A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

Abstract

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.

Figure 1. Response of sos6-1 plants to different salts and ion content measurement

(a) Four-day-old wild type and sos6-1 seedlings grown on germination media were transferred to media containing 0 or 50 mM NaCl and allowed to grow an additional 8 days. (b) Four-day-old wild type and sos6-1 seedlings grown on germination media were transferred to media containing 0, 50 or 100 mM NaCl and allowed to grow additional 30 days. (c) Response of sos6-1 plants to various levels of NaCl. The root growth was represented as proportion of growth compared with growth without NaCl (100%). (d) Response of sos6-1 plants to different levels of KCl. (e) Response of sos6-1 plants to various concentrations of LiCl. Error bars in (c–e) indicate standard deviation (n = 18). (f) Na⁺ content in sos6-1 plants as described (Zhu et al., 2007a). (g) K⁺ content in sos6-1 plants as described (Zhu et al., 2007a). (h) Sensitivity of sos6-1 seed germination to NaCl as described (Zhu et al., 2002). Error bars in (f–h) indicate standard deviation (n = 6). All experiments presented here and in the subsequent figures were performed at least three times and similar results were obtained.

Figure 2. sos6-1 plants are hypersensitive to general osmotic stress

(a) Growth of wild type and sos6-1 plants in the presence of 0 or 100 mM mannitol for 21 days. The seeds were first germinated on media containing 0 mM mannitol and then transferred to media containing 0 or 100 mM mannitol. (b) Quantification of growth (at the end of day 21; indicated by fresh weight) of wild type and sos6-1 in response to different levels of mannitol. (c) Seed germination and seedling development of wild-type and sos6-1 plants in media with low ψ_W imposed by PEG for 21 days. Seeds were directly germinated in agar plates with low ψ_W. (d) Quantification of growth of wild type and sos6-1 as shown in (c). Error bars in (b) and (d) indicate standard deviation (n = 60–80).

Figure 3. Content of soluble sugars and proline in sos6-1.

(a) Effect of sos6-1 mutation on soluble sugar levels. (b) Raffinose content of wild type and sos6-1 plants. (c) Sucrose content of wild type and sos6-1 plants. Error bars in (a–c) represent standard deviation (n = 16). (d) Effect of sos6-1 mutation on proline levels under low water-potential condition. Proline content was quantified as described (Verslues and Bray, 2004). Error bars represent standard deviation (n = 8).

Figure 4. Soil-grown sos6-1 plants are hypersensitive to NaCl treatment and dehydration conditions

(a) Two-week-old wild type and sos6-1 plants were treated with 0 or 150 mM NaCl and allowed to grow for additional 28 days. Plants were watered with solution containing 0 or 150 mM NaCl every 3 days. (b) Quantification of growth of wild type and sos6-1 at the end of salt treatment. Error bars represent standard deviation (n = 80–96). (c) Two-week-old wild type and sos6-1 plants grown in soil in the same pots were either well-watered or deprived of water for an additional 20 days. (d) Wild type and sos6-1 plants grown for 14 days when water was resumed. (e) Quantification of plants that were wilted when water was withdrawn (Wilted) and wilted plants that survived again when water was resumed (Survived). Error bars represent standard deviation (n = 74–90).

Figure 5. Detection of ROS and oxidative stress tolerance

(a) Detection of total ROS accumulation in seedlings with or without stress treatment. Fluorescence indicates the presence of ROS. (b) Nitroblue tetrazolium staining for superoxide radical. Blue color indicates the presence of superoxide. (c) Quantitative measurement of H₂O₂ levels in sos6-1 and wild type plants. Error bars indicate standard deviation (n = 12). (d) Tolerance of sos6-1 and wild type plants to methyl viologen as indicated by chlorophyll content. (e) Chlorophyll content in plants as shown in (d) determined as described (Lichtenthaler and Wellburn, 1983). Error bars represent standard deviation (n = 10).

Figure 6. Molecular cloning of SOS6.

(a) SOS6 was identified through map-based cloning. SOS6 is located on bacterial artificial chromosome clone F22D16. The numbers of recombinant and total chromosomes tested (in parentheses) and developed sslp markers are indicated. The structure of SOS6 and the positions of sos6-1 and sos6-2 mutations are also indicated. Positions are relative to the translation initiation codon. Closed boxes indicate the open reading frame and lines between boxes indicate introns. (b) Gene complementation of sos6-1 and tolerance of sos6–2 to NaCl and mannitol. (c) Quantification of seedling development as shown in (b). (d) Quantification of growth of wild type, sos6-1, Col-0 and sos6-2 as shown in (b). Error bars in (c) and (d) represent standard deviation (n = 90–110). (e) Tissue-specific expression of SOS6::GUS. (f) Validation of SOS6::GUS expression patterns by real-time PCR analysis (left panel) and SOS6 is not regulated by salt stress as revealed by northern blot analysis (right panel). EF1α gene was used as loading controls. Error bars indicate standard deviation (n = 8).