CURRENT CONCEPTS ON THE RELATIONSHIP
BETWEEN ABSCISIC ACID AND LEAF WATER STRESS
1
LEONIDAS P. PASSaS2
ABSTRACT- A review of the available information on the relationship between ABA (abscisic acid) and leaf water stress is presented. Major subjects focused on are: leaf ABA content and water stress, possible mechanisms o! ABA-induced stomatal closure, ABA effects as related toCO 2 and other plant hormones, ABA and proline accumulation, and ABA as related to fim "a!ter-effect" of water stress. Remarkable results and some contradictory data are commented on briefly. Finaily, the extension of present knowledge is analyzed and suggestions for further work are nade.
Index terms: ABA, stomata..
CONCEITOS CORRENTES SOBRE A RELAÇÃO ENTRE O ÁCIDO ABSCISICO E O DÉFICIT HÍDRICO FOLIAR
RESUMO - O presente trabalho constitui uma revisão bibliográfica sobre a relação entre o ácido absc(-sico (ABA) e o déficit hídrico foliar. Os principais assuntos enfocados são: teor foliar de ácido abscí-sico e déficit hidrico, possíveis mecanismos de indução de fechamento de estômatos pelo ácido abscÇ-sico, relação entre os efeitos do ácido abscisico e CO2 e outros hormônios vegetais, ácido absc(sico e acumulaçân de prolina. e relação entre o ácido abscisico e o 'efeito posterior" do déficit hi'drico. Co-mentam.se brevemente os resultadós marcantes e alguns dados contraditórios. Analisa-se a extensão do atual conhecimento no assunto. Apresentam-se sugestôes para futuros trabalhos.
Termos para indexação: estômatos.
IlNd:t.].1'I4tI.1
ABA (abscisic acid) is a growth inhibitor widely studied in plant growth and development, and recent reviews (Walton 1980, Milborrow 1981 and 1984) have covered various aspects of its biosyn-thesis, metabolism and function. It has been suggested that native ABA is derived from the 2-cis isomer of C1 5 aldehyde xanthoxin (Taylor & Bur-den 1972 and 1973), but most workers now beieve it is ultimately synthesized from MVA (mevalonic acid) (Milborrow 1981 and Creelman & Zeevaart 1984) in plastids of plant celis. However, questions still remain about the site 0f ABA biosynthesis, and some results indicate that ABA is synthesized in the cytoptasm (Hartung et ai. 1982), and is then stored in plastids, where it can be released during water stress (Waiton 1980).
li is sUll unclear how the further metabolism of ABA is related to lis activity as a growth regulator. There are suggestions that ABA is metabolized
1 Accepted for publication on March 21, 1985. 2
Eng. - Agr., M.Sc., EMBRAPA/tjnidade de Execução de Pesquisa de Ambito Estadual de Bento Gonçalves (UEPAE de Bento Gonçalves), Caixa Postal 130, CEP 95700 Bento Gonçalves, RS, Brazil.
outside the chloroplasts (Milborrow 1979) and I-Iartung et aI. (1980) hypothesize that the enzymes involved in ABA degradation occur in the cytoplasm. The major pathway of ABA metabolism is lis conversion into PA (phaseic acid) and DPA (dihydrophaseic acid) (Murphy 1984). In addition, ABA is conjugated to ABA-GE (abscisic acid-B-D--glicose ester) (Milborrow 1981).
Several problems are recognized in studies of ABA. First, naturaily occurring ABA is highiy (+) in optical rotation, whereas the synthesized material commonly applied to tissues is (±) racemic (Milborrow 1984). Because differences are known to exist in the functions of the two forms (Mil-borrow 1984), applied forms of ABA should be cleariy stated. Second, inherent problems are associated with the analysis of ABA leveis in tissues. ABA content was originally assayed with biological inhibitor studies, but resuits sometimes differed substantially from those obtained with physical methods such as GLC. Lastly, it is recognized that ABA must be partially purified before attempts are made to analyze it with the more recently favored methods such as HPLC (high-performance liquid chromatography and GLC (gas iiquid chromatography) (Milborrow 1984). A recent description of separation and Pesq. agropec. bras., Brasília, 20(10):1171-1182, out. 1985.
