ABSCISIC ACID Dr. Uttam Kumar Kanp

Contents: 1. Discovery of ABA 2. Chemical Nature 3. Bioassay of ABA 4.

Physiological roles of 5. Biosynthesis of AB A in Plants 6.

Degradation/Inactivation of AB A in Plants 7. Occurrence and Distribu tion of ABA in

Plants and 8. ABA Transport in Plant.

1. Discovery of ABA:

In 1963, a substance strongly antagonistic to growth was isolated by F. T. Add icott from young cotton fruits and named Abscisin II. Later on, this name was changed to Abscisic acid (ABA). The chemical name of abscisic acid whose structure is given in fig. 1.

Is [3-methyl 5-1′ (1′- hydrox y, 4′-oxy-2′, 6′, 6′-trimethyl-2- cyclohexane-l-yl)-cis, trans-2,4- penta-dienoic acid].

Eagles and Wareing (1963, 64), at the same time pointed out the presence of a substance in birch leaves (Betula pubescens, a decidu ous plant) which inhibited growth and induced dormancy of buds and, therefore, named it ‘dormin’. But, very soon as a result of the work of Cornfort h et. al. (1965), it

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

was found to be identical with absc isic acid.

2. Chemical Nature of ABA:

‹ Abscisic acid is a 15-C sesquiterpene compound (molecular formula C15H20O4) composed of

three residues and having a cyclohexane ring with keto and one hydroxyl group and a

side chain with a terminal car boxylic group in its structure.

‹ ABA resembles terminal portion of some such as and neoxanthin (see

Fig. 17.31) and appears to be a breakdown product of such carotenoids.

‹ Any change in its molecular structure results in loss of activity.

‹ ABA occurs in cis and trans -isomeric forms that are decided by orientation of -COOH group

around 2nd carbon atom in the molecule (Fig. 1.).

‹ Almost all naturally occurring ABA in plants exist in cis form that is biologically active and the

name absci sic and usually refers to this form.

‹ Trans-ABA is inactive form but can be inter convertible with cis - ABA.

‹ Due to the presence of a chiral centre (asymmetric carbon atom) at l’ -position, ABA also occurs

in two enantiomeric forms called S and R or (+) and (-) forms respectively which can be

distinguished from each other by their optical rotatory dispersion curves. In the S or (+) form,

which is dextrorotatory, the -OH at 1′ position faces below the plane of the ring and is shown by

dashed bond line while in R or ( -) form, which is laevorotatory, it faces above the plane of the

ring and is shown by a thick wedge shaped bond line (Fig. 2.).

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

2.

‹ Although both S and R enantiome rs of cis-ABA are biologically active, the R form is not active

in fast responses of ABA such as stomatal closure.

‹ Unlike cis and trans forms of ABA, the S and R forms are not inter- convertible in plant.

‹ Commercially available synthetic sample of ABA is a racemic mixture of equal amounts of

both S and R forms.

3. Bioassay of ABA:

A number of different bioassays have been developed to detect and estimate ABA in plant extracts which are based on,

‹ Acceleration of the abscission of cotyledonary petioles in explants of 14 day old cotton

seedlings;

‹ Inhibition of IAA-induced straight growth of Avena (oat) coleoptile;

‹ Inhibition of GA-induced synthesis of a -amylase in aleurone layer of germinating barley

(Hordeum);

‹ Inhibition of growth of duckweed Lemna minor.

‹ Inhibit ion of germination of isolated wheat (Triticum) embryos and,

‹ Stimulation of stomatal closure.

Of all above bioassays, the last one that is based on stimulation of stomatal closure has fast response of guard cells and is comparativ ely more sensitive. It can detect as low as 10-9 M ABA in plant

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

extracts.

Preliminary purification measures are however, necessary in all bioassays.

Physicochemical Methods:

The old bioassay methods have now been replaced with more accurate and reliable modern physicochemical methods of sep aration and quantification of the hormone. These include high performance liquid chromatography (HPLC) and gas chromatography (GC) which are then followed by mass spectrometry (MS) to pro vide proof of structure. Such methods can dete ct as low as 10-13g

ABA in plant extracts. Prelimina ry purification steps including thin layer chro- ma tography (TLC) are also required in such methods.

