As discussed above, most of the research underlying the view that ABA is an inhibitor of shoot growth during water stress has involved applications of ABA to non-stressed plants or correlations of growth inhibition to increased endogenous ABA levels during stress. Indeed, the early phase of the maize seedling studies described above (Fig. 6; Saab et al. 1990, 1992) remains the only demonstration of enhanced shoot growth in response to endogenous ABA deficiency at low water potentials. The apparent change in response to ethylene during shoot development in those experiments, from promotive to inhibitory, is consistent with reports that ethylene stimulates mesocotyl growth in some species (Suge 1971; Cornforth & Stevens 1973), whereas it is usually inhibitory to shoot growth of terrestrial plants at later stages of development (Abeles, Morgan & Saltveit 1992; Lee & Reid 1997; Hussain et al. 1999). The following sections summarize recent studies which suggest that restriction of ethylene production may be a common function of ABA and therefore that endogenous ABA may often act to maintain rather than inhibit shoot growth.
Reassessment of the role of ABA in shoot growth of tomato and Arabidopsis under well-watered conditions
Paradoxically to the long-standing view that ABA is generally inhibitory to shoot growth, it has been observed for over 30 years that ABA-deficient mutants are often shorter and have smaller leaves than the corresponding wild types, and that leaf and stem growth can be substantially restored by applying ABA (Imber & Tal 1970; Bradford 1983; Quarrie 1987). As already mentioned, in addition to reduced growth, ABA-deficient mutants are typically wilty even when the soil is well supplied with water. This results from high stomatal conductance and has also been shown to involve decreased root hydraulic conductance. These effects can also be prevented by applying ABA (Imber & Tal 1970; Tal & Nevo 1973; Koornneef et al. 1982; Bradford 1983). Accordingly, the inhibited shoot growth of ABA-deficient mutants of tomato and Arabidopsis has been attributed to shoot water deficits (Bradford 1983; Neill, McGaw & Horgan 1986; Nagel, Konings & Lambers 1994; Léon-Kloosterziel et al. 1996). The growth-promotive effect of applied ABA in these cases has therefore been assumed to result from improvement of plant water balance. Consequently, these findings have generally not been regarded as evidence against a direct inhibitory role of ABA in leaf and stem growth, although the observations have led some authors to question this view (Jones et al. 1987; Quarrie 1987; Taylor 1987; Zeevaart & Creelman 1988).
Alternatively, endogenous ABA may be required to maintain shoot growth of well-watered plants independently of its effect on plant water balance. Consistent with the finding that ABA restricts ethylene production in water-stressed maize seedlings, it was reported that under well-watered conditions, ethylene production was greater in shoots of the flacca (flc) mutant of tomato (Tal et al. 1979) and in whole plants of the aba1 mutant of Arabidopsis (Rakitina et al. 1994) than in the corresponding wild types. In flc, it was also shown that ethylene production could be reduced to normal levels with exogenous ABA. In addition, the ABA-deficient mutants of tomato exhibit morphological symptoms characteristic of excess ethylene such as leaf epinasty and adventitious rooting (Tal 1966; Nagel et al. 1994). Despite these observations, the possibility that ethylene is a cause of reduced shoot growth in ABA-deficient mutants was not considered until recently.
To distinguish between these possibilities, it is necessary to assess the growth of ABA-deficient mutants at the same plant water status as the wild type. If the inhibition of growth normally observed is caused by adverse water relations, then under such conditions growth should be restored or even enhanced relative to wild-type plants. In contrast, if endogenous ABA is required to maintain shoot growth independently of effects on plant water balance, then the mutants should remain smaller. Jones et al. (1987) grew flc and two other ABA-deficient mutants of tomato, notabilis (not) and sitiens (sit), under mist in a greenhouse, and observed that stem height became greater than in the wild type, consistent with ABA playing an inhibitory role in stem elongation. In contrast, later-formed leaves remained smaller and total leaf biomass remained substantially decreased. However, leaf water potentials also remained considerably lower than in the wild type, so interpretation of the role of ABA in leaf growth was not possible. Taylor (1987) reported that the inhibition of shoot growth in double mutants of flc, not and sit was partially alleviated when the plants were grown at high humidity, but information on plant water relations was not included so, again, full interpretation was not possible. In other studies in which flc was grown at high humidity, effects on growth were not reported (Puri & Tal 1977; Tal et al. 1979).
