Harmone Regulatory Pathway: ABA

1. SACHIN ARUNRAO TAJNE
Ph.D. first year
PMBB
IGKV, Raipur

2. Abscisic acid (ABA) is a 15-C weak acid
that was first identified in the early 1960s
by F.T. Addicott and his associates as a
growth inhibitor accumulating in abscising
cotton fruit (“abscisin II”) and leaves of
sycamore trees photoperiodically induced
to become dormant (“dormin”) by
Cornforth et al. (1965)
ABA has since been shown to regulate
many aspects of plant growth and
development including embryo maturation,
seed dormancy, germination, cell division
and elongation, floral induction, and
responses to environmental stresses such as
drought, salinity, cold, pathogen attack and
UV radiation.
Abscisic acid (ABA)

3. Functions of Abscisic Acid
Abscisic acid (ABA) is the major hormone that controls plants
ability to survive in a harsh, changing environment. When
challenged by water stress, plants increase their synthesis of ABA,
which triggers responses including stomatal closure to reduce
transpiration and expression of genes to produce osmoprotectants.
Inhibits shoot growth but will not have as much affect on roots or
may even promote growth of roots.
Induces seeds to synthesize storage proteins.
Inhibits the affect of gibberellins.
Has some effect on induction and maintanance of dormancy.
Induces gene transcription especially for proteinase inhibitors in
response to wounding which may explain an apparent role in
pathogen defense.

4. CHEMICAL COMPOSITION
ABA is a 15-C Sesquiterpene compound
Composed of three isoprene residues
Cyclohexane ring with keto, one hydroxyl group, a side chain
with a terminal carboxylic group
The orientation of carboxylic group at carbon 2 determines the cis
and trans isomers of ABA Cis-Abscisic acid (biologically active)
Trans-Abscisic acid (biologically inactive) Nearly all the naturally
occurring ABA is in the cis form

5. OCCURRENCE AND DISTRIBUTION
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
A 15-C compound called lunularic acid has been found in algae and
liverworts
ABA is synthesized in all types of cells that contain chloroplasts or
other plastids
It occurs predominantly in mature green leaves
ABA has been detected in all major organs or living tissues from
root caps to apical buds, Phloem sap, xylem sap and in nectar

6. THE ABA BIOSYNTHESIS PATHWAY
The first step that is more specific to the ABA biosynthesis pathway is
the epoxidation of zeaxanthin and antheraxanthin to violaxanthin, which
occurs in plastids. This step is catalyzed by a zeaxanthin epoxidase
(ZEP).
After a series of structural modifications, violaxanthin is converted to
9-cis-epoxycarotenoid. Oxidative cleavage of the major
epoxycarotenoid 9-cis-neoxanthin by the 9-cis-epoxycarotenoid
dioxygenase (NCED) yields a C15 intermediate, xanthoxin. This step
was considered the first committed step in the ABA biosynthesis
pathway.
The product xanthoxin is then exported to the cytosol, where it is
converted to ABA through a two-step reaction via ABA-aldehyde.
A short-chain alcohol dehydrogenase/reductase (SDR), encoded by
the AtABA2 gene, catalyzes the first step of this reaction and generates
ABA aldehyde.
ABA aldehyde oxidase (AAO) then catalyzes the last step in the
biosynthesis pathway.

7. ZEP- zeaxanthin epoxidase
NCED - 9-cis-epoxycarotenoid dioxygenase
SDR - short-chain alcohol dehydrogenase/reductase
AAO - ABA aldehyde oxidase

8. Mutations in either the aldehyde oxidase apoprotein or
molybdenum cofactor (MoCo) synthase (e.g. Ataba3 mutant) would
impair ABA biosynthesis.
The AtABA3 gene encodes a MoCo sulfurase that catalyzes the
sulfurylation of a dioxo form of MoCo to a sulfurylated mono-oxo
form .
This form of MoCo is required by aldehyde oxidase and xanthine
dehydrogenase for their activities.
Mutants have been isolated that cause defects in the conversion of
ABA aldehyde into ABA

9. ABA signaling
PYRABACTIN RESISTANCE (PYR)/REGULATORY
COMPONENT OF ABA RECEPTOR (RCAR) protein family
serves as ABA receptors.
PYR/RCAR proteins are a family of small proteins with a
steroidogenic acute regulatory lipid transfer domain. When ABA
binds to PYR/RCARs, the hormone moves into a pocket that is then
enclosed by a receptor conformational change.

