ABSTRACT
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.
Subject(s)
Calcium-Binding Proteins/genetics , Abscisic Acid/pharmacology , Amino Acid Sequence , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/growth & development , Brassica/genetics , Calcium-Binding Proteins/metabolism , DNA, Plant/chemistry , DNA, Plant/genetics , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Plant/drug effects , Lipid Metabolism , Molecular Sequence Data , Multigene Family , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seeds/drug effects , Seeds/genetics , Seeds/growth & development , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
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.
Subject(s)
ADP-Ribosylation Factors/metabolism , Arabidopsis/metabolism , GTPase-Activating Proteins/metabolism , Membrane Proteins/metabolism , Phospholipids/metabolism , ADP-Ribosylation Factors/genetics , Amino Acid Sequence , Arabidopsis/genetics , Binding Sites , Binding, Competitive , DNA, Complementary/chemistry , DNA, Complementary/genetics , Expressed Sequence Tags , GTPase-Activating Proteins/genetics , Gene Expression , Membrane Proteins/genetics , Molecular Sequence Data , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue Distribution , Zinc FingersABSTRACT
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.
