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Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis

¹ Zentrum für Molekularbiologie der Pflanzen, Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany
² Zentrum für Molekularbiologie der Pflanzen, Mikroskopie, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany
^* Author for correspondence (e-mail: gerd.juergens{at}uni-tuebingen.de )

View larger version (27K): [in a new window]   Fig. 1. Constructs for KNOLLE expression in transgenic plants. The constructs were cloned into the multiple cloning site of the pBar transformation vectors indicated (see Materials and Methods). Numbers indicate distances (in bp) from ATG (+1) of the KNOLLE gene. The arrow above the gene indicates the orientation of transcription. KNRescue (top), genomic DNA fragment containing the KNOLLE gene and fragments of the adjacent genes. Thiored., gene encoding thioredoxin-1-like protein (AF144387); VI, hypothetical gene possibly encoding a protein similar to violaxanthin de-epoxidase (CAB59211.1); AP3::KN1 (middle), AP3 promoter (pAP3) fused to a genomic DNA fragment with KNOLLE intron; p35S::KN (bottom), CaMV 35S promoter (p35S) of the pBAR-35S vector fused to a genomic DNA fragment without KNOLLE intron. Restriction sites: E, EcoRI; P, PstI; Sc, SacI; Sn, SnaI; X, XbaI. polyA, poly-adenylation site; L, left border of the T-DNA; R, right border of the T-DNA.

View larger version (30K): [in a new window]   Fig. 2. Protein blots of extracts from KNOLLE transgenic plants. Protein extracts were separated by SDS-PAGE and analysed by immunoblotting with anti-KN antiserum (see Materials and Methods). (A) Misexpression of KNOLLE from the flower-specific AP3 promoter. Lanes 1-4: total protein extracts of wild-type (wt) flowers (lane 1) and AP3::KN1 transgenic petals (lanes 2-4). Lanes 5-7: cell fractionation of pooled extracts (lanes 2-4). (B) Organ distribution of KNOLLE protein misexpressed from the CaMV 35S promoter. 35S, T3 transgenic plants homozygous for 35S::KN; wt, wild-type control. (C) Quantitative analysis of KNOLLE expression in 20 days old seedlings grown on different media. Lanes 1-3: 0.5x MS salts with 0%, 1%, 3% sucrose. Lanes 4-6: 1x MS salts with 0%, 1%, 3% sucrose. Upper panel: similar amounts of total protein were loaded. Lower panel: 35S::KN samples 1 and 3 were diluted 1:100 and 1:500; wild-type (wt) samples 2, 3 and 5 were not diluted. (D) Membrane integration properties of KNOLLE protein from T3 generation plants homozygous for the 35S::KN transgene. Protein extracts from leaves of 35S::KN plants (35S, top) or from flowers and siliques of wild-type control (wt, middle) were differentially centrifuged. The P100 fraction (lane 3) was resuspended with or without Triton X-100 followed by a second 100,000 g centrifugation (lanes 4-7). Plants heterozygous for the knolle mutant allele UU1319 (bottom) express normal KN protein (arrow) and a truncated KN protein without the membrane anchor that was not pelleted by 100,000 g centrifugation (arrowhead). Arrow: position of the 34 kDa KN protein; S10, supernatant of 10,000 g centrifugation; S100, supernatant; P100, pellet of 100,000 g centrifugation.

View larger version (60K): [in a new window]   Fig. 3. KNOLLE protein immunolocalisation in 35S::KN seedling roots. Seedlings were grown on 1% agar with 0.5x MS salts and 1% sucose. Dark-field images of wild-type root tip (B) and root differentiation zone (E) are shown as reference for the epifluorescence images of roots homozygous for the 35S::KN transgene (A,D,G) and of wild-type control roots (C,F,H). Broken lines delineate root tips (A,C) and root hairs and lateral surface of root (D,F). (A,C) Root tip with KN-labelled cell plates (arrowheads). KN is also expressed in the non-proliferating cells of the central cylinder in the transgenic root (A; see also D) but not in the wild-type root (C). (D,G,F,H) Differentiation zone of the root. KN is expressed in the central cylinder (cr) and in root hair tips (arrowheads) of transgenic roots (D,G) but not in wild-type control roots (F,H). (D,F) Brightness of images increased; (F) maximum brightness to visualise the wild-type root in the absence of the KN signal. (G,H) Original images; (H) wild-type root is not visible. (I-L) GUS staining (2 hours, blue colour) to visualise 35S promoter activity in 6-day-old 35S::GUS transgenic seedlings. (I) Seedling with high GUS activity in root tip, root hairs and tips of the cotyledons. Boxed areas are magnified in J (young root hair), K (central part of the root) and L (root tip). Scale bar: 50 µm.

