A genome-wide study of alternative splicing in Arabidopsis thaliana reveals that at least 42% of intron-containing genes are alternatively spliced, a significantly higher proportion than previously estimated. The study used Illumina RNA-seq to map the transcriptome at single-base resolution, identifying thousands of novel alternatively spliced mRNA isoforms. Alternative splicing events were found to be associated with specific tissue types, developmental stages, and environmental stresses. Many alternatively spliced transcripts contain premature termination codons (PTCs), which may be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway or regulated by the regulated unproductive splicing and translation (RUST) mechanism. The study also showed that abiotic stress treatments can significantly shift the relative abundance of PTC+ and reference isoforms in key regulatory genes. Intron retention was found to be a prevalent form of alternative splicing in plants, often generating PTC+ isoforms that may be regulated by NMD or RUST. The study confirmed that intron retention events are highly conserved among diverse plant species, including in essential regulatory genes like CCA1. The results suggest that NMD and RUST may be widespread in plants and play important roles in regulating gene expression. The study also identified numerous novel splice variants, including those with rare nonconsensus terminal dinucleotide splice signals. The findings highlight the importance of alternative splicing in increasing transcriptome and proteome diversity in plants and suggest that the extent of alternative splicing in plants has been significantly underestimated. The study used a combination of RNA-seq and cDNA data to identify and validate alternative splicing events, demonstrating the superior detection capability of RNA-seq over conventional cDNA/EST approaches. The results provide an unprecedented and unbiased evaluation of alternative splicing in Arabidopsis, the premier model plant. The study also showed that alternative splicing can be regulated by environmental stress, with specific stress treatments leading to dramatic shifts in the relative ratio of full-length versus PTC+ transcript variants. The findings suggest that alternative splicing plays a crucial role in plant gene expression regulation, similar to its role in animals. The study also identified several novel splice variants in SR splicing factors, including those with PTC+ isoforms that may be regulated by NMD or RUST. The results highlight the importance of alternative splicing in plant biology and suggest that further studies are needed to elucidate the detailed molecular mechanisms underlying the network of splicing factors and their transcript targets.A genome-wide study of alternative splicing in Arabidopsis thaliana reveals that at least 42% of intron-containing genes are alternatively spliced, a significantly higher proportion than previously estimated. The study used Illumina RNA-seq to map the transcriptome at single-base resolution, identifying thousands of novel alternatively spliced mRNA isoforms. Alternative splicing events were found to be associated with specific tissue types, developmental stages, and environmental stresses. Many alternatively spliced transcripts contain premature termination codons (PTCs), which may be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway or regulated by the regulated unproductive splicing and translation (RUST) mechanism. The study also showed that abiotic stress treatments can significantly shift the relative abundance of PTC+ and reference isoforms in key regulatory genes. Intron retention was found to be a prevalent form of alternative splicing in plants, often generating PTC+ isoforms that may be regulated by NMD or RUST. The study confirmed that intron retention events are highly conserved among diverse plant species, including in essential regulatory genes like CCA1. The results suggest that NMD and RUST may be widespread in plants and play important roles in regulating gene expression. The study also identified numerous novel splice variants, including those with rare nonconsensus terminal dinucleotide splice signals. The findings highlight the importance of alternative splicing in increasing transcriptome and proteome diversity in plants and suggest that the extent of alternative splicing in plants has been significantly underestimated. The study used a combination of RNA-seq and cDNA data to identify and validate alternative splicing events, demonstrating the superior detection capability of RNA-seq over conventional cDNA/EST approaches. The results provide an unprecedented and unbiased evaluation of alternative splicing in Arabidopsis, the premier model plant. The study also showed that alternative splicing can be regulated by environmental stress, with specific stress treatments leading to dramatic shifts in the relative ratio of full-length versus PTC+ transcript variants. The findings suggest that alternative splicing plays a crucial role in plant gene expression regulation, similar to its role in animals. The study also identified several novel splice variants in SR splicing factors, including those with PTC+ isoforms that may be regulated by NMD or RUST. The results highlight the importance of alternative splicing in plant biology and suggest that further studies are needed to elucidate the detailed molecular mechanisms underlying the network of splicing factors and their transcript targets.