1172
L.P. PASSOS
quantification of ABA PA and OPA by RPLC is
presented by Mapeili & Rocchi (1983). Alio,
several RIA (radioimmunossay) methods have
been described for ABA quantification (Walton
et ai. 1979, Weiler 1979 and 1980), including the
use of TLC (thin-iayer cliromatography) to test
the distribution of immunoreactiye material (Le
Page-Degivry & Bullard 1984). RIA methods have
beca recently reviewed by Weiler (1984) and
I-lanke (1984).
ABA was first isolated as au
abscission-accelera-ting substance in cotton frult (Ohicuma et ai.
1965) and also as an inhibitor of bud development
ia woody plants (Cornforth et ai. 1965). Ahhough
subsequent work lias shown that abscission in
leaves is normally regulated by ethylene rather
than ABA (Milborrow 1984), ABA is clearly
involved in many plant developmental processes
such as growth inhibition (Rehm & Cline 1973,
Noggle 1983 and Pilet 1983) and promotion
(Biumensfield & Gazit 1970, Milborrow 1974,
McWha & Jackson 1976 and Bradford 1983),
tissue permeability increase .(Glinka & Reinhold
1971), root geotropism (Kundu & Audus 1974),
seed dormancy (Wareing & Sounders 1971 and
Ackerson 1984) and germination (Karssen 1982),
fruit ripening (Coombe & Hale 1973), and
exogenously.induced fruit seedlessness (,Jackson
• Biundeil 1966), plant ccli cold hardiness (Chen
• Gusta 1983). Inhibition of trap closure in
Venus flytrap (Rondo & Yaguchi 1983) and
counteractive effect on stress-induced decrease of
root hydraullc conductance (Markhart et ai.
1979, Fiscus 1981 and Markhart 984). In
addition, Raschke (1982) believes he lias evidence
that ABA acts as a photosynthesis inhibitor. There
are also controversial information about ABA
effects on fiowering (Milborrow 1984) and
correlative inhibition of biid development (Walton
1980). Finaily, ABA is also known to have a major
role lii controlling responses of plaats to water
stress, and much of the current research and
controversy is focused on this arca.
Despite the extensive number of reports ou this
subject, studies ou the role 0f ABA ia plant
metabolic adjustment to wilt and prevention of
further water toss are stil at a relatively eariy
stage, mainly because there are many gaps ia the
knowledge of several aspects of ABA action
(Waiton 1980). Ia thisreview, 1 intend to provide
a general picture of the relationships of ABA to
leaf water stress and also to present thoughts on
the available data.
LEAF ABA CONTENT AND WATER STRESS
lnterest in the possible role of ABA in water stress
responses aí plants began when Little & Eidt (1968)
observed that exogenous applications of ABA led to
reduced transpiration rates iii leaves oí several woody
plants. Their Í'indings were extended to other plants 1w
Mittelheuser & Stevenick (1969) and by Jones &
Mans-field (1970). The role of ABA as a natural regulator oí
stomatal aperture was suggested by Wright (1969), who
noted an increase in "/3-inhibitor" in detached leaves oí
wheat foliowing a period of stress, and by Wright &
Ijiron (1969), who identiíied this inhibitor as being ABA
in brussel sprouts and aSso observed that increases in
ABA leveis during stress could be related to regaining of
leaf turgidity. Subsequent work confirmed these results
(Ortan & Mansfield 1974, Uehara et ai. 1975, Itai &
Meidner 1978a and Mansfield & Davies 1981). Further
evidence af the relation of ABA to stomatal
regu-lation was derived from the Flacca mutant of tomato,
which is unable to produce ABA nor doses stomata
(Imber & Tal 1970, Tal & Imber 1970, Bradford 1983
and Bradford et ai. 1983). This plant is constantly wilted
and as a result it is stunted in growth. In addition to
stomatal effects, stress-induced increases in ABA content
are considered to be responsible for proline accumulation
in some species (Aspinail etal. 1973 and Stewart 1980).
