Genes with abnormal splicing in sad1 are closely associated with stress response and transcriptional activation. (A) A two-dimensional view of the relationship between the genes with abnormal splicing and their functional annotations generated by the DAVID software. The top 50 functional annotations that were ordered by the enrichment scores were selected for the two-dimensional view, which indicates that genes with abnormal splicing were strikingly enriched (colored green) in the response-to-abiotic-stress category. (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 in sad1 are mostly up-regulated (colored red) by ABA, cold, drought and salt stress but less regulated by biotic stress (bacteria infection). (C) A network generated by Mapman indicates that genes with aberrant splicing in sad1 are involved in various stress response pathways, including hormone-signaling pathways, MAPK-signaling pathways and transcription regulation. (D) Validation of the intron retention in 10 stress-responsive genes by RT-PCR using the intron-flanking primers. The grey asterisks (*) denote the intron-retained splicing variants. ABA, abscisic acid; SA, salicylic acid; JA, jasmonic acid; sad1, sad1 mutant; WT, wild type; HSP, heat shock protein; MAPK, mitogen-activated protein kinase; ERF, ethylene response factor; bZIP, basic-leucine zipper; WRKY, WRKY transcription factor; DOF, DNA-binding with one finger; PR-proteins, pathogenesis-related proteins; R genes, (plant disease) resistance genes.