ABA Signal Perception and ABA Receptors - Abscisic Acid: Metabolism, Transport and Signaling - page 98

6.5 PYR/PYL/RCAR Proteins: Cytosolic ABA Receptors
Mediating a Central Signaling Pathway
6.5.1 START-Domain Proteins PYR/PYL/RCAR: Soluble
ABA Receptors Coupled with PP2Cs
Park et al. ( 2009 ) developed a chemical genetic strategy to screen candidate recep-
tors for ABA, which may bypass redundancy by inducing phenotypes not revealed
by conventional forward genetic approaches with single-locus mutation: A selec-
tive agonist can illuminate the function of one member of normally redundant
regulators or receptors (Cutler and McCourt 2005 ). In a forward genetic screen
for new signaling components or receptors for ABA, a synthetic ABA agonist spe-
cifically inhibiting seed germination, called pyrabactin, was used and allowed to
successfully isolate a PYRABACTIN RESISTANCE 1 mutant allele ( pyr1 ), which
shows pyrabactin insensitive phenotype in seed germination. PYR1 encodes a
member of the START-domain superfamily soluble ligand-binding proteins (Iyer
et al. 2001 ), and the gene family includes thirteen homologous genes similar to
PYR1 , named PYR - LIKE 1 to PYR - LIKE 13 ( PYL1 to PYL13 ) (Park et al. 2009 ).
The pyr1 mutant responds normally to ABA, suggesting that functional redundancy
from other family members could mask PYR1's role in ABA signal transduction.
Indeed, triple and quadruple mutants with genotypes pyr1 pyl1 pyl4 and pyr1
pyl1 pyl2 pyl4 , respectively, show significant ABA insensitivity in seed germina-
tion and seedling growth (Park et al. 2009 ). The quadruple mutant also shows ABA
insensitivity in ABA-induced stomatal closure (Nishimura et al. 2010 ) and ABA-
mediated transcriptional responses (Park et al. 2009 ). A more recent report showed
that a sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1,
PYL2, PYL4, PYL5, and PYL8, displays extremely strong ABA-insensitive phe-
notypes: It germinates and grows even on 100- ʼ M ABA, and its leaves are overly
sensitive to drought (Gonzalez-Guzman et al. 2012 ). Transgenic overexpression of
either PYL1 or PYL4 restores ABA sensitivity in the triple mutant, revealing the
functional specificity for ABA signaling (Park et al. 2009 ). These data illustrate the
power of the chemical genetic approach for sidestepping genetic redundancy.
Another group (Ma et al. 2009 ) used reverse genetic approaches to screen new
components of ABA signaling and identified, by a yeast two-hybrid system, an
interaction protein of ABI2 type 2C protein phosphatase that has been well char-
acterized as a key, negative, regulator of ABA signaling (Leung et al. 1994 , 1997 ;
and see Chap. 8 ). This interaction partner of ABI2 was named regulatory compo-
nent of ABA receptor 1 (RCAR1), which is identical to PYL9. Consistent with the
observations in the mutants of PYR / PYL genes (Park et al. 2009 ), downregulation
of RCAR1/PYL9 reduces, but upregulation of RCAR1/PYL9 enhances, ABA sen-
sitivity in the three major ABA responses in seed germination, vegetative growth,
and stomatal movement (Ma et al. 2009 ). Further ABA-binding assays through
hetero-nuclear single-quantum coherence nuclear magnetic resonance and isother-
mal titration calorimetry techniques showed that PYR1 and RCAR1/PYL9 bind
ABA (Ma et al. 2009 ; Park et al. 2009 ). PYR/PYL/RCAR proteins, upon ABA