Response of leaf growth to ABA in different ionic solutions

The effect of different feeding solutions on the growth of the detached shoots (distilled water- DI or artificial xylem solution - AX) was tested (Figure 2.2a). Control (not supplied with ABA) shoots showed a 25-30 % increase in LER over the first 4 hours of the assay. This was a common feature in many leaf elongation assays, although the magnitude of the increase was highly variable (compare Figures 2.2a, 2.3a). This response may be a recovery from any stresses imposed by excavation of the seedlings. A stable maximum LER was attained by control shoots between 4 and 8 hours. Although statistical comparisons (Student's unpaired t-test, P> 0.05) revealed no difference in LER between AX-fed and Di-fed controls at any time (as shown by Munns, 1992); the LER of AX-fed shoots was consistently 10 % higher than the LER of Di-fed shoots over the first 8 hours of the assay. Stimulation of growth by a dilute ion solution (10 mM KC1) has been previously reported in a leaf disc bioassay system (Van Volkenburgh and Davies, 1983). By 10 hours, a 35 % (DI) or 20 % (AX) decline in LER, relative to the stable maximum LER, had occurred. This decline was greater in Di-fed shoots, as observed in 2 subsequent replications of the same experiment (data not shown). For this reason, an artificial xylem solution was routinely used in all subsequent assays.

There was no change in LER of shoots fed 10"6M ABA (compared to control shoots) after 2 hours. By 4 hours, a 50-55 % reduction in LER had occurred, which was maintained for the duration of the assay. The timing of the steady-state phase of leaf growth inhibition for 10"6M ABA was consistent with Munns (1992).

o UL H Z o o o 5 CD z o HI u_ < in 1 0 0 80 60 40 20 9 10 5 6 7 8 2 3 4 0 1 T I ME ( h o u r s )

Figure 2.2: Effect of different feeding solutions on leaf elongation rate of detached barley (Hordeum vulgare L. cv. Klaxon) shoots of maintained at 25°C (a). Treatments were 10"^M ABA dissolved in distilled water

(A)

or artificial xylem solution (A) while controls were distilled water ( • ) or artificial xylem solution (O). Points are means ± S.E. of at least 7 shoots, (b) Leaf elongation of ABA-treated shoots expressed as a percentage of the controls for distilled water ( • ) or artificial xylem solution (O). Error bars have been omitted for clarity.

Expressing the results as a percentage of the controls (Figure 2.2b) revealed the attainment of a steady-state level of leaf growth inhibition after 4 hours. The decline in LER of the DW-fed controls resulted in a spurious value for the inhibition caused by ABA at 9 hours. When this point was disregarded, it was apparent that adding a dilute solution of ions to the feeding solution did not affect the response to ABA. This was supported by analysis of variance, which showed no nutrient x ABA interaction (see Appendix 2). This finding is in contrast to other workers who have found that incubation of epidermal strips in ions such as K+ (Wilson et al., 1978; Snaith and Mansfield, 1982b), Na+ (Jarvis and Mansfield, 1980) and C a ^ (De Silva et al., 1985) can increase (Ca++) or decrease (K+, Na+) stomatal sensitivity to ABA. Similarly, Van Volkenburgh and Davies (1983) found that the inhibitory effect of ABA on leaf disc growth was completely reversed when the discs were incubated in 50 mM KC1. These observations were given some support in the whole plant study by Schurr et a l (1992), who found significant correlations between stomatal sensitivity to ABA and concentrations of NO3" and C a ^ in the xylem sap of droughted sunflower plants. However, transpiration bioassays have shown no difference in stomatal response to ABA when leaves were incubated in either distilled water or 10 mM KNO3 (Munns and King, 1988). Therefore the results of Figure 2.2b, showing no difference in leaf elongation response to ABA in distilled water and a composite nutrient solution, are not entirely surprising. These results were confirmed with solutions of 0, 5 and 10 mM KC1 and KNO3, which also showed no interaction between nutrient concentration and leaf elongation response to ABA (data not shown). When 50 mM KC1 was substituted as a feeding solution in the leaf elongation assay, transpiration was inhibited by 30 % in comparison to shoots fed deionised water (data not shown), presumably due to osmotic effects. For this reason, the possibility of a KC1 x ABA interaction affecting leaf growth was not pursued.

However, such assays have employed tissues detached from plants grown under optimum nutrient supply. The stomata of leaves detached from nutrient stressed plants (Radin et al., 1982; Radin, 1984) are more sensitive to ABA; however the effect of different nutrient solutions on the leaf elongation response of such leaves to ABA has not been assessed. It is surmised that differences in stomatal response to ABA in epidermal peels in the presence of individual ions may be the result of an unrealistic assay situation; since feeding identical solutions via the transpiration stream, where the mesophyll has the possibility of controlling the flow of ions to the epidermis, elicits no difference in ABA response between distilled water and ion solutions (Munns and King, 1988). The correlations described in the study of Schurr et al. (1992) are believed to be merely correlations since changes in stomatal sensitivity will occur over the course of a drying cycle and could be related to an increase or decrease of any one of a number of xylem sap components (Gollan et al., 1992). In the absence of bioassay evidence to confirm the conclusions of the whole plant study, the interactions between specific nutrient ions and stomatal sensitivity to ABA, as demonstrated by Schurr et al. (1992), are best treated as correlations, and not cause and effect.

The possibility that the initial increase in LER (25-30 % over the first 4 hours) of control detached shoots (Figure 2.2a) may have affected the response to ABA was tested by supplying ABA to treatment shoots at either 0 hours (usual assay procedure) or after 4 hours when leaves had attained their maximum LER. No differences in the response to ABA (expressed as a percentage of control values) were demonstrated (data not shown). In all further experiments, ABA was supplied at 0 hours.

In another test of whether the assay procedure affected the response of leaf growth to ABA two assays were conducted using the same batch of plants, but excavation of 2 separate groups of plants was separated by 6 hours. No differences in the response to ABA (expressed as a percentage of control values) were demonstrated (data not shown), indicating that levels o f carbohydrate in the growing zone (assumed

to be much higher 6 hours further into the photoperiod) did not affect the ABA response. In all further experiments, plants were excavated circa 2 hours into the photoperiod.

In document Sensitivity of Gramineacous Leaf Growth to Abscisic Acid: Its Potential Importance to Leaf Growth of Plants in Drying Soil (Page 49-53)