The existence of an inverse correlation between stomatal conductance and xylem sap ABA concentration in plants experiencing soil compaction has been demonstrated using wild-type and ABA-deficient mutants of barley (Hordeum vulgare L. cv Steptoe and cv Az34; Mulholland et al., 1996a). However, the link between root-sourced ABA and compaction-induced reductions in leaf expansion proved more complex, as both genotypes exhibited reduced growth in severely compacted soil (1.7-g cm⁻³ bulk density), whereas significant genotypic differences were apparent at a subcritical bulk density of 1.6 g cm⁻³ (Mulholland et al., 1996a). In the latter treatment, the wild-type plants maintained uncompacted (1.1 g cm⁻³) rates of leaf expansion, while the ABA-deficient mutant exhibited reductions in growth comparable to plants grown in severely compacted soil (Mulholland et al., 1996a, 1996b). These results demonstrate that elevated xylem sap ABA concentrations were not responsible for the observed inhibition of shoot growth in the ABA-deficient mutant. Nor was any relationship between shoot growth and xylem sap ABA detected when the same genotypes were grown in columns containing horizons of differing bulk density, although stomatal conductance was again inversely correlated with xylem sap ABA concentration (Hussain et al., 1999). Munns (1992) previously questioned whether ABA is the only potential inhibitor of leaf expansion present in the xylem sap of wheat and barley plants grown in drying soil.

Increased ethylene production rates in impeded roots have been correlated with their characteristic short, thick root morphology and concurrent reductions in shoot growth (Kays et al., 1974; Sarquis et al., 1991; Morgan et al., 1993). As ethylene may act as a root-sourced chemical signal transported as its soluble precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), under anaerobic soil conditions (Jackson, 1997), it is possible that this hormone may also be involved in mediating shoot responses to soil compaction (He et al., 1996). The present study examined the role of ethylene in mediating shoot growth in tomato (Lycopersicon esculentum Mill.) plants experiencing compacted soil conditions. A split-pot approach enabled the root system to be divided between uncompacted (1.1 g cm⁻³) and compacted (1.5 g cm⁻³) soil; the majority of the roots grew unhindered in uncompacted soil, while the remainder experienced impeded growing conditions. This system enables responses to be examined in species such as tomato, which are particularly sensitive to compaction (Mulholland et al., 1999).

Isogenic ABA-deficient mutant and wild-type genotypes represent a powerful tool for studies of the role of ABA as a root-sourced signal regulating plant responses to compaction (Mulholland et al., 1996a, 1996b, 1999; Hussain et al., 1999). This approach was extended in the present study to examine the role of ethylene by using a genetically modified, low-ethylene genotype, ACO1_AS, and an isogenic wild-type cultivar (Ailsa Craig). Applications of ethephon were used to promote ethylene production, while the action of this gaseous plant growth regulator was blocked using silver ions applied as silver thiosulfate (STS). The objective was to test the hypothesis that ethylene has a central role in mediating plant responses to soil compaction.

Seeds of both genotypes were germinated on moist filter paper in foil-covered Petri dishes under the following experimental conditions: 25°C/20°C day/night temperature, 16-h photo- and thermoperiod, approximately 150 μmol m⁻² s⁻¹ photosynthetically active radiation, and 80% relative humidity). When the radicle was a few millimeters in length, the seedlings were transplanted into the loose soil covering the split-pot system; as they developed, their root systems became divided between the two underlying compartments. The columns were weighed and rewatered twice daily until 10 d after emergence (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). The roots in the compacted compartment of the 1.1/1.5-g cm⁻³ treatment were severed at 15 DAE for 20 plants of each genotype.

Ethephon, an ethylene-releasing agent, was supplied as a 400-μL L⁻¹ aqueous solution; this compound is hydrolyzed following delivery to the shoots, releasing ethylene (Maynard and Swain, 1963). A 1 mm STS solution was used to block the action of ethylene, as silver ions have been proposed to reduce the capacity of ethylene to interact with its receptors (Beyer, 1976); STS is readily absorbed and transported by plants (Morgan et al., 1993). Both solutions were supplied in place of water to the surface of the compartment containing compacted soil in the 1.1/1.5-g cm⁻³ treatment from 5 DAE; the volumes of solution required were determined gravimetrically as described above.

