Fully sequencing the cassava full-length cDNA library reveals unannotated transcript structures and alternative splicing events in regions with a high density of single nucleotide variations, insertions-deletions, and heterozygous sequences.

The primary transcript structure provides critical insights into protein diversity, transcriptional modification, and functions. Cassava transcript structures are highly diverse because of alternative splicing (AS) events and high heterozygosity. To precisely determine and characterize transcript structures, fully sequencing cloned transcripts is the most reliable method. However, cassava annotations were mainly determined according to fragmentation-based sequencing analyses (e.g., EST and short-read RNA-seq). In this study, we sequenced the cassava full-length cDNA library, which included rare transcripts. We obtained 8,628 non-redundant fully sequenced transcripts and detected 615 unannotated AS events and 421 unannotated loci. The different protein sequences resulting from the unannotated AS events tended to have diverse functional domains, implying that unannotated AS contributes to the truncation of functional domains. The unannotated loci tended to be derived from orphan genes, implying that the loci may be associated with cassava-specific traits. Unexpectedly, individual cassava transcripts were more likely to have multiple AS events than Arabidopsis transcripts, suggestive of the regulated interactions between cassava splicing-related complexes. We also observed that the unannotated loci and/or AS events were commonly in regions with abundant single nucleotide variations, insertions-deletions, and heterozygous sequences. These findings reflect the utility of completely sequenced FLcDNA clones for overcoming cassava-specific annotation-related problems to elucidate transcript structures. Our work provides researchers with transcript structural details that are useful for annotating highly diverse and unique transcripts and alternative splicing events.

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds.

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.