ABA accumuiation ia water-stressed leaves is accepted
as a genetic character associated with drought tolerance
(Larquê-Saavedra & Wain 1976, Quarrie & Sanes 1979,
Quarrie 1981 and Kirkham 1983), but many factors are
known to affect the extent of this increase. Variabie
resuits have been obtained because of differences in leaf
age (Quarrie & Henson 1981 and Comish & Zeevaart
1983) and, in intact piants, younger leaves show higber
capacity to accumulate ABA than older ones (Zeevaart &
Boyer 1984). Increases in ABA leveis are affected by
environmental factors such as temperature (Wright &
Hiron 1969) and iight intensity (Bengtson et al. 1978,
Rajagopal & Andersen 1980 and Henson 1983), and also
by duration and intensity of water stress (Wright 1977,
Pierce & Raschke 1980, Eze et ai. 1981, Henson & Quarrie
1981, Pierce & Raschke 1981 and Henson 1982). Under
stress, fieid-grown plants accumulate much less ABA than
pot-grown ones (Ilenson et al. 1981 and 1984). Finaily,
leaf ABA levei is a!so raised by warm ah, waterlogging
(Hiron & Wright 1973), salinity (Milborrow 1984), and
chilling (Rikin etal. 1976 and 1979, Chen etal. 1983 and
Markhart 1984).
CURRENT CONCEPTS ON THE RELATIONSHIP 1173
Wright & Huron (1969) observed that ABA leveis increased detectably within 2 hours after the onset of wilting and rapidly decreased foliowing rewatering. Similar rapid and reversible changes in ABA content of water stressed leaves have been subsequently reported (Iliron & Wright 1973, Bengtson et ai. 1977, Drffllng et ai. 1977, Ludiow et ai. 1980, Zeevaart 1980 and Pierce & Raschke 1981). Overali, foliar leels of ABA frequently use from about 20 ng/g initial fresh weight (1 x 10' M) to $00 ng/g (2 x 106 M) during wilting (Milborrow 1984). Fiowever, as shown on Tabie 1, intraspecific differences in ABA accumuiation in response to water stress are aiso found. There is evidence that the degree of stress affects time to increase ABA to maximai values, since Loveys & Krledemann (1973) observed that detached leaves of grapevines doubied ABA content in 15 minutes at a water potential of -1.5 MPa (megapascal), but iii intact plants the increase was siower (about •1.0 MPa of water potential) up to 6 days, when ABA content had increased 44 fold.
Many papers suggest that ABA content increases suddeniy when a threshold value of water potential is reached during water stress and that there are but srnail differences among several species in relation to the values of the threshold. Zabadal (1974) observed that ABA leveis remained low ia Arnbrosia artemisilfotia and Am-brada tnfida leaves until water potential decreased to a value between -1.0 and -1.2 MPa. At this point, ABA started accumuiating, and this accumuiation was proportional to further decreases ia water potential down to -2.4 MPa. However, the observed threshoid represents just 0.1 MPa in water potentiai decrease from unstressed conditions, and values like this are associated with errors in therrnocouple psychrometry so that the real value of water potential to cause ABA to increase in levei rernains te be established. Aiso, this ABA increase starts rapidly (less than 1 hour alter stress imposition) and other factors might be regulating ABA synthesis prior to detectable changes in water potential. Under similar conditions, subsequent work has obtained the sarne kind of results, such as between -0.8 and -LO MPa in excised leaves of maize and sorghum (Beardsell & Cohen 1975). between -1.0 and -1.2 MPa in seediings o! Douglas4ir (Blake & Ferrei 1977), between -0.7 and -0.9 MPa in bean leaves (Walton et ai. 1977 and Wright 1977) and between -1.0 and -1.2 MPa in fully and haif-expanded leaves o! Xanthium st,'uniarium (Cornisli & Zeevaart 1983).
