QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 January 2003
doi: 10.1242/jcs.00274

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bouquin, T. Articles by Mundy, J. Search for Related Content PubMed PubMed Citation Articles by Bouquin, T. Articles by Mundy, J. Social Bookmarking                         What's this?

The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth

Institute of Molecular Biology, Department of Plant Physiology, Øster Farimagsgade 2A, 1353K Copenhagen, Denmark

View larger version (46K): [in a new window]   Fig. 1. A nonsense mutation in a katanin p60 ortholog gene (AtKSS) is responsible for the lue1 phenotype. (A) Rescue of lue1 phenotype with the C3300-AtKSS transgene. (B) Wild-type (top) and lue1 (bottom) bioimaging. (C) lue1 bioimaging. (D) lue1 C3300-AtKSS bioimaging. (B-D) Bright field image (left), LUC in vivo image (center), superimposition of bright field and LUC images (right). (E) RNA blot analysis of AtKSS mRNA accumulation in lue1, wild-type transgenic Col0 (WT), Ler and the GA-deficient ga1-1 mutant (top). GA treatment (50 µM GA3) was applied to the ga1-1 mutant for 2 or 24 hours. Ethidium bromide staining of the nitrocellulose membrane after RNA blotting (rRNA, bottom).

View larger version (54K): [in a new window]   Fig. 2. The lue1 mutant exhibits altered cell elongation in response to GA. (A,B) GA treatment promotes flowering in lue1. (A) 35-day-old lue1 mutants control or sprayed with 10 µM GA3 or GA4 every 4 days. (B) Flowering time of WT and lue1 plants control or sprayed with 10 µM GA3 every 4 days until bolting. (C) WT and lue1 leaf elongation upon GA3 treatment. Petiole and blade measurements were performed on adult plants by selecting the longest leaf of control or GA3-treated WT and lue1 plants (n minimum=30).

View larger version (30K): [in a new window]   Fig. 3. Lue1 responses to ACC application are partially compromised. (A-E) Seedlings were grown in the dark on MS plates with or without 50 µM ACC. (A) 3-day-old WT and lue1 seedlings. Hook angle (B), hypocotyl thickness (C), hypocotyl length (D) and root length (E) of WT and lue1 seedlings (n minimum=40). (B-E) , WT control; , WT+ACC; , lue1 control; , lue1+ACC. (F,G) 4-day-old seedlings grown under light conditions on low nutrient medium plates with or without 50 µM ACC. (F) Hypocotyl length. (G) Root length.

View larger version (53K): [in a new window]   Fig. 4. Lue1 exhibits disorganized CMF and CMT. (A,B) Polarizing microscopy of WT and lue1 CMF orientation in different cell types. (A) CMF orientation in single pitted vessel cells of WT and lue1 showing maximum birefringence for rotation angles relative to main growth axis of 45° and 0°, respectively. This indicates a transverse orientation of CMF compared with the main growth axis in WT, whereas the average CMF orientation in lue1 is 45°. Bars represent 5 µm. (B) Deviation from transverse orientation of CMF in WT and lue1 cells (n minimum=20). (C-F) Confocal microscopy of CMT organization in WT and lue1 cells. Bars represent 10 µm. The microtubule-decorating GFP-MAP4 reporter was introduced in lue1 by crossing and CMT organization assessed in segregating F2 seedlings. WT epidermal root (C) and stomata (E) cells. Lue1 epidermal root (D) and stomata (F) cells.

View larger version (56K): [in a new window]   Fig. 5. The AtKSS-GFP-GUS protein fusion decorates CMT. (A) Schematic representation of the CaMV35S-AtKSS-GFP-GUS reporter construct introduced into Arabidopsis. (B) Ectopic expression of the AtKSS-GFP-GUS (AtKSS-G-G) protein fusion in Arabidopsis Col0 ecotype phenocopies the lue1 phenotype. (C) Differential interference contrast (DIC) reference images of D. (D-K) Confocal microscopy of AtKSS-G-G subcellular distribution. GFP fluorescence is encoded in the green channel. Bars represent 10 µm. (D,E) Root epidermis cells close to the root tip. (F,G) Root epidermis cells distal from tip. (H) Hypocotyl. (I,J) Transition zone between root and hypocotyl. (K) Stomata.

View larger version (128K): [in a new window]   Fig. 6. AtKSS protein interactions. (A) Prey proteins isolated from the yeast two-hybrid screen. (B,C) Yeast two-hybrid interactions. Growth of strains on minimal SD media lacking tryptophan (TRP) and leucine (LEU) (left). ß-galactosidase assay of a replica of the left panels (center). Growth of yeast strains on minimal SD media lacking TRP, LEU, adenine (ADE) and histidine (HIS) (right). (B) Fusion proteins expressed from the DNA-binding (BD) and activation (AD) domains: 1 (BD: AtKSS; AD: 1.38); 2 (BD: AtKSS; AD: 1.25); 3 (BD: AtKSS; AD: 1.25B2); 4 (BD: AtKSS; AD: KSN1); 5 (BD: AtKSS; AD: 1.86); 6 and 8 (positive controls, Clontech); 7 (negative control, Clontech). (C) Protein-protein interaction assays using empty BD vectors. Yeast strains 1 to 5 had the AD as in B but carried the empty pGBDT7 BD vector. Yeast strains 6 to 8 were as in B. (D) Co-immunoprecipitation of AtKSS and prey proteins (left panel) using in vitro methionine 35S-labelled translated proteins (right panel). Proteins were incubated in the presence of either anti-HA or anti-c-Myc antibodies. Protein complexes were pulled down using protein-G-coupled Dynabeads. Polypeptides for in vitro translation were: AtKSS (p60): PGABKT7-AtKSS; KTN p80.1 (p80): clone pGADT7-1.25B2; KSN1: clone pGADT7-1.52; LAMIN C: pGBKT7-Lam (Clontech). (E) Sequence alignment of the C-terminal regions of putative katanin p80 proteins. Amino acids residues conserved in at least five sequences are in black boxes, similar residues are in gray. At-1 to At-4, Arabidopsis AAB71474, CAC08339, AAD49999 and BAB09559. Os-1 and Os-2, rice BAB63574 and BAB91860. Hs-1, human XP_048046. Mm, mouse BAB26884. Xl-1, Xenopus laevis AAC25113. Sp-1, Strongylocentrotus purpuratus AAC09329. The consensus (cons.) is presented beneath the alignment. The underlined sequence represents the polypeptide encoded by clone 1.25B2.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter