Essay on Abscisic Acid
Abscisic Acid (ABA):-
> It is found in older leaves, apical buds and seeds.
> Both GA and ABA hormones work opposite to each other.
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> ABA inhibits the biosynthesis of the hormone GA.
1. Discovery of Abscisic Acid:-
- Torsten Hemberg (1940) reported that dormant potato tubers and buds of ash (Fraxinus excelsior) contained inhibitor (rather than stimulators) that blocked the effects of IAA. When the buds germinated, the amount of these inhibitors decreased.
- Eagles and Wareing (1963) isolated an inhibitor from the birch (Betula pubescens) leaves held under short day conditions. When this substance was reapplied to the leaves of birch seedlings, apical growth was completely arrested. As this substance induced dormancy, they named it as ‘dormin’.
- Cornus and Addicot (1963) isolated this compound from the shedding bolls of cotton plant.
- Ohkum (1965) isolated an inhibitor from cotton fruits and named it ‘abscisin II’.
- Cornforth (1965) demonstrated that both Dormin and Abscisin- II were chemically identical and given a common name Abscisic acid (ABA) because of its known effects on stimulating abscission.
2. Chemical nature of Abscisic Acid:- This phytohormone is a 15-carbon sesquiterpene and its synonyms are dormin and abscisin-II.
3. Biosynthesis of Abscisic Acid:- Two pathways for the biosynthesis of ABA have been identified:
a. By direct synthesis from mevalonic acid:-
> Direct synthesis of abscisic acid from mevalonic acid through farnesyl pyrophosphate has been demonstrated in many cases, especially in the water stressed tissues.
> The water stress increases Abscisic acid formation.
> The early reactions in the synthesis of abscisic acid are identical to those of gibberellins, sterols, carotenoids and other isoprenoid compounds.
Mevalonic acid→ Isopentenyl pyrophosphate→ Farnesyl pyrophosphate→ Abscisic acid
b. By indirect synthesis from the oxidation of carotenoids:-
> Indirect synthesis of abscisic acid takes place by the oxidation of carotenoids or oxidized carotenoids (xanthophyll such as violaxanthin or neoxanthin).
> The xanthophyll, violaxanthin has been known to be the precursor of ABA.
> Violaxanthin in synthesized from zeaxantin (also a 40-C xanthophyll) in a reaction that is catalyzed by the enzyme zeaxanthin epoxidase (ZEP).
> Violaxanthin is converted into 9’ –cis-neoxanthin which is then cleaved into a 15-C compound Xanthoxal (previously called Xanthoxin) and a 25-C epoxy aldehyde in the presence of the enzyme 9′-cis-epoxycarotenoid dioxygenase (NCED). (This enzyme can also catalyse cleavage of violaxanthin into xanthoxal and a 25-C allenic apoaldehyde).
> Xanthoxal (xanthoxin) is finally converted into ABA in cytosol via two oxidation steps catalysed by the enzymes aldehyde oxidases involving abscisyl aldehyde (and/or possibly xanthoxic acid) as intermediates.
> The enzymes aldehyde oxidases require Mo as cofactor.
> The initial steps of ABA biosynthesis take place in chloroplasts or other plastids while final steps occur in cytosol.
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4. Bioassay of Abscisic Acid:-
> ABA now ranks in importance with auxins, gibberellins and cytokinins as a controlling factor in physiological processes.
> ABA can decrease, overcome, reverse, counteract, and inhibit the responses of plant materials to each of the growth-promoting hormones.
> The choice of bioassay is dictated by the type of process believed to be influenced by the substance.
> The bioassay methods which have been extensively used in connection with ABA are:
a. Acceleration of Abscission in Excised Abscission Zones (Explants):-
- Addicott et al. who first detected ABA by its effect in promoting leaf abscission, used this action in a biological test.
- Test object was young cotton seedlings from which roots, stem tips and blades of cotyledons (seed leaves) were removed leaving an explant consisting of a section of stem to which the stumps of the petioles, i.e., leaf stalks were still attached.
- ABA as lanolin paste may be applied either to the proximal or to the distal end of the abscission zone.
- Time taken by the petiolar stump to abscise in response to the application of a definite concentration of ABA is taken as the standard.
b. Inhibition of Coleoptile Section Growth:-
- Inhibitors have usually been detected by their ability to reduce the extension growth of oat or wheat coleoptiles.
