The role of TaCHP in salt stress responsive pathways

Abstract

In our previous study, we found wheat TaCHP confers salt tolerance through regulating salt responsive signaling pathways. TaCHP possesses three divergent C1 domains that can specifically bind to phospholipid signaling molecule diacylglycerol (DAG) in animal cells, and most of proteins with this domain have kinase activity. Here, we found that TaCHP localizes both at cytoplasmatic membrane and in nuclei; it has no kinase activity but transcriptional activation activity, and the latter owes to C-terminal two C1 domains. TaCHP transcription was reduced by H2O2 application, but its ectopic expression in Arabidopsis improved both ROS production and scavenging capacity, and enhanced tolerance to H2O2 application. We suggest that TaCHP serve as both a transcription factor and a putative DAG binding protein to confer salt tolerance in part through improving ROS scavenging capacity; which is a component of the cross-talk machinery in the phospholipids-ROS-salt responsive signaling pathways.

Figure 1. TaCHP localizes at cytoplasmic membrane and in nuleus, with no protein kinase C (PKC) kinase activity but transcriptional activation activity. (A) GFP and TaCHP-GFP fused protein were transiently expressed in onion epidermis cells. (B) The fluorescent map of agrose gel electrophoresis of TaCHP in vitro PKC kinase assay. +: positive control; -: negative control; +1: band of peptide substrate with net charge +1; -1: band of PKC-phosphorylated peptide with net charge -1. (C) TaCHP transcriptional activation assay. C1C2, C2 and C2C3: truncated TaCHP fragments cover N-terminal C1 and C2 domains (1–128aa), C2 domain (83–117aa), and C2 and C3 domains (84–215aa), respectively. Bar in panel A: 150μm.

Figure 2. TaCHP is responsive to H₂O₂, and its overexpression in Arabidopsis improves both ROS production and scavenging capacity and enhances tolerance to H₂O₂. (A) Phenotypic analysis of Arabidopsis under the control and H₂O₂/ABA treatments. Three-d-old seedlings were transferred on 1/2MS agar plates containing none, 0.3mM H₂O₂ or 5uM ABA, and vertically grew for 2 weeks. C13: Arabidopsis line transformed with empty vector. BS, A5: Arabidopsis lines transformed with vector fused with TaCHP driven by CaMV 35S promoter. (B) Comparison of root length of (A). (C) Transcription level of TaCHP in roots of SR3 and JN177 seedlings at three-leaf-stage under 10 mM H₂O₂ treatment measured by RT-PCR, and single copy gene TaActin was used as internal reference. (D) Relative expression levels of genes encoding ROS production and scavenging enzymes of Arabidopsis measure by real-time PCR, which is defined as the ratio of expression level (single copy gene AtTublin as internal reference) to that of C13. (E) CAT and SOD activities of Arabidopsis. In (D and E), 2-week-old Arabidopsis seedlings were used. *: significant difference when compared with C13 using Student’s t-test (p < 0.05). Bars in (A) 1 cm.