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Key Message: Plasma membrane-localized AtAVT6D importing aspartic acid can be targeted to develop plants with enhanced osmotic and nitrogen-starvation tolerance. AtAVT6D promoter can be exploited as a stress-inducible promoter for genetic improvements to raise stress-resilient crops. The AtAVT6 family of amino acid transporters in Arabidopsis thaliana has been predicted to export amino acids like aspartate and glutamate. However, the functional characterization of these amino acid transporters in plants remains unexplored. The present study investigates the expression patterns of AtAVT6 genes in different tissues and under various abiotic stress conditions using quantitative Real-time PCR. The expression analysis demonstrated that the member AtAVT6D was significantly induced in response to phytohormone ABA and stresses like osmotic and drought. The tissue-specific expression analysis showed that AtAVT6D was strongly expressed in the siliques. Taking together these results, we can speculate that AtAVT6D might play a vital role in silique development and abiotic stress tolerance. Further, subcellular localization study showed AtAVT6D was localized to the plasma membrane. The heterologous expression of AtAVT6D in yeast cells conferred significant tolerance to nitrogen-deficient and osmotic stress conditions. The Xenopus oocyte studies revealed that AtAVT6D is involved in the uptake of Aspartic acid. While overexpression of AtAVT6D resulted in smaller siliques in Arabidopsis thaliana. Additionally, transient expression studies were performed with the full-length AtAVT6D promoter and its deletion constructs to study the effect of ACGT-N24-ACGT motifs on the reporter gene expression in response to abiotic stresses and ABA treatment. The fluorometric GUS analyses revealed that the promoter deletion construct-2 (Pro.C2) possessing a single copy of ACGT-N24-ACGT motif directed the strongest GUS expression under all the abiotic conditions tested. These results suggest that Pro.C2 can be used as a stress-inducible promoter to drive a significant transgene expression. [ABSTRACT FROM AUTHOR]

Zinc (Zn) and iron (Fe) deficiency are widespread among citrus plants, but the molecular mechanisms regarding uptake and transport of these two essential metal ions in citrus are still unclear. In the present study, 12 members of the Zn/Fe-regulated transporter (ZRT/IRT)-related protein (ZIP) gene family were identified and isolated from a widely used citrus rootstock, trifoliate orange (Poncirus trifoliata L. Raf.), and the genes were correspondingly named as PtZIPs according to the sequence and functional similarity to Arabidopsis thaliana ZIPs. The 12 PtZIP genes were predicted to encode proteins of 334-419 amino acids, harboring 6-9 putative transmembrane (TM) domains. All of the PtZIP proteins contained the highly conserved ZIP signature sequences in TM-IV, and nine of them showed a variable region rich in histidine residues between TM-III and TM-IV. Phylogenetic analysis subdivided the PtZIPs into four groups, similar as found for the ZIP family of A. thaliana, with clustered PtZIPs sharing a similar gene structure. Expression analysis showed that the PtZIP genes were very differently induced in roots and leaves under conditions of Zn, Fe and Mn deficiency. Yeast complementation tests indicated that PtIRT1, PtZIP1, PtZIP2, PtZIP3, and PtZIP12 were able to complement the zrt1zrt2 mutant, which was deficient in Zn uptake; PtIRT1 and PtZIP7 were able to complement the fet3fet4 mutant, which was deficient in Fe uptake, and PtIRT1 was able to complement the smf1 mutant, which was deficient in Mn uptake, suggesting their respective functions in Zn, Fe, and Mn transport. The present study broadens our understanding of metal ion uptake and transport and functional divergence of the various PtZIP genes in citrus plants. [ABSTRACT FROM AUTHOR]

• Hypoxanthine-guanine phosphoribosyltransferase (HGPT) occurs in both eukaryotic and prokaryotic organisms. However, the molecular and functional properties of plant HGPT are not well understood. • In this study, it was found that the putative HGPT proteins from dicot and monocot plant species exhibited significant identities to their homologs from other cellular organisms. Ectopic expression of the HGPTs from Arabidopsis, soybean or wheat complemented HGPT deficiency in the hpt1 mutant of Saccharomyces cerevisiae. • Recombinant Arabidopsis HGPT (AtHGPT) catalyzed both forward and reverse reactions in in vitro biochemical assays. The relative catalytic efficiency for the synthesis of guanosine monophosphate (GMP) was significantly greater than that for the production of guanine from GMP. Further investigations led to identification of the candidate residues that may form the pyrophosphate (PPi) binding loop in AtHGPT. AtHGPT expression level was dynamically regulated in Arabidopsis organs and during leaf development and senescence and seed germination. AtHGPT knockout mutant germinated more slowly than wild type control, whereas its overexpression mutant exhibited accelerated germination. • Collectively, the data suggest that functional HGPTs are expressed in higher plants. In Arabidopsis, HGPT plays an active role in the salvage of purine bases and its activity is required for efficient seed germination. [ABSTRACT FROM AUTHOR]

