Abscisic Acid: Biosynthesis and Methods | Plant Hormones

In this article we will discuss about Abscisic Acid. After reading this article you will learn about: 1. Biosynthesis of Abscisic Acid 2 . Responses of Plants to Abscisic Acid 3. Mode of Action 4. Bioassay Methods.

Biosynthesis of Abscisic Acid:

Two pathways have been suggested for the biosynthesis of abscisic acid. One involves the cleavage of a C₄₀ precursor, a xanthophyll carotenoid, and the other involves the direct formation from C₁₅ precursor, farnesyl pyrophosphate.

(a) Indirect or Xanthophyll Cleavage Pathway of Abscisic Acid:

The initial reactions of ABA biosynthesis take place in chloroplasts. The biosynthetic pathway begins with the C₅ isoprene unit, isopentenyl pyrophosphate (IpPP), and through a few steps leads to the synthesis of oxygenated carotenoid like C₄₀ xanthophyll, zeaxanthin, which is then converted to violaxanthin.

Subsequently, violaxanthin is converted to 9-cis-neoxanthin, which then undergoes cleavage to form C₁₅ compound xanthoxin, possibly outside the chloroplasts. Xanthoxin is a neutral growth inhibitor with ABA-like properties. In the last step, xanthoxin is converted to ABA aldehyde in the cytosol by the loss of epoxy group, which is finally oxidized to ABA. This pathway is predominant in higher plants.

(b) Direct or Isoprenoid Pathway of Abscisic Acid:

A direct pathway for ABA biosynthesis has been suggested which uses the initial steps of polymerization of isoprene units leading to the formation of a C₁₅ precursor farnesyl pyrophosphate, a sesquiterpenoid molecule. The C₁₅ ABA is directly synthesized from C₁₅ farnesyl pyrophosphate. The direct pathway occurs mainly in the fungi.

(c) ABA Inactivation:

Free ABA can be inactivated by oxidation, which involves hydroxylation of one of the 6-dimethyl groups forming 6-hydroxymethyl ABA (HMABA). This unstable intermediate undergoes rearrangement to be converted to phaseic acid (PA). PA may be reduced to 4-dihydrophaseic acid (DPA). It has been reported that PA is either inactive or shows weak activity. The other product DPA is, however, without any activity.

Inactivation of free ABA can also occur by conjugation to a monosaccharide like glucose. ABA- glucosyl ester (ABA-GE) is an example of such a conjugate that is inactive as a hormone. In contrast to free ABA that is localized in cytosol, ABA-GE migrates to vacuoles and may function as a storage form of ABA.

(d) ABA Transport:

ABA can be transported over long distances in plants via the phloem and xylem, and the movement may occur from mature leaves to shoot tips and roots. As indicated previously, ABA can move basipetally from the root cap to the meristem of roots. Contradictory results have been obtained in petiole explants which indicate that ABA movement shows either a slight basipetal polarity or it is non-polar.

ABA synthesized in the roots can be transported to the shoot via the xylem. ABA concentration in xylem sap of water-stressed plant rises several fold as compared to the well-watered plants. It is believed that ABA synthesized in the root serves to act as a signal conveying the water status of the drying soil to the leaves wherein the stomata are closed to check transpiration.

It has been noted that depending upon the increase in xylem sap pH, ABA undergoes redistribution in the leaf during water stress. Under normal conditions, the xylem sap is slightly acidic (pH 6.3) which is favourable for the mesophyll cells to take up un-dissociated and protonated form of ABA (ABAH).

The xylem sap becomes slightly alkaline (pH 7.2) during water stress causing dissociation of ABAH to free ABA. Since ABA does not readily cross membrane and enter into the mesophyll cells, it is expected that more ABA reaches the guard cells. Wilkinson and Davis have suggested that alkalization of the xylem sap serves as a root signal that promotes stomatal closure.

2. Responses of Plants to Abscisic Acid:

(i) Growth Inhibition:

ABA can antagonize the responses of plant materials to each of the growth-promoting hormones, viz., auxins, gibberellins and cytokinins.

