Figure 5.

Functional analysis of Gal83 Ser-26 phosphorylation mutants. A, Gal83 confers cell viability in the presence of high NaCl. Tomato protoplasts expressing GFP-Adi3 or the indicated Gal83-GFP constructs for 18 h were treated with 200 mm NaCl, and cell viability was determined by Evans blue staining over a 5.5-h time course. Values are averages of at least three independent experiments. Error bars represent se. Data analysis was carried out using Duncan’s multiple-range test. Samples with the same letter above the bars are not significantly different (P < 0.05). Protein expression detected by α-GFP western blot is shown on the right. B, SnRK1 substrate phosphorylation with the Gal83^S26D mutant and Adi3. Kinase-active and -inactive MBP-SnRK1 proteins were tested for phosphorylation of the SAMS peptide in combination with MBP-Snf4, MBP-Gal83, and MBP-Adi3 using [γ-³²P]ATP in in vitro kinase assays. Values are shown as pmol phosphate incorporated mg⁻¹ SnRK1 protein min⁻¹ and are averages of three independent experiments. Error bars represent se. Asterisks indicate significant increases (*) or decreases (**) in SAMS phosphorylation as compared with phosphorylation by SnRK1^T175D alone (Student’s t test, P < 0.01). The SDS-PAGE gel shows proteins put into the assay. C, Expression of the data in B as a percentage of the GST sample (column 13). All other information is as in B. D, SAMS phosphorylation by protoplast extracts expressing SlGal83. The indicated SlGal83-GFP proteins were expressed in protoplasts for 16 h, and an extract was made and tested for phosphorylation of SAMS as in B. Values are averages of three independent experiments, and error bars represent se. The asterisk indicates a significant decrease in SAMS phosphorylation as compared with the SlGal83 sample (Student’s t test, P < 0.01).