Since the increase in ABA concentrations appears to be generaily coincident with wiiting onset (Milborrow 1981), it has been suggested that turgor pressure is the criticai cornponent of water potential for ABA accumu-iation (Pierce & Raschke 1978). Beardsell & Cohen (1975) observed the beginning o! ABA raising ia water potential values probably corresponding to zero turgor for maize and sorghum. Pierce & Raschke (1978) report that the beginning of ABA accumuiation in fact occurred at
zero leaf turgor for cockiebur. Further papers confirm this observatiora (Pierce & Raschke 1980 and 1981). i-iowever, although there ís a proportionaiity between ABA leveis and water potential during wiiting, turgor pressure remains at zero while ABA content changes. Hence, turgor is uniikeiy to regulate ABA synthesis during • stress. Aiso, Henson et ai. (1984) noted very di!ferent leveis o! ABA at nearly the sarne value of turgor pressure in peari miiiet stressed ieaves. Other suggestion for the reguiation o! ABA synthesis under stress is given by Walton (1980), who beiieves that osmotic readjustment is the cause of poor correiation between ABA leveis and watcr potentiai in !ieid-grown wheat and good correiation in excised chamber-grown wheat ieaves, since Davies & Lasko (1978) report that ABA leveis in apple seediings, which undergo osmotic readjustment, appear to correiate better with lesf turgor than with water potential. This hypothesis iooks reasonabie, since piant reservoirs outside the ieaves could avoid the observation of a threshoid phenomenon for ABA accumuiation as noted to detached leaves. Other possibility is that, although ABA synthesis under stress conditions appears to be controiied by water potential as a whole, ABA transiocation in intact piants in response to stress might troubie fite observation of a weil-defined threshold value. In !act, there is evidence that ABA recircuiates in cockiebur piants, moving down the stem in the phloem and back up in the transpiration stream to the mature leaves (Zeevaart & Boyer 1984), yet ABA is present in sieve tube sap o! Lupinus albus (lload 1978).
POSSIBLE MECHANISMS OF ABA-INDUCED STOMATAL CLOSURE
Applied ABA initiates stomatal ciosure either in detached ar in attached leaves (Little & Lidt 1968 and Mitteiheuser & Stevenick 1969) and several groups of researchers have reported extremely rapid response times. Cummins et ai. (1971) and itai & Meidner (1978a) observed stomatal ciosure within 10 minutes and Kriedemann et ai. (1972) noted reductions iii aperture can be detected within 1 minute foiiowing applications o! ABA. These rapid response times have ied some workers te suggest that exogenously added or endogenous ABA must somehow act to alter the distribution of osmotically importaM soiutes of guard celis, and this could be by causing a reduction in Kt concentration (l-lorton & Moran 1972 and Squire & Mans!ield 1972), since potassium ions are reported as the major promoters of solute potential variations (Humble & Raschke 1971) because they migrate frorn the subsidiary ceiis into the guard celis when stomata open and return to subsidiary cells during ciosure (Raschke & Fellows 1971). 1-lowever, it has been aiso suggested that ABA can act through an expuision mechanism in the plasmalemma of the guard ceils (Raschke 1975a) and Mans!ieid & Daviês (1981) infer that the !ast response of stomata to '0w doses of ABA supports this view.
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CURRENT CONCEPTS ON THE RELATIONS1-Il?
1175
...
Other possibulity is that ABA acta on melabolie
e
coprocesses needed for interconversions between starch
N E E >
2
1' CI O
and malate (Mansfield
&Davies 1981). $1 lias been
OiD o V N C
O •
-
hypothesized that lhe frequent starch loss from
chloroplasts of guard cells during stomalal opening
E
(Meidner
&Mansfield 1968) is perhaps caused - by its
conversion into malale (Wilimer
&Rutter 1977 and
-Mansfield
&Davies 1981). Consequently, the result of
tN 0 N E>wW>o, o ,
o,
ABA action on stomata may bõ the inhibition of iC
:0N Q - N m
uptake and starch conversion (Mansfield
&Jones 1971
and Mansfield
&Davies 1981). ilowever, Dittrich
&2'
. .c
Mayer (1983) observed that regular closure o! stoniala
E
E E ois
a physicai process due to leaking of osmotically relevant
e)
C)4
0
.
ions, whereas the action o! ABA appeais to be reiated to
•5
ao.
a prolein-catalyzed process such as faciiitated diffusion,
E C a,
whlch opens additional leaks within the membrane.
