Department of Plant Physiology and Biophysics, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Akademicka 19, Lublin 20-033, Poland.
Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510 Japan.
Plant Cell Physiol. 2022 Feb 15;63(2):163-175. doi: 10.1093/pcp/pcab176.
The two-pore channel (TPC) family is widely conserved in eukaryotes. Many vascular plants, including Arabidopsis and rice, possess a single TPC gene which functions as a slow vacuolar (SV) channel-voltage-dependent cation-permeable channel located in the vacuolar membrane (tonoplast). On the other hand, a liverwort Marchantia polymorpha genome encodes three TPC homologs: MpTPC1 is similar to TPCs in vascular plants (type 1 TPC), while MpTPC2 and MpTPC3 are classified into a distinctive group (type 2 TPC). Phylogenetic analysis suggested that the type 2 TPC emerged before the land colonization in plant evolution and was lost in vascular plants and hornworts. All of the three MpTPCs were shown to be localized at the tonoplast. We generated knockout mutants of tpc1, tpc2, tpc3 and tpc2 tpc3 double mutant by clustered regularly interspaced short palindromic repeats/Cas9 genome editing and performed patch-clamp analyses of isolated vacuoles. The SV channel activity was abolished in the Mptpc1 loss-of-function mutant (Mptpc1-1KO), while Mptpc2-1KO, Mptpc3-1KO and Mptpc2-2/tpc3-2KO double mutant exhibited similar activity to the wild type, indicating that MpTPC1 (type 1) is solely responsible for the SV channel activity. Activators of mammalian TPCs, phosphatidylinositol-3,5-bisphosphate and nicotinic acid adenine dinucleotide phosphate, did not affect the ion channel activity of any MpTPCs. These results indicate that the type 1 TPCs, which are well conserved in all land plant species, encode the SV channel, while the type 2 TPCs likely encode other tonoplast cation channel(s) distinct from the SV channel and animal TPCs.
双孔通道（TPC）家族在真核生物中广泛保守。许多维管植物，包括拟南芥和水稻，都拥有一个单一的 TPC 基因，该基因作为位于液泡膜（液泡被膜）中的慢液泡（SV）通道-电压依赖性阳离子渗透性通道发挥作用。另一方面，一种苔类植物 Marchantia polymorpha 的基因组编码三个 TPC 同源物：MpTPC1 与维管植物中的 TPC 相似（1 型 TPC），而 MpTPC2 和 MpTPC3 被归类为一个独特的群体（2 型 TPC）。系统发育分析表明，2 型 TPC 出现在植物进化的陆地殖民化之前，并在维管植物和角苔中丢失。所有三种 MpTPC 都被证明位于液泡被膜上。我们通过成簇规律间隔短回文重复序列/Cas9 基因组编辑生成了 tpc1、tpc2、tpc3 和 tpc2 tpc3 双突变体的敲除突变体，并对分离的液泡进行了膜片钳分析。SV 通道活性在功能丧失突变体（Mptpc1-1KO）中被消除，而 Mptpc2-1KO、Mptpc3-1KO 和 Mptpc2-2/tpc3-2KO 双突变体表现出与野生型相似的活性，表明 MpTPC1（1 型）单独负责 SV 通道活性。哺乳动物 TPC 的激活剂，磷脂酰肌醇-3,5-二磷酸和烟酰胺腺嘌呤二核苷酸磷酸，都没有影响任何 MpTPC 的离子通道活性。这些结果表明，在所有陆地植物物种中都很好保守的 1 型 TPC 编码 SV 通道，而 2 型 TPC 可能编码不同于 SV 通道和动物 TPC 的其他液泡阳离子通道。
