Nuclear localization of the mutant protein phosphatase abi1 is required for insensitivity towards ABA responses in Arabidopsis

Abstract

SummaryABI1, a protein phosphatase 2C, is a key component of ABA signal transduction in Arabidopsis that regulates numerous ABA responses, such as stomatal closure, seed germination and inhibition of vegetative growth. The abi1-1 mutation, so far the only characterized dominant allele for ABI1, impairs ABA responsitivity in both seeds and vegetative tissues. The site of action of ABI1 is unknown. We show that there is an essential requirement for nuclear localization of abi1 to confer insensitivity towards ABA responses. Transient analyses in protoplasts revealed a strict dependence of wild-type ABI1 and mutant abi1 on a functional nuclear localization sequence (NLS) for regulating ABA-dependent gene expression. Arabidopsis lines with ectopic expression of various abi1 forms corroborated the necessity of a functional NLS to control ABA sensitivity. Disruption of the NLS function in abi1 rescued ABA-controlled gene transcription to wild-type levels, but also attenuated abi1-conferred insensitivity towards ABA during seed germination, root growth and stomatal movement. The mutation in the PP2C resulted in a preferential accumulation of the protein in the nucleus. Application of a proteosomal inhibitor led to both a preferential nuclear accumulation of ABI1 and an enhancement of PP2C-dependent inhibitory action on the ABA response. Thus, abi1-1 acts as a hypermorphic allele, and ABI1 reprograms sensitivity towards ABA in the nucleus.

The Nuclear Interactor PYL8/RCAR3 ofFagus sylvaticaFsPP2C1 Is a Positive Regulator of Abscisic Acid Signaling in Seeds and Stress

“…In a recent report, Moes et al (2008) described the nuclear localization of ABI1 wild-type and mutant proteins and the requirement of a functional nuclear localization sequence in order to regulate ABA sensitivity and ABA-dependent gene expression, and they suggested that ABI1 reprograms sensitivity toward ABA in the nucleus. Similarly, Sáez et al (2008) confirmed the interaction of HAB1 and SWI3B (an Arabidopsis homolog of the yeast SWI3 subunit of SWI/SNF chromatin-remodeling complexes) in the nucleus and suggested a direct involvement of HAB1 in the regulation of ABA-induced transcription.…”

Evolutionarily Conserved Regulatory Mechanisms of Abscisic Acid Signaling in Land Plants: Characterization of ABSCISIC ACID INSENSITIVE1-Like Type 2C Protein Phosphatase in the Liverwort Marchantia polymorpha

“…In contrast, gene products of At1g07430, AtPP2CA/AHG3, and ABI1 have NLS in the C terminus of the PP2C domain, while ABI2, HAB1, and HAB2 gene products do not have obvious NLS. The role of nuclear localization of group A PP2Cs is not clearly understood, but, recently, it has been shown that the basic amino acid cluster found in the C terminus of ABI1 directs nuclear localization when the abi1-1 mutation has been introduced, suggesting that its nuclear localization is responsible for causing a hypermorphic phenotype (Moes et al, 2008). The mode of regulation of MpABI1 by NLS in the N-terminal domain would be different from that of ABI1 with C-terminal NLS.…”

Abscisic acid sensor RCAR7/PYL13, specific regulator of protein phosphatase coreceptors

“…Arabidopsis protoplasts of the ABA-deficient aba2-1 mutant (10 5 protoplasts in 0.1 mL) were transfected with 5 μg DNA of reporter construct (pRD29B::LUC), 3 μg of p35S::GUS plasmid as a control for internal normalization of expression, and various levels of effector expression cassettes as indicated. The effector cassettes drive expression of RCAR and PP2C cDNA under control of the 35S promoter (20), and all constructs were verified by DNA sequence analysis. The total amount of transfected DNA per assay remained constant by supplementation with DNA of the empty effector cassette.…”

“…Preparation and analysis of Arabidopsis protoplasts were performed as described (20). Arabidopsis protoplasts of the ABA-deficient aba2-1 mutant (10 5 protoplasts in 0.1 mL) were transfected with 5 μg DNA of reporter construct (pRD29B::LUC), 3 μg of p35S::GUS plasmid as a control for internal normalization of expression, and various levels of effector expression cassettes as indicated.…”

“…Conserved amino acid residues in the RCAR molecule and a conserved tryptophan residue found in clade A PP2Cs coordinate the phytohormone by direct ionic interaction and/or water-mediated hydrogen bonds. ABA coreceptors present in the cytosol and nucleus (8,20,21) target protein kinases, transcription factors, and ion channels for ABAdependent regulation (22)(23)(24)(25)(26). The PP2Cs negatively regulate SNF1-related kinases such as OST1/SnrK2.6/SnrK2E, SnrK2.2/ SnrK2D, and SnRK2.3/SnrK2I (27,28).…”