spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online May 8, 2009
doi: 10.1242/10.1242/dev.033605

Development 136, 1909-1918 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liljegren, S. J.
Right arrow Articles by Ecker, J. R.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Liljegren, S. J.
Right arrow Articles by Ecker, J. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein

Sarah J. Liljegren1,2,3,*, Michelle E. Leslie2, Lalitree Darnielle1, Michael W. Lewis1, Sarah M. Taylor1, Ruibai Luo4, Niko Geldner3,5, Joanne Chory3,6, Paul A. Randazzo4, Martin F. Yanofsky7 and Joseph R. Ecker3

1 Department of Biology and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
2 Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
3 Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
4 Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
5 University of Lausanne, Department of Plant Molecular Biology, UNIL-Sorge, 1015 Lausanne, Switzerland.
6 Howard Hughes Medical Institute Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
7 Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA.


Figure 1
View larger version (101K):
[in this window]
[in a new window]

  		Fig. 1. Mutations in NEV prevent organ separation. (A) Scanning electron micrograph (SEM) of a wild-type Arabidopsis flower before organ separation (stage 13). The sepals, petals, stamens and gynoecium are colored purple, green, yellow and red, respectively. A few organs have been removed for clarity. (B) SEM of a wild-type flower after organ separation (stage 17). The remaining abscission zone (az) cells of each organ are colored as in A. (C) Floral organs remain attached in nev flowers (stage 17) compared with wild type (wt). (D,E) Cauline leaves in nev plants fail to detach after senescence (E), as compared with wild type (D). (F,G) Longitudinal sections of wild-type (F) and nev (G) flowers at the time of shedding (stage 16) stained with Toluidine Blue. Adjacent petal (p az) and sepal (s az) abscission zones at the base of each flower are indicated. In wild-type flowers, the remaining abscission zone cells have expanded, which does not occur in nev flowers in the absence of organ separation. Scale bars: 100 µm.

 

Figure 2
View larger version (56K):
[in this window]
[in a new window]

  		Fig. 2. NEV encodes an ARF GAP. (A) The At5g54310 locus, showing the sites and sequence alterations of characterized nev mutations. Exons are shown as boxes, and the translated regions corresponding to the ARF-GAP domain and the rest of the corresponding protein are indicated in black and gray, respectively. Point mutations are marked by arrows, and T-DNA insertions by arrowheads. (B) Sequence alignment of the ARF-GAP domain from NEV and related proteins from plants (OsI_025582, AGD15), yeast (Age2), worm (W09D10.1) and mouse (Smap1). Amino acids conserved between NEV and other proteins are shaded, and the four cysteine residues that constitute the zinc finger are indicated with asterisks. The sites of nev missense mutations that affect two of these cysteines and a crucial arginine residue are marked by arrows above the alignment. Characterized ARF-GAP proteins in the alignment include yeast Age2 (Zhang et al., 1998Go) and mouse Smap1 (Sato et al., 1998Go). Uncharacterized proteins with closely related ARF-GAP regions include OsI_025582, AGD15 (Vernoud et al., 2003Go) and W09D10.1, predicted from Oryza sativa, Arabidopsis and Caenorhabditis elegans sequences, respectively. (C) NEV promotes GTP hydrolysis of mammalian ARF1. The ARF-GAP activity of recombinant full-length NEV protein was measured using AGAP1, a mammalian ARF-GAP, as a positive control (inset graph). The percentage of GTP bound to Arf1 that was converted to GDP is presented. The data are the summary of two experiments. (D,E) NEV regulatory regions direct broad expression of β-glucuronidase in flowers and shoot inflorescence stems (D), and in developing leaves and vascular strands (E).

