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OsRab11 is Required for Resistance to Salt Stress in Rice

Jae Bok Heo

Department of Molecular Genetic Engineering, Dong-A University, Busan 604-714, Korea

Abstract

Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice

Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane (PM) and/or

vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression

levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment.

Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1,

whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na /H antiporter genes
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such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant

plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-

OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11(S28N) expression. These results indicate

that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.

Results

Fig. 1 Dominant negative OsRab11(S28N) mutant showed Fig. 2 Overexpressed-DN(S28N) inhibits the trafficking of PM Fig. 3 Trafficking of PM and vacuolar

hypersensitive phenotypes under high salt conditions. Na /H antiporter AtSOS1-GFP in Arabidopsis protoplasts. Na /H antiporters are impaired in OE-
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a Relative expression level of OsRab11under several a RT-PCR analyses of antiporter genes under high salt DN(S28N) transgenic plants.

stress conditions by real-time RT-PCR and western blot. conditions in WT and DN(S28N) plants. a-d Protoplasts isolated from WT were

Actin1 gene and rice α-tubulin protein were used as the b Complementation test of sos1-1 mutant plants. transformed with AtSOS1-GFP (a) or

internal controls, respectively. c Targeting of AtSOS1-GFP in protoplasts of complemented AtNHX2-GFP (c). VM indicates vacuole

b Expression of OsRab11 under 100 mM NaCl at different transgenic plants. Bar = 10 µm. membrane (tonoplast). Protoplasts

time points by real-time RT-PCR and western blot. d Targeting of AtSOS1-GFP in protoplasts co-expressing T7- expressing dominant negative

c WT, dominant negative OsRab11(S28N) (7-1) and sos1-1 DN(S28N). Bar = 10 µm. OsRab11(S28N) were transformed with

were grown on 1/2 MS medium for 14 days, and then e Trafficking efficiency of AtSOS1-GFP to the PM. The whole AtSOS1-GFP (b) or AtNHX2-GFP (d).

transferred to 1/2 MS Medium without NaCl or with NaCl population of transformed protoplasts was divided into two Bar = 10 µm.

(100 mM). groups based on GFP signal localization : protoplasts with e-f After incubating the cells for 36h, the

d Root elongation test at 100 mM NaCl in WT and the PM staining and cytosol staining pattern. A minimum of targeting efficiencies of the AtSOS1-GFP

overexpressed-OsRab11(6-1) plants. 100 transformed protoplasts in each group was counted. (e) or AtNHX2-GFP (f) were calculated as

e Comparison of root elongation at different NaCl Data are mean of three technical replicates. described in Fig. 2e.

concentrations among WT, DN(S28N) and sos1-1 plants. f Western blot analysis using monoclonal anti-T7 antibody .

f Comparison of root elongation at 100 mM NaCl in WT for confirming expression of T7-DN(S28N) and T7-OsRab11

and overexpressed-OsRab11(6-1) plants. proteins in protoplasts : lane 1 is from Fig. 2c and lane 2 is

from Fig.2d (upper) and Fig. 2e (lower).

Summary

1. OsRab11 is involved in the responses to several environmental stresses

2. Dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype under high salt conditions.

3. Dominant negative OsRab11(S28N) inhibits trafficking of PM and/or vacuolar antiporter proteins.

Taken together, OsRab11 plays a role in the trafficking of vacuolar and PM Na+/H+ antiporter proteins in plants.