Han Beibei, Tai Yuxin, Li Shuping, Shi Junmei, Wu Xueqing, Kakeshpour Tayebeh, Weng Jianfeng, Cheng Xianguo, Park Sunghun, Wu Qingyu
Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China.
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
Front Plant Sci. 2022 Jul 11;13:938839. doi: 10.3389/fpls.2022.938839. eCollection 2022.
Calcium (Ca) is an essential plant nutrient, and Ca/H exchangers (CAXs) regulate Ca 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) but not the full-length SlCAX1 was active in yeast, similar to Arabidopsis AtCAX1. Characterization of mutants generated by CRISPR-Cas9 confirmed the calcium transport ability of SlCAX1. Sequence alignment between SlCAX1, AtCAX1, AtCAX3, and the Ca/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) further increased its activity. The same truncation had a similar effect on Arabidopsis AtCAX1 and AtCAX3. Expression of full-length SlCAX1 and SlCAX1 in tomato plants confirmed the results. Our results suggest that SlCAX1 is critical for Ca 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 Ca partitioning to reduce the incidence of Ca-related physiological disorders in crops.
钙（Ca）是植物必需的营养元素，钙/氢交换体（CAXs）调节亚细胞区室之间的钙分配。拟南芥AtCAX1的活性受到其N端调节区域（NRR）的抑制，该区域最初被定义为前两个甲硫氨酸之间的序列。然而，这种NRR定义的准确性和NRR调节机制仍不清楚。在这里，我们以番茄SlCAX1为模型，重新定义了CAXs的NRR，并证明我们的新定义也适用于拟南芥AtCAX1和AtCAX3。与拟南芥AtCAX1类似，N端截短的SlCAX1（SlCAX1）而非全长SlCAX1在酵母中具有活性。通过CRISPR-Cas9产生的突变体的表征证实了SlCAX1的钙转运能力。SlCAX1、AtCAX1、AtCAX3与钙/氢反向转运蛋白YfkE之间的序列比对显示，SlCAX1没有第二个甲硫氨酸，YfkE在第一个跨膜结构域之前没有任何氨基酸残基。将SlCAX1的氨基酸残基截短至第一个跨膜结构域（SlCAX1）进一步提高了其活性。相同的截短对拟南芥AtCAX1和AtCAX3有类似的影响。在番茄植株中全长SlCAX1和SlCAX1的表达证实了这些结果。我们的结果表明，SlCAX1对钙稳态至关重要，第一个跨膜结构域之前的所有氨基酸残基均抑制CAXs的活性。我们对NRR的重新定义将有助于微调钙分配，以降低作物中与钙相关的生理紊乱的发生率。
