Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus

HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp

[EN] This work presents the isolation and the biochemical characterization of the Arabidopsis thaliana gene AtSgpp. This gene shows homology with the Arabidopsis low molecular weight phosphatases AtGpp1 and AtGpp2 and the yeast counterpart GPP1 and GPP2, which have a high specificity for dl-glycerol-3-phosphate. In addition, it exhibits homology with DOG1 and DOG2 that dephosphorylate 2-deoxy-d-glucose-6-phosphate. Using a comparative genomic approach, we identified the AtSgpp gene as a conceptual translated haloacid dehalogenase-like hydrolase HAD protein. AtSgpp (locus tag At2g38740), encodes a protein with a predicted Mw of 26.7 kDa and a pI of 4.6. Its sequence motifs and expected structure revealed that AtSgpp belongs to the HAD hydrolases subfamily I, with the C1-type cap domain. In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugars substrates (pH optima at 7.0 and K (m) in the range of 3.6-7.7 mM). AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate, as substrates. AtSgpp is ubiquitously expressed throughout development in most plant organs, mainly in sepal and guard cell. Interestingly, expression is affected by abiotic and biotic stresses, being the greatest under Pi starvation and cyclopentenone oxylipins induction. Based on both, substrate lax specificity and gene expression, the physiological function of AtSgpp in housekeeping detoxification, modulation of sugar-phosphate balance and Pi homeostasis, is provisionally assigned.We acknowledge Professors Montserrat Pages (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernandez (University Polytechnic of Valencia, Spain) for their warm support. We also thank the advice and provision of plasmid pSBETa by Dr. Florence Vignols and Yves Meyer (University of Perpignan, France); the computer software helps by Ramon Nogales-Rangel and Alexis Gonzalez-Policarpo; Eugenio Grau-Ferrando for kind advice and help for sequencing. This work was funded by the 10 month research contract MEC-FEDER to J.A.C.-M., 10 month research contract JAE-DOC to I.M.-S. and by the research project BIO2006-10138 from the MEC-FEDER of Spain to F.A.C.-M. In memoriam of Dr. Mari Cruz Cutanda-Perez.Caparrós Martín, JA.; Mccarthy Suarez, I.; Culiañez Macia, FA. (2013). HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp. Planta. 237(4):943-954. https://doi.org/10.1007/s00425-012-1809-5S9439542374Allen KN, Dunaway-Mariano D (2004) Phosphoryl group transfer: evolution of a catalytic scaffold. 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Arabidopsis thaliana Atrab28: a nuclear targeted protein related to germination and toxic cation tolerance

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.This work has been supported by grant BIO 2000-1562 from Plan Nacional de Investigación Científica y Desarrollo Tecnológico.Peer reviewe

The maize abscisic acid-responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals

The maize abscisic acid (ABA)-responsive rab17 mRNA and Rab17 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rab17 mRNA and Rab17 protein were found in all cells of embryo tissues. Synthesis of rab17 mRNA occurred initially in the embryo axis. As maturation progressed, rab17 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rab17 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rab17 and a nuclear localization signal (NLS) binding phosphoprotein, Nopp140, interaction of Rab17 with NLS peptides was studied. We found specific binding of Rab17 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rab17 was found to be dependent upon phosphorylation. These results suggest that Rab17 may play a role in nuclear protein transport.M.M.A., M.F., and A.B.J. were supported by predoctoral fellowships from Departament d’Ensenyament of the Generalitat de Catalunya, Comision lnterministerial de Ciencia y Tecnologia of the Spanish. Ministerio de Educacion y Ciencia, and Biotechnology Research for lnnovation Development and Growth in Europe of the European Communities, respectively. This work was supported by Grant No. B1091-0546 from Plan Nacional de Investigación Científica y Desarrollo Technológico to M.P.Peer reviewe

