alternative suggestion is that additional signal(s) are involved. A possible candidate is ethylene, whose production is known to increase when roots encounter compacted soil (Kays et al. 1974; Sarquis et al. 1991), and which has been shown to be a potent inhibitor of shoot elongation (Jackson 1993). As a relationship between ABA and ethylene in controlling the growth of maize roots in drying soil has been suggested (Spollen & Sharp 1994), it is possible that these two hormones may also participate in an antagonistic relationship mediating leaf expansion and shoot growth when roots encounter subcritical levels of soil compaction.

ACKNOWLEDGMENTS A.H. and B.J.M. gratefully acknowledge financial support from the UK Natural Environment Research Council.

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