Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance

Abstract

Intracellular vesicle trafficking performs essential functions in eukaryotic cells, such as membrane trafficking and delivery of molecules to their destinations. A major endocytotic route in plants is vesicle trafficking to the vacuole that plays an important role in plant salt tolerance. The final step in this pathway is mediated by the AtVAMP7C family of vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) that carry out the vesicle fusion with the tonoplast. Exposure to high-salt conditions causes immediate ionic and osmotic stresses, followed by production of reactive oxygen species. Here, we show that the reactive oxygen species are produced intracellularly, in endosomes that were targeted to the central vacuole. Suppression of the AtVAMP7C genes expression by antisense AtVAMP711 gene or in mutants of this family inhibited fusion of H2O2-containing vesicles with the tonoplast, which resulted in formation of H2O2-containing megavesicles that remained in the cytoplasm. The antisense and mutant plants exhibited improved vacuolar functions, such as maintenance of DeltapH, reduced release of calcium from the vacuole, and greatly improved plant salt tolerance. The antisense plants exhibited increased calcium-dependent protein kinase activity upon salt stress. Improved vacuolar ATPase activity during oxidative stress also was observed in a yeast system, in a DeltaVamp7 knockout strain. Interestingly, a microarray-based analysis of the AtVAMP7C genes showed a strong down-regulation of most genes in wild-type roots during salt stress, suggesting an evolutionary molecular adaptation of the vacuolar trafficking.

Fig. 1.

Expression of AtVAMP7C gene and its effect on Arabidopsis salt tolerance. (A Lower) Semiquantitative RT-PCR analysis of the AtVAMP711 expression in wild-type (wt) and transgenic Arabidopsis lines transformed with sense (lines 5 and 7) and antisense (2091 and 2095) constructs. Total RNA was prepared from 4-week-old plants. The actin2 gene served as RNA loading control. (A Upper) The AtVAMP711 gene and protein map, indicating the synaptobrevin domain (red bar) and the exons, is shown. (B) Salt tolerance of the AtVAMP711 transgenic lines. Ten-day-old seedlings were transferred from 1/2 MS plates to 1/2 MS medium supplemented with 200 mM NaCl. Pictures were taken 6 days after transfer. Similar results also were obtained with other antisense transgenic lines (2092 and 2096). (C) Graphical display of the Arabidopsis AtVAMP7c gene family during abiotic stresses. The data are from microarrays analysis from AtGenExpress-Salt Stress (roots of seedlings grown on agar plates). The experimental details can be accessed at the Botany Array Resource database (ref. ; http://bbc.botany.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi?pub=&dataset=atgenexp_stress).

Fig. 2.

Intracellular production of ROS in Arabidopsis seedlings during salt stress. (A) Visualization of NaCl-induced ROS production within a single cell along Z stack. Wild-type Arabidopsis seedlings were grown on agar plates in 1/2 MS medium. Ten-day-old seedlings were exposed to 200 mM NaCl solution for 10 min, and the production of ROS was assayed by confocal microscopy with a ROS-sensitive probe H₂DCFDA. Shown are four sections (0.5-μm-thick) along Z stack of a whole single representative cell from the elongation zone. Shown are layers from cell surface, cytosol, vacuole, and cytosol (image depth is indicated on the left). The bright-field (BF) image of the cell is shown at the bottom. (B–E) Simultaneous staining of intracellular membranes (FM 4-64, red) and ROS (H₂DCFDA, green). Wild-type (B and C) and AtVAMP711 antisense (line 2091) plants (D and E) were treated as in A. Pictures were taken 5 min after the salt treatment. (Scale bars of B and C also apply to D and E, respectively.). (F) Magnified view of megavesicles in antisense 2091 line double-stained with FM 4-64 and H₂DCFDA as described in B–E. (G and H) Single-membrane dye staining with FM 1-43 in seedlings of wild type (G) and antisense 2091 line (H) were treated as in A and stained for 10 min then analyzed by confocal microscopy. Similar results were obtained with FM 4-64 staining.