 

Figure 3
View larger version (92K):
[in this window]
[in a new window]

  		Fig. 3. NEV localizes to the trans-Golgi network (TGN) and endosomes. (A) NEV antiserum recognizes a ~55 kDa protein in wild-type flower extracts. As the nev-2 allele encodes a truncated protein lacking the C-terminal recognition sequence of the NEV antiserum, no protein is detected in nev-2 flower extracts. Ponceau Red staining was used as a protein-loading control. (B-O) Immunofluorescent localization of NEV and endomembrane markers in primary root epidermal cells of wild-type (B), nev (C) and transgenic marker (D-O) plants. In M-O, the primary roots of YFP-RabA1e plants were incubated for 1 hour with 100 µM BFA before fixation and immunofluorescent staining. (B,C) NEV antiserum detects NEV protein in punctate structures in wild-type cells (B). Background fluorescence is shown for nev-2 cells (C). (D-I) NEV localization is distinct from the Golgi (D-F), and shows more precise localization with YFP-VTI12, a marker of the TGN (arrows, G-I). Occasional spots labeled by NEV (arrowheads, G-I) do not co-localize with VTI12. (J-O) NEV also co-localizes with YFP-RabA1e, a novel endosomal marker proposed to localize to the recycling endosome (arrows, J-L). BFA treatment of the primary root causes both NEV and YFP-RabA1e to localize to BFA bodies (M-O). Whereas the localization of YFP-RabA1e is confined to the core of the BFA bodies, NEV is also found at the periphery (arrows, O). Individual channels: YFP markers, green; NEV, red. Scale bars: 5 µm.

 

Figure 4
View larger version (117K):
[in this window]
[in a new window]

  		Fig. 4. Mutations in NEV alter Golgi structure and location of the TGN and cause an accumulation of paramural vesicles. Transmission electron micrographs of cells at the base of wild-type and nev sepals at the time of shedding (stage 16). (A,B) Cup-shaped and circularized multilamellar structures are predominantly found in nev cells (B) instead of the typically flat Golgi cisternae of wild-type cells (A). Whereas the TGN is frequently observed (84%, n=19) near the Golgi (0.041±0.018 µm, n=16) in wild-type cells (A), these tubular-vesicular compartments are not clearly identifiable near the circularized structures of nev cells (B). (C,D) Whereas paramural vesicles are infrequently observed between the plasma membrane and cell wall of wild-type cells (C), numerous vesicles are frequently observed in large paramural bodies in nev cells (D). (E,F) Distribution of paramural vesicles in cells at the base of wild-type (E) and nev (F) sepals. Abscission zone regions are indicated by arrowheads, analyzed cells are colored light blue, and paramural bodies with more than 30 vesicles or 10-30 vesicles are indicated by pink and blue circles, respectively. cg, circularized multilamellar structures; cw, cell wall; g, Golgi cisternae; pm, plasma membrane; pmb, paramural body; t, TGN. Scale bars: 0.5 µm in A-D; 10 µm in E,F.

 

Figure 5
View larger version (70K):
[in this window]
[in a new window]

  		Fig. 5. The fruit and stems of nev flowers also show membrane trafficking defects. (A-C,E-H) Transmission electron micrographs of cells in the fruit wall and pedicel (stem) of wild-type and nev flowers (stage 16). (A-C) Circularized multilamellar structures are enriched in nev fruit and stem cells (C,D) by comparison with the linear Golgi cisternae of wild-type cells (A,D). Rarely, Golgi cisternae with a curved appearance were observed in wild-type cells (B,D). Vesicular structures resembling the TGN of wild-type cells (A) are often observed in the vicinity or inside the multilamellar structures of nev cells (C, arrow) (73%, n=15). (D) Frequency of Golgi with a wild-type appearance (G, purple) and circularized multilamellar structures (CG, blue) per cell in sections of nev and wild-type sepal AZ regions, fruit walls and pedicels. For each type of tissue analyzed, n (cells) ≥15. Statistical differences between nev and wild-type tissues are indicated by asterisks (Fisher's exact test, P<0.0001). (E-H) Large paramural bodies are found in nev fruit (F) and stem (H) cells. Paramural vesicles and paramural bodies were also observed in wild-type fruit (E) and stem (G) cells. cg, circularized multilamellar structures; g, Golgi cisternae; pmb, paramural body; pv, paramural vesicles; t, TGN. Scale bars: 0.5 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2009