Agriculture Reference
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factions from
Vicia faba
leaves and detected the binding of [
3
H] () ABA to mem-
brane-rich fractions. They showed that the binding bears the properties of binding
saturation, of which the dissociation constant is 3.5 10
−
8
M with more than one
binding sites in the membrane factions. Using guard cells isolated from the epider-
mis of
Valerianella locusta
, Hartung (
1983
) found that ABA could induce stomatal
closure at pH 8.0, where nearly all ABA molecules are present as the anion of ABA
(ABA
−
), and could not penetrate plasmalemma, which suggests that ABA could be
perceived by binding sites or receptors outside the plasmalemma and induce stomatal
closure. Hornberg and Weiler (
1984
), using guard cell protoplast of
Vicia faba
leaves,
provided new, compelling evidence for high-affinity [
3
H]-
cis
(
+
)ABA-binding sites,
which are located at the plasmalemma with high density and potentially mediate the
guard cell signaling in response to the extracellular ABA. The possible extracellu-
lar perception site for ABA in guard cells was repeatedly confirmed by several inde-
pendent groups in
Commelina
,
Vicia faba,
and
Arabidopsis
(Anderson et al.
1994
;
MacRobbie
1995
; Jacob et al.
1999
; Yamazaki et al.
2003
). Additionally, the percep-
tion sites of ABA in the plasma membrane were detected in the barley
(Hordeum vul-
gare
) aleurone protoplasts and
Arabidopsis
suspension cells (Gilroy and Jones
1992
,
1994
; Ritchie and Gilroy
1998
,
2000
; Jeannette et al.
1999
; Hallouin et al.
2002
).
In addition to these extracellular binding or perception sites at the cell surface,
the intracellular binding or perception sites were also detected, which may poten-
tially contribute to ABA signal perception within cells. Allan et al. (
1994
) synthe-
sized and microinjected caged ABA directly into
Commelina
guard cells, where
ABA was released internally and subsequently caused stomatal closure, suggest-
ing the presence of the intracellular perception sites. Meanwhile, Schwartz et al.
(
1994
) demonstrated that ABA could act from internal of guard cells to regulate
Commelina
stomatal closure, implying that there is an intracellular locus of ABA
perception. The coexistence of the intracellular and extracellular perception sites
for ABA in guard cells was consistently reported in
Commelina
by other groups
(Anderson et al.
1994
; MacRobbie
1995
).
ABA-binding sites were also studied in other plants with various tissues and
were detected at different subcellular fractions of different tissues, such as cyto-
solic fraction of wheat (Veliev
1991
), membrane fractions of rice seedling (Chen
et al.
1992
), maize roots (Chen and Zhu
1996
), the plasma membrane frac-
tion of
Arabidopsis
cells cultured in vitro (Pedron et al.
1998
), the mesocarp of
grape berry (Zhang et al.
1999
), and developing apple fruits (Zhang et al.
2001
).
All these studies support the presence of the ABA-binding proteins or receptors
for perceiving ABA signal in both the inside and outside of plant cells (Assmann
1994
; Finkelstein et al.
2002
; Verslues and Zhu
2007
).
6.2.2 Isolation of ABA-binding Proteins
Isolation of the ABA-binding proteins may be a major step toward identifica-
tion of ABA receptors by reverse genetic approaches. Based on the information
from the studies of the ABA-binding proteins and using affinity chromatography
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