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.

Temperature is one of the most critical environmental factors affecting survival, and thus species that inhabit different thermal niches have evolved thermal sensitivities suitable for their respective habitats. During the process of shifting thermal niches, various types of genes expressed in diverse tissues, including those of the peripheral to central nervous systems, are potentially involved in the evolutionary changes in thermosensation. To elucidate the molecular mechanisms behind the evolution of thermosensation, thermal responses were compared between two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) adapted to different thermal environments. X. laevis was much more sensitive to heat stimulation than X. tropicalis at the behavioral and neural levels. The activity and sensitivity of the heat-sensing TRPA1 channel were higher in X. laevis compared with those of X. tropicalis. The thermal responses of another heat-sensing channel, TRPV1, also differed between the two Xenopus species. The species differences in Xenopus TRPV1 heat responses were largely determined by three amino acid substitutions located in the first three ankyrin repeat domains, known to be involved in the regulation of rat TRPV1 activity. In addition, Xenopus TRPV1 exhibited drastic species differences in sensitivity to capsaicin, contained in chili peppers, between the two Xenopus species. Another single amino acid substitution within Xenopus TRPV1 is responsible for this species difference, which likely alters the neural and behavioral responses to capsaicin. These combined subtle amino acid substitutions in peripheral thermal sensors potentially serve as a driving force for the evolution of thermal and chemical sensation.Animals have evolved sophisticated physiological systems for sensing ambient temperatures, as fluctuations in environmental temperature significantly affect various biological processes. Species adapted to different thermal niches are likely to have acquired specific thermal sensitivities suitable for their respective habitats. Recent progress and understanding of the molecular mechanisms behind thermosensation has enabled us to elucidate the molecular basis for the evolution of thermosensation in the process of shifting thermal niches (1-3).In vertebrates, peripheral sensory neurons such as dorsal root ganglion (DRG) 3 and trigeminal ganglion neurons relay temperature information to the central nervous system. During the initiation of signal transduction, thermal stimuli are transduced into electrical signals and a subset of ion channels that are thermally activated plays crucial roles in this process. These ion channels belong to the transient receptor potential (TRP) ion channel superfamily and are called "thermoTRP" (1-3). Animals possess several kinds of thermoTRP channels, which have distinctive temperature ranges for activation. For example, TRPV1 is activated by heat (4 -7), whereas TRPM8 is activated by cold (1-3, 8).Temperature sensitivity among orthologous thermoTRP channels has changed duri...

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Temperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

Ambient temperature fluctuations are detected via the thermosensory system which allows animals to seek preferable thermal conditions or escape from harmful temperatures. Evolutionary changes in thermal perception have thus potentially played crucial roles in niche selection. The genus Xenopus (clawed frog) is suitable for investigating the relationship between thermal perception and niche selection due to their diverse latitudinal and altitudinal distributions. Here we performed comparative analyses of the neuronal heat sensors TRPV1 and TRPA1 among closely related Xenopus species (X. borealis, X. muelleri, X. laevis, and X. tropicalis) to elucidate their functional evolution and to assess whether their functional differences correlate with thermal niche selection among the species. Comparison of TRPV1 among four extant Xenopus species and reconstruction of the ancestral TRPV1 revealed that TRPV1 responses to repeated heat stimulation were specifically altered in the lineage leading to X. tropicalis which inhabits warmer niches. Moreover, the thermal sensitivity of TRPA1 was lower in X. tropicalis than the other species, although the thermal sensitivity of TRPV1 and TRPA1 was not always lower in species that inhabit warmer niches than the species inhabit cooler niches. However, a clear correlation was found in species differences in TRPA1 activity. Heat‐evoked activity of TRPA1 in X. borealis and X. laevis, which are adapted to cooler niches, was significantly higher than in X. tropicalis and X. muelleri which are adapted to warmer niches. These findings suggest that the functional properties of heat sensors changed during Xenopus evolution, potentially altering the preferred temperature ranges among species.

Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non-selective cation-permeable channel that has six transmembrane domains and cytoplasmic N- and C-termini. The N-terminus is characterized by an unusually large number of ankyrin repeats. Although the 3-dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature-dependent TRPA1 activation is unclear. We previously reported that extracellular Ca , but not intracellular Ca , plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca -dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat-activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca , rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat-evoked TRPA1 activation in the presence of extracellular Ca . These results suggest that neutralization of acidic amino acids by extracellular Ca is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation.