Figure 1.
Expression of AtCRT1a, CRT1b and CRT3.
Expression patterns of the AtCRT1a, CRT1b, and CRT3 genes assessed through promoter:GUS constructs. The GUS expression is indicated by blue colour. A to C. Six-day-old seedlings expressing GUS-constructs for AtCRT1a (A), CRT1b (B), and CRT3 (C). Root tip images for the different lines are represented as inserts. Arrows indicate expression in the root tips. D to F. Floral tissues from five week-old plants expressing GUS-constructs for AtCRT1a (D), CRT1b (E), and CRT3 (F). Arrows indicate GUS activity in the pollen. G to I. Two-day-old seedlings expressing GUS-constructs for AtCRT1a (G), CRT1b (H), and CRT3 (I). Arrows indicate GUS activity in the roots. J to L. Etiolated six-day-old seedlings expressing GUS-constructs for AtCRT1a (J), CRT1b (K), and CRT3 (L). Inserts indicate GUS expression in the cotyledons. M to O. Rosette leaves from four-week-old plants expressing GUS-constructs for AtCRT1a (M), CRT1b (N), and CRT3 (O). P and Q. Senescing leaves (from eight-week-old plants) expressing GUS-constructs for AtCRT1a (P), and CRT3 (Q).
Figure 2.
Stress induction of AtCRT1a, CRT1b, and CRT3.
A. Tunicamycin induction (10 µg/ml tunicamycin) of AtCRT1a, CRT1b, and CRT3 expression assessed by promoter:GUS constructs in five-day-old control seedlings and in seedlings treated with tunicamycin for 12 and 24 h, respectively. The GUS expression is indicated by blue colour, and by arrows. B. Co-expression network for AtCRT1a and CRT1b using the AraGenNet at http://aranet.mpimp-golm.mpg.de/aranet/ [44]. Brief annotations of genes are indicated in black boxes. Different colored edges indicate strength of transcriptional coordination. Green; mutual rank ≤10, Orange; mutual rank ≤20, Red; mutual rank ≤30. Low mutual rank indicates stronger co-expression relationships.
Figure 3.
Immuno-fluorescence of AtCRT1a, and CRT1b in Arabidopsis roots.
A. Western blot of microsomal membrane fractions from Arabidopsis cell suspensions using antibodies against a full-length maize CRT [33], and against AtCRT1a, CRT1b, and CRT3 peptides, respectively. A putative band for CNX recognized by the full-length maize CRT antibody is indicated by arrow. Twenty µg total protein was loaded per lane. B. Schematic model depicting potential N-linked glycosylation sites in respective Arabidopsis CRT. C to F. Root sections from 14-day-old Arabidopsis seedlings corresponding to the early elongation zone, probed with AtCRT1a, and CRT1b antibodies. C and D. Root sections probed with specific peptide antibodies for AtCRT1a (C), and AtCRT1b (D), and with pre-immune sera used as control (E and F). Scale bars = 70 µm. White arrows indicate root cap. G to J, Root sections from 14-day-old Arabidopsis seedlings corresponding to the upper elongation zone, probed with AtCRT1b antibodies (G and H), and with pre-immune sera used as control (I and J). atr; atrichoblasts, tr; trichoblasts, px; protoxylem, pp; protophloem, rc; root cap. Scale bars = 25 µm. K and L. AtCRT1a, and CRT1b expression in roots in six-day-old seedlings as assessed by GUS activity (blue colour).
Figure 4.
Subcellular localization of AtCRT1a, CRT1b, and CRT3.
Root sections of Arabidopsis incubated with AtCRT1b (A and B), and CRT1a (C) peptide antibodies. A. AtCRT1b antibody labeling is indicated by white arrowheads. Scale bar = 0.2 µm. Insert show enlarged area of immunogold labeled area. Scale bar = 50 nm. B. The AtCRT1b and callose antibody labeling are indicated by white, and black arrowheads, respectively. Scale bar = 50 nm. C. AtCRT1a and callose antibody labeling is indicated by white, and black arrowheads, respectively. Scale bar = 100 nm. PD; Plasmodesmata, CW; Cell wall. Insert show enlarged area of immunogold labeled area; white arrow heads CRT1a, and black arrow heads Callose. Scale bar = 20 nm D. Transient expression of AtCRT3 tagged with a CFP in tobacco leaves. Fluorescence is indicated in light-blue, and chloroplasts in red. Scale bar = 500 nm. Arrow indicates nuclear envelope.
Figure 5.
AtCRT3 complementation of crt−/− mouse fibroblasts.
A. Immunoblot analysis of AtCRT3 expressed in crt−/− mouse fibroblasts. The PVDF membranes were probed with anti-HA-tag antibodies. Twenty µg total protein was loaded per lane. B to E. Immuno-labeling of AtCRT3 expressed in CRT-deficient mouse cells (line AtCRT3–25) with anti-HA-tag (AtCRT3; B), anti-PDI (ER-marker; C), and DAPI (nuclear-marker; D). E. Overlay, the yellow color indicates identical localization of ER-marker, and AtCRT3. Scale bar = 25 µm. F. Comparison of amino acid sequences of AtCRT1a, CRT1b and CRT3 and human CRT1 and CRT2 (GenBank accession no. AAC49695, AAK74014, AAC49697, AAA51916, NP_659483, respectively). The black rectangles represent the amino acids residues important for CRT function. G. ER Ca2+ content measurements in different mouse fibroblast lines. Total cellular Ca2+ content was determined by incubation with 55 µCi 45Ca2+ followed by addition of thapsigargin. H. Measurements of bradykinin (BK)-induced Ca2+ release in different mouse fibroblast lines. Mouse fibroblasts were loaded with the fluorescent Ca2+ indicator Fura-2 followed by stimulation with BK. Typical traces showing cytosolic Ca2+ levels before and after addition of BK, and thapsigargin (TG). The experiments were carried out using a Ca2+-free medium. I. ΔCa2+ after addition of BK from (H). Wild-type; Mouse fibroblast containing CRT, CRT def control; CRT-deficient cell line, AtCRT3 and AtCRT3m; CRT-deficient cell lines complemented with AtCRT3 constructs. Results represent the average ± SE of three independent experiments.