View larger version (97K): [in a new window]   Fig. 4. KNOLLE protein immunolocalisation in developing root hairs of 35S::KN seedlings. Seedlings were grown on 1% agar with 0.5x MS salts and 1% sucrose. (A) Dark-field, (B) epifluorescence microscopy, (C-Q) optical sections, confocal laser-scanning microscopy; n, nucleus; pm, plasma membrane. (A-M,O-Q) 35S::KN, (N) wild-type control. (B,C,E-G,N) Anti-KN antiserum (Cy3, red) and DAPI (blue) staining. (D,H-M) Double labelling with anti-KN antiserum (Alexa-m488, green) and anti-PM-ATPase monoclonal antibody (Cy3, red) and DAPI (blue) staining. (O-Q) Membrane labelling of live seedling roots with lipophilic dye FM4-64. The surface of root hairs and the root is delineated by broken lines in (E-G,K-O,Q). (A-D) Overviews. (A) Seedling root with root hairs of increasing age. (B-D) KN accumulation in root hairs: (B) young root hair tip (boxed) of similar age as those in (E,H-J); (C) old root hairs; boxed area is enlarged in G and similar region is shown in Q; (D) old root hairs; boxed area is shown in (K-M). Surface of root hairs is delineated by white lines in (C,D). (E-G) KN accumulation in young (E), mid-age (F) and old (G) transgenic root hairs. (H-M) Co-localisation of KN and PM-ATPase in young (H-J) and old (K-M) root hairs. (H,K) KN staining, (I,L) PM-ATPase staining, (J,M) overlays; pink colour in J is due to additional DAPI nuclear signal. Note co-localisation of the two antigens in old root hair (K-M), which is partly collapsed owing to fixation-induced plasmolysis. (N) Young root hairs of wild-type control showing no KN signal; compare with B,E. (O-Q) Endomembrane staining of young (O), mid-age (P) and old (Q) root hairs. Compare (O,P) with strong KN accumulation in root hair tips (E,H) filled by vesicles, ER and Golgi (see Fig. 5K,L). In old root hairs (Q), FM4-64 staining resembles the KN staining (C,G).

View larger version (142K): [in a new window]   Fig. 5. Subcellular immunolocalisation of KNOLLE protein in root cells of 35S::KN seedlings. (A-C) Confocal laser-scanning microscopy of dividing cells in wild-type root tip stained for KN (Cy3, red) and DAPI (false colour, green) to show the temporal and spatial dynamics of KN relocalisation during cytokinesis; cells are delineated by broken lines. (A) Prophase: KN patches, presumably Golgi. (B) Telophase: KN in plane of cell division and nearby patches. (C) Late phase of cytokinesis: KN-positive cell plate extends to the parental cell wall, only few patches remain. (D-L) 35S::KN transgenic root cells. (D-F) Semi-thick cryo-sections through the central cylinder of root. (D,E) Cross section: (D) KN immunofluorescence; (E) phase contrast of D; cells close to the phloem show strong signals (D) at the cell surface and inside the cells (asterisks, D; arrowheads, E) point to the xylem. (F) KN immunofluorescence of longitudinal section: the plasma membrane (arrowhead, pm) and Golgi-like intracellular structures (arrow, g) are labelled. (G-I,L) Ultrathin cryo sections of root (G-I) and root hair (L) labelled with anti-KN immunogold. Cryo sections were required because the anti-KNOLLE antiserum does not detect the integral membrane protein KNOLLE on conventional EM sections after chemical fixation (Lauber et al., 1997). Cryosections do not preserve membrane structures very well. (G) Root cell adjacent to xylem cell (overview). Gold labelling within areas delineated by dashed or dotted lines is highlighted by yellow (internal staining) or red (plasma membrane) asterisks to visualise gold label that is not readily detectable at this low magnification. g, Golgi; m, mitochondrion; pm, plasma membrane. (H) Higher magnification of upper boxed area in G rotated 90° clockwise showing KN immunogold label at the plasma membrane (pm), Golgi (g) and post-Golgi area. m, mitochondrion. (I) KN immunogold labelling of Golgi (g) and trans-Golgi (t) compartments. (J,K) Ultrathin section of chemically fixed and embedded root hair. (K) Higher magnification of boxed area in J: root hair tip filled with rough endoplasmic reticulum (er) and numerous electron-dense vesicles (v). (L) Strongly KN-labelled vesicles (v) and trans-Golgi network (t) from root hair tip. Scale bar: 20 µm in D-F; 250 nm in H,I,K,L.

View larger version (29K): [in a new window]   Fig. 6. KNOLLE protein expression in a kn-X37-2 mutant seedling carrying the 35S::KN transgene. (A) Cartoon of knolle mutant seedling. Boxed area is shown in B. (B) Root hairs stained for KNOLLE protein (green) and nuclei (DAPI, blue). Arrows indicate KNOLLE protein accumulation in root hair tips.

View larger version (175K): [in a new window]   Fig. 7. KNOLLE mRNA expression pattern in 35S::KN transgenic embryos and seedlings. In situ analysis using KN antisense probe. Embryos 8 µm, seedlings 17.5 µm sections. Unless stated otherwise, 35S::KN material is homozygous for the transgene. (A-H) embryos, (I-U) seedlings. (A) Wild-type, torpedo-stage embryo. (B) 35S::KN, torpedo-stage embryo. (C) Wild-type, bent-cotyledon stage embryo. (D) 35S::KN, bent-cotyledon stage embryo carrying only one copy of the transgene. Note slight KNOLLE mRNA overexpression in cotyledons. (E-H) 35S::KN, bent-cotyledon stage embryo. KNOLLE mRNA is overexpressed in cotyledons. (E) Overview; (F) higher magnification of area boxed in E — expression in pre-vasculature of cotyledon (arrowheads). (G) Overview; (H) higher magnification of area boxed in G — arrowheads indicate cells with stronger signals resembling the patchy mRNA pattern observed in wild type. (I) Wild-type, root with root hairs. (J) 35S::KN, root with root hair. KN mRNA is accumulated in central part of the root. (K) 35S::KN, lateral root primordium with strongly labelled cells (asterisks) and young root hair (arrowhead) show mRNA accumulation. cot, cotyledon. Scale bars: 50 µm.