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Publikationen - Molekulare Signalverarbeitung

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Publikation

Gross, H. B.; Dalebout, T.; Grubb, C. D.; Abel, S.; Functional detection of chemopreventive glucosinolates in Arabidopsis thaliana Plant Sci. 159, 265-272, (2000) DOI: 10.1016/S0168-9452(00)00354-X

Natural isothiocyanates, derived from glucosinolates by myrosinase-catalyzed hydrolysis, are potent chemopreventive agents that favorably modify carcinogen metabolism in mammals by inhibiting metabolic activation of carcinogens and/or by inducing carcinogen-detoxifying enzymes. Methylsulfinylalkyl isothiocyanates are potent selective inducers of mammalian Phase 2 detoxification enzymes such as quinone reductase [NADP(H):quinone-acceptor oxidoreductase, EC 1.6.99.2]. Members of the Cruciferae family, including the model plant species Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates. We have adapted a colorimetric bioassay for quinone reductase activity in Hepa 1c1c7 murine hepatoma cells as a versatile tool to rapidly monitor methylsulfinylalkyl glucosinolate content in A. thaliana leaf extracts. Using wild type plants and mutant plants defective in the synthesis of 4-methylsulfinylbutyl glucosinolate (glucoraphanin), we have demonstrated that A. thaliana (ecotype Columbia) is a rich source of Phase 2 enzyme inducers and that methylsulfinylalkyl glucosinolates, predominantly glucoraphanin, account for about 80% of the quinone reductase inducer potency of Columbia leaf extracts. We have optimized leaf extraction conditions and the quinone reductase bioassay to allow for screening of large numbers of plant extracts in a molecular genetic approach to dissecting glucosinolate biosynthesis in A. thaliana.
Publikation

Colón-Carmona, A.; Chen, D. L.; Yeh, K.-C.; Abel, S.; Aux/IAA Proteins Are Phosphorylated by Phytochrome in Vitro Plant Physiol. 124, 1728-1738, (2000) DOI: 10.1104/pp.124.4.1728

Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived transcription factors that are induced as a primary response to the plant growth hormone IAA or auxin. Gain-of-function mutations in Arabidopsis genes,SHY2/IAA3, AXR3/IAA17, andAXR2/IAA7 cause pleiotropic phenotypes consistent with enhanced auxin responses, possibly by increasing Aux/IAA protein stability. Semidominant mutations shy2-1D,shy2-2, axr3-1, and axr2-1induce ectopic light responses in dark-grown seedlings. Because genetic studies suggest that the shy2-1D andshy2-2 mutations bypass phytochrome requirement for certain aspects of photomorphogenesis, we tested whether SHY2/IAA3 and related Aux/IAA proteins interact directly with phytochrome and whether they are substrates for its protein kinase activity. Here we show that recombinant Aux/IAA proteins from Arabidopsis and pea (Pisum sativum) interact in vitro with recombinant phytochrome A from oat (Avena sativa). We further show that recombinant SHY2/IAA3, AXR3/IAA17, IAA1, IAA9, and Ps-IAA4 are phosphorylated by recombinant oat phytochrome A in vitro. Deletion analysis of Ps-IAA4 indicates that phytochrome A phosphorylation occurs on the N-terminal half of the protein. Metabolic labeling and immunoprecipitation studies with affinity-purified antibodies to IAA3 demonstrate increased in vivo steady-state levels of mutant IAA3 in shy2-2 plants and phosphorylation of the SHY2-2 protein in vivo. Phytochrome-dependent phosphorylation of Aux/IAA proteins is proposed to provide one molecular mechanism for integrating auxin and light signaling in plant development.
Publikation

Chen, D. L.; Delatorre, C. A.; Bakker, A.; Abel, S.; Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana Planta 211, 13-22, (2000) DOI: 10.1007/s004250000271

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants.
Publikation

Abel, S.; Nürnberger, T.; Ahnert, V.; Krauss, G.-J.; Glund, K.; Induction of an Extracellular Cyclic Nucleotide Phosphodiesterase as an Accessory Ribonucleolytic Activity during Phosphate Starvation of Cultured Tomato Cells Plant Physiol. 122, 543-552, (2000) DOI: 10.1104/pp.122.2.543

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2′:3′-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970–976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2′:3′-cyclic NMP to 3′-NMP and the 3′:5′-cyclic isomers to a mixture of 3′-NMP and 5′-NMP. Specific activities of the enzyme are 2-fold higher for 2′:3′-cyclic NMP than for 3′:5′-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared −Pi culture medium as a source of 3′-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.
Publikation

Abel, S.; Nguyen, M. D.; Theologis, A.; The PS-IAA4/5-like Family of Early Auxin-inducible mRNAs in Arabidopsis thaliana J. Mol. Biol. 251, 533-549, (1995) DOI: 10.1006/jmbi.1995.0454

The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts inArabidopsis thaliana. The corresponding genes, IAA1 and IAA14, are homologs of PS-1AA4/5 and PS-IAA6 from pea, AUX22 and AUX28 from soybean, ARG3 and ARG4from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cyclohexamide. Cyclohexamide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cyclohexamide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 μM IAA. However, individual genes respond differentially between 10 nM and 5μM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2, and aux1.The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spaces vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative βαα-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic β-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.
Publikation

Abel, S.; Theologis, A.; A polymorphic bipartite motif signals nuclear targeting of early auxin-inducible proteins related to PS-IAA4 from pea (Pisum sativum) Plant J. 8, 87-96, (1995) DOI: 10.1046/j.1365-313X.1995.08010087.x

The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS‐IAA4/5 and PS‐IAA6 , that encode short‐lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS‐IAA4 and PS‐IAA6 using progressive deletion analysis and site‐directed mutagenesis is reported. A C‐terminal SV40‐type NLS is sufficient to direct the β‐glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS‐IAA4 and PS‐IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR‐X(24–71)‐(RSXRK)/(RK/RK). Both basic elements of this motif in PS‐IAA4, (KR‐X41‐RSYRK), function interdependently as a bipartite NLS. However, in PS‐IAA6 (KKNEKKR‐X36‐RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40‐type monopartite NLS. The spacer‐length polymorphism, X(24–70), in respective bipartite NLS peptides of several PS‐IAA4‐like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS‐IAA4‐like proteins supports the proposed integrated consensus of NLS.
Publikation

Abel, S.; Theologis, A.; Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression Plant J. 5, 421-427, (1994) DOI: 10.1111/j.1365-313X.1994.00421.x

An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β‐glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)‐mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin‐mediated induction of chloramphenicol acetyl‐transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin‐inducible PS‐IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.
Publikation

Abel, S.; Oeller, P. W.; Theologis, A.; Early auxin-induced genes encode short-lived nuclear proteins. Proc. Natl. Acad. Sci. U.S.A. 91, 326-330, (1994) DOI: 10.1073/pnas.91.1.326

The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses.
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