Figure 1 Influence of compaction treatment and excision of the roots in the compacted compartment of the 1.1/1.5-g cm−3 split-pot treatment (A) and treatment with STS and ethephon (eth) (B) on the water potential of the youngest expanded leaf in wild-type (more ...)

Figure 3 Influence of compaction treatment and excision of the roots in the compacted compartment of the 1.1/1.5-g cm−3 split-pot treatment on stomatal conductance (A), xylem sap ABA concentration (B), and ethylene evolution (C) from leaf tissue in wild-type (more ...)

Figure 4 Influence of STS and ethephon (eth) on leaf area (A) and root dry weight (B) in wild-type (cv Ailsa Craig) and transgenic (ACO1AS) genotypes of tomato grown in 1.1/1.5-g cm−3 split-pot soil columns. A, Black columns, split-pot; white columns, (more ...)

Figure 2 Influence of compaction treatment and excision of the roots in the compacted compartment of the 1.1/1.5 g cm−3 split-pot treatment on leaf area (A), shoot dry weight (B), and root dry weight (C) in wild-type (cv Ailsa Craig) and transgenic (ACO1 (more ...)

Excision of the roots at 15 DAE in the compacted compartment of the 1.1/1.5-g cm⁻³ treatment of cv Ailsa Craig (split-cut treatment) promoted a recovery of shoot growth (Fig. 2, A and B). Leaf area and shoot dry weight were, respectively, 58% and 62% of the uncompacted control values at 15 DAE, but increased to approximately 75% by 20 DAE, when the values for both variables were significantly greater than in plants with intact root systems (split-pot treatment). During the ensuing 10-d period, leaf area and shoot dry weight in split-cut plants reached approximately 90% of the uncompacted control values, whereas equivalent plants with intact root systems showed no recovery.

Shoot growth in 1.1-g cm⁻³ control plants of ACO1_AS was comparable to the wild-type genotype, cv Ailsa Craig, and was again greatly reduced in the uniformly compacted 1.5-g cm⁻³ treatment, as in cv Ailsa Craig (P < 0.001; Fig. 2, A and B); thus, leaf area and shoot dry weight were 58% and 49%, respectively, of the uncompacted control values at 10 DAE, but declined to 30% and 14%, respectively, over the ensuing 20-d period. However, the response of shoot growth in the 1.1/1.5-g cm⁻³ treatment contrasted sharply with that of wild-type plants, as leaf area in ACO1_AS was unaffected relative to 1.1-g cm⁻³ control plants at all harvests. Excision of the roots in the compacted compartment of the 1.1/1.5-g cm⁻³ treatment at 15 DAE had no effect on leaf area (Fig. 2A). The responses of the ACO1_AS therefore clearly differed from the wild-type control plants.

Root dry weight was evenly distributed between the two compartments in the uniform 1.1- and 1.5-g cm⁻³ treatments, although the values were consistently much lower in the 1.5-g cm⁻³ treatment of both genotypes (P < 0.001; Fig. 2C). In the 1.1/1.5-g cm⁻³ treatment, root growth was lower in the compacted compartment than in the uncompacted compartment for both genotypes, particularly in ACO1_AS. Excision of the roots in the 1.5-g cm⁻³ compartment also promoted genotypic differences in root growth; thus, total root dry weight in split-cut plants of cv Ailsa Craig was comparable to plants in the split-pot treatment, indicating that compensatory growth occurred in the uncompacted compartment (Fig. 2C). In contrast, total root dry weights for split-cut ACO1_AS plants were 69% to 75% of the equivalent values for the split-pot treatment between 20 and 30 DAE (P < 0.001), suggesting that they were unable to compensate fully for the loss of the roots in the 1.5-g cm⁻³ compartment by increasing growth in the 1.1-g cm⁻³ compartment.

Both genotypes exhibited comparable trends for stomatal conductance (Fig. 3A), which declined slightly between 10 and 12 DAE in the uniform 1.1-g cm⁻³ treatment and then remained relatively constant for the remainder of the experiment. In the 1.5-g cm⁻³ treatment, stomatal conductance decreased sharply in both genotypes between 10 and 12 DAE (P < 0.001), then declined more gradually until 14 DAE, before remaining almost constant for the rest of the observation period. The values for the 1.5-g cm⁻³ treatment were significantly lower than for the 1.1-g cm⁻³ treatment after 12 DAE in both genotypes (P < 0.001), while those for ACO1_AS were consistently, but not significantly, higher than in cv Ailsa Craig.

