Genes with increased splicing efficiency in SAD1-OE plants are related to stress response and overexpression of SAD1 improves salt stress tolerance. (A) A two-dimensional view of the functional annotation of genes with increased splicing efficiency in SAD1-OE. The top 50 functional annotations that were ordered by the enrichment scores were selected for two-dimensional view, which indicates that genes with increased splicing efficiency were strikingly enriched (green) in the response-to-abiotic-stress pathways. (B) A heatmap was generated by mapping the genes enriched at the response-to-abiotic-stress pathways to the microarray database using Genevestigator. The heatmap indicates that genes with abnormal splicing are closely associated with stress responses and are up-regulated (red) by the indicated stresses. (C) Increased salt tolerance in seedlings overexpressing SAD1. Twelve-day-old seedlings on the regular ½ Murashige and Skoog (MS) medium were transferred to ½ MS media supplemented with the indicated concentrations of NaCl. The pictures were taken four days after the transfer. (D) Percentage of green leaves of seedlings on 200 mM NaCl media. Two-week-old seedlings grown on ½ MS media were transferred to ½ MS medium plates supplemented with 200 mM NaCl and incubated for five days before counting the number of green leaves or yellow and bleached leaves. A total 36 seedlings for each genotype were counted. Data are averages and standard deviations. Averages with different letters are statistically different (P <0.01, t-test). (E) Morphology of 28-day-old wild-type, sad1 and transgenic plants (SAD1-OE) that were subjected to four-day treatment with 400 mM NaCl solution. Also shown at the bottom are pictures of the damaged inflorescent stem and leaf seen in the wild type compared to undamaged ones in the SAD1-OE. ABA, abscisic acid; sad1, sad1 mutant; SAD1-OE, plants over-expressing wild-type SAD1 in the sad1 mutant background; WT, wild type.