The CLC proteins in Arabidopsis have been studied in

The CLC proteins in Arabidopsis have been studied in some detail, and a total of seven genes (named AtCLCa-AtCLCg) have been identified (Lv et al., 2009). It is interesting that AtCLCa-AtCLCd and AtCLCg define a distinct phylogenetic branch that is closely related to eukaryotic CLCs, while the other two members of the AtCLC family, AtCLCe and AtCLCf, are related to prokaryotic CLCs (Marmagne et al., 2007). Further, AtCLC proteins are localized to various cellular compartments, including vacuolar membranes (AtCLCa-AtCLCc and AtCLCg), Golgi vesicles (AtCLCd and AtCLCf), and thylakoid membranes (AtCLCe) (Lv et al., 2009). AtCLCc-AtCLCe and AtCLCg are reported to possess a selectivity filter that favors the transport of Cl− over other anions (De Angeli et al., 2009; Jossier et al., 2010; Nguyen et al., 2016). A comprehensive analysis of the CLC gene family in trifoliate orange was reported recently (Wei et al., 2015). Six PtrCLC genes were identified, and the gamma-Glu-Cys receptor of all of these genes was dramatically induced in response to NaCl stress. Tobacco is an economically important plant throughout the world. The concentration of Cl− in tobacco leaves has an important influence on tobacco quality. However, little research has been conducted about chloride transport in tobacco. This report details a comprehensive, genome-wide inventory of the CLC gene family in tobacco. Seventeen CLC genes were identified briefly. We determined the genomic location, the gene structure, and the phylogenetic relationships of each CLC gene. The expression patterns of these genes in various tissues and in response to salt stress treatment were also analyzed. The results of our study establish a foundation for further investigation of the functions of the NtCLC genes in tobacco.
Methods
Results
Discussion Previous studies of CLC genes in plants have established that these genes encode chloride channels/transporters (Teakle and Tyerman, 2010; Zifarelli and Pusch, 2010). Although the CLC genes of Arabidopsis have been extensively studied, efforts to catalog the genome-wide identification of CLC genes in other plant species have been limited. In this study, a total of seventeen CLC genes were identified in the tobacco genome. The relatively high number of NtCLC members in tobacco (compared to 7 in Arabidopsis, 10 in rice, and 6 in trifoliate orange) could mean that there may be an uncharacterized functional role for these proteins that is particularly important for the growth and/or metabolism of tobacco. Thus, it will be interesting to further characterize the specific functions of the various NtCLCs in the future. Previous studies detail the expressions of the CLC genes in various plant species. In Arabidopsis, AtCLCb is expressed strongly in the roots (Lv et al., 2009), and the AtCLC members in clade 1 exhibit a higher expression than those in clade 2 in other tissues. In trifoliate orange, PtrCLC6 displays a leaf-specific expression pattern, and all of the PtrCLCs show a relatively low expression in the roots (Wei et al., 2015). Our observations that all of the expressed NtCLC genes had a low expression in the roots are thus consistent with previous reports. In the present study, no NtCLC gene was expressed exclusively in a single tissue, and none of the tissues expressed only a single NtCLC gene. This suggests that the functions of the NtCLC genes are diversified, but they likely also overlap at least partially, as has been speculated for Arabidopsis (Lv et al., 2009). It is worth noting that NtCLC6 and NtCLC10 showed a largely stem-specific expression pattern. Since the transport of some anions from the roots to the leaves requires transport through stems, it will be informative to characterize the role of these two genes in anion transport in the future. In addition, NtCLC8 and NtCLC15 showed largely bud- and pistil-specific expression patterns, respectively, suggesting that these two genes may have distinct functions in these tissues. Previous studies report that both AtCLCe (Marmagne et al., 2007) and PtrCLC6 showed leaf-specific expression patterns. Our results revealed that there was no CLC gene in tobacco that exhibited leaf-specific expression. Our phylogenetic tree showed that NtCLC9 was related to AtCLCe, and our expression analysis showed that NtCLC9 had a relatively high expression in the stems, leaves, and buds. Therefore, it is clear that the expression patterns of CLC members are diverse in different plant species.