Thies AB et al. (2022), A Rhesus channel in the coral symbiosome membra...

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Sci Adv 2022 Mar 11;810:eabm0303. doi: 10.1126/sciadv.abm0303.

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A Rhesus channel in the coral symbiosome membrane suggests a novel mechanism to regulate NH3 and CO2 delivery to algal symbionts.

Thies AB , Quijada-Rodriguez AR , Zhouyao H , Weihrauch D , Tresguerres M .

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Reef-building corals maintain an intracellular photosymbiotic association with dinoflagellate algae. As the algae are hosted inside the symbiosome, all metabolic exchanges must take place across the symbiosome membrane. Using functional studies in Xenopus oocytes, immunolocalization, and confocal Airyscan microscopy, we established that Acropora yongei Rh (ayRhp1) facilitates transmembrane NH3 and CO2 diffusion and that it is present in the symbiosome membrane. Furthermore, ayRhp1 abundance in the symbiosome membrane was highest around midday and lowest around midnight. We conclude that ayRhp1 mediates a symbiosomal NH4+-trapping mechanism that promotes nitrogen delivery to algae during the day-necessary to sustain photosynthesis-and restricts nitrogen delivery at night-to keep algae under nitrogen limitation. The role of ayRhp1-facilitated CO2 diffusion is less clear, but it may have implications for metabolic dysregulation between symbiotic partners and bleaching. This previously unknown mechanism expands our understanding of symbioses at the immediate animal-microbe interface, the symbiosome.

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	Fig. 1. Functional characterization of total ammonia (Tamm) and CO2 transport by Acropora yongei Rhesus protein (ayRhp1).(A) Effect of [NH3] on Tamm uptake rate in Xenopus oocytes expressing ayRhp1. Control Tamm uptake rates have been subtracted. Data show means ± SEM of six to eight oocytes; the letters denote significant differences [one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test; pH 6.5 versus pH 7.5, P < 0.0001; pH 7.5 versus pH 8.5, P = 0.0222; pH 6.5 versus pH 8.5, P < 0.0001]. (B) Michaelis-Menten Tamm uptake kinetics calculated from the data shown in (A) (black dots to the left of the dotted line). Apparent Jmax = 51.93 ± 1.45 pmol Tamm min−1 and Km = 7.14 ± 0.91 μmol Tamm liter−1. The red triangle indicates Tamm uptake rate obtained in a solution with 10 mM Tamm at pH 7.5 (175 μM NH3 and 9.825 mM NH4+) (i.e., similar [NH3] to the previous data point, but ~10-fold higher [NH4+]). (C) Functional characterization of CO2 transport by ayRhp1. Xenopus oocytes expressing ayRhp1 (ayRhp1) display a higher rate of CO2 release than control oocytes after equal CO2 preloading. Data show means ± SEM of n = 8, 25 oocytes per n; ** denotes significant differences (Welch’s t test; P = 0.0019).
	Fig. 2. Immunolocalization of Acropora yongei Rhesus protein (ayRhp1).(A) Overview of A. yongei tissues; the boxes indicate regions of interest shown at higher magnification below, and the white arrowhead indicates ayRhp1-labeled calcifying cells. (B1) Apical membrane of columnar cells in the oral epidermis. (C1) Desmocyte with intense signal in its apical region. (D1) Alga-containing gastrodermal cells. (B2, C2, and D2) Corresponding bright-field differential interference contrast images; the white arrowheads mark corresponding locations in (B), (C), and (D). Coral and algal nuclei are shown in blue, and ayRhp1 immunofluorescence is shown in green. Several algal nuclei are marked with asterisks in (B1) and (D1) for clarity. This coral was sampled at midday. sw, seawater; co, coelenteron; sk, skeleton. Scale bars, 20 μm.
	Fig. 3. Confocal Airyscan immunolocalization of Acropora yongei Rhesus protein (ayRhp1) in the oral epidermis, desmocytes, and calcifying cells.(A1–3) ayRhp1 on the apical membrane of columnar cells in the oral epidermis. (B1–3) Desmocyte with intense ayRhp1 signal in its apical region. (C1–3) Calcifying cells displaying ayRhp1 signal on membranes and in the cytosol. Corresponding areas between panels are marked with arrowheads. [(A1) to (C1)], [(A2) to (C2)], and [(A3) to (C3)] show ayRhp1, Na+/K+-ATPase (NKA), and 4′,6-diamidino-2-phenylindole (DAPI) signals, ayRhp1 signal alone, or NKA signal alone, respectively. Nuclei (DAPI) are shown in blue, ayRhp1 in green, and the NKA in purple. Scale bars, 5 μm.
	Fig. 4. Confocal Airyscan immunolocalization of Acropora yongei Rhesus protein (ayRhp1) in alga-containing coral cells.(A1) Cells displaying ayRhp1 in the symbiosome membrane of tissue sections. (B1) Cells displaying nonsymbiosomal ayRhp1 of tissue sections. (A2 and B2) Higher magnification of the region denoted by the white boxes in (A1) and (B1). (A3 and B3) Three-dimensional renderings of (A2) and (B2). Corresponding areas between (A2) and (B2) and (A3) and (B3) are marked. Nuclei are shown in blue, ayRhp1 in green, and the NKA in purple. Notice the separation (A1–3) or colocalization (B1–3) of ayRhp1 and NKA corresponding to symbiosomal or nonsymbiosomal ayRhp1 localizations, respectively. Host nuclei are denoted with an asterisk, and algal nuclei with arrowheads. Scale bars, 5 μm (A1 and B1), 0.5 μm (A2 and B2), and 0.5 μm (A3 and B3). (C) Percentage of total alga-containing A. yongei host cells with symbiosomal ayRhp1 over a diel cycle. Data show means ± SEM. n = 3 per time point, 50 cells per n, 900 cells total. The asterisks indicate significant differences with the 1300-hour time point (two-way repeated-measures ANOVA followed by Dunnett’s posttest; P < 0.01; *P < 0.0001).
	Fig. 5. Model of the coral nitrogen concentrating mechanism in alga-containing coral host cells.(1) Coral mitochondria produce NH3 and CO2. (2) Cytosolic CA catalyzes CO2 hydration into H+ and HCO3−. (3) H+ and (4) HCO3− are moved into the symbiosome space by VHA and an unidentified HCO3− transporter, respectively. Inside the symbiosome, H+ and HCO3− dehydrate into CO2, which diffuses into and is photosynthetically fixed by the alga. (5) NH3 diffuses via the A. yongei Rhesus protein (ayRhp1) into the symbiosome space, where it is immediately protonated and trapped as NH4+. An unidentified algal transporter imports NH4+ into the alga, where it is assimilated. Some CO2 also diffuses via ayRhp1 back into the host cell’s cytosol, where it is rehydrated and transported back to the symbiosome space. ATP, adenosine 5′-triphosphate.

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Allison, Corals concentrate dissolved inorganic carbon to facilitate calcification. 2014, Pubmed