Expression and cellular localization of Atrab28 during Arabidopsis embryogenesis

The maize abscisic acid (ABA)-responsive gene rab28 has been shown to be ABA-inducible in embryos and vegetative tissues, expression being mostly restricted to vascular elements during late embryogenesis. In the course of an expressed sequence tags (ESTs) programme, we have isolated an Arabidopsis thaliana gene, Atrab28, encoding the orthologue of maize rab28. The Atrab28 cDNA is 1090 bp long, including a poly(A)+ stretch, and encodes a polypeptide of 262 amino acids. Atrab28 antibody against the recombinant protein recognizes a polipeptide of about 30 kDa and pI 6, in close agreement with the predicted molecular mass and pI. As for maize rab28, expression studies with Atrab28 revealed high specificity for embryo tissues, transcription being stimulated by the transcriptional activator abi3. In contrast, Atrab28 was not induced in vegetative tissues by ABA, osmotic stress or dehydration. The expression of Atrab28 mRNA and the accumulation of Atrab28 protein was largely restricted to provascular tissues of mature embryos and in the seed coat outer tegument and embryo and silique epidermis, as revealed by in situ hybridization and immunocytochemistry with anti-Atrab28 antibodies.This work was supported by a joint programme between the CNRS (Centre National de la Recherche Scientifique) and the CSIC (Consejo Superior de Investigaciones Científicas), the European Associated Laboratory between Perpignan and Barcelona in Plant Molecular and Cellular Biology. In addition, both Perpignan and Barcelona groups are members of the European project ‘Characterising and engineering abscisic acid action’ (Biotech BIO4-CT96-0062). Work in Barcelona was supported in part by the European Communities Biotech Programme, as part of the Project of Technological Priority and by grant BIO94-0750 from Plan Nacional de Investigatión Científica y Desarrollo Tecnológico. Work in Perpignan was supported by CNRS grants URA 565 and UMR 5545.Peer reviewe

Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus

Gel filtration analyses of native SPDS aminopropyltransferases.

<p>Total protein extracts from <i>Arabidopsis</i> cell suspensions were size fractionated with two different chromatography columns: HiPrep S300 (A) and Superose 6 (B) and tested for immunoblotting with affinity purified anti-SPDS2 antibodies to estimate the apparent molecular size of native SPDS complexes that resulted approximately the size of a dimer.</p

Histological immunolocalisation of SPDS aminopropyltransferases in <i>Arabidopsis</i>.

<p>Paraffin-embedded sections of different organs of <i>A. thaliana</i> were incubated with anti-SPDS antibodies and an avidin-biotin-peroxidase detection system. Dark brown staining indicates SPDS antibody-specific reaction. Either transverse or oblique sections are shown for: (a) wild-type developed flower, (b) embryo sac, (c) stamen, (d) sepal, (e) wild-type flower primordia, (f) receptacle, (g) gynoecium (i) mature silique, (j, k) embryo, (m, n, o) leaf, (q, r, s) stem, and (u, v, w) root. Specificity of the signal is shown by using preimmune serum as control reactions in (h, l, p, t, x). Abbreviations: C, cotyledon; Ca, cambium; Ch, chloroplast; Co, cortex; E, epidermis; Em, embryo sac; En, peripheral endosperm; G, gynoecium; I, inner integument; N, nucleus; P, pith; Pa, palisade mesophyll; Pe, petal; Ph, phloem; R, radicle; S, embryo; Sc, seed coat; Se, sepal; Si, silique; Sp, stigmatic papillae; St, stamen; Sy, style; Ue, upper epidermis; V, vascular bundle; X, xylem. Bar = 100 µm in (a, e, i, m, q, u); 25 µm in (j, n, r, v); 10 µm in (b, c, d, f, g, h, k, l, o, p, s, t, w, x).</p

Subcellular localization of aminopropyltransferases as GFP fusion proteins in <i>N.benthamiana</i>.

<p>Translational fusion constructs of aminopropyltransferases to GFP, both at the N-terminus and the C-terminus were transiently expressed in <i>N. benthamiana</i> by agroinfiltration together with a viral nuclear marker fused to mRFP, and analysed with a laser-scanning confocal fluorescence microscope. GFP and mRFP fluorescence spectrum are shown in left and middle column panels. Merged visible and fluoresecent signals are shown in the right column panel. Scale bars: 40 µm.</p