21.1.

5 ABSCISIC ACID MEDIATES RESPONSE TO WATER STRESS

Plants generally respond to acute water deficits by closing their stomata in order to match
transpirational water loss from the leaf surface with the rate at which water can be resupplied by
the roots. Since the discovery of ABA in the late 1960s, it has been known to have a prominent role
in stomatal closure during water stress. In fact, ABA has long been recognized as antitranspirant
because of its capacity to induce stomatal closure and thus reduce water loss through transpiration.
ABA accumulates in water-stressed (that is, wilted) leaves and exogenous application of ABA is a
powerful inhibitor of stomatal opening. Furthermore, two tomato mutants, known as flacca and
sitiens, fail to accumulate normal levels of ABA and both wilt very readily. The precise role of ABA in
stomatal closure in water-stressed whole plants has, however, been difficult to decipher with
certainty. This is because ABA is ubiquitous, often occurring in high concentrations in nonstressed
tissue. Also, some early studies indicated that stomata would begin to close before increases in ABA
content could be detected. According to current thinking, the initial detection of water stress in
leaves is related to its effects on photosynthesis. Inhibition of electron transport and
photophosphorylation in the chloroplasts would disrupt proton accumulation in the thylakoid lumen
and lower the stroma pH. At the same time, there is an increase in the pH of the apoplast
surrounding the mesophyll cells. The resulting pH gradient stimulates a release of ABA from the
mesophyll cells into the apoplast, where it can be carried in the transpiration stream to the guard
cells (Figure 21.4).

As noted above, wilted leaves accumulate large quantities of ABA. In most cases, however,
stomatal closure begins before there is any significant increase in the ABA concentration. This could
be explained by the release of stored ABA into the apoplast, which occurs early enough and in
sufficient quantitythe apoplast concentration will at least doubleto account for initial closure.
Increased ABA synthesis follows and serves to prolong the closing effect. Stomatal closure does not
always rely on the perception of water deficits and signals arising within the leaves. In some cases it
appears that the stomata close in response to soil desiccation well before there is any measurable
reduction of turgor in the leaf mesophyll cells. Several studies have indicated a feed-forward control
system that originates in the roots and transmits information to the stomata. In these experiments,
plants are grown such that the roots are equally divided between two containers of soil (Figure
21.5A). Water deficits can then be introduced by withholding water from one container while the
other is watered regularly. Control plants receive regular watering of both containers. Stomatal
opening along with factors such as ABA levels, water potential, and turgor are compared between
half-watered plants and fully watered controls. Typically, stomatal conductance, a measure of
stomatal opening, declines within a few days of withholding water from the roots (Figure 21.5B), yet
there is no measurable change in water potential or loss of turgor in the leaves. In experiments with
day flower (Commelina communis), there was a significant increase in ABA content of the roots in
the dry container and in the leaf epidermis (Figure 21.6). Furthermore, ABA is readily translocated
from roots to the leaves in the transpiration stream, even when roots are exposed to dry air. These
results suggest that ABA is involved in some kind of early warning system that communicates
information about soil water potential to the leaves.
21.1.6 OTHER ABSCISIC ACID RESPONSES

There is recent evidence that ABA may also have a role in lateral or secondary root
development. The initiation and development of lateral roots is known to be primarily under the
control of auxin, but lateral root development can be inhibited by ABA if the hormone is applied
during early stages of lateral root development, before the lateral root meristem becomes organized.
Earlier studies also indicated an impact of exogenous ABA on flower formation under certain
conditions, but the data was equivocal. In particular, no causal relationship could be established
between endogenous ABA levels and flowering behavior. However, the prospect of a role for ABA in
flowering has been revived recently with the discovery that, under conditions that would normally
delay flowering, ABA-deficient mutants of Arabidopsis produce flowers somewhat earlier than
wildtype plants. This observation suggests that endogenous ABA may normally inhibit or delay
flowering in Arabidopsis. Further support comes from the discovery that a gene (FCA) previously
known to be involved in controlling the time of flowering also has the properties of an abscisic acid
receptor. We will take a closer look at this receptor in the next section and the role of the FCA gene
in flowering in Chapter 25.

21.1.7 ABA PERCEPTION AND SIGNAL TRANSDUCTION

ABA perception and signaling appears to be particularly complex and, although its
metabolism and physiology ABA perception and its subsequent signal chain have remained elusive.
As noted earlier, ABA is a weak acid. As such it is likely to exist in both the protonated and
unprotonated forms in the relatively acidic environment of the apoplast. In the protonated state it
may diffuse across the plasma membrane and react with an intracellular receptor or, in the
unprotonated form, it may remain outside the cell to be sensed by a site on the plasma membrane.
Indeed, experiments employing impermeable ABA derivatives and/or microinjection of ABA into
cells have indicated multiple ABA receptors at multiple locations. Over the last 20 years, methods
that have normally been employed to identify hormone receptors have proven relatively
unsuccessful in the search for ABA receptors. A more recent approach has made use of antigen-
antibody reactions with what are called antiidiotypic antibodies. In this method, antibodies raised
against ABA are themselves used as antigens to raise a second group of antibodiesthe anti-
idiotypic antibodiesthat have binding characteristics similar to ABA. Thus, any protein that binds
with the anti-idiotypic antibodies could be a putative ABA receptor. The anti-idiotypic antibodies
were then used to screen the proteins encoded by a complimentary approach led to the
identification of ABAP1, a protein that is located in the plasma membrane of barley aleurone cells
and that specifically and reversibly binds ABA in vitro. Since the discovery of ABAP1, at least three
other putative ABA receptors have been identified. One is a chloroplast protein Magnesium
Protoporphyrin-IX Chetalase H subunit (CHLH, also known as ABAR). The second is a soluble,
flowering-time control protein FCA isolated from Arabidopsis. Based on similarity of amino acid
sequence, FCA is homologous to the barley protein ABAP1. FCA interacts with another protein (FY)
to regulate the processing of functional mRNA (see Chapter 25 for the role of FCA in flowering).

