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doi: 10.1242/10.1242/dev.00441

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Isolation of COV1, a gene involved in the regulation of vascular patterning in the stem of Arabidopsis

¹ School of Biological Sciences, The University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK
² Department of Forest Genetics and Plant Physiology, SLU, Umeå SE-901 83, Sweden

View larger version (104K): [in a new window]   Fig. 1. Hand-cut sections of stems from wild-type (A,B), cov1-1 (C) or cov1-2 (D) plants stained with Toluidine Blue and viewed under brightfield illumination (A), or stained with Aniline Blue and viewed under UV light (B-D). Xylem and interfascicular cells are identified by their blue-stained walls when stained with Toluidine Blue, or by their blue fluorescence when stained with Analine Blue. Phloem cells produce a distinctive yellow fluorescence when stained with Analine Blue. VB, vascular bundle; Ph, phloem; Xy, xylem; If, interfascicular region; Dif, differentiation in the interfascicular region.

View larger version (133K): [in a new window]   Fig. 2. Hand-cut sections from wild-type (A,C,E) and cov1-1 (B,D,F) plants. Sections were taken from four-week-old plants at different positions down the stem: just below the apical meristem (A,B); a quarter of the way down (C,D); and halfway down (E,F). Sections were stained with Aniline Blue and viewed under UV light. Additional differentiation of vascular bundles in the interfascicular region and unconnected to the normally patterned vascular bundles can be seen in the cov1-1 sections (thick white arrows in B,D). There is also evidence of lateral expansion of vascular bundles or additional differentiation of vascular tissue in close proximity to the normally patterned vascular bundles (open arrows in B,D). VB, vascular bundle; If, interfascicular region; Ph, phloem; Xy, xylem.

View larger version (114K): [in a new window]   Fig. 3. (A) Wild-type, cov1-1 and cov1-2 plants were grown side by side under continuous light and photographed at four weeks of age. (B) Although the leaves of both mutant alleles showed a wrinkled phenotype, the vein patterning was similar to wild-type though slightly asymmetric in both cov1-1 and cov1-2.

View larger version (20K): [in a new window]   Fig. 4. (A) Map-based cloning of COV1 based on data obtained from recombinants identified in the testcross screen. The number of recombinants identified between COV1 and closely linked markers are shown in brackets. Subclones used to determine complementation are shown as unbroken black lines. The cov1-1 mutant was partially complemented with pGDP1 and fully complemented with pGDP6 and pGDP7. Other genes in the region are indicated by numbers (1, At2g20160; 2, At2g20150; 3, At2g20140; 4, At2g20130; 5, At2g20110). The intron-exon structure and position of COV1 and the closely related gene LCV1 are also shown (bottom). The predicted translation initiation sites are indicated by arrows. (B) The predicted topology of COV1. The protein contains three predicted membrane-spanning domains; the N-terminal end of the protein is predicted to be intracellular and the C-terminal end is predicted to be in the wall. The mutation in cov1-1 (*1) is in exon 3, which forms part of the first membrane-spanning domain and results in an amino acid change from proline to serine (P76S). The mutation in cov1-2 (*2) is located in exon 4, which is predicted to lie just after the second membrane domain and results in an amino acid change from glycine to arginine (G130R).

View larger version (119K): [in a new window]   Fig. 5. (A) Morphology of four-week-old plants of cov1-1, cov1-1 transformed with pGDP7 and wild-type Ler grown side by side, demonstrating that pGDP7 complements the mutation. Sections from the base of the stem of a four-week-old cov1-1 plant (B) and a cov1-1 plant transformed with pGDP7 (C), stained with Aniline Blue and viewed under UV light. In both A and C, cov1-1 plants transformed with pGDP7 are indistinguishable from the wild type.

View larger version (86K): [in a new window]   Fig. 6. Comparison of COV1 with its closest homologue in Arabidopsis (LCV1) and homologues from rice and two bacterial species (Mesorhizobium loti and Agrobacterium tumefaciens). Black boxes indicate regions in which more than half the residues are identical and grey boxes indicate conserved residues. Dashes have been introduced to optimise the alignment.

View larger version (12K): [in a new window]   Fig. 7. COV1 is expressed in all tissues and at a much higher level than LCV1 (n3). The expression levels of COV1 and LCV1 in various tissue was determined using quantitative PCR and are presented as a percentage of the actin control. Values are mean±s.e.m.

View larger version (21K): [in a new window]   Fig. 8. (A) wild-type Ler, cov1-1, axr1-3 and cov1-1:axr1-3 double mutant plants were grown vertically on agar plates containing increasing concentrations of 2,4-D; root lengths were measured after 7 days (n12). (B) Auxin levels were measured in various regions of the inflorescence stems of wild-type Ler and cov1-1 plants (n3). The only regions where there were significant differences were in the base of the stems and in the hypocotyl.