11. DEVELOPMENTAL REGULATION OFABA BIOSYNTHESIS
Seed maturation and germination expose the young embryo to
dramatic osmotic stresses. ABA is the major factor that is required to
escort the embryo upon entering and exiting its quiescent state.
ABA in developing seeds can either be derived from maternal
tissues or be synthesized de novo in the embryo.
Studies in Arabidopsis suggest that during seed development, there
appear to be two peaks of ABA accumulation.
The first one occurs about halfway during seed development
(approximately 10 d after pollination). This ABA is likely to be
derived from maternal tissues.
ABA at this stage promotes the synthesis of storage proteins.
The second peak with less significant ABA accumulation (about
one-third of the first peak) is from biosynthesis in the embryo and
may activate the synthesis of LEA (Late Embryogenesis Abundant)
proteins that prepare the embryo for desiccation.
This peak of ABA also initiates seed dormancy.

12. Soluble sugars, osmotic stress,
and ABA itself are likely to be
the signals that activate ABA
biosynthesis in developing
seeds.
Maize Viviparous1 (Vp1) gene - an ortholog of
the ABI3 of Arabidopsis

13. ABA added to the culture medium
inhibits precocious germination.
Further evidence for the role of ABA in
preventing precocious germination has
been provided by genetic studies of
vivipary.
In maize, several viviparous mutants
have been selected in which the embryos
germinate directly on the cob while still
attached plant.
Several of these mutants are ABA-
deficient (vp2, vp5, vp7, vp9, vp14); one
is ABA-insensitive (vp1)
ABA inhibits precocious germination and vivipary
precocious germination in
ABA- deficient vp14 mutant
of maize

14. ABIOTIC STRESS REGULATION OF ABA BIOSYNTHESIS
The environmental conditions that most dramatically activate ABA
biosynthesis are drought and salt stress.
Increased ABA levels under these abiotic stresses result mainly from
increased de novo biosynthesis.
Abiotic stresses such as drought and salt activate the biosynthetic
genes , probably through a Ca2+ dependent phosphorelay cascade.
ABA feedback stimulates the expression of the biosynthetic genes,
which is also likely through a Ca2+ dependent phosphoprotein cascade.
Among the biosynthetic genes, NCED is strongly upregulated by
stress

16. Drought stress treatments were shown to induce NCED expression in maize
(Tan et al., 1997), tomato (Burbidge et al., 1999), bean (Phaseolus
vulgaris; Qin and Zeevaart, 1999), Arabidopsis (Luchi et al., 2001), cowpea
(Vigna unguiculata; Luchi et al., 2000), and avocado (Persea
americana; Chernys and Zeevaart, 2000).
ZEP genes in roots were clearly regulated by drought stress. Its transcript
levels increased severalfold after drought stress both in tobacco and tomato
plants (Audran et al., 1998; Thompson et al., 2000a).
The SDR gene is expressed constitutively at a relatively low level and is not
induced by drought stress. Rather, its expression is enhanced by sugar. Sugar
levels also vary diurnally and are influenced by abiotic stress.
In fact, with the exception of AtSDR1, whose expression appears not to be
regulated by stress (Cheng et al., 2002; Gonzalez-Guzman et al., 2002), all the
other ABA biosynthetic genes are up-regulated by drought and salt stress.

17. ABA closes stomata in response to water stress
ABA closes stomata in response to water stress
Stomatal closing can also be caused by ABA synthesized in the roots
and exported to the shoot

18. SELF-REGULATION OF ABA BIOSYNTHETIC GENES
•Inhibition of its own biosynthesis
ABA 8′-hydroxylase, which catalyzes the first step of ABA degradation, was stimulated by
exogenous ABA. Studies with transgenic tobacco overexpressing NCED gene showed that
ABA overproduction correlated with the overaccumulation of the catabolite, phaseic
acid. ABA negatively regulate ABA accumulation by activating its catabolic enzymes
under non-stressful conditions.

