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.

SUMMARYThe 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.

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 Polyisoprenoid alcohols together with sterols and quinone side chains constitute three main branches of terpene products originating from farnesyl diphosphate (FPP) 4 (1). These linear five-carbon unit polymers are divided into two groups, i.e. polyprenols and dolichols, according to the hydrogenation status of the ␣-terminal isoprene unit (dolichol structure is shown in Fig. 1). In cells, polyprenols and dolichols are always found as mixtures of prenologues, and data collected so far show polyprenols to be typical for bacteria and plants, whereas dolichols are generally attributed to animals and yeast (2). Nevertheless, it should be remembered that dolichols are the predominant form in some plant organs like roots (3). Data on the occurrence and functions of polyisoprenoids are summarized in recently published reviews (4, 5). The formation of the polyisoprenoid chain, starting from the -end of the molecule (Fig. 1), proceeds in a biphasic manner with farnesyl-diphosphate synthase responsible for the synthesis of the all-trans-FPP (three isoprene units of -t 2 structure, t stands for trans-isoprene unit), and its further elongation by cis-prenyltransferase. The latter enzyme, cloned from several prokaryotic and eukaryotic organisms (see Refs. 6, 7 and references therein), including Arabidopsis thaliana (8,9) and Hevea brasiliensis (10), utilizes isopentenyl diphosphate (IPP) for elongation of FPP up to the desired chain length, thus producing a family of polyprenyl diphosphates (n isoprene units of -t 2 -c n-3 structure, c stands for cis-isoprene unit), which are subsequently converted to polyprenols or dolichols according to the "tissue-specific requirements" by a still unknown mechanism.In plant cells two pathways are known to produce IPP utilized by numerous enzymes to finally give more than 50,000 different isoprenoid structures, the mevalonate pathway (MVA) and the mevalonate-independent methylerythritol phosphate pathway (MEP) (for reviews, see Refs. 11-13). Both pathways are compartmentalized as follows: the MVA in the cytoplasm to provide sterols, the many sesquiterpenes, and the prenyl chains of ubiquinones, and the MEP one in the plastids Tables 1 and 2

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