Regulation of Polar Auxin Transport by AtPIN1 in Arabidopsis Vascular Tissue
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Went F. W., Rec. Trav. Bot. Neerl. 25, 1 (1928);
van der Weij G. H., ibid. 31, 810 (1934).
; T. L. Lomax G. K. Muday P. H. Rubery in Plant Hormones Physiology Biochemistry and Molecular Biology P. J. Davies Ed. (Martinus Nijhoff Kluwer Dordrecht The Netherlands 1995) pp. 509–530.
T. Sachs Pattern Formation in Plant Tissues (Cambridge Univ. Press Cambridge 1991); R. Aloni in Plant Hormones Physiology Biochemistry and Molecular Biology P. J. Davies Ed. (Martinus Nijhoff Kluwer Dordrecht The Netherlands 1995) pp. 531–546.
Jacobs M., Gilbert S. F., ibid. 220, 1297 (1983).
. The heterozygous Atpin1::En134 mutant (M 1 ) whose M 2 progeny segregated 3:1 for wild-type and mutant phenotypes was identified in generation S 6 [
]. Backcrosses of heterozygous Atpin1::En134 plants with wild type expressed the mutant phenotype only in the F 2 generation.
L. Gälweiler et al. data not shown.
Crosses between the heterozygous transposon insertional mutants yielded ∼25% mutant phenotypes in the F 1 generation indicating allelism. Using En-1– and AtPIN1 -specific primers we amplified the transposon-flanking DNA in the Atpin1::En111 Atpin1::En134 and Atpin1::En349 alleles by PCR and then sequenced it. The sequences were identical with AtPIN1 sequences showing independent En-1 insertions.
Plant DNA sequences flanking the 5′ end of En-1 in the Atpin1::En134 allele were cloned by a ligation-mediated PCR technique [
] with En-1 – and linker-specific oligonucleotides after Csp6 I 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 λ 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: ( AtPIN1 cDNA) and ( AtPIN1 genomic DNA).
By screening the CIC YAC library {[
]; 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 .
Repeating auxin transport measurements with stem segments we confirmed the reduction of polar auxin transport in pin-formed mutants (6 7) and found a reduction of polar auxin transport in Atpin1::En134 mutants as well.
; M. D. Marger und
GenBank accession numbers of homologous clones in Arabidopsis thaliana are as follows: ACC002311 ( EIR1 ) ( AtPIN2 cDNA) ( AtPIN2 genomic DNA) ACC003979 and .
. AtPIN2 () and EIR1 () were independently isolated and represent the same genetic locus.
GenBank numbers of AtPIN1 homologous rice clones are as follows: ( REH ) and .
To generate AtPIN1-specific polyclonal antibodies we ligated a Rsa I fragment of the AtPIN1 cDNA encoding the antigenic peptide of AtPIN1 from amino acid 155 to 408 into the bacterial expression vector pQE-31 (Qiagen). This expression construct encoded a recombinant fusion protein with an NH 2 -terminal His 6 tag. After expression in Escherichia coli SG13009 the recombinant protein was affinity purified on a Ni 2+ –nitrilotriacetic acid column as described by the Quiaexpressionist manual (Qiagen) and checked by SDS–polyacrylamide gel electrophoresis [
]. After immunization of rabbits (Eurogentec Ougrée Belgium) the polyclonal antiserum was affinity purified against the recombinant AtPIN1 peptide [
Gu J., Stephenson G., Iadarola M. J., Biotechniques 17, 257 (1994);
] and diluted to a final protein concentration of 0.22 mg/ml. In protein immunoblot analysis the affinity-purified anti-AtPIN1 detected specifically the recombinant AtPIN1 peptide in bacterial extracts as well as a 67-kD protein in microsomal membrane fractions from A. thaliana [
F. M. Ausubel et al. Current Protocols in Molecular Biology (Green Wiley New York 1993).
The 3′ En-1 probe DNA was generated by PCR with the En-1 –specific primers En 7631 (5′-TCAGGCTCACATCATGCTAGTCC-3′) and En 8141 (5′-GGACCGACGCTCTTATGTTAAAAG-3′). In Southern blot analysis this PCR product hybridized to the 3′ ends of Xba I–digested En-1 DNA detecting fragments of 1.98-kb En-1 DNA plus flanking plant DNA.
Single-letter abbreviations for amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
. To check for equal RNA loading we rehybridized the Northern blots with ribosomal protein large subunit 4 ( RPL4 ) and ubiquitin carrier ( UBC ) probes.
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 4× standard saline citrate (SSC) containing 5 mM dithiothreitol (DTT) (10 min room temperature) 2× SSC containing 5 mM DTT (30 min room temperature) and 0.2× 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.
Inflorescence axes of 3- to 4-week-old Arabidopsis wild-type and mutant plants (grown in a greenhouse at 18° to 24°C with 16 hours of light) were cut and fixed in ice-cold methanol/acetic acid (3:1). Paraffin embedding sectioning and mounting were done as described (22). Antibody incubation and immunohistochemical staining was performed as described [
] with the following modifications: 8-μm cross sections and 30-μm longitudinal sections of inflorescence axes were incubated with affinity-purified anti-AtPIN1 [(18) 4°C overnight] diluted 1:100 in buffer [3% (w/v) milk powder in phosphate-buffered saline (PBS) pH 7.4]. Incubation with secondary antibodies coupled to fluorescein isothiocyanate (FITC) or alkaline phosphatase (Boehringer Mannheim 1:100) was done at room temperature for 2 to 3 hours. After antibody incubation washing was performed three times (10 min) with PBS containing 0.2% Tween 20. For hand sectioning stem segments were fixed in 4% paraformaldehyde diluted in MTSB (50 mM piperazine ethanesulfonic acid 5 mM ethylene glycol tetraacetic acid 5 mM MgSO 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 histological 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.
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.
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.
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.