Amino Acid Substitutions in Loop C of Arabidopsis PIP2 Aquaporins Alters the Permeability of CO2

Abstract

The transport of CO₂ across biomembranes in plant cells is essential for efficient photosynthesis. Some aquaporins capable of CO₂ transport, referred to as 'COOporins', are postulated to play a crucial role in leaf CO₂ diffusion. However, the structural basis of CO₂ permeation through aquaporins remains largely unknown. Here, we show that amino acids in loop C are critical for the CO₂ permeability of Arabidopsis thaliana PIP2 aquaporins. We found that swapping tyrosine and serine in loop C to histidine and phenylalanine, which differ between AtPIP2;1 and AtPIP2;3, altered CO₂ permeability when examined in the Xenopus laevis oocyte heterologous expression system. AlphaFold2 modelling indicated that these substitution induced a conformational shift in the sidechain of arginine in the aromatic/arginine (ar/R) selectivity filter and in lysine at the extracellular mouth of the monomeric pore in PIP2 aquaporins. Our findings demonstrate that distal amino acid substitutions can trigger conformational changes of the ar/R filter in the monomeric pore, modulating CO₂ permeability. Additionally, phylogenetic analysis suggested that aquaporins capable of dual water/CO₂ permeability are ancestral to those that are water-selective and CO₂-impermeable, and CO₂-selective and water impermeable.

Keywords: Arabidopsis thaliana; CO2 transport; PIP2 aquaporin; Xenopus laevis; monomeric pore.

Figure 1

CO₂‐and water‐permeability of Arabidopsis PIP2 aquaporins. (A) Phylogenetic tree of Arabidopsis PIP proteins. Phylogenetic tree was generated by ClustalW with Neighbour Joining method using the full‐length amino acid sequences. The Arabidopsis Gene Identifier codes are AtPIP1;1, At3g61430; AtPIP1;2, At2g45960; AtPIP1;3, At1g01620; AtPIP1;4, At4g00430; AtPIP1;5, At4g23400; AtPIP2;1, At3g53420; AtPIP2;2, At2g37170; AtPIP2;3, At2g37180; AtPIP2;4, At5g60660; AtPIP2;5, At3g54820; AtPIP2;6, At2g39010; AtPIP2;7, At4g35100; AtPIP2;8, At2g16850. (B) Raw recording of cytosolic pH of water and AtPIP2;1 cRNA‐injected X. laevis oocytes by the micro‐pH electrode impalement method. Arrows indicate where the perfusion with 6.5 mM CO₂/H₂CO₃‐containing solution initiated. (C) Diffusion coefficient of CO₂ (P _CO2) of the cell membrane of X. laevis oocytes injected with water (n = 44) or cRNAs of either HvPIP2;1 (n = 8), AtPIP2;1 (n = 25), AtPIP2;2 (n = 27), AtPIP2;3 (n = 23), AtPIP2;4 (n = 23), AtPIP2;5 (n = 24), AtPIP2;6 (n = 22), AtPIP2;7 (n = 22) or AtPIP2;8 (n = 23). (D) Diffusion coefficient of water (P _f) of the cell membrane of X. laevis oocytes injected with water (n = 56) or cRNA (10 ng/50 nL) of HvPIP2;1 (n = 8), AtPIP2;1 (n = 30), AtPIP2;2 (n = 30), AtPIP2;3 (n = 31), AtPIP2;4 (n = 30), AtPIP2;5 (n = 32), AtPIP2;6 (n = 31), AtPIP2;7 (n = 30) or AtPIP2;8 (n = 29). P _f was measured by the oocyte swelling assay. Different letters show significant difference (α = 0.05) by Tukey–Kramer multiple comparison test.

Figure 2

Involvement of loop C in the CO₂ permeability of AtPIP2;1. (A) Schematic illustration of chimeric proteins. 1N‐3C, 3‐1‐3 and 1‐3‐1 indicate PIP2;1 (N)‐PIP2;3(C), PIP2;3‐PIP2;1‐PIP2;3 and PIP2;1‐PIP2;3‐PIP2;3, respectively. (B) Diffusion coefficient of CO₂ (P _CO2) of the cell membrane of X. laevis oocytes injected with water as the control (n = 17), and AtPIP2;1 cRNA (n = 24), AtPIP2;3 (n = 25), and the chimeric constructs [1N‐3C (n = 9), 3‐1‐3 (n = 8) and 1‐3‐1 (n = 11)]. (C) Diffusion coefficient of water (P _f) of the cell membrane of X. laevis oocytes injected with water as control (n = 38), AtPIP2;1 cRNA (n = 41), AtPIP2;3 (n = 39) and the chimeric constructs, 1N‐3C cRNA (n = 9), 3‐1‐3 cRNA (n = 21) and 1‐3‐1 cRNA (n = 17). Twenty‐five nanograms of cRNA were injected per oocyte for the oocyte swelling assay. Different letters indicate significant difference (α = 0.05) by the Tukey–Kramer multiple comparison test. (D) Immunoblot showing AtPIP2;1 and AtPIP2;3 protein accumulation in the crude membrane fraction of X. laevis oocytes. Crude membrane extract from five oocytes was loaded to a well. (E) Coomassie Brilliant Blue (CBB) staining serves as the loading control. Regular Range Protein Marker (PM1500, ExcelBAND, SMOBIO Technoloty Inc., New Taipei, Taiwan). Images of (D) and (E) are representative images from three biological replicates giving essentially the same results. (F) Localization of AtPIP2:1 and AtPIP2;3 in X. laevis oocytes injected with either water, AtPIP2;1 cRNA or AtPIP2;3 cRNA. A fluorescent image of a sliced PIP2;1, PIP2;3‐expressing oocyte and water‐injected oocytes. Exposure = 1 s. Sensitivity = +6 dB. Bar = 50 µm.

