13.6—
Actions of Abscisic Acid

13.6.1—
Introduction

Abscisic acid (ABA) is a plant hormone which now ranks in importanance with the auxins, gibberellins, cytokinins, and ethylene. Interest in the physiology and chemistry of ABA has grown greatly since the structure was established in 1965. During the 1950's and early 1960's a number of laboratories were engaged in research on growth-inhibiting substances. ABA was first isolated from cotton plants and was named abscisin II by a team from Addicott's laboratory (Ohkuma et al., 1963). During the same year Wareing's research team (Eagles & Wareing, 1963) isolated an active substance from Acer leaves which they named dormin. Abscisin II and dormin are the same substance which is now called abscisic

Figure 13.8
Structure of abscisic acid.

acid (Fig. 13.8). Plant tissues of all ages appear to synthesize and inactivate ABA. The number of various plant responses affected by ABA is very large. Generally, the physiological processes are related to senescence or abscission and growth retardation or inhibition. ABA appears to act as an abscission accelerating hormone in many fruits and leaves. Furthermore, it also tends to induce dormancy in some woody plants. ABA has been shown to move from the leaves to the apical bud to bring about a dormant condition. In potato, the

levels of inhibitors, including ABA, decrease during the quiescent period prior to renewed growth. ABA in extremely low concentration, moreover, prolongs the dormancy of excised potato buds.

Recently, ABA has been shown to be involved in the responses of many plants to stress conditions. As the ABA concentration increases in the leaves the stomata close. In this way, it appears that ABA is directly involved in the opening and closing of the stomata, thus regulating the rate of transpiration. Through this mechanism, ABA appears to protect the plant through conditions of water stress or drought.

At present the mechanism of action of ABA is not clearly understood. However, the available evidence indicates that ABA affects transcription as shown by reduced activity of chromatin-associated RNA polymerases. In other cases the mechanism of action of ABA appears to involve regulation of the translation of long-lived messenger-RNA, whilst its effects on stomata probably involve the regulation of membrane permeability.

13.6.2—
Role of ABA in Dormancy

One of the lines of research which led to the isolation of ABA from sycamore leaves was a change in the growth inhibitory activities in extracts of tree seedlings grown on long- or short-days. An increase in content of the growth inhibitory material in leaves was noted during the late summer and early autumn (Phillips & Wareing, 1958). Furthermore, the idea that bud dormancy in potato tubers may be caused by growth inhibiting substances had been suggested by Hemberg (1949). The original correlation of inhibitory material in leaves grown under short- and long-days and the induction of dormancy were based on measurements obtained by bioassay techniques. Recently, Lenton et al. (1973) have attempted to repeat these determinations using gas chromatography to compare the amounts of ABA present. They found that transferring birch, red maple or sycamore plants to short-days had no effect on ABA content. The importance of a balance between growth promoters and growth inhibitors has been stressed frequently. Tinklin and Schwabe (1970) have found considerably more inhibitor in bud scales than in the bud axis of blackcurrant. In these experiments the ABA concentrations were correlated with the degree of dormancy and the levels were reduced by treatments which encouraged bud break.

The most convincing evidence suggesting that ABA induces dormancy is the production of turions in Lemna polyrrhiza. When ABA is added to the medium under conditions that allow continuous growth, it not only inhibits growth but causes production of the dense, dormant, fronds known as turions (Stewart, 1969).

ABA is a potent inhibitor of seed germination, and its presence as a major growth inhibitor in dormant seeds of many species has cast it in the role of the maintainer of seed dormancy. ABA has been isolated from seeds of many genera of higher plants, and seeds of an equally large number of species have

been prevented from germination by soaking in ABA solutions. When seeds and fruit parts are separated, it is usually found that the concentration of ABA in the fruit is about 5 to 10 times greater than in the seed (Milborrow, 1974).

Lipe and Crane (1966) found that ABA in peach seeds decreased during stratification. Obviously, the part played by endogenous growth promoting compounds and the balance between them and ABA during the breakage of dormancy needs to be explored in more detail. Certainly, the balance between the two kinds of regulators is important. ABA is not an irreversible inhibitor since one of its most striking features is the facility with which it can be leached out of treated seeds thereby allowing the resumption of growth.

