The proteomic response of the reef coral Pocillopora acuta to experimentally elevated temperatures

Abstract

Although most reef-building corals live near the upper threshold of their thermotolerance, some scleractinians are resilient to temperature increases. For instance, Pocillopora acuta specimens from an upwelling habitat in Southern Taiwan survived a nine-month experimental exposure to 30°C, a temperature hypothesized to induce stress. To gain a greater understanding of the molecular pathways underlying such high-temperature acclimation, the protein profiles of experimental controls incubated at 27°C were compared to those of conspecific P. acuta specimens exposed to 30°C for two, four, or eight weeks, and differentially concentrated proteins (DCPs) were removed from the gels and sequenced with mass spectrometry. Sixty unique DCPs were uncovered across both eukaryotic compartments of the P. acuta-dinoflagellate (genus Symbiodinium) mutualism, and Symbiodinium were more responsive to high temperature at the protein-level than the coral hosts in which they resided at the two-week sampling time. Furthermore, proteins involved in the stress response were more likely to be documented at different cellular concentrations across temperature treatments in Symbiodinium, whereas the temperature-sensitive host coral proteome featured numerous proteins involved in cytoskeletal structure, immunity, and metabolism. These proteome-scale data suggest that the coral host and its intracellular dinoflagellates have differing strategies for acclimating to elevated temperatures.

Fig 1. Two-dimensional gel electrophoresis of proteins from representative control and high- temperature coral samples after two, four, and eight weeks of treatment exposure.

Encircled and labeled protein spots were 1) determined to be differentially concentrated between temperature treatments within a sampling time with image analysis software, 2) removed from the gels, 3) purified, 4) digested with trypsin, and 5) submitted for sequencing by nano-liquid chromatography+mass spectrometry as described in the main text and the S1 File. The isoelectric point (pI) and molecular weight (in kilodaltons [kDa]) were the 1^st and 2^nd dimensions, respectively. Only labeled spots were removed; in certain cases at the four- and eight-week sampling times, matched (i.e., same pI and molecular weight) spots have been encircled in gels of both treatments to emphasize their differential concentrations, yet they were not sequenced for reasons discussed in the main text. “C2” and "H2" = control and high-temperature samples at the two-week sampling time, respectively. “C4” and "H4" = control and high-temperature samples at the four-week sampling time, respectively. “C8” and "H8" = control and high-temperature samples at the eight-week sampling time, respectively.

Fig 2. Pie graphs showing breakdown of differentially concentrated proteins (DCPs) between control (C2) and high (H2) temperature samples of the two-week sampling time.

Three and six protein spots were found to be more highly concentrated in the C2 (i.e., C>H) and H2 (H>C) proteomes, respectively, and 13 C>H and 25 H>C DCPs were identified from these nine spots. When the ratio of host/Symbiodinium (Sym) DCPs in panels a and c was significantly lower (z-test, p<0.05) than the Pocillopora acuta/Sym mRNA ratio of 1.9 (i.e., a 65/35% host coral/Sym ratio), the word “host” has been underlined. When the cellular process “stress response” was over-represented in the proteomes of the host coral (g-i) and Sym (j-l) relative to the host coral (6% of the P. acuta host transcriptome) and Sym (4% of the Sym transcriptome) transcriptomes [29], respectively (2-sample proportion test, p<0.05), a bar has been inserted over the term “stress response”.

Fig 3. Pie graphs showing breakdown of differentially concentrated proteins (DCPs) between control (C4) and high (H4) temperature samples of the four-week sampling time.

Four and four protein spots were found to be more highly concentrated in the C4 (i.e., C>H) and H4 (H>C) proteomes, respectively, and 8 C>H and 4 H>C DCPs were identified from these eight spots. When the host:Symbiodinium (Sym) ratio was significantly higher (z-test, p<0.05) than the P. acuta:Sym mRNA ratio of 1.9±0.4 (std. dev.), a bar has been inserted over the word “host” in (a-c). Likewise, when a cellular process was over-represented in the proteomes of the host (g-i) and Sym (j-l) relative to the host coral and Sym transcriptomes [29], respectively (2-sample proportion test, p<0.05), a bar has been placed over the category name.

Fig 4. Pie graphs depicting breakdown of differentially concentrated proteins (DCPs) between control (C8) and high (H8) temperature samples of the eight-week sampling time.

Two and six spots were more highly concentrated in the C8 (i.e., C>H) and H8 (H>C) proteomes, respectively, and 6 C>H and 19 H>C DCPs were identified from these eight spots. When the host:Symbiodinium (Sym) DCP ratio was significantly lower (z-test, p<0.05) than the P. acuta: Sym mRNA ratio of 1.9±0.4 (std. dev.), a bar has been inserted under the word “host” in (a-c).

Fig 5. Compartmental and functional breakdown of all differentially concentrated proteins (DCPs).

Across the nine, eight, and eight differentially concentrated/uniquely synthesized protein spots removed from representative gels of the two-, four-, and eight-week sampling times, respectively, 75 DCPs were uncovered; upon counting five proteins sequenced at multiple time points only once, 37 and 23 of the 70 unique DCPs were found to be of host coral (Pocillopora acuta) and Symbiodinium (Sym) origin, respectively. When a cellular process was over-represented in the differentially concentrated proteomes of the host coral (g-i) and Sym (j-l) relative to the host coral and Sym transcriptomes [29], respectively (2-sample proportion test, p<0.05), a bar has been inserted above the category name.

Fig 6. Summary of the dataset.

The number of differentially concentrated proteins (DCPs) was plotted over time for both the host coral (“H”) and Symbiodinium (“S;” Sym) for proteins documented at higher concentrations at high temperature (red icons) and those over-expressed at the control temperature (blue icons). The sizes of the icons are proportional to the host/Sym DCP ratio at each sampling time (listed at the top of the plot). The upper and lower hatched lines represent the total number of DCPs at each sampling time for the host coral and Sym, respectively. In (b) the sizes of the bubbles are proportional to the number of DCPs involved in each cellular process for both the host coral (b-1) and Sym (b-2). Purple bubbles represent those cellular processes for which one protein was over-expressed at high temperature, whereas another was documented at higher concentrations at the control temperature. This annotation is not used for the Sym “stress response” category at the two-week sampling time (b-2), in which 1 and 4 DCPs were more highly concentrated by the control and high-temperature samples, respectively. Underlined functional categories in b-2 reflect processes that were also temperature-sensitive in the host coral compartment (b-1).