Protein Phosphatase Activity of Abscisic Acid Insensitive 1 (ABII) Protein from Arabidopsis Thaliana

Abstract

Mutations at the ABIl (abscisic acid insensitive 1) locus of the plant Arabidopsis thaliana cause a reduction in sensitivity to the plant hormone abscisic acid. The sequence of ABZl predicts a protein composed of an N-terminal domain that contains motifs for an EF-hand Caz+-binding site, and a Cterminal domain with similarities to protein serinekhreonine phosphatases 2C. We report here two sets of experimental evidence that indicate that ABIl has typical protein phosphatase 2C activity. First, expression of the ABIl C-terminal domain partially complemented the temperature-sensitive growth defect of a Saccharomyces cerevisiae protein phosphatase 2C mutant. Second, recombinant proteins that contained the ABIl C-terminal domain displayed in vitro phosphatase activity towards 'ZP-labelled casein, and this activity displayed Mg2+ or Mn'+ dependence and okadaic acid insensitivity typical of protein phosphatases 2C. Characterisation of recombinant proteins that contained various portions of ABIl indicated that the putative EF-hand motif is unlikely to mediate Caz+ regulation of the ABIl phosphatase activity at physiological CaZ+ concentrations, and may represent an EF-hand analogue rather than an EF-hand homologue. The abil-l mutation appeared to cause significant reduction in the phosphatase activity of ABII. These results are discussed in relation to the dominant phenotype of abil-1 over the wild-type allele in plants, and to the possible role of ABIl in abscisic acid signalling.

The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction.

“…The abi7-7 and abi2-7 mutations did indeed similarly entail a marked decrease in the PP2C activity of the A611 (Bertauche et al, 1996) and AB12 (Figure 48) proteins, respectively. Furthermore, the mutant GST-abi2 fusion protein required higher Mg2+ concentrations with respect to its optimal activity compared with wild-type GST-A612 ( Figure 4C), suggesting that the Gly-168-to-Asp substitution in abi2-1 effectively decreases divalent cation affinity.…”

“…A cDNA for the abi2-7 mutant gene was created by site-directed mutagenesis of the wild-type pcABI2 cDNA, as described previously (Bertauche et al, 1996), by using the mutagenic oligonucleotide primer 5'-GCCATGACGGTTCTCAG-3', creating a G-to-A change at nucleotide 553 in pcAB12. A positive clone (pcabi2-I) was verified by sequencing and retained for subsequent use.…”

“…In guard cells, ABI1 appears to relay, together with counteracting protein kinases, ABA signals to stomatal regulation in that defects in stomatal closure caused by the abi7-7 mutant gene can be partially suppressed by kinase antagonists (Armstrong et al, 1995). The abi7-7 mutation, converting Gly-180 to Asp in the PP2C domain, leads to a marked decrease in the phosphatase activity of ABll (Bertauche et al, 1996). However, this mutation behaves genetical1y;depending on the particular phenotypes, as either fully dominant or semidominant (Koornneef et al, 1984;Finkelstein, 1994b).…”

“…In seed, both mutations reduce dormancy and the sensitivity of germination to the inhibitory effects of ABA (Koornneef et al, 1984;Finkelstein and Somerville, 1990). In vegetative tissues, both mutants display ABA-resistant seedling growth, abnormal stomatal regulation, and defects in various ABA-induced morphological and molecular responses (Koornneef et al, 1984;Finkelstein and Somerville, 1990;Schnall and Quatrano, 1992;Finkelstein, 1994b;Gosti et al, The cloning and characterization of the ABl7 gene (Leung et al, 1994;Meyer et al, 1994) revealed that it encodes a protein composed of a nove1 N-terminal segment and a C-terminal domain that is an active protein serine/threonine phosphatase 2C (PP2C) (Bertauche et al, 1996). In guard cells, ABI1 appears to relay, together with counteracting protein kinases, ABA signals to stomatal regulation in that defects in stomatal closure caused by the abi7-7 mutant gene can be partially suppressed by kinase antagonists (Armstrong et al, 1995).…”

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

“…A single point mutation in the abi1-1 mutant significantly reduces PP2C activity [29,30], and genome-wide gene expression profiling indicates that ABA gene regulation is impaired [45]. Although recombinant fusion proteins containing either the whole ABI1 protein or only the phosphatase domain display in vitro phosphatase activity towards 32 P-labelled casein [46,47]; the in vivo targets have not yet been identified.…”