To assess whether the reduced leaf and stem growth of well-watered flc and not are attributable to water deficits, in a recent study plants were grown under controlled-humidity conditions in a growth chamber, such that the leaf water potentials of the mutants were equal to or higher than those of wild-type plants throughout development (Sharp et al. 2000). Most parameters of shoot growth remained markedly impaired; for example, total leaf area in flc was 48% less than in the wild type (Fig. 8a). Root growth was also greatly reduced. Consistent with the observation of Jones et al. (1987), the stems of the mutants initially elongated more rapidly than those of the wild types; however, this effect was not sustained, and the mutants became shorter at later stages of development. Shoot growth substantially recovered when wild-type levels of ABA were restored by treatment with exogenous ABA (Fig. 8a,b), even though the experimental strategy prevented improvement in leaf water potential. The ability of applied ABA to increase growth was greatest for leaf expansion, which was restored by 75%. The ethylene evolution rate of growing leaves was doubled in flc compared with the wild type (Fig. 8c), and treatment with STS to inhibit ethylene action partially restored leaf, stem and root growth (Sharp et al. 2000).
Figure 8. Total leaf area (a, 35 d after emergence), leaf ABA content (b, 21 d) and leaf ethylene evolution rate (c, 21 d) of wild-type (WT) tomato (cv. Rheinlands Ruhm), flacca (flc), flacca spray control (sc), and flacca sprayed with 10 µm ABA daily from day 9. Spray control plants were sprayed with deionized water containing the same concentrations of ethanol and Tween 20 as in the ABA solution. All plants were well watered, and were grown under controlled-humidity conditions so that throughout development, leaf water potentials of untreated flacca were equal to or higher than those of the wild type, and leaf water potentials of flacca treated with ABA were equal to or lower than those of the flacca spray control. Within each panel, bars with different letters are significantly different at the 0·05 (leaf area, ABA) or 0·1 (ethylene) level. (Modified from Sharp et al. 2000.)
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Similar results have been obtained in preliminary studies of Arabidopsis. When grown at the same leaf water potentials as well-watered wild-type plants throughout development, total leaf area of the aba2 mutant (ABA-deficient) remained greatly inhibited, and was restored by about 50% in a double mutant of aba2 and etr1 (ethylene-resistant) (M.E. LeNoble, W.G. Spollen & R.E. Sharp, unpublished results). The leaf ethylene evolution rate of aba2 was approximately twice that of the wild type under these conditions.
These results demonstrate that (a) normal levels of endogenous ABA are required to maintain shoot development, particularly leaf expansion, in well-watered tomato and Arabidopsis plants independently of effects on plant water balance, and (b) the impairment of leaf and shoot growth caused by ABA deficiency is at least partly attributable to increased ethylene production.
It should be noted that these findings do not negate the necessity of avoiding differences in plant water status among genotypes in future studies of the effects of ABA manipulation on growth. Differences in water relations, when they do occur, might also contribute directly or indirectly to growth responses. It will be particularly critical (and challenging) to extend the strategy of circumventing variation in plant and also soil water status between control and ABA-deficient (or ABA-overproducing) genotypes to address the function of ABA accumulation in the responses of plant growth to soil drying.
