Figure 1.
Characteristics of SYT2 gene in Arabidopsis thaliana.
Schematic structure of Arabidopsis synaptotagmin 2 gene (SYT2, At1g20080). Solid boxes are exons, lines between the boxes are the introns. The start codon ATG and the stop codon TGA are marked. Triangle indicates the T-DNA insertion site in syt2-1 (SALK_135307) mutant. (B and C) Amino acid sequence alignment of the C2A (C) and C2B (D) domain of SYT1 and SYT2 in Arabidopsis and of mouse Syt1 and Syt2 using the multiple alignment program of Vector NTI Suite 7 (Invitrogen). Asterisks indicate the amino acids involved in calcium binding.
Figure 2.
Subcellular localization of SYT2 in Arabidopsis.
(A and B) Transient expression of SYT2-GFP in leaf epidermis cells of tobacco (A) and Arabidopsis (B) shows punctate structures in the cytoplasm. Autofluorescence of chloroplasts appears as golden structures (B). Arrows indicate the punctate structures of SYT2-GFP. Bars = 20 µM. (C–F) Expression of SYT2-GFP in stably transformed root hairs (C) and root tip cells (D–F). (E), High-magnification image of root cells in the inset in (D). Arrows indicate the punctate structures of SYT2-GFP. Bars = 20 µM.
Figure 3.
SYT2-GFP does not colocalize with FM4-64-positive compartments.
(A–I) Confocal sections of root cells labeled with FM4-64 (red) at room temperature and incubated for 30 min (A–C), 60 min (D–F) and 120 min (G–I). Arrows indicate the punctate structures of SYT2-GFP (green); arrowheads denote vacuolar membranes. Bars = 20 µM. (J–R) Root cells containing VHA-a1-GFP (J–L), ARA6-GFP (M–O) and ARA7-GFP (P–R) were labeled with FM4-64 at room temperature and Confocal sections were taken after a 30-min incubation. Arrows indicate overlapping spots of GFP (green) and FM4-64 (red) fluorescence. Arrowheads indicate that GFP fluorescence did not overlap with that of FM4-64. Bars = 20 µM.
Figure 4.
Bars = 10 µm. (A) Protein gel blot of SYT2 in wild-type (WT), syt2-1, and SYT2-GFP overexpressing plants (SYT2-GFP1 and SYT2-GFP2 are different lines). The blots were probed with polyclonal anti-SYT2 (Right) and monoclonal anti-GFP (Left) antibodies to detect the SYT2 and GFP-tagged proteins, respectively. The expected sizes of the proteins are indicated. The bottom images show Coomassie Brilliant Blue staining (CBB) as loading controls. (B) RT-PCR analysis of SYT1 and SYT2 in syt2-1 and wild-type plants. Actin served as a control. (C and D) Localization of SYT2 in root cells of wild-type plants. Root tissues of Arabidopsis grown on ½ MS solid medium for 3–4 days were prepared for immunolabeling with normal rabbit serum (as a control) (C), or anti-SYT2 antibody as the primary antibody (D) and fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG as the secondary antibody. (E) Double-labeling with anti-SYT2 and anti-GFP antibodies in root cells of SYT2-GFP-overexpressing seedlings. Anti-SYT2 and anti-GFP antibodies were labeled with tetramethylrhodamine-5-isothiocyanate (TRITC)-labeled anti-rabbit IgG and FITC-labeled anti-rat IgG, respectively. Arrows indicate the overlap of green and red fluorescent signals. (F) Double-labeling with anti-SYT2 and anti-GFP antibodies in root cells containing Golgi marker ST-YFP. Anti-SYT2 and anti-GFP antibodies were used as in (E). Arrows indicate the overlap of green and red fluorescence signals. (G–L) Immuno-gold labeling and electron microscopic observation showed that SYT2 was located on Golgi apparatus in root tip cells of Arabidopsis. (G and H) Electron microscopic observation showed that SYT2 was located mainly on Golgi apparatus in root tip cells of wild type plants. (H) High-magnification image of Golgi apparatus in the inset in (G). (I and J) Immuno-gold labeling of Golgi apparatus in root tip cells of SYT2-overexpressing plants. (J) High-magnification image of Golgi apparatus in the inset in (I). (K and L) Control section, incubated with the secondary antibody alone, did not show gold particles on Golgi apparatus. G: Golgi apparatus. Bars: 2 µm (G, I,); 50 nm (H, J, K, L).
Figure 5.
Co-expression of SYT2 and HYGR in Arabidopsis induced hypersensitivity to hygromycin B.
(A) Reverse transcriptase-PCR (RT-PCR) analysis of HYGR expression in wild type (WT), HYGR, syt2-1/HYGR and SYT2/HYGR plants. Actin was used as a control. (B) Response of wild-type (WT), HYGR, syt2-1/HYGR and SYT2/HYGR plants to hygromycin B. Seeds were germinated on ½ MS medium supplemented with indicated concentrations of hygromycin B and grew for 7 days before images were taken. SYT2/HYGR1 and SYT2/HYGR2 were different lines that were simultaneously transformed with SYT2-GFP and HYGR genes. (C) Measurement of the length of roots and shoots for seedlings treated as described for (B). Values are the means ± SD of 30–40 seedlings from three independent experiments.
