RESUMO
The recent discovery of a variety of receptors has led to new models for hormone perception in plants. In the case of the hormone abscisic acid (ABA), which regulates plant responses to abiotic stress, perception seems to occur both at the plasma membrane and in the cytosol. The cytosolic receptors for ABA have recently been identified as complexes between protein phosphatases 2C (PP2C) and regulatory components (RCAR/PYR/PYL) that bind ABA. Binding of ABA to the receptor complexes inactivates the PP2Cs, thereby activating the large variety of physiological processes regulated by ABA. The Arabidopsis genome encodes 13 homologues of RCAR1 and approximately 80 PP2Cs, of which six in clade A have been identified as negative regulators of ABA responses. In this study we characterize a novel member of the RCAR family, RCAR3. RCAR3 was identified in a screen for interactors of the PP2Cs ABI1 and ABI2, which are key regulators of ABA responses. RCAR3 was shown to repress ABI1 and ABI2 in vitro, and to stimulate ABA signalling in protoplast cells. RCAR3 conferred greater ABA sensitivity to the PP2C regulation than RCAR1, whereas stereo-selectivity for (S)-ABA was less stringent with RCAR3 as compared with RCAR1. In addition, regulation of the protein phosphatase activity by RCAR1 and RCAR3 was more sensitive to ABA for ABI1 than for ABI2. Based on the differences we have observed in transcriptional regulation and biochemical properties, we propose a model whereby differential expression of the co-receptors and combinatorial assembly of the receptor complexes act in concert to modulate and fine-tune ABA responses.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calorimetria , Proteínas de Transporte , Dicroísmo Circular , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas Fosfatases/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Proteína Fosfatase 2C , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
The plant hormone abscisic acid (ABA) acts as a developmental signal and as an integrator of environmental cues such as drought and cold. Key players in ABA signal transduction include the type 2C protein phosphatases (PP2Cs) ABI1 and ABI2, which act by negatively regulating ABA responses. In this study, we identify interactors of ABI1 and ABI2 which we have named regulatory components of ABA receptor (RCARs). In Arabidopsis, RCARs belong to a family with 14 members that share structural similarity with class 10 pathogen-related proteins. RCAR1 was shown to bind ABA, to mediate ABA-dependent inactivation of ABI1 or ABI2 in vitro, and to antagonize PP2C action in planta. Other RCARs also mediated ABA-dependent regulation of ABI1 and ABI2, consistent with a combinatorial assembly of receptor complexes.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas , Germinação , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Raízes de Plantas/crescimento & desenvolvimento , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Mutação Puntual , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Estereoisomerismo , Regulação para CimaRESUMO
Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.