Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue

Science

Volumn 282, Issue 5397, 1998, Pages 2226-2230

  Gälweiler, Leo ^a   Guan, Changhui ^a   Müller, Andreas ^a   Wisman, Ellen ^b   Mendgen, Kurt ^c   Yephremov, Alexander ^b   Palme, Klaus ^a  

^a Max Delbruck Laboratorium der Max Planck Gesellschaft   (Germany)

^b MAX PLANCK INSTITUTE FOR PLANT BREEDING RESEARCH   (Germany)

^c UNIVERSITY OF KONSTANZ   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

AUXIN; CARRIER PROTEIN; GENE PRODUCT;

ARABIDOPSIS; ARTICLE; GENE EXPRESSION; GENE MUTATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PHENOTYPE; PLANT SEED; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN TRANSPORT; REGULATORY MECHANISM; SIGNAL TRANSDUCTION; SOUTHERN BLOTTING; TISSUE DIFFERENTIATION; VASCULAR TISSUE;

AMINO ACID SEQUENCE; ARABIDOPSIS; ARABIDOPSIS PROTEINS; BIOLOGICAL TRANSPORT; BLOTTING, NORTHERN; CLONING, MOLECULAR; CONTIG MAPPING; DNA TRANSPOSABLE ELEMENTS; GENES, PLANT; INDOLEACETIC ACIDS; MEMBRANE PROTEINS; MEMBRANE TRANSPORT PROTEINS; MOLECULAR SEQUENCE DATA; MUTAGENESIS, INSERTIONAL; PHENOTYPE; PHTHALIMIDES; PLANT ROOTS; PLANT STEMS; PROTON-MOTIVE FORCE;

ARABIDOPSIS; ARABIDOPSIS THALIANA; BACTERIA (MICROORGANISMS); EMBRYOPHYTA; EUKARYOTA; SPERMATOPHYTA;

EID: 0032545346     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5397.2226     Document Type: Article

References (45)

