Summary• LHY/CCA1 genes play a key role in the plant circadian clock system and are highly conserved among plant species. However, the evolutionary process of the LHY/ CCA1 gene family remains unclear in angiosperms. To obtain details of the phylogeny of these genes, this study characterized LHY/CCA1 genes in a model woody plant, Populus tree.• The evolutionary process of angiosperm LHY/CCA1 genes was elucidated using three approaches: comparison of exon-intron structures, reconstruction of phylogenetic trees and examination of syntenic relationships. In addition, the molecular evolutionary rates and the expression patterns of Populus LHYs were analyzed.• Gene duplication events of Populus LHYs and Arabidopsis LHY/CCA1 had occurred independently by different chromosomal duplication events arising in each evolutionary lineage. Populus LHYs were under purifying selection by estimating substitution rates of these genes. Further, Populus LHYs conserved diurnal expressions in leaves and stems but the transcripts of LHY2 were more abundant than those of LHY1 in Populus plants.• This study uncovered phylogenetic relationships of the LHY/CCA1 gene family in angiosperms. In addition, the transcript abundance and the evolutionary differences between Populus LHY1 and LHY2 imply that Populus LHY2, rather than LHY1, may have a major role in the Populus clock system.

BackgroundPlant circadian clocks regulate many photoperiodic and diurnal responses that are conserved among plant species. The plant circadian clock system has been uncovered in the model plant, Arabidopsis thaliana, using genetics and systems biology approaches. However, it is still not clear how the clock system had been organized in the evolutionary history of plants. We recently revealed the molecular phylogeny of LHY/CCA1 genes, one of the essential components of the clock system. The aims of this study are to reconstruct the phylogenetic relationships of angiosperm clock-associated PRR genes, the partner of the LHY/CCA1 genes, and to clarify the evolutionary history of the plant clock system in angiosperm lineages.ResultsIn the present study, to investigate the molecular phylogeny of PRR genes, we performed two approaches: reconstruction of phylogenetic trees and examination of syntenic relationships. Phylogenetic analyses revealed that PRR genes had diverged into three clades prior to the speciation of monocots and eudicots. Furthermore, copy numbers of PRR genes have been independently increased in monocots and eudicots as a result of ancient chromosomal duplication events.ConclusionsBased on the molecular phylogenies of both PRR genes and LHY/CCA1 genes, we inferred the evolutionary process of the plant clock system in angiosperms. This scenario provides evolutionary information that a common ancestor of monocots and eudicots had retained the basic components required for reconstructing a clock system and that the plant circadian clock may have become a more elaborate mechanism after the speciation of monocots and eudicots because of the gene expansion that resulted from polyploidy events.

Nociceptive receptors enable animals to sense tissue-damaging stimuli, thus playing crucial roles in survival. Due to evolutionary diversification, responses of nociceptive receptors to specific stimuli can vary among species. Multispecies functional comparisons of nociceptive receptors help elucidate their evolutionary process and molecular basis for activation. The transient receptor potential ankyrin 1 (TRPA1) ion channel serves as a nociceptive receptor for chemical and thermal stimuli that is heat-activated in reptiles and frogs while potentially cold-activated in rodents. Here, we characterized channel properties of avian TRPA1 in chicken. Chicken TRPA1 was activated by noxious chemicals that also activate TRPA1 in other vertebrates. Regarding thermal sensitivity, chicken TRPA1 was activated by heat stimulation, but not cold, thus thermal sensitivity of avian TRPA1 does not coincide with rodent TRPA1, although both are homeotherms. Furthermore, in chicken sensory neurons, TRPA1 was highly coexpressed with TRPV1, another nociceptive heat and chemical receptor, similar to mammals and frogs. These results suggest that TRPA1 acted as a noxious chemical and heat receptor, and was coexpressed with TRPV1 in the ancestral terrestrial vertebrate. The acquisition of TRPV1 as a novel heat receptor in the ancestral terrestrial vertebrate is likely to have affected the functional evolution of TRPA1 regarding thermal sensitivity and led to the diversification among diverse vertebrate species. Additionally, we found for the first time that chicken TRPA1 is activated by methyl anthranilate (MA) and its structurally related chemicals used as nonlethal bird repellents. MA-induced responses were abolished by a TRPA1 antagonist in somatosensory neurons, indicating that TRPA1 acts as a MA receptor in chicken. Furthermore, TRPA1 responses to MA varied among five diverse vertebrate species. Utilizing species diversity and mutagenesis experiments, three amino acids were identified as critical residues for MA-induced activation of chicken TRPA1.