Although ABA is present even in rapidly growing organs, normal growth is attributed to its less than optimal concentrations to produce an inhibitory effect which is also thought to be masked by the presence of growth-promoting phytohormones. Presence of ABA in inhibited dormant lateral buds points to the role of ABA as a correlative inhibitor.

(ii) Growth Promotion:

At very low concentration (viz., 10^-9 M), ABA has been found to promote some growth processes like parthenocarpic seed development, rooting of cuttings, soybean callus growth in presence of kinetin and frond number of duckweed (Lemna polyrhiza).

(iii) Water Stress:

There is an abrupt rise in ABA content in leaves of many plants as the water potential falls below (-) 1.0 MPa (mega Pascal) which is equivalent to (-) 10.0 bars. It has also been shown that exogenous ABA initiates stomatal closure when applied to intact leaves or isolated epidermal strips.

These observations have led to the hypothesis that ABA is involved in regulating stomatal aperture in water-stressed plants. It is now clear that rises in endogenous levels of ABA in leaves can readily inhibit stomatal opening and such inhibition plays an important part in water conservation mechanism.

(iv) Drought Resistance and Desiccation Tolerance:

Since endogenous ABA regulates stomatal opening in response to water stress, it plays a positive role in drought resistance. In drought-resistant cultivars of maize and sorghum, more ABA has been found to accumulate.

In developing seeds, ABA promotes the synthesis of proteins involved in desiccation tolerance. During late stages of seed development, membranes and other cellular components are damaged by desiccation. At the same time, high levels of endogenous ABA promote an accumulation of specific mRNAs, which encode late-embryogenesis abundant (LEA) proteins involved in desiccation tolerance.

(v) Root Geotropism:

Experiments have indicated that root cap is the source of growth- inhibitory substances formed in response to gravity. These results have led to the hypothesis that when roots are maintained for a horizontal position, i.e., subjected to gravitropic stimulus, ABA produced in the root cap moves basipetally to the growing part of the root and accumulates in the lower half of the root causing a geotropic response.

(vi) Seed Development and Germination:

During the development of a variety of seeds, ABA levels rise sharply and then decline.

The highest concentration of ABA in the embryo occurs in many seeds at a time when their dry weight is increasing rapidly. High ABA levels during late embryogenesis cause an accumulation of storage proteins in the developing seeds either by regulating the translocation of sugars and amino acids to the seeds or by promoting protein synthesis.

The germination of most non-dormant seeds can be inhibited by exogenous ABA. Activities of various enzymes which rise during germination appear to be specifically inhibited by ABA. Likewise, ABA also inhibits the synthesis of a-amylase and other hydrolases in aleurone layers of cereal grains.

(vii) Dormancy:

Attempts to determine the factors which induce bud dormancy in trees led to the discovery of ABA.

Exogenous ABA has been shown to induce bud dormancy in woody plants where it proves to be an effective growth inhibitor. In dormant seeds it is present in high concentrations which declines when the seeds are given treatments which break dormancy. These observations have led to the hypothesis that ABA is involved in the induction and maintenance of dormancy.

ABA at high levels can maintain the mature embryo in a dormant state until the environmental conditions are favourable for growth. It is well known that seed dormancy is an important factor in the adoption of plants to un-favourable conditions.

(viii) Fruit Growth and Flowering:

Ripening fruits include the richest sources of ABA, yet the application of ABA to fruits has little effect on the process of ripening.

Ripening grape berry is an exception where ABA has the capacity to hasten the ripening and colouring of the fruit. ABA application in very low concentration has a slight promoting effect on flower growth. High ABA inhibits or delays flowering in a number of plants but this effect is probably a reflection of its inhibitory effect on growth.

3. Mode of Action in Abscisic Acid:

(i) 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).

(ii) 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.

(iii) 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.

4. Bioassay Methods 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:

(i) 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.

(ii) 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.

(iii) 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.

(iv) 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.

(v) Bioassay for Detecting 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.