Recent studies have shown that
Recent studies have shown that some CLC genes participate in plant salt tolerance and have speculated that they function by mediating Cl− transport across the tonoplast. For instance, AtCLCc, a vacuolar anion transporter, is shown to be involved in the regulation of stomatal movement and to contribute to salt tolerance responses (Jossier et al., 2010). AtCLCg is localized to the vacuolar membrane, and atclcg knockout mutants have a decreased OG-L002 sale phenotype when they are grown under treatment with NaCl or KCl (Nguyen et al., 2016). The GmCLC1 gene from soybean encodes a chloride ion transporter and is regulated by pH under salt stress (Li et al., 2006; Wong et al., 2013). The transcription of ZmCLCd from maize is up-regulated under salt treatment, and the overexpression of ZmCLCd in Arabidopsis confers tolerance to salt stress (S. Wang et al., 2015). Our qPCR expression analysis demonstrated that almost all of the NtCLC genes with detectable expression showed varied transcriptional responses to salt stress. To briefly recap, after treatment with 300 mM NaCl for 7 d, only NtCLC2, NtCLC3 and NtCLC12 showed obvious changes in both the roots and shoots. These results indicated that these three NtCLC genes seemed likely to participate in tobacco responses to salt stress. This supposition was further supported by our phylogenetic analysis, which showed that NtCLC2 was related to AtCLCc, and NtCLC3 or NtCLC12 was related to AtCLCg. In addition, these two Arabidopsis genes are confirmed to be involved in response to salt stress (Jossier et al., 2010; Nguyen et al., 2016). In addition, it was striking that the expression of NtCLC3, NtCLC13 and NtCLC15, in the shoots, increased >10-fold under salt stress. Combined with the phylogenetic analysis, NtCLC3 was close to NtCLC12, and NtCLC13 was close to NtCLC15. Finally, we chose three NtCLC genes, NtCLC2, NtCLC3 and NtCLC13, for the genetic experiments using VIGS. The Cl− concentrations were measured, and we found that NtCLC2 and NtCLC13 might participate in chloride transport or metabolism. Previously, GmCLC1 was reported to enhance salt tolerance by regulating chloride accumulation in soybean (Wei et al., 2016). In this study, we noticed that the Cl− accumulated in the NtCLC13-silenced plants. Whether NtCLC13 shares the same function as GmCLC1 is interesting. Moreover, the Cl− concentrations were reduced in the NtCLC2-silenced plants, and thus, it was valuable to detect whether NtCLC2 was related to the inhibition of chloride transport. It is known that large amounts of chloride accumulation have many adverse effects on the quality of tobacco leaves (Ashkan et al., 2014), and the NtCLC2 gene might be a biotechnological tool with great potential for engineering low-chloride tobacco. Our study establishes that the identified NtCLC genes likely play crucial roles in tobacco responses to salt stress, but the particular mechanisms by which the NtCLC genes function in these processes will require further exploration and experimental confirmation.
Conclusions In this study, we identified and characterized seventeen members of the CLC gene family in tobacco. A phylogenetic analysis indicated that the NtCLC genes were divided into two clades, as was reported for other plant species. The results of our gene structure and conserved motif analysis were consistent with the relationships revealed by our phylogenetic analysis. Twelve of the seventeen NtCLC genes were expressed, and CLC gene expression was detected in all of the examined tissues, with some obvious tissue-specific patterns for genes such as NtCLC6 and NtCLC8. Under salt stress, some of the NtCLC genes showed a significant and tissue-specific induction of expression. Therefore, it seems highly likely that these NtCLC genes may function in tobacco responses to salt stress. Specifically, the concentration of Cl− in the NtCLC2- and NtCLC13-silenced plants showed a significant decrease or increase compared to the control plants, which indicated that these two genes might play important roles in chloride transport or metabolism in tobacco. The results of our study provide the basis for the further functional characterization of the NtCLC genes and suggest several interesting hypotheses about the cellular and biochemical mechanisms by which CLCs contribute to salt stress responses in plants.