The Protein Phosphatase AtPP2CA Negatively Regulates Abscisic Acid Signal Transduction in Arabidopsis, and Effects of abh1 on AtPP2CA mRNA  

Abstract

To identify new loci in abscisic acid (ABA) signaling, we screened a library of 35STcDNA Arabidopsis (Arabidopsis thaliana)-expressing lines for ABA-insensitive mutants in seed germination assays. One of the identified mutants germinated on 2.5 mM ABA, a concentration that completely inhibits wild-type seed germination. Backcrosses and F 2 analyses indicated that the mutant exhibits a dominant phenotype and that the ABA insensitivity was linked to a single T-DNA insertion containing a 35STcDNA fusion. The inserted cDNA corresponds to a full-length cDNA of the AtPP2CA gene, encoding a protein phosphatase type 2C (PP2C). Northern-blot analyses demonstrated that the AtPP2CA transcript is indeed overexpressed in the mutant (named PP2CAox). Two independent homozygous T-DNA insertion lines, pp2ca-1 and pp2ca-2, were recovered from the Arabidopsis Biological Resource Center and shown to lack full-length AtPP2CA expression. A detailed characterization of PP2CAox and the T-DNA disruption mutants demonstrated that, whereas ectopic expression of a 35STAtPP2CA fusion caused ABA insensitivity in seed germination and ABA-induced stomatal closure responses, disruption mutants displayed the opposite phenotype, namely, strong ABA hypersensitivity. Thus our data demonstrate that the PP2CA protein phosphatase is a strong negative regulator of ABA signal transduction. Furthermore, it has been previously shown that the AtPP2CA transcript is down-regulated in the ABAhypersensitive nuclear mRNA cap-binding protein mutant abh1. We show here that down-regulation of AtPP2CA in abh1 is not due to impaired RNA splicing of AtPP2CA pre-mRNA. Moreover, expression of a 35STAtPP2CA cDNA fusion in abh1 partially suppresses abh1 hypersensitivity, and the data further suggest that additional mechanisms contribute to ABA hypersensitivity of abh1.

The unique mode of action of a divergent member of the ABA-receptor protein family in ABA and stress signaling

“…Our data showed that the germination of OE-PYL13 #1 was 56% under 0.5 µM ABA treatment and 14% under 1 µM ABA treatment ( Figure 4E). Consistently, the phenotype of pyl13 mutants is in the same pattern as PP2CA overexpression lines [34], whose seedling establishment is hyposensitive to ABA ( Figure 4F). However, the phenotype of pyl13 mutants is very weak compared with OE-PP2CA transgenic lines, indicating that there are functional redundancy between PYL13 and other PYLs in their inhibition of PP2CA.…”

“…The PYL13 triple mutant Q38K/ F71L/T135N lost the ABA-independent induction of the RD29B-LUC basal expression ( Figure 4C), suggesting that it is these sequence variations that confer ABAindependent inhibition of PP2CA, HAI2, and HAI3. Moreover, the phenotype of PYL13 overexpression lines is similar to that of pp2ca-knockout mutants [34], whose seed germination is hypersensitive to ABA ( Figure 4E). According to previous reports, the germination of pp2ca-1 mutant was about 45% under 0.5 µM ABA treatment and completely inhibited by 1 µM ABA on day 5 after plating [34].…”

“…Moreover, the phenotype of PYL13 overexpression lines is similar to that of pp2ca-knockout mutants [34], whose seed germination is hypersensitive to ABA ( Figure 4E). According to previous reports, the germination of pp2ca-1 mutant was about 45% under 0.5 µM ABA treatment and completely inhibited by 1 µM ABA on day 5 after plating [34]. Our data showed that the germination of OE-PYL13 #1 was 56% under 0.5 µM ABA treatment and 14% under 1 µM ABA treatment ( Figure 4E).…”

“…Overexpression of AtPP2CA resulted in ABA insensitivity while knockout of AtPP2CA caused ABA hypersensitivity during seed germination [34]. Given the strong inhibition by PYL13 on PP2CA activity in vitro and in vivo, we investigated whether PP2CA function in planta may be impacted by PYL13 overexpression.…”

Enhancement of Abscisic Acid Sensitivity and Reduction of Water Consumption in Arabidopsis by Combined Inactivation of the Protein Phosphatases Type 2C ABI1 and HAB1

“…Currently, at least four Arabidopsis (Arabidopsis thaliana) PP2Cs, ABI1, ABI2, PP2CA, and HAB1 (formerly named AtP2C-HA), are known to regulate ABA signaling. Evidence on their role as negative regulators of ABA signaling has been provided by genetic approaches (Gosti et al, 1999;Merlot et al, 2001;Tahtiharju and Palva, 2001;Gonzalez-Garcia et al, 2003;Leonhardt et al, 2004;Saez et al, 2004;Kuhn, et al, 2006;Yoshida et al, 2006). For instance, the recessive T-DNA insertion mutant hab1-1 shows ABAhypersensitive inhibition of seed germination and enhanced ABA-mediated stomatal closure (Leonhardt et al, 2004;Saez et al, 2004).…”