13.6.3—
Effects on Abscission

In the last few years several hundred experiments have been carried out in which plants, and parts of plants, of many genera have been treated in a variety of ways with ABA. It is quite unusual for any report to show clearly that the hormone controls abscission of leaves, which leads to the conclusion that exogenously applied ABA has little effect on leaf abscission. Nevertheless, ABA was first isolated by following its abscission-accelerating activity in petiolar stumps of cotton explants. The growth inhibitory action of this factor was believed to be responsible for the premature abscission of immature young lupin fruits (Cornforth et al., 1966). Consequently, the abscission-accelerating effect of ABA is well documented and extensively discussed (Milborrow, 1974).

Many of the experiments reporting leaf abscission have been carried out on tree crops using extremely high concentrations of ABA and often near the end of the growth season in attempts to cause abscission of fruits. The observed stimulation of leaf abscission may be an indirect effect of non-physiological high concentrations which stimulate ethylene production (Edgerton, 1971). For example, Cooper and Henry (1968) treated orange trees with sprays of 500 µg ml–l of ABA in summer and winter. The summer treatment caused leaves to develop colour and fall, but the winter treatments had no such effects. Olive trees were found to suffer some leaf abscission in one experiment but no effect was observed in another.

It appears, therefore, that ABA is not closely involved in the regulation of leaf abscission. The test system in which it has a stimulatory action consists of isolated petiolar explants containing presumptive abscission zones and maturing leaves nearing the end of their life. Even this tissue requires application of abnormally high concentrations of ABA to manifest an effect.

The role of ABA on fruit abscission is more certain. The early work of Van Steveninck (1959) showed that the inhibitor now known as ABA was intimately involved in the abortion of the young immature fruit of lupin. Application of ABA to mature peach, olive, apple and citrus fruits accelerated abscission and the effect of ABA was also marked on young grape flowers and berries (see Milborrow, 1966).

13.6.4—
Effects of ABA on Wilting

Wright (1969) found that when cut shoots were wilted there was an increase in the concentration of the so-called b -inhibitor. He went on to identify this inhibitor as ABA and defined the conditions under which the increase occurred. A water loss of about 10% in total fresh weight caused approximately 40-fold increase in ABA content while further water loss had no additional effect. Wright and Hiron (1969) have reported that other stress conditions, such as waterlogging, caused a similar rise in ABA, but they point out that such treatments cause wilting by reducing the efficiency of water uptake. The surprising feature of the increase in ABA is the rapidity with which it occurs. The content in turgid bean leaves has been calculated from the results of Wright and Hiron (1969). They show that ABA content increases from a normal level of 6 µg kg–1 fresh weight to 7 µg kg^–1 within 7 minutes after blowing a dry and warm air stream across the plants. This level increases to 33 µg kg^–1 within 25 minutes and 68 µg kg^–1 within 45 minutes. In other experiments they show that in wheat leaves the ABA content increased from 23 to 171 µg kg^–1 fresh weight within 4 hours of wilting. The ABA content of bean leaves remained at 67 µg kg^–1 while they were kept waterlogged for 5 days. The implications of these data have yet to be explored in detail, but the observations offers a feasible explanation of the reduced growth of crops suffered during drought.

The extra ABA is probably formed by synthesis rather than by release from a precursor or conjugate because much more labelled mevalonate was incorporated into ABA by a sample of leaves that had been fed and then wilted than similar leaves which were kept moist during the entire experiment. Furthermore, the presence of 40 times the amount of a precursor or conjugate probably would have been detected by extraction and bioassay techniques. Many types of experiments have shown dramatic increases in ABA content on wilting in french beans, brussel sprouts, sugar cane, wheat, avocado spinach, cotton, peas and tomatoes (see Milborrow, 1974).

Perhaps the best information supporting a direct involvement in the direct closure of the stomata is afforded by a wilty tomato mutant produced by X-irradiation (Imber & Tal, 1970). The shoots of this plant contain one-tenth the amount of ABA contained in the normal variety. Applications of 0.1 to 10 µg of ABA caused a rapid and progressive reduction in transpiration rate of leaves and leaf discs. With 10 µg ABA ml^–1 the stomata closed in darkness.