The conclusion with well-watered tomato and Arabidopsis that endogenous ABA is required to maintain shoot growth contrasts with the many examples in the literature where ABA has caused shoot growth inhibition when applied to well-watered plants. The explanation for this difference might be simply explained by suggesting that the effects of supplemental ABA in well-watered plants are non-physiological. However, Bacon et al. (1998) published compelling evidence that the normal levels of ABA in well-watered barley can cause leaf growth inhibition. They showed that the leaf elongation rate of wild-type plants declined as the pH of artificial xylem sap fed to the leaves was increased, but that this response did not occur in the ABA-deficient mutant Az34 unless a normal well-watered concentration of ABA was supplied in the sap. (The pH-induced reduction in leaf elongation is suggested to result from increased accumulation of ABA in the apoplast.) This result is difficult to reconcile with the inhibited growth of the ABA-deficient mutants of tomato and Arabidopsis. It is possible that whole plant ABA-deficiency leads to an overriding inhibitory effect on growth of increased ethylene production. However, the above-described experiments with water-stressed maize seedlings indicate that, for root growth at least, this is not the case. In ABA-deficient seedlings in which ethylene evolution was restored to the normal water-stressed rate by treatment with AOA, root growth recovered but only to the rate of the control seedlings (Fig. 4). In other words, preventing the effect of ABA-deficiency on ethylene production did not uncover a growth-promotive effect of ABA deficiency.
Resolution of the differing conclusions on the role of ABA in leaf growth of well-watered plants from the studies of barley (Bacon et al. 1998) and of tomato and Arabidopsis will require further investigation.
Role of ABA in shoot growth response to compaction
Plants growing in compacted soil often exhibit reduced shoot growth. Evidence that endogenous ABA plays a role in shoot growth maintenance rather than inhibition in compaction-stressed barley was reported by Mulholland et al. (1996a, b). Leaf growth of plants growing in compacted soil was more inhibited in Az34 than in the wild type, and the evidence suggested that changes in leaf water relations were not the cause. Recent studies from the same group demonstrated that ethylene was a major cause of the inhibition of shoot growth in tomato plants grown with their root system divided between pots of uncompacted and compacted soil (Hussain et al. 1999, 2000). As shown in Fig. 9c, ethylene production increased greatly in wild-type plants under compaction. This response was almost fully prevented in the ethylene-deficient transgenic ACO1AS (ACC oxidase antisense; Hamilton, Lycett & Grierson 1990), as was the inhibition of leaf growth (Fig. 9a). The increase in ethylene production in the wild type occurred despite an increase in xylem sap ABA concentration (Fig. 9b). Interestingly, when supplemental ABA was supplied to the wild type, leaf ethylene production was partly reduced and leaf growth was partially restored. The same ABA treatment had a minimal effect on growth or ethylene evolution in ACO1AS, indicating that the effect of ABA on growth in the wild type was likely an indirect effect via inhibition of ethylene production. These results indicate that the normal increase in ABA in compaction-stressed plants was insufficient to fully prevent excess ethylene production in the leaves.
Figure 9. Total leaf area (a), xylem sap ABA concentration (b), and leaf ethylene evolution (c) of wild-type (cv. Ailsa Craig) and ACO1AS transgenic tomato plants at 21 d after emergence. Plants were well watered, and were grown in a split-pot system in which either both compartments contained uncompacted soil or one compartment contained uncompacted soil and the other contained compacted soil. The compartment containing compacted soil was supplied either with water or with 100 nm ABA (compacted + ABA) twice daily from day 5. (Modified from Hussain et al. 2000.)
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In addition, Hussain et al. (2000) attempted to use the ABA-deficient not mutant to demonstrate that the increase in endogenous ABA in the wild type helped to maintain leaf growth during compaction by limiting the production of ethylene. However, ethylene production was similar and inhibition of shoot growth was actually less in the mutant than in the wild type in compacted relative to non-compacted plants. Interpretation of these findings was confounded, however, because growth of the mutant was already substantially inhibited in uncompacted control plants. The inhibition in the control plants was associated with, and probably caused by, an already-increased rate of leaf ethylene evolution, consistent with the results for flc shown in Fig. 8. The problem of impaired growth of ABA-deficient control plants is discussed further below.