.
.2
- -Most of lhe evidence leads lo the possibility that the
EE -
ABA required lo dose stomates is formed in lhe
a . t 0
,
—
.-
mesophyll aI the onset of wilting and traasferred, actively
..-
- c° °
or by diffusion, to the guard cells (Milborrow 1981). The
.c
.c
ai - - E2
fact lhat a considerable amount o! ABA in leaves is
.c EIocaled in the mesophyli (Singh et ai. .1979 and Weiler
0
2
Og .
8
et ai. 1982), especially iii the chloroplasts (I-Ieilmann et aI.
.1980), is an indication of lhis possibilily. Also, there are
suggestions lhat ABA transiocation from chloropiasts mb
LO
LO
cyboplasm during darkness is caused by a reduclion o! the
w
pllofsbroma (Heilmann et ai. 1980 and Cowan et aL
o
1982), and that ABA release depends only on intraceilular
p11 gradients (1-lartung cl ai. 1983).
Although the potential role o! ABA as a reguiator
a
a
of stomatal aperture is supported by a variely. of
experimenta, several sludies have shown that changes iii
E •
- -
ABA content in leaf tissues are zot aiways correlated
E
o
- ° 'with stoinatal movemenls. llsiao (1973) cites many
papers which indicate that ABA levei changes lagged
E E c c . .
c
behind stomata opening following lhe reief of stress, and
Beardseli
&Cohen (1975) noled that increases in stomatal
a, CO LO O)N- .
resistance precede increases in ABA leveis o! slressed
('11 Co
maize and sorghum leaf tissues. Also, Loveys (1977)
-
N
found that no ABA was synthesized when broad bean
o
epidermis was wilted. More recentiy, Raschke (1982) has
LO
-: cq D
e?
asmbled several reporls thal casl doubl on the existence
se
< E o o o e) g LO O9
o
of a simple, straight forward relation o! leal ABA to
e
e
stomatal conductance.
The possibilily o! ABA compaxtmenlalion in leal
tissues can be presented for ekpiamning the discrepancy.
'Since slomala normaliy comprise less lhan
5%of lhe celis
o,found in the epidermis (Milborrow 1984), ABA leveis in
E
lhe mesophyll or even lii the epidermis need not to
E
.reflecl the levei acting on lhe guard celis. This possibilily
was suggesled ia eariy, sludies by
.Wallon
&Sondheimer
(1972) who found no relalionship of ABAto growth rales
j
-e .
of excised bean axis. Cummins (1973) suggests the
E
existance o! ABA compartmentation because he observed
stomatal reopening ia a short period of time alter stopping
2
E
additions o! ABA to the transpiralion stream. Raschke
&o, s s . .
Zeevaarl (1976) suggest ABA compartmentation in ieaves
1176 L.P..PASSOS
of cocklebur as a reson for no correiation between ABA leveis and stoinatal conductance ia plants under different
environments. Actually,
ir
there were ABAcompartmen-tation in fite presented time-course studies, and the ABA
type related to stomatal ciosure changed rapidily,
ir
thebackground were high, that type would not be detectable. Recently, Bray & Zeevaart (1983) observed na irreversible conjugation of ABA to ABA-GE in cockiebur which Lcd to fite finding that ABA-GE but not ABA is sequestered in the vacuole, where it is met.abolically inaccessible.
