RESUMO
We have previously identified a rice gene encoding a 27 kDa protein with a single Ca2+-binding EF-hand and a putative membrane anchor. We report here similar genes termed caleosins, CLO, in other plants and fungi; they comprise a multigene family of at least five members in Arabidopsis (AtClo1-5). Northern hybridization demonstrated that AtClo2-4 mRNAs levels were low in various tissues, while AtClo1 mRNA levels were high in developing embryos and mature seeds. Analysis of transgenic Arabidopsis plants expressing the GUS reporter under control of the AtClo1 promoter showed strong levels of expression in developing embryos and also in root tip cells. Antibodies raised against AtCLO1 were used to detect caleosin in cellular fractions of Arabidopsis and rapeseed. This indicated that caleosins are a novel class of lipid body proteins, which may also be associated with an ER subdomain.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Brassica/genética , Proteínas de Ligação ao Cálcio/metabolismo , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Arabidopsis/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Fatores de Ribosilação do ADP/genética , Sequência de Aminoácidos , Arabidopsis/genética , Sítios de Ligação , Ligação Competitiva , DNA Complementar/química , DNA Complementar/genética , Etiquetas de Sequências Expressas , Proteínas Ativadoras de GTPase/genética , Expressão Gênica , Proteínas de Membrana/genética , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Dedos de ZincoRESUMO
Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde oxidases (AtAO1, 2, 3) were isolated. An aldehyde oxidase is the last step in abscisic acid (ABA) biosynthesis. AtAO1 is mainly expressed in seeds and roots which might reflect that it is involved in ABA biosynthesis.
Assuntos
Aldeído Oxirredutases/genética , Proteínas de Arabidopsis , Arabidopsis/enzimologia , Molibdênio , Aldeído Oxidase , Aldeído Oxirredutases/classificação , Aldeído Oxirredutases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , DNA de Plantas , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Xantina Desidrogenase/metabolismoRESUMO
A cDNA corresponding to an mRNA which accumulates in germinating rice seeds in response to the phytohormone abscisic acid was isolated by differential hybridization. Northern blotting indicated that the mRNA also accumulates in vegetative tissues in response to treatment with abscisic acid and to osmotic stress. Sequencing identified a major open reading frame encoding a novel protein of 27.4 kDa. The identity of the open reading frame was confirmed by comparing the translation products of cellular, hybrid-selected, and in vitro transcribed RNAs and by immunoprecipitation. Western blotting of cellular extracts indicated that the protein is associated with microsomal or membrane fractions. Data base searches indicated that it contains a conserved Ca(2+)-binding, EF-hand motif and that related proteins are similarly expressed in Arabidopsis thaliana. A fusion protein purified from Escherichia coli containing the putative EF-hand region was shown to bind Ca2+ in blot binding assays. These data identify a novel gene family encoding proteins involved in the response of plants to abscisic acid and osmotic stress.