Immunoassay Methods:

Like other plant hormones (aux ins, gibberellins and cytokinins), highly sens itive and specific immunoassays are also applied for quantification of ABA which can detect as low as 10-13g ABA in partly purified or crude extracts. In immunoassay, specific anti hormonal antibodies are obtained by injecting the hormone (in this case ABA) in mice or rabbit. These antibodies are then used to react with the hormone in a cuvette assa y.

4. Physiological Role s of Abscisic Acid (ABA):

1. Stomatal Closing: The role of ABA in causing stomatal closure in plants undergoing water -

stress is now widely recognised. It has been suggested by various workers that in response to

the water-stress, the permeability of the chloroplast membranes of mesophyll cells to ABA is

greatly increased.

As a result, the ABA synthesized and stored in mesophyll chloroplasts diffuses out into the cyt oplasm. It then moves from one mesophyll cell to another through plasmodesmata and finally

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

reaches to the guard cells where it causes closing of stomata. Fresh biosynthesis of ABA continues in mesophyll chloroplasts during period of water -stress.

When wate r potential of the plant is restored (i.e., increased), the movement of ABA into the guard cells stops. ABA disappears from the guard cells a little later. The application of exogenous ABA to leaves of normal plants causes closing of stomata within a few m inutes. It has been suggested that ABA causes closing of stomata by inhibiting the ATP-mediated H +/K + ions exchange pumps in guard cells.

2. Bud Dormancy: Inducing bud dormancy in some temperate zone trees such as birch (Betula

pubescens), Acer, Fraxinus etc. ; Inducing dormancy of seeds which require stratification (i.e.,

exposure to low temp, for germination);

3. Process of tuberization;

4. Senescence of leaves;

5. Fruit ripening;

6. Abscission of leaves, flowers and fruits

7. Increasing the resistance of temperate zone p lants to frost injury,

8. Inhibition of GA-induced synthesis of a -amylase in aleurone layers of germinating barley;

9. Inhibition of precocious germination and vivipary and

10. Increase in root : shoot ratio at low water potentials .(The role of ABA in causing absc ission of

leaves, flowers and fruits is controversial.

11. ABA may stimulate abscission of organs only in a few species that too probably through

increase in production of ethylene. The primary hormone causing abscission is not ABA but

ethylene).

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

5. Biosynthesis of ABA in Plants: Extensive studies done by research ers with ABA deficient mutants of tomato, Arab idopsis and other plants have clearly shown that ABA is synthesized in higher plants not from simple terpenoid precursors directly through 15-C farnesyl diphosphate (FPP), but indirectly through pathway as breakdown product of 40-C xanthophyll such as violaxanthin or neo xan thin (Fig. 3).

3.

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

The initial steps of ABA biosynthesis take place in chloroplasts or other plastids while final steps occur in cytosol.

Violaxanthin is synthesized from (also a 40 -C xanthophyll) in a reaction that is catalysed by the enzyme zeaxanthin epoxidase (ZEP). This enzyme is encoded by ABA 1 locus of

Arabidopsis.

Violaxanthin is converted into 9 ′-cis-neoxanthin. The latter 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 xanthox al 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.

6. Degradation/Inactivation of ABA in Plants:

There are two ways in which ABA is degraded or inactivated in plants (Fig. 4).

Oxidation to Phaseic Acid:

This is the main route of degradation of ABA in plants. ABA is o xidised to phaseic acid (PA) with a subsequent reduction of the keto group on the cyclohexane ring to form dihydro phaseic acid

(DPA).

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

4.

In some cases, DPA may be further metabolized to form 4 ′-glucoside of DPA.

Both dihydrophaseic acid and its glucosid e are metabolically inactive. Phaseic acid is also inactive or exhibits greatly reduced activity in most bioassays but not in all. Like ABA, phaseic acid (PA) can also induce stomatal closure in some species and is as effective in inhibiting GA induced synthesis of a-amylase in aleurone layers of germinating barley.

