Requirement of extracellular Ca2+ binding to specific amino acids for heat-evoked activation of TRPA1.

KEY POINTS: We found that extracellular Ca2+ , but not other divalent cations (Mg2+ and Ba2+ ) or intracellular Ca2+ , is involved in heat-evoked activation of green anole (ga) TRPA1. Heat-evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca2+ . Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+ is important for heat-evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. ABSTRACT: 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 Ca2+ , but not intracellular Ca2+ , plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+ -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 Ca2+ , 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 Ca2+ . These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation.

W14/15 esterase gene haplotype can be a genetic landmark of cultivars and species of the genus Gentiana L.

We have identified multiple alleles for a single gene termed W14/15. This gene encodes closely related but not identical proteins W14 and W15 that accumulate in overwinter buds of Gentiana triflora (Takahashi et al. in Breed Sci 56:39-46, 2006; Hikage et al. in Mol Genet Genomics 278:95-104, 2007). In this study, structural analysis of the W14/15 gene was carried out for 21 different gentian lines/cultivars consisting of 5 different species, to survey species- or line/cultivar-specific haplotypes. Within the samples examined, multiple variant forms were found. Those were categorized into seven major types (type I-VII) and ten subtypes based on the presence of three short insertion/deletion sites, three RFLP sites, and several SNP sites. Each line/cultivar had a distinct set of W14/15 gene variants for an allelic pair. Phylogenetic analysis showed that the W14/15 alleles cluster into groups that are characteristic of gentian species, i.e., G. triflora, G. scabra, G. pneumonanthe, G. septemfida and an unknown species other than the former four. In addition, within the same gentian species, different sets of haplotypes were found. Thus, the W14/15 alleles provide useful landmarks to resolve phylogenies of the genus or section Gentiana, as well as to analyze pedigree and breeding history of the cultivars derived from those Gentiana sp.

Molecular phylogeny and expression of poplar circadian clock genes, LHY1 and LHY2.

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 exon­intron 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.

Structure and allele-specific expression variation of novel alpha/beta hydrolase fold proteins in gentian plants.

Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to alpha/beta hydrolase fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles (W14/W15), one carried the W15 genes in both alleles (W15/W15), and others were homozygous of W14 (W14/W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These observations were discussed in relation to winter hardiness of the gentian plants.