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17. Plant DNA sequences flanking the 5′ end of En-1 in the Atpin1::En134 allele were cloned by a ligation-mediated PCR technique [P. R. Mueller and B. Wold, Science 246, 780 (1989); M. Frey, C. Stettner, A. Gierl, Plant J. 13, 717 (1998)] with En-1- and linker-specific oligonucleotides after CspS 1 restriction of genomic DNA and ligation of compatible linker DNA. The isolated flanking DNA was used as a probe to screen a cDNA library, prepared from suspension cells, for homologous clones that were then used to screen a genomic library of A. thaliana. The γ libraries were prepared from the ecotype Columbia and provided by the Arabidopsis DNA Centre, Cologne. Sequence analysis of the longest AtPIN1 cDNA (2276 base pairs) identified an open reading frame encoding 622 amino acids. An in-frame stop codon located upstream to the first ATG suggested that the cDNA encodes a full-length protein. GenBank accession numbers are as follows: AF089084 (AtPIN1 cDNA) and AF089085 (AtPIN1 genomic DNA).
18. By screening the CIC YAC library {[F. Creusot et al., Plant J. 8, 763 (1995)]; provided by the Arabidopsis DNA Centre, Cologne} with a radiolabeled AtPIN1 probe, we identified a contig consisting of the overlapping clones CIC6H1, CIC12G10, CIC12H9, and CIC9C4. Physical mapping was performed with the server http://cbiLhumgen.upenn.edu/∼atgc/physical-mapping.
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41. Segments of inflorescence axes of 3- to 4-week-old A. thaliana ecotype Columbia (grown in a greenhouse at 18° to 24°C, with 16 hours of light) were fixed, paraffin embedded, and analyzed by in situ hybridization as described (22), with the following modifications. To generate AtPIN1-specific RNA probes, we inserted the Bgl II-Hind III fragment of the AtPIN1 cDNA (base pairs 602 to 1099) into the Bam HI-, Hind III-cleaved vector pBluescript SK- (Stratagene), generating pin23HX. After linearizing pin23HX (Hind III for antisense and Xba I for sense transcription), we performed in vitro transcription and digoxigenin labeling using the DIG RNA Labeling Kit (Boehringer Mannheim). The RNA hybridization was performed overnight at 42°C with a probe concentration of 30 ng per 100 μl. The slides were then washed with 4x standard saline citrate (SSC) containing 5 mM dithiothreitol (DTT) (10 min, room temperature), 2x SSC containing 5 mM DTT (30 min, room temperature), and 0.2x SSC containing 5 mM DTT (30 min, 65°C). After blocking with 0.5% blocking agent (Boehringer Mannheim), we detected signals using anti-digoxigenin (1:3000, Boehringer Mannheim) coupled to alkaline phosphatase followed by a nitroblue tetrazolium, brome-chloro-indolyl phosphate staining reaction.
42. 4, pH 7.0), treated with 2% Driselase (Sigma, in MTSB, 0.5 hour), and permeabilized with 10% dimethylsulfoxide and 0.5% NP-40 (in MTSB, 1 hour). After hand sectioning with razor blades, antibody incubation was performed as described above. Alkaline phosphatase staining reactions were carried out for several hours to overnight, and the results were analyzed microscopically. Fluorescent signal analysis was performed with a confocal laser scanning microscope (Leica DMIRBE, TCS 4D with digital image processing) with a 530 ± 15 nm band-pass filter for FITC-specific detection and a 580 ± 15 nm band-pass filter for autofluorescence detection. For histotogical signal localization both images were electronically overlaid, resulting in red autofluorescence and green-yellow AtPIN1-specific fluorescence. DIC images were generated to determine the exact cellular signal localization. Controls with preimmune serum and secondary antibodies alone yielded no specific signals. Tissue orientation of the longitudinal stem sections was determined with the help of residual traces of lateral leaves and by cutting stem segments apically and basally with different angles. Polar signal localization was also obvious in cells in which the immunostained cytoplasm was detached from the basal cell wall (9). The AtPIN1 localization results were reproduced by several experiments.
43. Tissue was frozen with an HPM 010 high-pressure instrument (Balzers, Liechtenstein) and processed as described [K. Mendgen, K. Welter, F. Scheffold, G. Knauf-Beiter, in Electron Microscopy of Plant Pathogens, K. Mendgen and K. Lesemann, Eds. (Springer-Verlag, Heidelberg, 1991), pp. 31-42]. Substitution was performed in acetone at -90°C. embedding in Unicryl (British Biocell, Cardiff), and polymerization at 4°C. Ultrathin sections were incubated with primary antibodies [1% preimmune serum or affinity-purified anti-AtPIN1 (18)], diluted 1:10 with buffer [1% (w/v) bovine serum albumin (BSA) and 0.1% BSA-C, in TBS (10 mM tris(hydroxymethyl)aminomethane-HCL, 150 mM NaCl, pH 7.4)], for 3 hours, followed by incubation with a secondary antibody [10 nm gold coupled to goat antibody to rabbit immunoglobulin G (Biotrend, Köln, Germany)], diluted 1:20 with buffer, for 1 hour at 20°C. Sections were stained with uranylate and lead citrate and examined with an Hitachi H-7000 electron microscope.
44. Plants were grown in vitro as described (6), fixed, paraffin-embedded, and deparaffinated as described (22). Cross sections (10 μm) of inflorescence axes were analyzed microscopically. Anatomical studies with pin-formed plants gave similar results.
45. We thank P. Huijser for help with the confocal microscopic analysis, H. Vahlenkamp for electron microscopy, C. Koncz for comments on the manuscript, and H. Saedler and J. Schell for continuous support and help. Funded by the European Communities' BIOTECH program and by the Deutsche Forschungsgemeinschaft "Arabidopsis" program.