Conjugation as Glucosides:

Free ABA can also be inactivated in plants by covalent conjugation with some simple sugar molecule such as glucose to form ABA -p-D- glucosyl ester (ABA-GE). The latter accumulates in vacuole.

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

7. Occurrence and Distribution of ABA in Plants:

Abscisic acid (ABA) is a ubiquitous plant hormone in vascular plants. In bryophytes, it has been found in mosses but not in liverworts. Some fungi synthesize ABA as secondary metabolite. ABA has not been detected in any algae. However, a 15-C organic compound called lunularic acid has been found in algae and liverwort s that appears to be possible functional equivalent of ABA in these plants.

Within the plant, ABA has been detected in all major organs or living tissues from root caps to apical buds such as roots, stems, buds, leaves, fruits and seeds and also in phloem and xylem sap and in nectar.

ABA is synthesized in all types of cells that contain chloroplasts or other plastids. It occu rs predominantly in mature green leaves.

Most plant tissues contain ABA in conc. of 20-100 ng per g fresh weight, but hig her conc. of 10 µg and 20 µg per g fresh weight have been reported in avocado fruit pulp and dormant buds of cocklebur (Xanthium spp.) respectively.

The concentration of ABA in spe cific plant tissues varies greatly at different developmental stages or in response to environmental cond itions especially water stress. For instance, in developing seeds

ABA conc. may increase 100 fold within a few days and decline as the seed ma tures. Similarly, under water stressed conditions, ABA level may increase 50 fold in the leaves within a few hours and declines to normal when plant water potential is restored.

The concentration of ABA in plant tissue i s regulated by:

(i) Its synthesis, (ii) Degradation, (iii) Compartmentation and (iv) Transport.

In plant, ABA predominantly occurs in its free form but it may also occur in conjugated form as

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

glycoside with some simple sugar molecule such as glucose forming ABA-β-D-glucosyl esters.

ABA is biologically inactive in its conjugated or bound form.

8. ABA Transport in Plant:

The transport of endogenous ABA at various stages of growth in higher plants is not very clear.

However, experiments made with 14C-labelled ABA have shown that, Externally applied ABA is able to get into tissues rapidly and is distributed freely in all directions within the plant; Cell to cell transport of ABA is slow and non -polar; ABA is present in phloem and xylem sap and is most probably tran s located throughout the plant through vascular tissue; ABA synthesized in root cap moves basipetally into the central vascular tissue;

There is some evidence of lateral movement of ABA in root tips in response to gravity stimulus where it causes asymmetric inhibition of growth resulting in geotropic curvature. i. Redistribution of ABA among plant cell compartments is controlled by pH gradient across the membranes. At low pH (6.3 or less) ABA exists in protonated or un-dissociated for m (ABAH) which can readily cross most cell memb ranes. At higher pH (7.2 or more), ABA exists in dissociated form

(ABA) that is impermeant and cann ot cross the membranes easily. ABA in proto na ted form tends to diffuse from a compartment with low or acidic pH into a compartment with high or alkaline pH. At higher pH, ABAH dissociates into ABA – and is trapped. ii. ABA is known to be transported in plant mostly in its free form.

However, ABA can also be transported to some extent in its conjugated form as ABA -β-D-glucosyl ester.

REFERENCES: 1. Plant Physiology by S.N. Pandey and B. K. Sinha. Forth edition.

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID

2. Applied Plant Physiology by Arup Kumar Mitra (2014). 3. Fundamentals of Plant Physiology by Dr. V. K. Jain, 2017. (S. Chand). 4. Studies in Bot any (Vol. 2); D. Mitra, J. Guha and S. K. Chowdhuri. Moulik Library (2006). 5. https://www.biologydiscussion.com/plant -physiology-2/plant-hormones/Abscisic acid.

This information, including the figures, are collected from the above references and will be used solely for academic purpose.

BOTANY: SEM – V, PAPE R-C12T: PLANT PHYSIOLOGY, UNIT -5: PLANT GROWTH REGULATORS – ABSCISIC ACID