The discussed points lead to fite feeling that there are stili some aspects to be clarified. It seems that crucial questions are the biosynthesis and (ate of ABA as related to stomatal movements, fite rapidity of stomatal closure as related to ABA leveis, and ABA compartmentation in the leat Fotentially, they can be resolved with newer
techniques for
"m
:1w" determinations of ABA. Weiier(1984) presents a significant approach on the methods that have been reported for quantitative analysis of ABA ia plants RIA and ELISA (Enzyme-iinked immuno-sorbenf assay). Many liznitations of methods used in the past are litely to be sources of error. While antisera were
first raised against (R, S)-ABA coupied
via
C 1, it was laterfound that this resulted ia the production of predominan-tly anti-(R)-ABA antibodies 4 rendering the assay of (5)--ABA rather imprecise, being this problem overcome by the use of (S)-ABA-imznunogens. Nevertheicss, the free and conjugatcd ABA, in turn, were also highly reactive with ABA-amides. To solve this problem, Weiler in(ers that it has been used ABA-C 4 4yrosylhydrazone-substi-tuted immunogens, which ieads to little orno interference from conjugates. Finaily, the recent use of a mAB-RIA (monoclonal antibody) technique allows not to have any significant cross-reaction against compounds such as xanthoxin, PA, tWA, ABA-B-D-giuco pyranosyl ester, or 2-trans- and (R)-ABA. Also, it aliows fite determination of even traces of the physiologically active form of ABA ia plant extracts. Conclusively, it seems that the use of mAB-RIA technique for the quantification of active ABA (2-cis(S)-ABA) ia plant material could propitiate a clue for those questions. Major perspectives appear to be the determination of the actual ABA levei needed to induce stomatai closure and, if it is the case, the real ceilular levei of ABA binding proteins required by fite plant to dose stomata.
ABA EFFECTS AS RELATED Toco2 AND OTHER PLANT HORMONES
la addition to light, stomatal aperture in unstressed
plants is mediated by CO2 leveis and pH values, and some
controversy exists regarding the possibie relation of ABA to CO 2 (Zeiger 1983). Raschke (1975a) showed that ABA and CO2 interact in regulating stomatal closure. However, Mansfieid (1976) found that ABA and CO 2 influences on stomata are additive rather than interactive, Pesq. agropec. bras.,Brasflia, 20(10):1171-1182, out. 1985.
and Itai & Meidner (1978b) infer that ABA and CO2 do not interact because they probably act at different sites.
Altliough it has been observed that ABA-affected stomatal closure depends upon CO2 presence (Raschke 1975b) and that intraceiluiar CO2 leveis decrease after ABA is supplied to detached leaves (Cummins et ai.
1971). Snaith & Mansfield (1982) noted that ABA dependepcy on CO2 is cancelled by exogenous IAA (indoieacetic acid) at high concentrations. More recently, Eamus & Wiison (1984) also observed associated effects of ABA, IAA and CO2, whiclt were more remarkable under iow temperature: ABA was found to have no effect upon stomatal closure iii the presence of CO2 when IAA was added. A Iast aspect on this issue is that, although not reiated to ABA, severai natural and synthetic cytokinins have promoted stomatal opening ia Anthepho-rapubescen: (Jewer & Incoil 1980).
The reasons for the different conciusions on 1mw ABA and CO 2 affect stomata still have to be established - Walton (1980) calls attention to such things as differences
ia
experimental techniques and result interpretation.However, the determination of the sites of ABA and CO2 action seems to be imperative. Complementarily,
the influence ofexogenouslAAuponABA-CO 2 associated
effects ieads to fite need of measurements of endogenous IAA ia studies on stomatal movements and the search of interactive effects of fite two plant hormones on stomatal behavior. Lastly, it is worth verifying whether ABA is related to other piant hormones that can modulate stomatal movements, sucli as cytokinins.