19. •Stimulation of it’s own biosynthesis
ZEP, AAO3, and MCSU (MoCo sulfurase ) in Arabidopsis are all up-
regulated by ABA, in addition to being regulated by stress.
Exogenous ABA significantly enhanced the expression of these genes
(Xiong et al., 2001). Moreover, these genes appear also to be regulated by
endogenous ABA.
In addition, AtNCED3 transcript levels under drought and salt stress
treatments were significantly reduced in the ABA-deficient
mutants los5 and los6 as compared with those in wild-type seedlings,
demonstrating that ABA is required for full activation of At-NCED3 by
osmotic stress (Xiong et al., 2002)

20. SAD1 (supersensitive to ABA and drought 1) is a signaling component
which mediate this self-regulation of ABA biosynthetic genes.
Further characterization of sad1 found that the mutant was defective in
the self-regulatory loop because the sad1 mutation impairs ABA
regulation of the AAO3 and MCSU genes (Xiong et al., 2001).
Both gene products are required for the last step of ABA biosynthesis,
i.e. the conversion of ABA aldehyde to ABA.

21. DIFFERENTIAL REGULATION OFABA BIOSYNTHETIC GENES
Genes involved in ABA biosynthesis exist either as a single copy or a
gene family, and the family members may be subjected to differential
regulation.
For Arabidopsis, ZEP, MCSU, and SDR are single-copy genes,
whereas NCED and AAO belong to gene families.
Those belonging to gene families, each member is regulated
differently by stresses. In addition, they may be expressed in a tissue-
and developmental stage-specific manner. For example, AtAAO3 was
expressed in leaves but not in the root and less in seeds.

22. THE RATE-LIMITING STEP IN THE BIOSYNTHESIS PATHWAY
Because the ABA biosynthesis pathway involves multiple gene
products, there could be a rate-limiting step in the pathway.
Finding this limiting step is important for genetic manipulation of the
pathway.
It was recognized generally that the step catalyzed by NCED, i.e. the
oxidative cleavage of neoxanthin, is rate limiting (Tan et al., 1997; Qin
and Zeevaart, 1999). This may be valid in leaves, where most studies on
ABA biosynthesis are concerned.
Consistent with this prediction, constitutive or inducible overexpression
of the NCED genes resulted in increased ABA biosynthesis and reduced
transpiration water loss (Luchi et al., 2001; Qin and Zeevaart, 2002;
Thompson et al., 2000).
Experimental evidence indicated that even overexpressing the AtZEP
gene, whose product catalyzes the least rate-limiting step considered in
the pathway, could result in an increased stress gene induction in
Arabidopsis seedlings (Xiong et al., 2002).

23. Furthermore, overexpression of NtZEP led to increased seed dormancy
and delayed seed germination in tobacco (Frey et al., 1999)
NCEDs are either not upregulated or weakly up-regulated by ABA may
cause the NCED step to become rate limiting late in ABA biosynthesis in
leaves. As a consequence, regulating NCED genes may have a more
significant impact on overall ABA biosynthesis.

24. References
•Xiong, L. and Zhu, J.K., 2003. Regulation of abscisic acid
biosynthesis. Plant physiology, 133(1), pp.29-36.
•Milborrow, B.V., 2001. The pathway of biosynthesis of abscisic acid in
vascular plants: a review of the present state of knowledge of ABA
biosynthesis. Journal of experimental botany, 52(359), pp.1145-1164.
•Tuan, P.A., Kumar, R., Rehal, P.K., Toora, P.K. and Ayele, B.T., 2018.
Molecular mechanisms underlying abscisic acid/gibberellin balance in
the control of seed dormancy and germination in cereals. Frontiers in
plant science, 9.
•Raghavendra, A.S., Gonugunta, V.K., Christmann, A. and Grill, E.,
2010. ABA perception and signalling. Trends in plant science, 15(7),
pp.395-401.