- Since such growth is stimulated by auxins, a common practice has been to add a standard amount of IAA to the test solution and to observe the reduction by inhibitor of growth stimulated by auxin (Rothwell and Wain, 1964).
- Wheat seeds are thoroughly washed, soaked for 3-4 hours and then germinated in dark humid chamber for 72 hours.
- When the coleoptiles are about 3 cm in length, they are cut at the bases, placed vertically with the tips upward in specimen tubes containing distilled water.
- The tubes are then placed in a light-tight box, first incubated at 37°C for one hour and then kept in cold at 4°C for 24 hours. This pretreatment has been found to minimise the residual endogenous auxin content.
- After this, the sub-apical 6 mm lengths of coleoptiles 2 mm below the apices are cut with razor blades.
- Such coleoptile sections are placed in Petri dishes with ABA to be assayed, standard IAA, 3 per cent sucrose in phosphate buffer (0.006 M, pH 5.2).
- These are incubated at 25°C for 20 hours and the lengths measured accurately.
c. Retardation of Growth of Cultures of Small Aquatic Plants:-
- This assay has been described by Van Overbeek et al.
- Cultures of Lemna minor (duckweed) are the sensitive materials to respond to ABA. Therefore, sterile cultures of L. minor grown under constant fluorescent light and constant temperature are used as bioassay materials.
- Growth is vegetative by budding and is determined as increase of fresh weight.
- ABA concentration as low as 1 part per billion (ppb) causes detectable inhibition.
d. Barley, Rice and Wheat Endosperm Bioassay:-
- Paleg (1960) studied GA-induced production of a-amylase in barley and the subsequent release of reducing sugars into the medium.
- ABA inhibits the production of a-amylase which is triggered by GA in isolated aleurone layers or de-embryonated seeds.
- Seeds are cut, embryo-containing portion discarded, placed in vials with test solutions (GA + ABA) and antibiotic Streptomycin sulphate to prevent bacterial growth.
- After incubation for a definite period, reducing sugars are assayed.
- Instead of measuring reducing sugar in the medium, α-amylase may be directly assayed.
- Since enzyme synthesis is a stage closer to the primary site of action of GA and ABA in this system than is sugar release, the authors regard this an advantage in itself.
e. Detection of Antitranspirant Activity:-
- A greatly improved method has been described for the bioassay of ABA and other compounds that possess antitranspirant activity.
- The stomatal responses are observed on pieces of isolated epidermis of Commelina sp. immersed in small volumes of solution containing the compounds to be assayed.
- It is possible to obtain linear responses to ABA concentrations over the range 10-7 to 10-10M in citrate buffer at pH 5.5.
- The extent of stomatal closure in response to a definite concentration of ABA is the basis for this assay and it is possible to detect as little as 26 picogram (pg) of ABA present in the medium.
- Lack of interference from other regulators is a feature unique to this assay.
5. Physiological effects of Abscisic Acid:-
i. Seed and bud dormancy:- Its main function is to maintain seed dormancy. Abscisic acid induces dormancy of buds towards the approach of winter. Abscisic acid accumulates in many seeds during maturation and apparently contributes to seed dormancy.
ii. Senescence:- ABA acts as a general inducer of senescence (Thimann). The onset of senescence is correlated with stomatal closure. The ABA content of aging leaves increases markedly as senescence is initiated.
iii. Abscission:- It is the process of shedding old or unwanted organs such as leaves, flower, floral organs, and fruits. ABA promotes abscission through ethylene.
iv. Flowering:- In long-day plants, the effect of gibberellins on flowering is counteracted by ABA, which accumulated in the leaves during the short winter days. This ABA acts as inhibitor of flowering in long-day plants. On the other hand ABA induces flowering in short-day plants.
v. Starch hydrolysis:- The GA-induced synthesis of a-amylase and other hydrolytic enzymes in barley aleurone cells is inhibited by abscisic acid. This inhibition can be reversed by increasing the amount of GA supplied.
vi. Geotropism:- ABA controls geotropic responses of roots. It stimulates positive geotropism in roots.
vii. Stress response:- It also close the stomata of the leaves in dry conditions. Hence it is also called stress hormone.
6. Applications of Abscisic Acid:-
i. Antitranspirant:- Application of minute quantity of abscisic acid to leaves shall reduce transpiration to a great extent through partial closure of stomata. It conserves water and reduces the requirement of irrigation. Photosynthesis is reduced to a lesser extent (Transpiration 56%: Photosynthesis 14%).
ii. Flowering:- It is useful in introducing flowering in some short day plants kept under un-favourable photoperiods.
iii. Rooting:- Use of abscisic acid promotes rooting in many stem cuttings.
iv. Dormancy:- Abscisic acid can be used in prolonging dormancy of buds, storage organs and seeds.