ABA AND PROLINE AcCUMULATION
During water deficit, many species change.their content
of free amino acids, being the increase
ia
prolineconcen-tration fite most remarkable response (Aspiaau & Paleg 1981). Aspinall et ai. (1973) observed ABA-índuced increases ia proline leveis ia bariey and Loilum temulen-tuA and Stewart (1980) noted that proline leveis began
to rise following a treatment with ABA
ia
cuts of barieyleaves. This evidence, along with the observation of
accumulation of both endogenous proline and ABA
ia
leaves of dark-grown wheat seedlings during water stress (Bengtson et ai. 1978), suggests that proline biosynthesis
ia
response to stress is mediated by ABA. However,Aspiaall & Paieg (1981) cite some studies
ia
whichtobacco plants showed increases
ia
proline and ABAleveis during water stress, but did not accumulate proline
ia
response to applied ABA:The evidence is not ciear yet about what is causing the increase ia proline ievels during water deficit. The
relevance of changes
ia
ABA concentration or of anyother plant hormone in those reported proline iacreases remains to be verified. Moreover, because there are molecular differences between natural and synthetic ABA, iavestigations on native ABA effects upon proline
CURRENT CONCEPTS ON THE RELATIONSHIP 1177
accumuiation ralhei lhan exogenous ABA appiicatións, are necessary. In fact, if ABA piays a role in this phenomenon in leaves, il should begin lo elevate before proline accumulales foliowing water slress imposition, and a patlern iike lhis was been observed ia grape berries of vines subjected lo saiinily (Downton & Loveys 1978).
ABA AS RELATED TO THE AFTER.EFFECT OF WATER STRESS
In many species, a period of water stress also resulls in an "afler-effect" on slomatal aperlure, whereby slomala will not open fuily for hours or even days afler the relief of stress (Dirffling cl ai. 1977). Also, lhe recovering ia turgor is much fasler than the normalization of melaboiic events (Benglson eI ai. 1978). Allaway & Mansfield (1970) propose that lhis effecl may be caused by an inhibitor of slomatal aperlure, and il has been pointed oul that the after-effect of water stress is due lo a siow ABA levei decline afiei lhe rehef of slress (Wilimer 1983). in fact, Hiron & Wright (1973) observed a maintainance of both high ABA levei and leal resislance ia dwarfbean seediings alter rehydralion. Drffling eI ai. (1974) report a correlation belween lhe deiay in slomata opening and lhe siow decrease in ABA levei following rehydralion of pea seediings. Furlher, D6rffling et ai. (1977) noled a direcl correiation of duralion of after-effect to ABA levei in pea, sunfiower and broad bean plants, and conciuded lhat lhe jncrease in ABA concentration is lhe primary cause of lhe phenomenon, also because appiied ABA lo slressed ieaves lransferred lo water caused stomata lo remam ciosed.
Fiowever, there are some disagreemenls on this vieoinl, since results have shown lhat leaf ABA and slomala recovery do nol correlate weii (Wilimer 1983). Yet it has been noled ia some studies thal ABA concentration decrease 100 fast lo be related lo slomala
recovery (Loveys & Kriedemann 1973 and Beardseii & Coben 1975). Moreover, Benglson eI al.(1977) found no direcl reiationship belween lhe after-effecl and leal ABA contenl, since rehydrated wheal piants showed a re-allamning of pre-stress ABA leveis aI lhe sarne lime (3 hours) for lhree differenl durations of water stress and, hence, differenl ABA leveIs during slress. Finaily, 1-lenson (1981) observed, alter lhe relief of waler slress ia peari miflel, lhal ABA conlenl decreased wilhin 3 hours ia delached leaves, and within 8 hours in intact plants, bul slomatal conductance increased shorlly in bolh cases, suggesling lhat lhe proionged inhibilion of slornatal opening after rehydralion is aol entireiy due lo conlinued high leveis of buik leal ABA. -
I-lypolheses have been nade lo explain how the afler-effecl of waler stress could occur. Since lhe overail conlent o! ABA accumuialed during slress is much higher than the amounl necessary to affecl stomalal movemenls, Bengtson cl ai. (1977) infer thal lhe existence o! an
indirecl relalionship between lhe "extra" ABA and lhe duralion of lhe afler-effect is possibie. liórffling et ai. (1977), aiming aI resuils obtained wilh broad beans, argue lhe foilowing possibilities: lhe sensilivity of guard celis lo ABA is reduced by proionged stress or enhanced ABA levei consequenlly readering the lhreshoid levei reialive to concenlralion and duration; lhe reduction of the levei of ABA aI ils aclive sile in guard celis may be accornplished by planl processes other lhan degradation; and ABA removai into slorage sites where il cannol ad on lhe stomata may be involved. Benglson cl ai. (1978) argue thal lhe after-effect might be hnked lo chiorophyli metabolism lhrough ABA or some of its metaboiites. Raschkc (1982) presenls the following possibiiities ABA removai from lhe vicinity of lhe receplor Siles in lhe guard ceils is slower than lhe one from the resl of lhe tissue; after-effect is a refleclion of stress-induced enzyme degradalion instead of a phenomenon related lo ABA; and PA causes stomalal narrowing. Although PA has been reporlcd as having virlualiy no growlh-inhibilion activily (Tineili et ai. 1973), lhe possibilily lhal degradation producls of ABA can piay a role in lhe afler-effedl is supported by Milborrow (1981), since Kriedemann et ai.
