Functional analysis of TaCLC-e-3AL and the identification of interacting proteins in wheat (Triticum aestivum L.)

Objectives This study aimed to elucidate the mechanism of TaCLC-e-3AL involvement in response to low nitrogen stress by analyzing the function and identifying the interacted proteins of TaCLC-e-3AL.

Methods The amino acid sequence of Arabidopsis thaliana AtCLC-e was used as a reference sequence, and three genes, TaCLC-e-3AL (TraesCS3A02G253600), TaCLC-e-3B (TraesCS3B02G285500) and TaCLC-e-3DL (TraesCS3D02G254500), were obtained from wheat genome database by BlastP, and their structures and phylogenetic relationships were then analyzed. The TaCLC-e-3AL-p1300-GFP fusion was transformed into wheat protoplasts to analysis the subcellular localization characteristics of TaCLC-e-3AL. Transgenic Arabidopsis thaliana experiments were employed to determine the function of TaCLC-e-3AL. The interactors of TaCLC-e-3AL were identified by yeast two-hybrid system screening.

Results The TaCLC-e-3AL gene was cloned from wheat, and bioinformatics analysis indicated that the encoded protein of TaCLC-e-3AL gene contained 11 transmembrane domains, and showed the highest homology with TuCLC-e in Triticum urartu. TaCLC-e-3AL gene was highly expressed in leaves and stems of wheat, and several light, hormone and stress response elements were detected in its promoter region using the method of cis-acting elements analysis. Subcellular localization in protoplasts showed that TaCLC-e-3AL was mainly expressed in chloroplasts. When TaCLC-e-3AL was overexpressed in Arabidopsis thaliana under low nitrogen condition, the transgenic plants stored more NO₃⁻ and maintained stable NO₃⁻/Cl⁻ homeostasis, the root length and fresh weight were not impacted significantly. The screening results of the yeast two-hybrid system showed that TaCLC-e-3AL interacted with aquaporin, voltage-dependent anion channel and other proteins, indicating the possible involvement of TaCLC-e-3AL in drought stress response, photosynthesis, signal transduction and other biological processes.

Conclusions The wheat chloride ion channel protein TaCLC-e-3AL is located in the chloroplasts. The overexpression of TaCLC-e-3AL in Arabidopsis thaliana could raise NO₃⁻-N storage and thereby keep the normal NO₃⁻/Cl⁻ homeostasis in plants under low nitrogen stress, so the TaCLC-e-3AL is thought regulating the synergistic transport of Cl⁻ and NO₃⁻. Through interaction with aquaporin, voltage-dependent anion channel and other proteins, TaCLC-e-3AL participates in response to drought stress, photosynthesis, and ion stress in wheat.