Research Article

Regulation of Polar Auxin Transport by AtPIN1 in Arabidopsis Vascular Tissue

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Science  18 Dec 1998:
Vol. 282, Issue 5397, pp. 2226-2230
DOI: 10.1126/science.282.5397.2226

Figures

  • Figure 1

    Phenotypic and Southern blot analysis of the transposon insertional mutantAtpin1::En134. (A) The most obvious phenotypic aspect of the homozygous mutant represents the naked, pin-forming inflorescence with no or just a few defective flowers. (B) Atpin1::En134 seedlings showed frequently aberrant cotyledon positioning or triple cotyledons. (C) A mutant cauline leaf exhibited abnormal vein branching resulting in the appearance of fused twin or triple leaves. Unusually, the leaf and “pin”-forming axillary shoot have formed in opposite positions. (D) Drastically fasciated inflorescence of an aged mutant. (E) Southern blot analysis of a segregatingAtpin1::En134 mutant population. The M2 progeny of the heterozygousAtpin1::En134 mutant showed 3:1 segregation for wild-type and mutant phenotype plants (8). The cetyltrimethylammonium bromide method (23) was used to isolate genomic DNA from plants showing the mutant (22, 27, 25 28) and wild-type (12, 43, 45, 46, 47, 52, 56, 60, 75, 78, 79) phenotype and from ecotype Columbia (Col) plants lacking En-1 insertions. After Xba I digestion, the DNA was separated on a 0.8% agarose gel (2 μg per lane), transferred to a Nylon membrane and hybridized with a32P-labeled 3′-end probe of the En-1 transposon (24). Only one fragment of 2.3 kb in length (marked by an arrow) was commonly detected in all 12 tested homozygousAtpin1::En134 mutants and in 15 heterozygous plants (not all are shown), indicating cosegregation with theAtpin1::En134 allele. Size bars represent 25 mm (A), 2.5 mm (B), and 10 mm [(C) and (D)].

  • Figure 2

    Structural analysis of AtPIN1alleles and of the deduced AtPIN1 amino acid sequence. (A) Structure of the AtPIN1 gene (drawn to scale), with black boxes representing exons and mapped En-1 insertion sites in the independent mutant alleles Atpin1::En111(111), Atpin1::En134 (134), andAtpin1::En349 (349). Numbers in brackets show base pair positions. The positions of the translational start (ATG) and termination codons (TGA) of the predicted open reading frame are depicted. Nucleotide sequences flanking both ends of theEn-1 transposon in Atpin1::En134 show the disruption of the coding sequence at codon 45 (F). The duplication of nucleotide triplets (TTT) is characteristic forEn-1 insertion sites (25). (B) Amino acid sequence (26) deduced from the AtPIN1 cDNA (accession number AF089084). (C) Hydropathy analysis of AtPIN1. The hydropathy plot was generated with the Lasergene software (DNAstar, Madison, Wisconsin) and the method of Kyte and Doolittle with a window size of nine amino acids (27).

  • Figure 3

    AtPIN1 gene expression analysis. (A and B) Northern blot analysis. Total RNA from different organs and plants were isolated and northern blot analysis was performed (15 μg of total RNA per lane) with a32P-radiolabeled AtPIN1 (base pairs 602 to 1099) probe (28). In (A) various A. thaliana ecotype Columbia organs were analyzed: cotyledons (lane 1), flowers (lane 2), roots (lane 3), rosette leaves (lane 4), seedlings (lane 5), inflorescence axes (lane 6), and siliques (lane 7). In (B) different allelic Atpin1 mutants were analyzed: heterozygousAtpin1::En134 (lane 1), homozygousAtpin1::En134 (lane 2), homozygouspin-formed (lane 3), heterozygous pin-formed (lane 4), homozygous Atpin1::En349 (lane 5), heterozygous Atpin1::En349 (lane 6), and wild-type Columbia (lane 7). The RNA was prepared from inflorescence axes of each genotype. (C to E) In situ hybridization analysis of the AtPIN1 gene expression in wild-type inflorescence axes. Stem segments of plants were fixed, paraffin embedded, cross sectioned (8 μm), and probed with either antisense [(C) and (E)] or sense (D), digoxigenin-labeled, in vitro–transcribed AtPIN1 RNA. The AtPIN1transcript signals were indirectly visualized with the help of alkaline phosphatase–conjugated secondary antibodies (29). (E) is a magnified section of a vascular bundle of (C).AtPIN1-specific staining (red) is localized in cambial and xylem tissues. (F and G) Immunocytochemical localization of AtPIN1 protein in cross sections of inflorescence axes. Stem segments of wild-type plants were fixed, paraffin embedded, sectioned (8 μm), and incubated with affinity-purified polyclonal anti-AtPIN1. Bound anti-AtPIN1 was visualized with the help of alkaline phosphatase–conjugated secondary antibodies (18, 30). AtPIN1-specific staining (purple) was found in cambial and in young and parenchymatous xylem cells (G). Size bars represent 100 μm [(E) and (G)] and 200 μm [(C), (D), and (F)].

