Highly diversified molecular evolution of downstream transcription start sites in rice and Arabidopsis.

Alternative usage of transcription start sites (TSSs) is one of the key mechanisms to generate gene variation in eukaryotes. Here, we show diversified molecular evolution of TSSs in remotely related flowering plants, rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), by comprehensive analyses of large collections of full-length cDNAs and genome sequences. We determined 45,917 representative TSSs within 23,445 loci of rice and 35,313 TSSs within 16,964 loci of Arabidopsis, about two TSSs per locus in either species. The nucleotide features around TSSs displayed distinct patterns when the most upstream TSSs were compared with downstream TSSs. We found that CG-skew and AT-skew were clearly different between upstream and downstream TSSs, and that this difference was commonly observed in rice and Arabidopsis. Relative entropy analysis revealed that the most upstream TSSs had retained canonical cis elements, whereas downstream TSSs showed atypical nucleotide features. Expression patterns were distinguishable between upstream and downstream TSSs. These results indicate that plant TSSs were generally diversified in downstream regions, resulting in the development of new gene expression patterns. Furthermore, our comparative analysis of TSS variation between the species showed a positive correlation between TSS number and gene conservation. Rice and Arabidopsis might have evolved novel TSSs in an independent manner, which led to diversification of these two species.