Although Arabidopsis thaliana does not produce phytosiderophores (PS) under Fe deficiency, it contains eight homologs of the metal–PS/metal–nicotianamine (NA) transporter ZmYS1 from maize. This study aimed to investigate whether one of the closest Arabidopsis homologs to ZmYS1, AtYSL2, is involved in metal–chelate transport. Northern analysis revealed high expression levels of AtYSL2 in Fe-sufficient or Fe-resupplied roots, while under Fe deficiency transcript levels decreased. Quantitative real-time polymerase chain reaction (PCR) and analysis of transgenic plants expressing an AtYSL2 promoter::β-glucuronidase gene further allowed the detection of down-regulated AtYSL2 gene expression under Zn and Fe deficiency. In contrast to ZmYS1, AtYSL2 did not mediate metal–PS or metal–NA transport in yeast mutants defective in Cu or Fe uptake, nor did AtYSL2 mediate Fe(II)–NA-, Fe(III)–NA- or Ni(II)–NA-inducible currents when assayed by two-electrode voltage clamp in Xenopus oocytes. Moreover, truncation of the N-terminus to remove putative phosphorylation sites that might trigger autoinhibition did not confer functionality to AtYSL2. A direct growth comparison of yeast cells transformed with AtYSL2 in two different yeast expression vectors showed that transformation with empty pFL61 repressed growth even under non-limiting Fe supply. We therefore conclude that the yeast complementation assay previously employed does not allow the identification of AtYSL2 as an Fe–NA transporter. Transgenic plants expressing an AtYSL2 promoter::β-glucuronidase gene showed expression in root endodermis and pericycle cells facing the meta-xylem tubes. Taken together, our investigations support an involvement of AtYSL2 in Fe and Zn homeostasis, although functionality or substrate specificity are likely to differ between AtYSL2 and ZmYS1. [ABSTRACT FROM AUTHOR]

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues. [ABSTRACT FROM AUTHOR]

Most isoprenylated proteins are α-carboxyl-methylated. However, despite numerous studies linking protein isoprenylation in plants to cell cycle control, meristem development, and phytohormone signaling, α-carboxyl methylation of isoprenylated plant proteins has not been characterized in detail. Here, we report the cloning of a prenylcysteine α-carboxyl methyltransferase gene ( AtSTE14) from Arabidopsis thaliana. AtSTE14 restores fertility and enzymatic activity to a ste14 mutant of Saccharomyces cerevisiae, confirming its identity as a bona fide prenylcysteine α-carboxyl methyltransferase gene. Furthermore, the presence of AtSTE14 transcripts in various Arabidopsis organs suggests a ubiquitous role for the AtSTE14 protein in plant growth and development. These results demonstrate that Arabidopsis thaliana possesses a functional prenylcysteine α-carboxyl methyltransferase involved in post-isoprenylation protein processing. [ABSTRACT FROM AUTHOR]

It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA ( AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35–41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg2+ ions for full activity. [ABSTRACT FROM AUTHOR]

Mutations at the ABII (abscisic acid insensitive 1) locus of the plant Arabidopsis thaliana cause a reduction in sensitivity to the plant hormone abscisic acid. The sequence of ABII predicts a protein composed of an N-terminal domain that contains motifs for an EF-hand Ca2+-binding site, and a C-terminal domain with similarities to protein serine/threonine phosphatases 2C. We report here two sets of experimental evidence that indicate that ABI1 has typical protein phosphatase 2C activity. First, expression of the ABI1 C-terminal domain partially complemented the temperature-sensitive growth defect of a Saccharomyces cerevisiae protein phosphatase 2C mutant. Second, recombinant proteins that contained the ABI1 C-terminal domain displayed h z vitro phosphatase activity towards 32P-labelled casein, and this activity displayed Mg2+ or Mn2+ dependence and okadaic acid insensitivity typical of protein phosphatases 2C. Characterisation of recombinant proteins that contained various portions of ABI1 indicated that the putative EF-hand motif is unlikely to mediate Ca2+ regulation of the ABI1 phosphatase activity at physiological Ca2+ concentrations, and may represent an EF-hand analogue rather than an EF-hand homologue. The abil-1 mutation appeared to cause significant reduction in the phosphatase activity of ABI1. These results are discussed in relation to the dominant phenotype of abil-1 over the wild-type allele in plants, and to the possible role of ABI1 in abscisic acid signalling. [ABSTRACT FROM AUTHOR]

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed. [ABSTRACT FROM AUTHOR]

Summary Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2-1 and atmrs2-2. Atmrs2-1, but not atmrs2-2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2-1 and yeast MRS2 but not in AtMRS2-2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2-1. A differing, non-mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the psi-blast algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild-type levels in the yeast mutant on complementation with atmrs2-1. [ABSTRACT FROM AUTHOR]