Abstract
The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two closely related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis (Arabidopsis thaliana) BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion (Allium cepa) cells and agroinfiltration in tobacco (Nicotiana benthamiana) as green fluorescent protein fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by bimolecular fluorescence complementation assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA hypersensitivity in Arabidopsis seeds and, consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl, or paclobutrazol, which are not inhibiting conditions to the wild type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses.
Highlights
The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds
We previously reported the cloning of FsPP2C1, a functional PP2C expressed in beechnut (Fagus sylvatica), that was up-regulated upon ABA addition in seeds and early seedlings (Lorenzo et al, 2001)
We have previously reported the isolation and functional characterization of FsPP2C1 as a plant type 2C protein phosphatase, with all the conserved features of the catalytic domain of these proteins (Lorenzo et al, 2001), which acts as a negative regulator of ABA responses in seeds (Gonzalez-Garcıa et al, 2003)
Summary
The functional protein phosphatase type 2C from beechnut (Fagus sylvatica; FsPP2C1) was a negative regulator of abscisic acid (ABA) signaling in seeds. Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA response genes. Double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses. GTG1 and GTG2 bind ABA in vitro, and double mutants lacking both proteins exhibit ABA hyposensitivity They suggest that GTG proteins are a new type of G protein that function as membrane-localized receptors of ABA and mediate ABA responses in vivo. An ABA-specific-binding protein, ABAR/CHLH (for putative abscisic acid receptor/ Mg-chelatase H subunit), that mediates ABA signaling as a positive regulator in Arabidopsis, has been found to bind ABA using a newly developed ABA-affinity chromatography technique and a [3H]ABA-binding assay, supporting that ABAR/ CHLH may act as an intracellular ABA receptor (Wu et al, 2009)
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