Plasma Membrane-Type Aquaporins from Marine Diatoms Function as CO2/NH3 Channels and Provide Photoprotection

Abstract

Aquaporins (AQPs) are ubiquitous water channels that facilitate the transport of many small molecules and may play multiple vital roles in aquatic environments. In particular, mechanisms to maintain transmembrane fluxes of important small molecules have yet to be studied in marine photoautotrophic organisms. Here, we report the occurrence of multiple AQPs with differential cellular localizations in marine diatoms, an important group of oceanic primary producers. The AQPs play a role in mediating the permeability of membranes to CO₂ and NH₃ In silico surveys revealed the presence of five AQP orthologs in the pennate diatom Phaeodactylum tricornutum and two in the centric diatom Thalassiosira pseudonana GFP fusions of putative AQPs displayed clear localization to the plasma membrane (PtAGP1 and PtAQP2), the chloroplast endoplasmic reticulum (CER; PtAGP1 and PtAQP3), and the tonoplast (PtAQP5) in P. tricornutum In T. pseudonana, GFP-AQP fusion proteins were found on the vacuole membrane (TpAQP1) and CER (TpAQP2). Transcript levels of both PtAQP1 and PtAQP2 were highly induced by ammonia, while only PtAQP2 was induced by high (1%[v/v]) CO₂ Constitutive overexpression of GFP-tagged PtAQP1 and PtAQP2 significantly increased CO₂ and NH₃ permeability in P. tricornutum, strongly indicating that these AQPs function in regulating CO₂/NH₃ permeability in the plasma membrane and/or CER. Cells carrying GFP-tagged PtAQP1 and PtAQP2 had higher nonphotochemical quenching under high light relative to that of wild-type cells, suggesting that these AQPs are involved in photoprotection. These AQPs may facilitate the efflux of NH₃, preventing the uncoupling effect of high intracellular ammonia concentrations.

Figure 1.

Comparison of AQP sequences from diatoms with those of different AQP families. A, Phylogenetic tree of AQP family proteins derived from animals (black), land plants (deep green), cyanobacteria (deep blue), fungi (purple), bacteria (light blue), archaea (gray), green algae (light green), red algae (thin red line), diatoms (thick red line), and other eukaryotic algae (brown; Supplemental Table S1). PtAQP1 to PtAQP5, TpAQP1, and TpAQP2 are indicated with red text. Diatom clades are colored as follows: green algae-type AQP, light green; GIP, light blue; red algae-type AQP, orange; LIP, yellow. The phylogenetic tree was constructed using the maximum-likelihood method based on the Whelan and Goldman evolutionary model (Whelan and Goldman, 2001). Clades with greater than 50% bootstrap support (using 1,000 replicates) are indicated at the nodes of the tree. The branch lengths represent the distance between sequences in the number of substitutions per site as indicated by the scale bar. B, Schematic of the structure of specific regions of AQPs in human AQP1 (HsAQP1) and diatom AQPs (PtAQP1–PtAQP5, TpAQP1, and TpAQP2). Black boxes indicate transmembrane (TM) domains, red boxes indicate NPA motifs, and green boxes indicate ar/R residues.

Figure 2.

Subcellular localization of PtAQPs and TpAQPs. From left, bright field, Hoechst-stained nucleus (blue) and autofluorescence of chlorophyll (red), EGFP signal (green), and merged image. A, Images of P. tricornutum heterologously expressing EGFP-tagged PtAQP1, PtAQP2, PtAQP3, or PtAQP5. B, Images of T. pseudonana heterologously expressing EGFP-tagged TpAQP1 and TpAQP2. Bars = 10 µm (A) and 5 µm (B).

Figure 3.

Gene expression levels of PtAQPs and TpAQPs. The responses of PtAQP and TpAQP transcript levels to CO₂ and nitrogen were quantified by RT-qPCR. Transcript levels are shown for PtAQP1, PtAQP2, PtAQP3, PtAQP4, or PtAQP5 in P. tricornutum and TpAQP1 or TpAQP2 in T. pseudonana grown in 1% CO₂ (high CO₂/NO₃⁻; black bars), 0.04% CO₂ (air/NO₃⁻; white bars), with the addition of 1.8 mm NH₄Cl (air/NH₃; cyan bars) or 18 µm NaNO₃ (air/low NO₃⁻; magenta bars). The transcripts of GapC2 in P. tricornutum and Act in T. pseudonana were used as constitutive internal controls. A, PtAQPs in P. tricornutum. B, TpAQPs in T. pseudonana. All data are means ± sd (n = 3). Welch's t tests were performed between the air/NO₃⁻ condition as the reference and all treatment conditions (*, P < 0.05; **, P < 0.01; ***, P < 0.001; and n.s., not significant).

Figure 4.

MIMS ¹⁸O exchange analysis with P. tricornutum wild-type (WT) cells and transformant cells heterologously expressing PtAQP1 or PtAQP2 under dark conditions. A, Time-dependent changes in the concentrations of extracellular ¹³C¹⁸O¹⁸O (blue circles), ¹³C¹⁸O¹⁶O (orange circles), and ¹³C¹⁶O¹⁶O (gray circles) were monitored with MIMS. The addition of P. tricornutum cells (0 min) induced immediate ¹⁸O exchange. B, The CO₂ influx rate constants (f_c; cm³ s⁻¹) of PtAQP1G#3 and PtAQP1G#4 (gray bars) and PtAQP2G#5 and PtAQP2G#12 (black bars), grown under high (1%) CO₂ or air (0.04% CO₂), were compared with those of PtWT (white bars) grown under the same conditions. Values are means ± sd (n = 4–9). Statistical significance was determined using a Student t test (**, P < 0.01 and n.s., not significant).

Figure 5.

NH₃ efflux in diatom cells expressing PtAQP1 or PtAQP2. A, Time course of changes in NH₃ concentration in the external medium of cell cultures of PtG (white circles), PtAQP1G#3 (gray circles), and PtAQP2G#5 (black circles) grown in 0.04% CO₂. NH₃ concentration was monitored from the start of high-light illumination (1,500 μmol m⁻² s⁻¹) for 120 min. B, Maximum rates of NH₃ efflux in cell cultures of PtG (white bar), PtAQP1G#3, PtAQP1G#4, and PtAQP1G#6 (gray bars), and PtAQP2G#3, PtAQP2G#5, and PtAQP2G#12 (black bars). Student's t tests were performed between PtG and PtAQPnGs (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Figure 6.

Pulse amplitude modulator (PAM) assays in diatom cells expressing PtAQP1 or PtAQP2. A, NPQ of cell cultures of PtG (white circles), PtAQP1G#3 (gray circles), and PtAQP2G#5 (black circles) with a spectrum of actinic light from 7 to 1,100 μmol m⁻² s⁻¹. B, NPQ of PtG (white bars), PtAQP1G (#3, #4, and #6; gray bars), and PtAQP2G cells (#3, #5, and #12; black bars) exposed to an actinic light intensity of 1,100 μmol m⁻² s⁻¹ in the absence (left) or presence (right) of 2 mm NH₄Cl. All values are means ± sd (n = 3). Student's t tests were performed between PtG and PtAQPnGs (**, P < 0.01 and ***, P < 0.001).