Fig. 3.

Vacuolar functioning of wild-type and ΔVamp7 yeast treated with H₂O₂. (A) Saccharomyces cerevisiae BY4741 (wt) and BY4741ΔVamp7 (mutant) strains were stained in 10 μM FM 4-64 for 20 min, washed twice, and analyzed by confocal microscopy as described in Materials and Methods. Bright-field images are presented. Arrows indicate central vacuoles. (B) Formation of pH gradients across the tonoplast of isolated vacuole by V-ATPase activity. Wild-type (BY4741) Saccharomyces cerevisiae and ΔVamp7 mutant strains (in the same background) were treated with 9 mM H₂O₂ or water (control), and acidification of the vacuole (fluorescence quenching, Q) was determined as described in Materials and Methods. The collapse of the ΔpH across the tonoplast membrane was done by addition of 50 mM K⁺ and 5 μM nigericin (white arrows). (C) Vacuoles were isolated from wild-type yeast and treated with 80 μM H₂O₂ for 15 min or with water (control). Acidification of the vacuole (fluorescence quenching) was measured as described above. Where indicated, DTT was added immediately after treatment with H₂O₂.

Fig. 4.

Changes in vacuolar pH during salt stress in Arabidopsis root cells. (A) The intracellular pH in wild-type seedlings was analyzed by using the carboxy-SNARF probe, which shifts its emission from green to red when alkalinized (24). Confocal microscope images were taken in the root-hair zone cells after 0, 3, and 18 h from exposure to 0.2 M NaCl (see Table 1). Merged images of simultaneous emission of green and red filters are given (570 nm and 655 nm, respectively). (Insets) Inserted graphs indicate relative fluorescence (y axis) of each filter along the gray longitudinal line of the cells (x axis) shown and quantified by ImagePro program. (B) Percentage of alkalized vacuoles in wild-type and AtVAMP711C antisense (2091) lines during salt stress. Shown is a representative triplicate experiment, n = 10, ± SE. (Insets) Representative green/red merged images of wild-type and antisense seedlings after 18 h from the beginning of stress.

Fig. 5.

Dynamics of vacuolar Ca²⁺ in root cells during salt stress. (A) Confocal microscope images of intracellular Ca²⁺ fluorescence by using Fluo-4-AM dye in the root-hair zone of wild-type cells after 0, 1, and 20 h after treatment with 0.2 M NaCl. A bright-field image is attached to each fluorescent image at the right. Arrows indicate nuclei. Dye loading is described in Materials and Methods. (B) Quantification of the translocation of vacuolar Ca²⁺ into the cytosol 10 h after beginning of stress in wild-type (wt) and AtVAMP711 antisense (2091) seedlings. Confocal Z sections (1-μm-thick) of each vacuole were projected to quantify the whole-vacuolar Fluo-4-AM intensity. (Left) Z sections, projections, and fluorescence measurements were done with ImagePro (n = 25 ± SE). Black bars are from salt-treated seedlings, and white bars are control. (Right) Ca²⁺ staining in representative vacuoles at 10 h. Note the vacuolar shrinkage. (C) The CDPK activity was assayed by in-gel kinase method in roots of 3-week-old wild-type and AtVAMP711 antisense (2091) seedlings. Plants were treated with 0.2 M NaCl for 3 h. Kinase activity was assayed in protein extracts in the presence of 1 nM CaCl₂ or 2.5 mM EGTA as described in ref. . Numbers indicate mass in kDa. (D) Semiquantitative RT-PCR analysis of AtCDPK1 (At1g18890) and AtCDPK2 (At1g35670) expression during salt stress (200 mM NaCl for 2 h) in roots of wild-type (wt) and AtVAMP711 antisense (2091 and 2095) lines. Total RNA was prepared from 21-day-old roots of 20 plants in each treatment. The actin2 gene served as loading control.