  • Figure 4

    AtPIN1 immunolocalization in longitudinalArabidopsis tissue sections. (A to F) Indirect immunofluorescence analysis by laser scanning confocal microscopy. Stem segments of plants were fixed, sectioned, and incubated with polyclonal anti-AtPIN1 (18). Bound anti-AtPIN1 was indirectly visualized with the help of fluorescent (FITC) secondary antibodies (30). The immunofluorescent cells (green-yellow signals) formed continuous vertical cell strands in vascular bundles (A). The AtPIN1 signals are found at the basal end of elongated, parenchymatous xylem cells in the neighborhood of vessel elements, which are distinguished by secondary cell wall thickening structures (C). The red tissue autofluorescence [(A), (C), (E), and (F)] and comparison with the corresponding differential interference contrast (DIC) images [(B) and (D)] facilitated the histological localization of the AtPIN1-specific signals. The arrows point to the AtPIN1-specific fluorescence at the basal end of the xylem cells (C) or to the corresponding positions in the DIC image (D). They also indicate the direction of polar auxin transport in the tissue studied. In (C) two fluorescent signals of three cells forming a vertical cell strand are shown. The upper signal is found at the basal end of the cell extending out of the top of the picture. The cell underneath is fully shown in vertical extension, also fluorescently labeled at its basal end. The fluorescent signal of its basally contacting cell is not shown, because its basal end is out of the picture. A longitudinal hand section of an Arabidopsis stem is shown in (E). AtPIN1 immunofluorescence is primarily localized to the basal side of the cells extending slightly up the lateral walls. A control with a longitudinal section from the Atpin1::En134 mutant is shown in (F). No AtPIN1-specific fluorescent signals were detected. (G) Ultrathin tissue sections were incubated with the polyclonal anti-AtPIN1 and gold-coupled secondary antibodies and examined with an electron microscope (18, 31). Gold grains (marked by arrows) were detected only in one membrane of two contacting cells and were absent at the opposite plasma membrane. ep, epidermis; co, cortex; cw, cell wall; cy, cytoplasm; pm, plasma membrane; pi, pith; v, vessel; vb, vascular bundle. Size bars represent 25 μm [(C), (E), and (F)], 100 μm (A), and 0.1 μm (G).

  • Figure 5

    Analysis of vascular patterning inAtpin1::134 mutants (32). Inflorescence of a wild-type Columbia Arabidopsis plant (A), anAtpin1::En134 mutant (B), and a wild-type plant (C), grown in the presence of auxin transport inhibitor NPA (15 μM). Cross sections were cut as indicated by arrows in (A), (B), (C). The sections presented were cut just above the first cauline leaf (1, 4, 7) and directly below the first (2, 5, 8) and second cauline leaves (3, 6, 9). Arrows on the cross sections (5, 6, 8, 9) indicate the position of the leaves above. Abnormal xylem proliferation was observed in the inflorescence axis below cauline leaves, adjacent to the leaf attachment site. The diameters of the stem sections are ∼1 to 2 mm.

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