Figure 6.
Subcellular localization of HYGR-GFP and secretion of HYGR in transgenic Arabidopsis.
(A) Confocal image of transgenic root cells showed that HYGR-GFP was localized on the cell surface (arrows). Bar = 20 µm. (B and C) Confocal image of transgenic leaf showed that HYGR-GFP (green) was primarily expressed in the leaf-tip zone cells (arrows). Autofluorescence of chloroplasts appear as red structures. Bar = 100 µm. (D) Confocal image showed that HYGR-GFP was localized in both cell wall (arrows) and cytoplasm (arrowheads). The root cells were plasmolysed with 0.8 M mannitol for 1 h. Bar = 20 µm. (E) Control image of non-transgenic root cells taken at the same laser intensity and exposure time as that in (A) and (D). Inset is the reduced bright field image. Bar = 30 µm. (F) Non-transgenic protoplasts (WT) and protoplasts stably expressing HYGR-GFP were incubated for 5 h at 23°C. The protoplasts lysates (P) and medium (M) proteins were subjected to protein gel blot with anti-HYGR, anti-GFP and anti-tubulin antibody, respectively. (G) The response of HYGR secretion to BFA treatment. Protoplasts stably expressing HYGR-GFP were incubated for 5 h with (+) BFA or without (−) BFA at 23°C. The protoplasts lysates and medium proteins were separated by SDS-PAGE and immunoblotted with anti-HYGRand anti-tubulin antibody, respectively. (H) The effectiveness of bredeldin A (BFA) was demonstrated by the variation of acid phosphatase (AcPase) activities. Protoplasts were incubated in the absence (▵, ▴) or presence (□, ▪) of BFA. At the indicated periods during incubation, the protoplast was separated from the medium by centrifugation. AcPase activities in the medium (▴, ▪) and protoplasts fractions (▵, □) were determined. Note that the partial inhibition of the activities of AcPase after BFA treatment.
Figure 7.
Accumulation of HYGR in syt2-1 plants.
(A–C) Expression of HYGR-GFP in syt2-1 caused the fluorescent accumulation in cytoplasm. In magnified pictures punctate structures were found (B and C; Bars = 100 µm). Bar = 20 µm (A). (D–F) Confocal image showed that HYGR-GFP was brightly accumulated in punctate structures in cytoplasm (arrowheads) and in vacuoles. The cells were plasmolysed with 0.8 M mannitol for 1 h. V: vacuole. Bars = 20 µm. (G) Protein gel blot of HYGR-GFP in wild-type (WT), syt2-1, and HYGR-GFP plants. The blots were probed with anti-GFP (upper) and anti-tubulin (lower) antibodies to detect GFP-tagged proteins and tubulin, respectively. The positions of molecular weight markers are indicated. (H–K) Immuno-gold labeling and electron microscopic observation showed that HYGR was detected on the cell wall in root tip cells of HYGR-GFP-expressing Arabidopsis plants. (H and I) Concentrated gold particles near/on the cell wall. (J and K) The distribution of gold particles on the cell wall. (K) The magnification image of the inset in (J). CW: Cell wall. Bars: 500 nm (H, J), 100 nm (I), 200 nm (K). (L–O) Immuno-gold labeling of HYGR in the root tip cells of syt2-1 plants expressing HYGR-GFP. (L–N) HYGR was detected in/on vacuoles. (O) No signals were found in the cell wall. CW: Cell wall. V: Vacuole. Bars: 500 nm (L), 200 nm (M, O), 100 nm (N). (P and Q) Immuno-gold labeling using anti-HYGR antibody in the root tip cells of HYGR-GFP-expressing plants (P) and non-transgenic plants (Q). CW: Cell wall. V: Vacuole. Bars: 200 nm (P), 100 nm (Q).
Figure 8.
Response of SYT1-overexpressing plants to hygromycin B.
(A) Reverse transcriptase-PCR analysis of SYT1 transcripts in wild-type (WT) and syt2-1 plants after hygromycin B treatments for 0, 3 and 15 h. Actin was used as a control. (B) Growth of wild-type (WT), HYGR, syt1-2/HYGR and SYT1/HYGR plants under hygromycin B treatments. Seeds were germinated on ½ MS medium supplemented with indicated concentrations of hygromycin B and grew for 7 days before images were taken. SYT1/HYGR1 and SYT1/HYGR2 were different lines that were simultaneously transformed with SYT1 and HYGR genes. (C) Measurement of length of roots and shoots for seedlings treated as described for (B). Values are the means ± SD of 30–40 seedlings from three independent experiments.
Figure 9.
Hypothetical model summarizing function of SYT2 in trafficking of the unconventional proteins (UPs) in Arabidopsis.
(1) UPs were synthesized on the free ribosomes in the cytoplasm and then secreted into the cell wall (CW). Golgi apparatus-localized SYT2 is involved in their secretion. (2) When SYT2 gene gets knocked out, a proportion of the UPs trafficks through prevavuole compartment (PVC) en route to the vacuoles. SYT1 is probably involved in the redirectional trafficking of UPs in the syt2-1 mutant. (?) indicates the speculated role of SYT1 in the syt2-1 plants.