(1975) observed an inhibitory effect of PA on
photosynlhesis and a recovery paltern of CO 2 uptake weli behind lhe patlern of Iranspiration foliowing rewatering of water-slressedgrapevine ieaves. Furlherrnore, a comparlmentation phenomenon involving ABA or any of ils melabolites in leal lissues could explain lhe alter--effecl, since comparlmentaiized active ABA-iike compounds couid be avaiiabie lo mainlain stomalal closure in spile of lhe relief of water stress. Finally, thc investigalion of differences among species as to lhe concenlration o! ABA-receptor molecuies couid iead to lhe finding of bolh ABA actual levei needed lo affect slomala and lhe causes o! extra ABA synlhesis.
CONCLUSIONS
1. There is direcl evidence lhal leaf ABA accumulalion is rapid and reversibie following lhe onset of waler slress and Ihal cultural and environmenlai factors influence lhe patlern of ABA levei increases. Also, 11 is clear lhat slight decreases in waler polenlial iead lo ABA accumulalion and lhat there is a proporlionality between this increase in ABA levei and the decrease in waler polenliai. However, the evidence for a waler polenliai . threshold is aol convincing yel. Nor is lhe suggeslion thal turgor pressure is lhe critica1 component for increases ia leaf ABA conlenl,
1178
LJ'..PASSOS
2, ABA cause rapid stomatal ciosure, and the
mechanisms of this action appear to be more
extensive than a single induction of decreases in
1C concentration or inhibition ofstarch conversion
into maiate in guard celis - a catalized process
might be promoting membrane leakage. Besides,
some results showing no correlation of ABA with
stomatal movements appear to indicate that ABA
effects upon stomata do not follow a simple and
straight forward relationship. In fact, ABA effects
are associated with CO 2 leveis and affected by
exogenous IAA. Also, some species show increases
of both ABA and proline during stress, and one
might be reiated to each other. In addition, the
occurrence aí ABA compartmentation iii lcd
tissues could contribute for the obtained conflicting
data regarding ABA-regulated stornatal aperture
and afier-effect of water stress.
3. Some aspects are stii unclear such as how
water potential couid reguiate ABA accumuiation,
how to explain the fast stomatal ciosure as due to
ABA, and whether the after-effect of water stress
is a consequence ora cause of remaining high ABA
leveis in icaves.
4. Methodological limitations probabiy are
causative of controversial data, and newer
techniques such as mAB-R!A might provide
adequate ABA analysis in time-course experiments.
S. Suggestions for further work are: the
investi-gation of stomatal behavior as being regulated by
more than one singie factor such as ABA by
examining whether ABA is reiated to endogenous
IAA or any other plant horinone that can,modulate
stomatal aperture such as cytokinins, the verifying
whether endogenous ABA induces increases in
proline content, the determination ofthe role and
fate of ABA metaboiites in relation to water stress,
and the search for ABA putative receptors and
binding sites in leaf tissues.
ACKNOWLEDGMENTS
To Dr. Kaoru Marsuda, Department of
Mole-cular and Ceiiuiar Biology, University of Arizona,
who gave advice and provided helpful comments
during organizing and writing this review.
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