The third putative receptor is a membrane-localized G-protein coupled receptor (GPCR)

identified as GCR2. The simple fact that these proteins bind ABA in vitro,

however, does not prove they are true receptors. It

still needs to be demonstrated that loss-of-function

or gain-of-function mutants alter ABA functions in a

predictable manner.

The signal chain for ABA effects, both upstream and

downstream from the hormone, is a subject of intensive

study. The apparently complex interactions between

abiotic signals, receptors, second messengers, and

ABA-induced gene transcriptionlet alone crosstalk

with other signalsmake it difficult to assemble a

definitive scheme. Still, a number of components

are beginning to fall into place. Most of the recent

progress has been made through newly discovered

ABA-insensitive gene mutations and can be summarized

in the following points.

1. There appears to be rapid turnover of ABA in both

stressed and unstressed plants, but the events that

sense abiotic stress and initiate ABA accumulation

remain unknown.

2. Ca2+ appears to be an important part of the ABA

signal chain, especially in stomatal guard cells.

Ca2+ mediates ABA-induced turgor adjustments

by activating plasma membrane anion channels

(Figure 21.7).

3. The promoter region of some genes contains a

sequence called the ABA response element (ABRE).

Transcription factors known as ABA response element

binding factors (ABFs) bind to this promoter

region to regulate the activity of many ABA-induced

genes. These genes include putative protective proteins

such as enzymes required for the synthesis

of osmolytes or compatible solutes that help the

plant adapt to water stress (Chapter 13), and transcription

factors that in turn regulate other changes

in gene expression

4. A number of ABA-insensitive (abi) mutants have

been identified. At least three insensitive mutants,

abi3, abi 4, and abi 5, impair only seed germination

and early seedling development. All three wildtype

genes (ABI3, 4, 5) encode transcription factors that

are expressed mainly in seeds, suggesting that the

role of ABA in seeds requires gene transcription.

5. A number of ABA-activated protein kinases that

positively regulate ABA responses have been identified.

In addition, ABI1 and ABI2 are protein phosphatases

that negatively regulate ABA responses. So,

protein phosphorylation events are clearly important

in ABA signaling.

It will no doubt take some time to sort out all of

these components and those yet to be discovered and

construct a clear model of the signaling chains for various

ABA-mediated responses.
FIGURE 21.7 A simplified schematic illustrating the coordination of ion pumps

by ABA and Ca2+ during closure of stomatal guard cells. ABA is perceived by an

unknown receptor (ABA R) that transmits the ABA signal to inward-rectifying calcium

channels via a membrane-associated protein kinase. The kinase is antagonized

by a protein phosphatase (PP). ABA also stimulates the release of Ca2+ from internal

stores such as the endoplasmic reticulum (ER), possibly mediated by phospholipid

signaling and/or G protein. The increased cytosolic Ca2+ concentration inhibits K+

in

channels and opens both K+

out and anion channels (A). The result is a net loss of

ions from the guard cell, followed by a loss of water and turgor, and closure of the

stomatal pore.

FIGURE 21.5 (A) An experimental setup for

testing the effects of desiccated roots on

ABA synthesis and stomatal closure. Roots

of a single plant are divided equally between

two containers. Water supplied to one container

maintains the leaves in a fully turgid

state while water is withheld from the second

container. Withholding water from the

roots leads to stomatal closure, even though

the leaves are not stressed. (B) Stomatal closure

in a split-root experiment. Maize (Zea

mays) plants were grown as shown in (A). Control

plants (open circles) had both halves of the

root system well-watered. Water was withheld

from half the roots of the experimental plants

(closed circles) on day zero. Stomatal opening,

measured as leaf conductance, declined in

the plants with water-stressed roots. (B from

Blackman, P. G., W. J. Davies. 1985. Journal

of Experimental Botany 36:3948. Reprinted

by permission of The Company of Biologists,

Ltd.)
FIGURE 21.6 Effect of air drying on the ABA content

of Commelina communis root tips. Root tips were air

dried to the relative water contents shown in the upper

curve. Lower curve shows the dramatic increase in ABA

content as the fresh weight decreases. (From Zhang,

J., W. J. Davies. 1987. Journal of Experimental Botany

38:20152023. Reprinted by permission of The Company

of Biologists, Ltd.)

FIGURE 21.4 ABA movements in the apoplast. ABA synthesized

in the roots is carried to the leaf mesophyll cells

(heavy arrows) in the transpiration stream (light arrows).

ABA equilibrates with the chloroplasts of the photosynthetic

mesophyll cells or is carried to the stomatal guard

cells in the apoplast.