Redefining the N-Terminal Regulatory Region of the Ca2+/H+ Antiporter CAX1 in Tomato

Beibei Han; Yuxin Tai; Shuping Li; Junmei Shi; Xueqing Wu; Tayebeh Kakeshpour; Jianfeng Weng; Xianguo Cheng; Sunghun Park; Qingyu Wu

doi:10.3389/fpls.2022.938839

Redefining the N-Terminal Regulatory Region of the Ca²⁺/H⁺ Antiporter CAX1 in Tomato

Front Plant Sci. 2022 Jul 11:13:938839. doi: 10.3389/fpls.2022.938839. eCollection 2022.

Authors

Beibei Han¹, Yuxin Tai², Shuping Li^{1

3}, Junmei Shi⁴, Xueqing Wu¹, Tayebeh Kakeshpour⁵, Jianfeng Weng², Xianguo Cheng¹, Sunghun Park⁵, Qingyu Wu¹

Affiliations

¹ Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China.
² Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
³ College of Resources and Environmental Sciences, China Agricultural University, Beijing, China.
⁴ College of Land and Environment, Shenyang Agricultural University, Shenyang, China.
⁵ Department of Horticulture and Natural Resources, Kansas State University, Manhattan, KS, United States.

Abstract

Calcium (Ca²⁺) is an essential plant nutrient, and Ca²⁺/H⁺ exchangers (CAXs) regulate Ca²⁺ partitioning between subcellular compartments. AtCAX1 activity is inhibited by its N-terminal regulatory region (NRR), which was initially defined as the sequence between the first two methionines. However, the accuracy of this NRR definition and the NRR regulatory mechanism remain unclear. Here, using tomato SlCAX1 as a model, we redefined the NRR of CAXs and demonstrated that our new definition is also applicable to Arabidopsis AtCAX1 and AtCAX3. The N-terminal-truncated SlCAX1 (SlCAX1^Δ39) but not the full-length SlCAX1 was active in yeast, similar to Arabidopsis AtCAX1. Characterization of slcax1 mutants generated by CRISPR-Cas9 confirmed the calcium transport ability of SlCAX1. Sequence alignment between SlCAX1, AtCAX1, AtCAX3, and the Bacillus subtilis Ca²⁺/H⁺ antiporter protein YfkE revealed that SlCAX1 does not have the 2nd methionine and YfkE does not have any amino acid residues in front of the first transmembrane domain. Truncating the amino acid residues up to the first transmembrane of SlCAX1 (SlCAX1^Δ66) further increased its activity. The same truncation had a similar effect on Arabidopsis AtCAX1 and AtCAX3. Expression of full-length SlCAX1 and SlCAX1^Δ66 in tomato plants confirmed the results. Our results suggest that SlCAX1 is critical for Ca²⁺ homeostasis and all the amino acid residues in front of the first transmembrane domain inhibit the activity of CAXs. Our redefinition of the NRR will facilitate fine-tuning of Ca²⁺ partitioning to reduce the incidence of Ca²⁺-related physiological disorders in crops.

Keywords: CAX; CRISPR; NRR; calcium; tomato.