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Published on November 4, 2009; 10.1104/pp.109.146381

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Received August 19, 2009
Accepted October 30, 2009

THE NUCLEAR INTERACTOR PYL8/RCAR3 OF THE Fagus sylvatica FsPP2C1 IS A POSITIVE REGULATOR OF ABA SIGNALING IN SEEDS AND STRESS

Xandra Saavedra , Abelardo Modrego , Dolores Rodriguez , Mary Paz Gonzalez-Garcia , Luis Sanz , Gregorio Nicolas , and Oscar Lorenzo *

Departamento de Fisiologia Vegetal, Centro Hispano-Luso de Investigaciones Agrarias, Facultad de Biologia, Universidad de Salamanca, C/ Rio Duero 12, 37185 Salamanca, Spain

* Corresponding author; email: oslo{at}usal.es.

The functional protein phosphatase type-2C (PP2C) FsPP2C1 from beechnut (Fagus sylvatica L.) was a negative regulator of ABA signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two close-related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis 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 cells and agroinfiltration in Nicotiana as GFP 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 BiFC 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 to 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.






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