Stomatal conductance in the 1.1/1.5-g cm⁻³ treatment was generally similar to that in the uniform 1.5-g cm⁻³ treatment of both genotypes, although there was some evidence that conductance decreased more slowly between 12 and 16 DAE (Fig. 3A); conductance remained low for the remainder of the experiment. Conductance began to increase shortly after the roots in the compacted compartment were excised at 15 DAE, and was significantly greater (P < 0.001) than in equivalent split-pot plants of cv Ailsa Craig and ACO1_AS by 16 and 18 DAE, respectively. Both genotypes exhibited full recovery to the uncompacted control values by 20 DAE, and these were then maintained for the remainder of the observation period (Fig. 3A). Thus, conductances typical of uncompacted plants were restored in the 1.1/1.5-g cm⁻³ treatment by severing the roots growing in compacted soil, thereby preventing the export of root-sourced signal(s) to the shoot.

The reduced stomatal conductances in the 1.5-g cm⁻³ and 1.1/1.5-g cm⁻³ treatments of both genotypes were reflected by higher xylem sap ABA concentrations (P < 0.001; Fig. 3B). ABA concentrations in both genotypes were approximately double those for uncompacted control plants at 10 DAE, increased by 45% and 24%, respectively, at 20 DAE, and declined again by 30 DAE, although this decrease was significant only in the 1.5-g cm⁻³ treatment (P < 0.001). Severing the roots in the compacted compartment of the 1.1/1.5-g cm⁻³ treatment at 15 DAE promoted a significant decrease in xylem sap ABA (P < 0.001) during the ensuing 5-d period relative to intact split-pot plants in both genotypes; by 30 DAE, the values for split-cut plants were identical to those for uncompacted control plants. The values for control plants were closely comparable in both genotypes.

As expected, ethylene evolution was lower in ACO1_AS than in cv Ailsa Craig (Fig. 3C), particularly in plants which encountered compacted soil; ethylene evolution rates for the 1.5-g cm⁻³ treatment of cv Ailsa Craig were 3- to 5-fold greater than in uncompacted control plants (P < 0.001). The values for the 1.1/1.5-g cm⁻³ treatment of cv Ailsa Craig were significantly lower than in the 1.5-g cm⁻³ treatment at 15, 25, and 30 DAE (P < 0.01), but were consistently 2- to 5-fold greater than in uncompacted control plants. Excision of the roots in the compacted compartment greatly reduced ethylene evolution in cv Ailsa Craig, with the values at 20, 25, and 30 DAE being 64%, 39%, and 33%, respectively, of those for equivalent plants with intact root systems (P < 0.01).

Stomatal conductance followed a generally similar pattern in both genotypes (Fig. 5A), decreasing significantly (P < 0.001) by 16 DAE in the split-pot and STS treatments of cv Ailsa Craig to reach minimum values of 121 and 136 mmol m⁻² s⁻¹, respectively. Treatment of cv Ailsa Craig with ethephon promoted a more rapid decline in conductance, with a significant decrease being apparent by 14 DAE (P < 0.001). Conductance then remained almost constant in all treatments, although the values for ethephon-treated plants were generally slightly lower than in the 1.1/1.5-g cm⁻³ and STS treatments. ACO1_AS plants exhibited similar responses except that the decline in conductance was slightly delayed relative to wild-type plants in all treatments. As in cv Ailsa Craig, conductance was slightly lower in ethephon-treated plants than in the other treatments.

Figure 5 Influence of STS and ethephon (eth) on stomatal conductance (A), xylem sap ABA concentration (B), and ethylene evolution (C) from leaf tissue in wild-type (cv Ailsa Craig) and transgenic (ACO1AS) genotypes of tomato grown in 1.1/1.5-g cm−3 split-pot (more ...)

