ABA Signal Perception and ABA Receptors - Abscisic Acid: Metabolism, Transport and Signaling - page 88

combined with carefully designed affinity probe, two types of ABA-binding pro-
teins have been successfully purified from the epidermis of broad bean ( Vicia
faba ) and embryos of Brassica napus , respectively (Zhang et al. 2002 ; Nyangulu
et al. 2005 ). The first ABA-binding protein was purified from the epidermis of
broad bean ( Vicia faba ) leaves via the ABA-affinity chromatography where ABA
molecules were coupled through their carboxyl groups at C-1 to the free amino
groups located in the spacer arm of EAH-Sepharose 4B, which was used to 'catch'
ABA-binding protein from total proteins (Zhang et al. 2002 ). An ABA-binding
protein with a molecular mass of 42 kD and an isoelectric point of 4.86 was iso-
lated, which was shown to bind ABA with saturability, reversibility, high affinity,
stereo-specificity for the physiologically active ( + )ABA. In addition, evidence for
the potential receptor nature of the purified ABA-binding protein was provided,
in which the phospholipase D activity induced by ABA in guard cell protoplasts
(Wang 1999 ; Ritchie and Gilroy 1998 , 2000 ; Jacob et al. 1999 ) of broad bean
leaves in relation to the ABA-binding protein was measured (Zhang et al. 2002 ).
The 42-kD ABA-binding protein was further used to identify possible ABA recep-
tor subsequently (see following description). The second ABA-binding protein
was isolated with a different strategy by considering that the alterations of ABA
molecule at the C-1 or C-4' site may have effect on the affinity for ABA receptors
(Nangulu et al. 2005 ). A bioactive and biotinylated ABA probe ( + )-17, in which
the C-1 or C-4' site of ABA remains unmodified, was synthesized. A membrane-
associated ABA-binding protein was purified successfully using this biotinylated
ABA as an affinity probe (Nangulu et al. 2005 ).
6.3 Mg-chelatase H Subunit (CHLH): A Receptor
for ABA in Chloroplast Membrane
6.3.1 CHLH/ABAR Is an ABA-binding Protein
As described above, Zhang et al. ( 2002 ) purified an ABA-binding protein (ABAR,
for putative ABA receptor) via affinity chromatography from broad bean, accord-
ing to which a reverse genetic approach was made to identify a possible receptor
for ABA. The sequencing results of the ABAR protein led to cloning, from broad
bean leaves, of a complementary DNA fragment encoding the carboxy-terminal
half of about 770 amino acids of the putative H subunit of the magnesium proto-
porphyrin-IX chelatase (Mg-chelatase). The yeast-expressed products of both the
cDNA fragment of broad bean and the full-length cDNA of the Arabidopsis CHLH
were shown to bind ABA specifically with the [ 3 H]-ABA-binding assay, and the
ABA-binding properties of the Arabidopsis CHLH meet all criteria of the ligand-
receptor binding (Shen et al. 2006 ; Wang and Zhang 2008 ; Wang et al. 2011 ).
Further, the second line of evidence was provided for the ABA-binding ability of
the Arabidopsis CHLH/ABAR with a newly developed ABA-affinity chromatogra-
phy technique, which is based on the same principle as that of the technique used
for purification of the ABA-binding protein (Zhang et al. 2002 ; Wu et al. 2009 ;