Volume 12, Issue 17, Pages (September 2002)

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Volume 12, Issue 17, Pages 1519-1523 (September 2002) Functional Analysis of the Tubulin-Folding Cofactor C in Arabidopsis thaliana Victor Kirik, Jaideep Mathur, Paul E. Grini, Irene Klinkhammer, Klaus Adler, Nicole Bechtold, Michel Herzog, Jean-Marc Bonneville, Martin Hülskamp Current Biology Volume 12, Issue 17, Pages 1519-1523 (September 2002) DOI: 10.1016/S0960-9822(02)01109-0

Figure 1 Molecular Characterization of the POR Gene (A) A schematic presentation of the POR gene and the T-DNA insertion. (B) Expression analysis by RT-PCR in different organs. L, leaf; R, root; F, flower; S, stem. The expression level of elongation factor 1 (EF1) was used as a control to standardize the RNA concentrations. (C) The amino acid sequence comparison of POR (AtTFC-C) and the human cofactor C (hTFC-C) is shown. Identical amino acids are highlighted in dark gray, and similar amino acids are highlighted in light gray. (D) A Northern blot showing POR gene expression in wild-type (Ws) and the por-T1 mutant. Note that the POR transcript is much longer in por-T1 as compared to wild-type. 26S rRNA is shown as a loading control. The position of the RNA size marker (Promega) is indicated on the left. (E) RT-PCR analysis showing that the T-DNA-borne 35S-driven BAR coding region and the POR coding region are on the same transcript. Lane 1: por-T1 mutant cDNA, primer in the BAR gene (bar-s2) and in the POR gene (eav-as1); lane 2: wild-type cDNA, same primer as in lane 1; lane 3: por-T1 cDNA, primer in the 35S promoter (35S-k14) and in the POR gene (eav-as1); lane 4: por-T1 mutant genomic DNA, same primer as in lane 3. Current Biology 2002 12, 1519-1523DOI: (10.1016/S0960-9822(02)01109-0)

Figure 2 Cell Biological Analysis of por-T1 Mutants (A) Transgenic plants expressing GFP:POR under the control of the 35S promoter. A fluorescent micrograph of a trichome displaying ubiquitous GFP staining in the cytoplasm and the nucleus. (B) Microtubule organization in a wild-type trichome as revealed by GFP:MAP4, which decorates microtubules in vivo. Note that branches exhibit longitudinally oriented cortical microtubules (arrow). (C) A por-T1 mutant trichome in which cortical microtubules are oriented transversally in a trichome branch (arrow). (D) A light micrograph of dark-grown wild-type (right) and por-T1 seedlings (left). Note that plants were exposed to light for 2 days to allow rosette leaf formation and to enable recognition of mutant plants by their trichome phenotype. (E) An agarose impression of a hypocotyl of a dark-grown wild-type seedling. (F) An agarose impression of a hypocotyl of a dark-grown por-T1 seedling. Note the bloated cells. (G) Microtubule organization in hypocotyl cells in a dark-grown GFP:MAP4 wild-type plant. Microtubules are coaligned. (H) Microtubule organization in hypocotyl cells of a por-T1 GFP:MAP4 plant. Note that bloated cells have randomly organized microtubules (arrow), whereas less-affected cells have a more regular microtubule network. Current Biology 2002 12, 1519-1523DOI: (10.1016/S0960-9822(02)01109-0)

Figure 3 Phenotypic Analysis of the por-T1 Mutant (A) por-T1 mutant (left) and wild-type (right) plants. (B) An optical section of a wild-type embryo sac whole-mount preparation. Ec, egg cell; cc, central cell; ii, inner integumentes; v, vacuole. (C) An optical section of a por-T1 mutant embryo sac whole-mount preparation. The embryo sac is missing (arrow). (D) Scanning electron microscopy (SEM) micrographs of a wild-type trichome. (E) An SEM micrograph of mutant por-T1 trichomes. (F) A section of a wild-type leaf. (G) A section of a por-T1 mutant leaf. Note the bloated cells (asterisks). (H) Light micrographs of a por-T1 mutant epidermal cell with incomplete cell walls (arrow). (I) DAPI staining of the same cell that is shown in (H). Note that the cell contains three nuclei (arrow). (J) A light micrograph of an enlarged cell in the por-T1 mutant. (K) SYTO-25 (Molecular Probes) staining of the same cell that is shown in (J). Note that the nucleus is extremely enlarged (arrow). Current Biology 2002 12, 1519-1523DOI: (10.1016/S0960-9822(02)01109-0)