Xylem sap ABA concentrations again exhibited similar trends in both genotypes, increasing between 10 and 20 DAE (P < 0.001; Fig. 5B), when maximum values ranged from 154 to 184 μmol m⁻³ in cv Ailsa Craig and 143 to 176 μmol m⁻³ in ACO1_AS and declining by 30 DAE in all treatments of both genotypes; the maximum values therefore coincided with the observed reductions in stomatal conductance. The values for the 1.1/1.5-g cm⁻³ and STS treatments were comparable in both genotypes, and were generally slightly greater in ethephon-treated plants, reflecting their lower stomatal conductances.

Ethylene evolution was invariably much lower in ACO1_AS than in cv Ailsa Craig and increased with time in all treatments of cv Ailsa Craig and ethephon-treated plants of ACO1_AS (P < 0.001; Fig. 5C). Treatment with ethephon significantly increased ethylene evolution in both genotypes (P < 0.001), although the values for ACO1_AS were consistently lower than in cv Ailsa Craig (P < 0.001). Treatment with STS reduced ethylene evolution in cv Ailsa Craig (P < 0.001), particularly during the latter stages of the experiment, but had no detectable effect on ACO1_AS.

The split-pot system used here permitted investigation of the role of ethylene in mediating the impact of soil compaction on shoot and root growth in tomato, as described previously for ABA in barley (Hussain et al., 1999) and tomato (Mulholland et al., 1999). This system enables the effects of compaction to be examined in species particularly sensitive to impeded soil conditions by enabling most of the root system to be grown in uncompacted soil, while a smaller proportion penetrates the compartment containing compacted soil. The quantity of root produced in the compacted compartment was clearly sufficient to elicit significant growth and stomatal responses.

As leaf water potential was not affected in either genotype (Fig. 1), the observed growth and stomatal responses to compaction and treatment with ethephon or STS were apparently initiated by root-sourced chemical signals, as opposed to hydraulic signals. Stomatal conductance was inversely correlated with xylem sap ABA concentration (Figs. 3 and 5), in agreement with previous studies of the impact of water stress (Griffiths et al., 1996), suggesting that ABA transported from the impeded roots was responsible for inducing the observed stomatal responses in both genotypes. The ABA-deficient tomato mutant flacca has previously been shown to be incapable of regulating stomatal aperture effectively in a similar split-pot system, emphasizing the role of root-sourced ABA in coordinating stomatal responses to compacted soil conditions (Hussain et al., 1999; Mulholland et al., 1999). It therefore appears that a key role of root-sourced ABA in plants experiencing compacted soil conditions is to reduce stomatal conductance, thus limiting transpiration and permitting leaf water potential to be maintained at unstressed levels (Davies and Zhang, 1991).

The use of the transgenic low-ethylene ACO1_AS and isogenic wild-type genotypes proved invaluable in establishing a role for ethylene in mediating growth responses to compaction. Thus, although shoot growth was similar in both genotypes in the uniform 1.1- and 1.5-g cm⁻³ treatments, significant genotypic differences were apparent in the 1.1/1.5-g cm⁻³ treatment, which provided a subcritical level of compaction stress comparable to that reported previously for barley grown in uniform 1.6-g cm⁻³ soil columns (Mulholland et al., 1996a). Leaf area and shoot dry weight increased almost linearly between harvests in both genotypes, but were reduced to a greater extent in the 1.1/1.5-g cm⁻³ treatment of cv Ailsa Craig than in ACO1_AS relative to equivalent uncompacted control plants (Fig. 2, A and B). These effects occurred in the absence of detectable effects on leaf water potential both in the present (Fig. 1) and previous compaction studies (Mulholland et al., 1996a, 1999; Hussain et al., 1999).

The 1.1/1.5-g cm⁻³ treatment therefore promoted intriguing genotypic differences in which the wild-type, cv Ailsa Craig, exhibited greatly reduced shoot growth, while the low-ethylene genotype, ACO1_AS, was able to maintain uncompacted control growth rates (Fig. 2, A and B). Excision of the roots in the 1.5-g cm⁻³ compartment at 15 DAE promoted a recovery of shoot growth in cv Ailsa Craig from 20 DAE, whereas shoot growth in ACO1_AS was unaffected. These results demonstrate that root-sourced signal(s) other than ABA were responsible for the growth responses induced when cv Ailsa Craig encountered compacted soil, as xylem sap ABA concentrations were closely comparable in both genotypes yet differing genotypic responses were observed (Figs. 2 and 3). To our knowledge, this is the first reported comparison of ABA levels in the wild-type cv Ailsa Craig and ACO1_AS genotypes of tomato, and the results clearly demonstrate the potency of the antisense strategy for modifying the synthesis of specific hormones without affecting the levels or stress responses of others. Such approaches offer a powerful tool for studies of the importance of specific hormones in regulating the stress physiology of plants.

