ABA Signal Perception and ABA Receptors - Abscisic Acid: Metabolism, Transport and Signaling - page 89

Wang et al. 2011 ). The C-terminal half, but not N-terminal half of ABAR/CHLH,
was shown to bind ABA with this system (Wu et al. 2009 ). However, the ABA-
affinity technique was questioned because the carboxyl group at the C-1 locus of
ABA, which is required for the activity of ABA, was altered (Cutler et al. 2010 ).
This may possibly induce some errors in purification and analysis of ABA-binding
proteins. In regard to this question, Wu et al. ( 2009 ) suggested that the specific
binding ability to a protein may depend on the whole structure of ABA, and the
ABA molecule with the C-1-carboxyl group bound may interact with ABA-binding
protein, though the binding intensity may be reduced. This postulation was sup-
ported by the multiple controls that showed specificity and reliability of ABA bind-
ing to this affinity chromatography column (Wu et al. 2009 ). Also, we tested the
functionality of the ABA molecule with the fixed C-1-carboxyl group. Given that
ABA-Sepharose-4B is too big to permeate into cells and its bioactivity could not be
tested, we coupled ABA to a small molecule methotrexate to fix the C-1-carboxyl
group of ABA, forming an ABA derivate ABA-methotrexate (ABA-MTX), which
may enter cells to test the bioactivity of the C-1-carboxyl group-blocked ABA.
The results showed that ABA-MTX inhibits seed germination, seedling growth,
and promote the interaction between an ABA receptor member PYL5 and a type-
2C protein phosphatase member HAB1 (Ma et al. 2009 ; Park et al. 2009 ; Santiago
et al. 2009b ) as ABA does, though the activity is decreased (Q. Xin, X.F. Wang,
and D.P. Zhang, unpublished data), which strongly suggests that blocking the C-1-
carboxyl group of ABA does not result in loss of ABA bioactivity, supporting thus
the reliability of the ABA affinity chromatography based on ABA-Sepharose-4B
(Zhang et al. 2002 ; Wu et al. 2009 ; Wang et al. 2011 ).
Several lines of genetic evidence through transgenic manipulation consistently
support the ABA-binding data (Wu et al. 2009 ), in which expression of the ABA-
binding domains, C-terminal-truncated proteins of the Arabidopsis ABAR/CHLH,
results in ABA hypersensitivity in all the major ABA responses including seed
germination, seedling growth, and stomatal movement, but expression of the
N-terminal-truncated proteins of ABAR/CHLH, non-ABA-binding domains,
induces only limited ABA hypersensitivity in either seed germination or seedling
growth (Wu et al. 2009 ). These data reveal that the C-terminal half not only cov-
ers the core ABA-binding domains, but also is a core functional region to mediate
ABA signaling.
The third line of evidence for the binding interaction of the Arabidopsis
ABAR/CHLH with ABA comes from a test with the surface plasmon resonance
(SPR) system, by which Du et al. ( 2012 ) showed that ABAR/CHLH binds ABA
with a saturation curve typical for receptor-ligand binding. The ABA-binding
data with the SPR system are easily reproduced, but the detected ABA affinity to
ABAR/CHLH is low (disassociation constant Kd ≈ 20 ʼ M) in comparison with
our previously detected high ABA affinity (about 30 nM) with the 3 H-labeled
ABA-binding assay (Shen et al. 2006 ; Wu et al. 2009 ; Wang et al. 2011 ). This
is likely due to technical limitations of the SPR system to test the interaction
between a huge (about 150 kDa), hydrophobic CHLH protein and a small ligand
ABA (Du et al. 2012 ).