7. Mode of Action of Abscisic Acid:-
a. Regulation of Gene Expression and Enzyme Synthesis:-
> ABA has inhibitory effects on protein synthesis. The effect of ABA on protein synthesis appears to be selective since it has been shown to affect directly the synthesis of those proteins whose synthesis is under hormonal control.
> The best example of such translational control is the inhibition by ABA of the GA-promoted synthesis of α-amylase and other hydrolases like protease and ribonuclease in barley aleurone layers.
> Ho and Varner suggested that the inhibition of a-amylase synthesis by ABA is due to an effect on translation, since they found that ABA still inhibited the formation of α-amylase at 12 h, a time when RNA synthesis inhibitor cordycepin had no longer any effect.
> They have postulated that ABA might de-repress a regulator gene or interact with a regulator RNA protein species to inhibit the translation of α-amylase mRNA.
> ABA has regulatory effects at the levels of both transcription and translation. The expression of numerous genes is stimulated by ABA under various stress conditions. Such conditions include heat shock, low temperature and salinity stress as well as the period of seed maturation.
> It is well known that gene activation is mediated by DNA-binding proteins, which acts as transcription factors. ABA has been shown to stimulate the transcription of the genes, which encode these binding proteins.
> Distinct DNA segments have been identified that are involved in stimulation of transcription by ABA, thought to be ABA-response elements (ABAREs). Contrary to the stimulation of gene expression, ABA is also involved in repression of gene transcription.
> The common example is ABA-induced repression of barley a-amylase gene which is expressed by GA. In this case, a few DNA elements which mediate ABA-induced gene repression are quite similar to the gibberellin response elements (GAREs).
b. Stomatal Complexes and ABA:-
> Turgor changes within the guard cells regulate stomatal aperture. Such turgor changes are caused by movement of K+, H+, Cl– ions and the synthesis, metabolism and movement of organic anions, particularly malate. The role of ABA in these movements and synthesis is still a matter of conjecture.
> One important factor in regulating guard-cell turgor is thought to be the operation of an active H+/K+ exchange process. ABA inhibits K+ uptake into the guard cells and also proton release. The fungal toxin fusicoccin (FC) overcomes the effect of ABA on the stomata by stimulating H+/K+ exchange. ABA may also affect the distribution of malate.
> During stomatal closure in dark, malate leaks from the epidermal strips of Commelina communis, Vicia faba floating on water. Addition of ABA increases both the rate of closure and the rate of malate leakage.
> Malate serves as a source of protons for H+/K+ exchange process during opening and malate leakage from guard cells during closure is required to reduce turgor. ABA would inhibit H+/K+ exchange and promote specific leakage of malate, thus inhibiting opening and promoting closure.
c. Membrane Depolarization by Increasing Ca2+ and pH of Cytosol:-
> Although stomatal closure is mediated by a reduction in guard cell turgor pressure caused by massive outward movement of K+ and anions (CI– and malate) from the cell, ABA also induces a net influx of positive charge.
> ABA has been shown to induce an increase in cytosolic Ca2+ concentration by activation of calcium channels leading to membrane depolarization. Cytosolic calcium concentration is further increased by ABA by causing release of calcium from an internal store like vacuole, and this increase is sufficient to cause stomatal closing.
> In addition to increasing cytosolic Ca2+, ABA causes alkalization of the cytosol. The increase in cytosolic pH triggers the voltage-gated K+ efflux channels to open resulting in K+ loss and stomatal closure.
> The inhibition of plasma membrane H+-ATPase has also been held responsible for membrane depolarization. ABA inhibits the plasma membrane proton pump and favours depolarization in an indirect manner. It is presumed that the increase in Ca2+ concentration and pH of the cytosol in the presence of ABA results in the inhibition of H+ -ATPase.
> ABA not only causes stomatal closure by activating outward ion channels but also prevents stomatal opening. Under normal conditions, the inward K+ channels are open when the membrane is polarized by the proton pump and K+ ions get inside through H+/K+ exchange causing stomatal opening. In this case, ABA inhibits inward K+ channels through an increase in Ca2+ and pH, thus preventing stomatal opening.