Our results support the hypothesis that the impaired capacity of ACO1_AS to convert ACC to ethylene permitted shoot growth to continue at uncompacted rates in the 1.1/1.5-g cm⁻³ treatment because endogenous ethylene levels remained below a critical threshold. Thus, the genotypic differences in growth observed in the 1.1/1.5-g cm⁻³ treatment were reflected by the significantly greater ethylene evolution in cv Ailsa Craig compared with ACO1_AS (Figs. 3 and 5), even though evolution rates were comparable when both genotypes were grown in uncompacted soil. Leaf area and shoot dry weight were inversely correlated with ethylene evolution, suggesting that the greater ethylene production of wild-type plants in the 1.1/1.5-g cm⁻³ treatment contributed to the observed reduction in shoot growth. Comparisons may be drawn with the subcritical compaction stress imposed on barley by uniformly compacted 1.6-g cm⁻³ soil (Mulholland et al., 1996a, 1996b). These studies demonstrated that the inability of the ABA-deficient mutant, Az34, to synthesize and export ABA to the shoot at wild-type rates restricted its capacity to maintain leaf expansion at uncompacted levels during subcritical compaction stress (Mulholland et al., 1996a), and that treatment with synthetic ABA induced phenotypic reversion to wild-type growth rates (Mulholland et al., 1996b). Subsequent studies with barley (Hussain et al., 1999) provided evidence that an additional root-sourced signal, possibly ethylene, was involved in reducing shoot growth in the ABA-deficient mutant at the subcritical bulk density and in both genotypes during severe compaction stress; it was concluded that the growth responses induced may depend on the balance between endogenous ABA and ethylene levels.

In sharp contrast to the 1.1/1.5-g cm⁻³ split-pot system, the uniform 1.5-g cm⁻³ treatment provided a critical level of stress that greatly reduced shoot and root growth in both genotypes of tomato (Fig. 2), possibly by a mechanism that does not directly involve either ethylene or ABA. Thus, although ethylene evolution was significantly greater in the uniform 1.5-g cm⁻³ treatment of ACO1_AS than in uncompacted control plants at 30 DAE (P < 0.001; Fig. 3C), this effect became apparent only after significant reductions in root and shoot growth had occurred. The hypothesis that increased delivery of root-sourced ABA was responsible for the inhibition of shoot growth in plants growing on severely compacted soil may be discounted, as previous studies have shown that comparable reductions in shoot growth are induced in ABA-deficient mutants of both tomato and barley (Mulholland et al., 1996a, 1999; Hussain et al., 1999). Another, as-yet-unidentified mechanism, perhaps involving changes in nutrient uptake or imbalances in hormones other than ethylene and ABA, may therefore act to inhibit shoot growth when the entire root system encounters severely compacted soil.

The possibility that ABA is the only root-sourced signal produced in response to soil compaction, but has the secondary effect of invoking increased ACC synthesis within the shoot, may also be discounted. Under this scenario, ethylene production and consequent reductions in leaf expansion and shoot growth would be greater in wild-type plants, whereas the limited ability of ACO1_AS to convert ACC to ethylene would result in the maintenance of shoot growth. However, previous studies have shown that ethylene production is greater in ABA-deficient mutants of tomato in than in wild-type plants, even though xylem sap ABA concentrations are much lower (Hussain, 1998). Furthermore, similar studies of wild-type and ABA-deficient mutants of barley revealed that the increases in xylem sap ABA concentration induced by subcritical compaction stress had the contrary effect of reducing ethylene evolution from leaf tissue (Hussain, 1998).