13.6.5—
Affects of ABA on Enzyme Activities

The ability of plant hormones to affect directly the activity of enzymes has not been extensively investigated. When an effect has been reported it is smaller than would be expected if the hormone specifically regulates at that site. Hemberg (1967) reported that ABA can inhibit a -amylase in vitro, but as the enzyme used was extracted from fungi the significance as related to higher

plants requires additional investigation. ABA has been shown to complex with fungal a -amylase and thereby change its physical properties. Saunders and Poulson (1968) found a slight stimulation of invertase activity at 10^–7 M ABA and slight inhibition at 5 × 10^–7 M and above. Again the significance is difficult to assess since a fungal enzyme was used.

13.6.6—
Effects of ABA on Nucleic Acid Synthesis

The first observations of the effects of ABA on nucleic acids were made by Van Overbeek et al., (1967) using Lemna. The incorporation of radioactive phosphate (³² P) into nucleic acid fractions was inhibited by ABA but reversed by benzyladenine. The primary site of action of ABA cannot be deduced with certainty because of the time scale and the complexity of the responses. Subsequent work has indicated that DNA synthesis is almost certainly not the primary target of ABA (Villiers, 1968). This has been clearly shown to be the case in dry wheat embryos by Chen and Osborne (1970) who found that protein synthesis commenced from imbibition and was inhibited at 6 hours by ABA, whereas RNA synthesis, as measured by the incorporation of ³ H-uridine, was not measurable until 12 hours. In the same experiments the incorporation of ³ H-thymidine into DNA was measurable after 24 hours. Experiments by Pearson and Wareing (1969) have shown that chromatin-directed RNA polymerase activity was slightly inhibited by 0.26 m g ml^–l of ABA when added to the grinding medium. However, when ABA was added to the purified chromatin little or no effect was noted. Bex (1972) also reports that ABA has no effect on the binding between nucleohistones and DNA as measured by their melting point measurements. Schwartz (1971) has shown that ABA can alter the balance of alcohol dehydrogenase in maize, but the inhibitor was added during the growth of the cells. Thus, while it appears that ABA has small and random effects on nucleic acid biosynthesis in a number of plants, it is reasonably clear that the hormone has little or no direct effect on the synthesis of nucleic acids at the level of the genome.

13.6.7—
The Involvement of Abscisic Acid in Messenger-RNA Translation

Ihle and Dure (1972) investigated the appearance of various enzyme activities during the germination of cotton seeds and embryos. They showed that protease and iso-citrate lyase appear to be synthesized de novo during germination utilizing messenger-RNA which had been transcribed much earlier when only about 60% completion of embryogenesis had been reached. The translation of these messenger-RNAs during the last 40% of embryogenesis was apparently prohibited by the presence of ABA diffusing into the embryo from the ovule wall. The mode of action of ABA in maintaining translation inhibition appears to

involve RNA synthesis as judged by the fact that the ABA inhibition was inhibited by the presence of actinomycin D. Translation of the required messenger-RNAs for the germination enzymes may be induced prematurely by simply dissecting the ovules from the plant, suggesting that the breakage of the connection between the ovule and the mother plant may be required for the in vivo induction of translation. This hypothesis is further substantiated by the observation that, in vivo, the connection between the ovule and the placenta is normally severed at 60% completion of embryo-genesis.

The results coming from Dure's laboratory are the only ones at present which show that ABA may have a direct effect on the translation of messenger-RNA pre-existing in the embryo tissue.

13.6.8—
Summary

Even though ABA was first found in cotton plants and dormant sycamore leaves, it seems reasonably clear that this hormone has little or no effect on either the abscission of leaves or the dormancy of buds. It appears that the most likely physiological action of ABA on plants is to control the abscission zone in the fruit pedicel and thereby allow abscission to take place and cause fruit drop. Secondly, ABA very definitely appears to be involved in stomatal closure in response to stress conditions, particularly drought. In terms of a more biochemical approach it appears that ABA has little effect on various enzyme activities. Those enzymes studied show only small enhancement, or in some cases, inhibition, by ABA. Generally it has been shown that ABA inhibits the synthesis of various nucleic acids.

Again, one must be drawn to the conclusion that ABA has little effect directly on nucleic acid synthesis at the genome level. The most excitingp aproach to abscisic acid action appears to be that found in the cotton embryo where it has been shown to inhibit the translation of pre-existing messenger-RNAs found in the developing embryos. Even this response probably involves more complex processes than a direct inhibitory effect, since actinomycin D inhibits the ABA-induced inhibition. These data possibly imply that a suppressor molecule has to be formed to bring about effects.