Figure 6.
Cell adhesion in AtCRT1a and CRT3 expressing crt−/− mouse fibroblasts.
A. Relative value of normal, and focal contact, phenotype with regards to the total number of the cells of a 25 mm cell culture dish. B to E. Phase contrast images of various cell lines after 16 h growth. Inserts display magnified part of image. Wild-type; Mouse fibroblast containing CRT, CRT def control; CRT-deficient cell line, AtCRT1a and AtCRT3; CRT-deficient cell lines complemented with AtCRT1a and AtCRT3 constructs, respectively. Results represent the average ± SE of three independent experiments.
Figure 7.
Atcrt1b single, and Atcrt1a crt1b double mutant characterization.
A. Schematic representation of approximate localization of the T-DNA lines for Atcrt1b. B. Western blot of microsomal proteins of Atcrt1b-1 single, and Atcrt1a crt1b double mutants using maize CRT antibodies (1∶10 000; upper panel), and AtCRT1b antibodies (1∶2000; lower panel). Twenty µg total protein was loaded per lane. C to E. Images (D and E), and fresh weight measurements (C) of 14-day-old seedlings grown on control MS media (D), and MS media supplemented with 0.1 µg/ml tunicamycin (E), respectively. C. Values are indicated as percent compared to wild-type on control media (left), and to wild-type on tunicamycin supplied media (right). From left, wt, Atcrt1b single, and Atcrt1a crt1b double mutants. Scale bars = 5 mm. SE = standard error (n = 20). F and G. Image (F), and hypocotyl length measurements (G) of 6-day-old etiolated seedlings grown on MS media. From left, wt, and Atcrt1a crt1b double mutants. SE = standard error (n = 20).
Figure 8.
Complementation of the Atcrt1b single, and Atcrt1a crt1b double mutants with AtCRT1a, or CRT3.
A. Expression of AtCRT1a, and AtCRT1b in the different mutant combinations assessed by semi-quantitative RT-PCR. ACTIN was used as control. B. Western blot using the peptide antibodies against AtCRT3 to assess production of AtCRT3 in the 35S:AtCRT3 transformed mutant lines. Twenty µg total protein was loaded per lane. C to F. Assessment of complementation of Atcrt1b single and Atcrt1a crt1b double mutants by AtCRT1a or CRT3 under the control of a 35S promoter, respectively. C. Graph of fresh-weight for 14-day-old wt, Atcrt1b single, and Atcrt1a crt1b double mutant seedlings, and of mutant seedlings over-expressing AtCRT1a or CRT3, respectively, grown on MS medium. The graph depicts one out of three experimental repeats. D. Images of typical 14-day-old wt, Atcrt1b single, and Atcrt1a crt1b double mutant seedlings, and of mutant seedlings over-expressing AtCRT3 grown on MS medium. Scale bars = 5 mm. E. Expression of AtCRT1a in Atcrt1b, or in Atcrt1b transformed with AtCRT1a under a 35S promoter. ACTIN was used as control. F. Western blot using the maize CRT antibody to assess production of AtCRT1a in the 35S:AtCRT1a transformed Atcrt1a crt1b double mutant lines. Twenty µg total protein was loaded per lane. G. Images of typical wt, and of Atcrt1b mutants that were complemented with AtCRT1a under the control of a 35S promoter. Scale bars = 5 mm.
Figure 9.
PAMP-induced responses in Atcrt mutants.
A. Schematic representation of approximate localization of the T-DNA line for Atcrt3. B. Western blot of Atcrt3 using AtCRT3 peptide antibodies (1∶2000). C. Anthocyanin content in 6-day-old wt and mutant seedlings was determined after incubation for three days with, or without, 100 mM sucrose, and PAMPs as indicated on the right. D and E. PAMP-induced oxidative burst in 4-week-old wt and mutant seedlings. Leaf discs derived from 4-week-old plants were treated with PAMPs as described in Saijo et al. (accepted elsewhere). F. Western blot analysis of 4-week-old plant leaves. Microsomal membrane fractions were produced for the different mutants, and subjected to immuno-blot analysis with the indicated antibodies. efr1 and fls2 indicate seedlings mutated in EFR and FLS2, respectively. Ten µg total protein was loaded per lane.
Figure 10.
AtCRT3 co-expression relationships.
Co-expression network for AtCRT3 using the AraGenNet at http://aranet.mpimp-golm.mpg.de/aranet/ [44]. Brief annotations of genes are indicated in black boxes. Different colored edges indicate strength of transcriptional coordination. Green; mutual rank ≤10, Orange; mutual rank ≤20, Red; mutual rank ≤30. Low mutual rank indicates stronger co-expression relationships.