The original hypothesis that genotypic differences in ethylene production were responsible for the observed reduction of growth in the 1.1/1.5-g cm⁻³ split-pot treatment of cv Ailsa Craig was substantiated by the experiments in which ethephon and STS were supplied. Treatment with ethephon increased ethylene evolution and reduced leaf area in both genotypes, whereas limiting ethylene action by blocking its binding sites with silver ions partially restored leaf expansion in cv Ailsa Craig (Figs. 4A and 5C). Thus, a partial restoration of uncompacted growth rates was achieved simply by blocking the physiological activity of the increased ethylene levels induced by compaction in the absence of any change in the soil conditions experienced by the plants. Treatment with STS significantly reduced ethylene evolution in cv Ailsa Craig, perhaps by eliminating the induction of autocatalytic ethylene production by stress (Sfakiotakis et al., 1996); this may have further contributed to the impact of silver ions in alleviating the symptoms of compaction. As STS-treated plants grown under a range of compaction treatments exhibited no significant effects on root growth relative to control plants treated with water (Fig. 4B; Hussain, 1998), the possibility that the restoration of shoot growth in STS-treated plants of cv Ailsa Craig resulted from an inhibition of root growth and subsequent reallocation of assimilates to support shoot growth may be discounted. Furthermore, a similar stimulation of shoot growth would have been expected in STS-treated ACO1_AS plants if reallocation of available assimilates was responsible for the observed growth responses; however, no detectable effects were observed relative to control plants treated with water.

Genotypic differences were also apparent for root biomass, which was evenly distributed between both compartments in the uniform 1.1- and 1.5-g cm⁻³ treatments, but reduced in the compacted compartment of the 1.1/1.5-g cm⁻³ system in both genotypes (Fig. 2). Root dry weight was also reduced in the uncompacted compartment of the split-pot treatment of cv Ailsa Craig relative to 1.1-g cm⁻³ control plants, suggesting that root growth may have been limited by reductions in assimilate availability resulting from the greatly reduced leaf area. Thus, the significantly greater ethylene levels in the wild-type genotype may have been responsible for reducing root growth in the uncompacted compartment in addition to shoot growth. In contrast, the absence of any reduction of root growth in the 1.1-g cm⁻³ compartment of the 1.1/1.5-g cm⁻³ split-pot treatment of ACO1_AS relative to uncompacted control plants reflected its ability to maintain leaf growth, and hence assimilate supplies, at near control levels. Wild-type plants showed a marked increase in root biomass in the uncompacted compartment following excision of the roots in the compacted compartment at 15 DAE, with the result that total root biomass was slightly greater in split-cut than in split-pot plants at 25 and 30 DAE (Fig. 2C); this increase in root biomass reflects the concurrent recovery of leaf area and hence assimilate production. In contrast, root biomass in the uncompacted compartment was comparable for split-pot and split-cut plants of ACO1_AS, reflecting the absence of significant compensatory root growth or any promotion of shoot growth following excision of the roots in the compacted compartment. These results suggest that the observed root growth responses are intimately linked to effects on shoot growth and assimilate availability.

Leaf expansion and shoot growth in plants growing on compacted soil were more closely correlated with endogenous ethylene levels than with xylem sap ABA concentration, whereas root-sourced ABA appeared to function as a signal regulating stomatal conductance, as reported previously (Davies et al., 1994; Mulholland et al., 1996a, 1999; Hussain et al., 1999). The use of the ethylene-deficient transgenic ACO1_AS clearly demonstrated the constraining influence of ethylene on shoot growth when roots encounter compacted soil. Excision of roots in the compacted compartment of the 1.1/.1.5-g cm⁻³ treatment confirmed that a root-sourced signal was responsible for limiting leaf area and shoot growth, while artificially increasing ethylene production by applying ethephon or limiting its perception using silver ions induced phenotypic reversion in ACO1_AS and cv Ailsa Craig, respectively. Although its concentrations and fluxes have yet to be measured, it appears likely that ACC may have a role as a potential root-sourced signal, as this ethylene precursor is readily transported in the transpiration stream and ACO1_AS would be insensitive to changes in its concentration (English et al., 1995). The uniformly high bulk density in the 1.5-g cm⁻³ treatment provided a critical compaction stress in both genotypes, which greatly reduced root and shoot growth, demonstrating that the ability of genotypic differences in endogenous ethylene levels to ameliorate the adverse effects of compaction is lost when the entire root system encounters severely compacted soil.

Financial support from the UK Natural Environment Research Council and the Biotechnology and Biological Science Research Council is gratefully acknowledged.