Figure 3

Exploration of the critical amino acid residues for determining CO₂ permeability in the loop C. (A) Amino acid alignment of the loop C of AtPIP2;1 and AtPIP2;3. Residues highlighted with grey boxes indicate the dissimilar residues. (B) Schematic illustration of single amino acid substitution mutant proteins on the 3‐1‐3 backbone. (C) Diffusion coefficient of CO₂ (P _CO2) of the cell membrane of X. laevis oocytes injected with water (n = 13), single amino acid mutants, 3‐1‐3 ^Y150H cRNA (n = 5), 3‐1‐3 ^T152V cRNA (n = 8), 3‐1‐3 ^R153N cRNA (n = 7), 3‐1‐3 ^S160F cRNA (n = 7) and 3‐1‐3 ^S166N cRNA (n = 9). (D) Diffusion coefficient of water (P _f) of the cell membrane of X. laevis oocytes injected with water (n = 7), single amino acid mutants, 3‐1‐3 ^Y150H cRNA (n = 7), 3‐1‐3 ^T152V cRNA (n = 8), 3‐1‐3 ^R153N cRNA (n = 8), 3‐1‐3 ^S160F cRNA (n = 8) and 3‐1‐3 ^S166N cRNA (n = 9). Twenty‐five nanograms of cRNA were injected per oocyte for the oocyte swelling assay. Three‐dimensional structures of AtPIP2;1 (E) and AtPIP2;3 (F) modelled by AlphaFold2 multimer2. Only a monomer is visualized. The loop C is shown in green. The amino acid residues involved in the difference in CO₂‐permeability between AtPIP2;1 and AtPIP2;3: tyrosine‐150/histidine‐147 and serine‐160 and phenylalanine‐157 are illustrated by blue and orange spheres, respectively.

Figure 4

Tyrosine and serine residues in the loop C are critical for determining CO₂ selectivity in AtPIP2;1. (A) Schematic illustration of amino acid substitution in AtPIP2;1 and AtPIP2;3. (B) Diffusion coefficient of water (P _CO2) of the cell membrane of X. laevis oocytes injected with water (n = 12), AtPIP2;1 cRNA (n = 18), AtPIP2;3 cRNA (n = 14), and the amino acid‐substituted constructs, AtPIP2;1 ^Y150H cRNA (n = 15), AtPIP2;1 ^S160F cRNA (n = 16) and AtPIP2;3 ^{H147Y‐F157S} cRNA (n = 17). (C) Diffusion coefficient of water (P _f) of the cell membrane of X. laevis oocytes injected with water (n = 6), AtPIP2;1 cRNA (n = 7), AtPIP2;3 cRNA (n = 7), AtPIP2;1 ^Y150H cRNA (n = 7), AtPIP2;1 ^S160F cRNA (n = 8) and AtPIP2;3 ^{H147Y‐F157S} cRNA (n = 8). Twenty‐five nanograms of cRNA were injected per oocyte for the oocyte swelling assay. Different letters indicate significant difference (α = 0.05) by Tukey–Kramer multiple comparison test.

Figure 5

Conformational change of sidechains between AtPIP2;1, AtPIP2;1^Y150H and AtPIP2;1^S160F modelled by AlphaFold2. (A) NPA boxes are shown by sticks. Blue and red sticks indicate AtPIP2;1 and AtPIP2;1^Y150H, respectively. (B) Four amino acid residues comprising the ar/R motif. Phenylalanine‐51, histidine‐216 and threonine‐223 are indicated by turquoise and orange in AtPIP2;1 and AtPIP2;1^Y150H, respectively. Arginine‐231 is indicated by blue and red, respectively. AtPIP2;1^Y160H is designated as AtPIP2;1^YH. (C–E) The serine and phenylalanine residues that are substituted in AtPIP2;1 and AtPIP2;1^S160F are shown by turquoise and orange, respectively. Arginine‐231 and lysine‐243 are shown in blue (AtPIP2;1) and red (AtPIP2;1^S160F). The main peptide chains of AtPIP2;1 are shown in grey. The main peptide chains of AtPIP2;1^Y150H and AtPIP2;1^S160F are shown in pale yellow.