Significance of oxygen transport through aquaporins

Abstract

Aquaporins are membrane integral proteins responsible for the transmembrane transport of water and other small neutral molecules. Despite their well-acknowledged importance in water transport, their significance in gas transport processes remains unclear. Growing evidence points to the involvement of plant aquaporins in CO₂ delivery for photosynthesis. The role of these channel proteins in the transport of O₂ and other gases may also be more important than previously envisioned. In this study, we examined O₂ permeability of various human, plant, and fungal aquaporins by co-expressing heterologous aquaporin and myoglobin in yeast. Two of the most promising O₂-transporters (Homo sapiens AQP1 and Nicotiana tabacum PIP1;3) were confirmed to facilitate O₂ transport in the spectrophotometric assay using yeast protoplasts. The over-expression of NtPIP1;3 in yeasts significantly increased their O₂ uptake rates in suspension culture. In N. tabacum roots subjected to hypoxic hydroponic conditions, the transcript levels of the O₂-transporting aquaporin NtPIP1;3 significantly increased after the seven-day hypoxia treatment, which was accompanied by the increase of ATP levels in the apical root segments. Our results suggest that the functional significance of aquaporin-mediated O₂ transport and the possibility of controlling the rate of transmembrane O₂ transport should be further explored.

Figure 1. Immunoblot probed with the anti-myoglobin antibody and the anti-human aquaporin 1 antibody.

(A) The yeast total proteins were immunoblotted with the primary anti-myoglobin antibody. (B) The yeast total proteins were immunoblotted with the primary anti-human aquaporin 1 antibody.

Figure 2. Indirect immunofluorescence of paraffin-embedded yeast cells of HsAQP1 strain after the incubation with the primary anti-aquaporin 1 monoclonal antibody and the fluorescein-conjugated secondary antibody.

(A) HsAQP1 strain under blue light excitation. (B) HsAQP1 strain in bright field. (C) Mock strain under blue light excitation. (D) Mock strain in bright field. The length of bars is 10 μm.

Figure 3. ∆A₅₄₁/∆A₆₀₀ of yeast protoplasts after the first 90 s with 2 times of 30 s aeration.

Asterisks indicate statistically significant difference with the mock strain (P values shown in the table below) (ANOVA, Tukey’s test, P ≤ 0.05, n = 19 ± SE).

Figure 4. ∆A₃₁₉/∆A₃₄₁ of yeast protoplasts after 5 min with 5 times of 30 s aeration.

Asterisks indicate statistically significant difference with the mock strain (P values shown in the table below) (ANOVA, Tukey’s test, P ≤ 0.05, n = 6 ± SE).

Figure 5. O₂ consumption of yeast strains over 1000 s.

(A) Respiration rates in HsAQP1, NtPIP1;3, AtPIP1;2, and mock strain (control). (B) Time for total O₂ consumption in HsAQP1, NtPIP1;3, AtPIP1;2, and mock strain (control). Immediately after air was supplied to the yeast suspension in N₂-bubbled SD-L-U + glucose medium to reach the saturation concentration of soluble O₂ of 235 μmol L⁻¹, the decrease of O₂ concentration in yeast suspension was monitored and logged per second using an O₂ microsensor. Asterisks indicate statistically significant difference with the mock strain (P values shown in the table below) (ANOVA, Tukey’s test, P ≤ 0.05, n = 6 ± SE).

Figure 6. Transcript abundance of tobacco plasma membrane intrinsic proteins (PIPs) after exposure to well-aerated and hypoxic conditions.

Relative transcript abundance of selected PIPs in (A) leaves and (B) roots after 2 days of exposure, and in (C) leaves and (D) roots after 7 days of exposure. Transcript abundance of PIPs was measured by the standard curve method in qRT-PCR assay, with normalization against geometric mean of that of the two reference genes, EF1-α and L25. Asterisks indicate significance difference in gene expression between well-aerated and hypoxic treatments on the same day; P values for the comparisons between day two and day seven are listed in the table below (ANOVA, Tukey’s test, P ≤ 0.05, n = 6 ± SE).

Figure 7. ATP levels in leaves and basal (BR), central (CR) and apical (AP) root segments of tobacco plants subjected to hypoxic and well-aerated conditions.

(A) ATP levels after 2 days of the treatments. (B) ATP levels after 7 days of the treatments. ATP level was determined by detecting bioluminescence in Luciferase/Luciferin reaction. Asterisks indicate significant differences in ATP levels between well-aerated and hypoxic treatments in the same tissue on the same day; P values for the comparisons between day two and day seven are listed in the table below (ANOVA, Tukey’s test, P ≤ 0.05, n = 6 ± SE).