Roots of maize (Zea mays L.) seedlings continue to grow at low water potentials that cause complete inhibition of shoot growth. In this study, we have investigated the role of abscisic acid (ABA) in this differential growth sensitivity by manipulating endogenous ABA levels as an altemative to extemal applications of the hormone. An inhibitor of carotenoid biosynthesis (fluridone) and a mutant deficient in carotenoid biosynthesis (vp 5) were used to reduce the endogenous ABA content in the growing zones of the primary root and shoot at low water potentials. Experiments were performed on 30 to 60 hour old seedlings that were transplanted into vermiculite which had been preadjusted to water potentials of approximately -1.6 megapascals (roots) or -0.3 megapascals (shoots). Growth occurred in the dark at nearsaturation humidity. Results of experiments using the inhibitor and mutant approaches were very similar. Reduced ABA content by either method was associated with inhibition of root elongation and promotion of shoot elongation at low water potentials, compared to untreated and wild-type seedlings at the same water potential. Elongation rates and ABA contents at high water potential were little affected. The inhibition of shoot elongation at low water potential was completely prevented in fluridone-treated seedlings during the first five hours after transplanting. The results indicate that ABA accumulation plays direct roles in both the maintenance of primary root elongation and the inhibition of shoot elongation at low water potentials.A major reason behind the slow progress in the area of crop adaptation to drought is the insufficient basic understanding of the regulation of growth responses to water stress. When water is limited, shoot growth in many species is more inhibited than root growth (24), and in some cases, the absolute biomass of roots has been shown to increase relative to wellwatered controls (12,23). In maize, roots continue to grow at low water potentials that cause complete inhibition of shoot growth (25,30). The role of the hormone ABA in the differential growth responses of the primary root and shoot of maize to low water potentials is the subject of this paper.ABA accumulates to high concentrations in tissues ofplants

Previous work showed that primary root elongation in maize (Zeamays L.) seedlings at low water potentials was severely inhibited when accumulation of abscisic acid (ABA) was decreased using either fluridone or the vp5 mutant to inhibit carotenoid (and ABA) biosynthesis. The objective of this study was to confirm that the inhibition of root elongation resulting from these treatments was indeed attributable to the decrease in ABA levels. Seedlings were transplanted after germination to vermiculite at water potentials of −1.6 or − 0.3 MPa. ABA was mixed at various concentrations with the vermiculite to test whether the effects of fluridone and the vp5 mutation on root elongation could be overcome. In both treatments, restoration of ABA levels in the root apical 10 mm, which encompassed the elongation zone, resulted in recovery of root elongation rate at both −1.6 MPa and −0.3 MPa. Analysis of the spatial distribution of elongation rate showed that the recovery of overall root elongation resulted from restoration of the profile of elongation, and did not involve over-promotion of local elongation rate at any position. The recovery of root elongation was shown to be independent of effects of ABA status on shoot growth. When ABA was applied at high water potential, such that levels of ABA in the root tip reached those associated with maintenance of elongation at low water potentials, root elongation was inhibited. Thus, the response of root elongation to bulk tissue ABA content varied with the tissue water status. The results confirm that accumulation of ABA is required for the maintenance of maize primary root elongation at low water potentials.

Seedlings of maize (Zea mays L. cv WF9 x Mol7) growing at low water potentials in vermiculite contained greatly increased proline concentrations in the primary root growth zone. Proline levels were particularly high toward the apex, where elongation rates have been shown to be completely maintained over a wide range of water potentials. Proline concentration increased even in quite mild treatments and reached 120 millimolal in the apical millimeter of roots growing at a water potential of -1.6 megapascal. This accounted for almost half of the osmotic adjustment in this region. Increases in concentration of other amino acids and glycinebetaine were comparatively small. We have assessed the relative contributions of increased rates of proline deposition and decreased tissue volume expansion to the increases in proline concentration. Proline content profiles were combined with published growth velocity distributions to calculate net proline deposition rate profiles using the continuity equation. At low water potential, proline deposition per unit length increased by up to 10-fold in the apical region of the growth zone compared to roots at high water potential. This response accounted for most of the increase in proline concentration in this region. The results suggest that osmotic adjustment due to increased proline deposition plays an important role in the maintenance of root elongation at low water potentials.apex, resulting in a shorter growth zone. Root radial expansion was also inhibited. In the succeeding paper (18), hexose was shown to make the major contribution to osmotic adjustment in basal locations. This was due primarily to the growth inhibition in that region, because hexose deposition rates were calculated to decrease rather than increase (18). In contrast, hexose (and the other measured solutes, sucrose and potassium) accounted for little of the osmotic adjustment in the apical region, where elongation was fully maintained despite very low 0,. These results indicated that other solutes must be preferentially deposited in that region. Because cells close to the apex are only slightly vacuolated, our objective in this paper was to examine the contributions of proline and glycinebetaine to osmotic adjustment. These compounds have been suggested to act as cytoplasmic solutes, which are compatible at high concentrations with metabolism (25, 28). We show that proline accounts for as much as 50% ofthe osmotic adjustment in the apical region and that this response involves a dramatic increase in the rate ofproline deposition, expressed per unit root length or volume.

When excised second leaves from 2-week-old barley (Hordeum rulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 03 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABAtreated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.Both ABA and proline accumulate in response to drought stress in a number ofplants (3), and in barley, the two compounds have been measured in the same experiments (2). Proline accumulates in response to salt stress in a number of plants and barley is a well-studied example (3,5,15,16). Application of ABA induces proline accumulation in Hordeum and Lolium leaves (4, 12). The lack of proline accumulation in response to ABA treatment has been reported for spinach, Pennisetum thyphoides, (10) tobacco, and sunflower leaves (3) and after several attempts we have not observed ABA-induced proline accumulation in bean leaves (C. R. Stewart and G. Voetberg, unpublished results). The metabolic processes leading to proline accumulation under all these treatments are similar (5,11,12 include increased proline synthesis and inhibition of proline utilization by both oxidation and protein synthesis.In pursuing the goal of understanding metabolic and cellular phenomena that lead to stress-induced proline accumulation, we have been interested in determining the relation, if any, between proline and ABA accumulations. Is the accumulation of ABA required for proline accumulation and can we identify other requisite processes such as the suggested (17) subcellular redistribution of solutes? The fact that ABA does not ind...

Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA)-treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt-shocked leaves was observed both when benzyladenine was added at the beginning ofor after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA-treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level.