Rubisco Expression in the Dinoflagellate Symbiodinium sp. Is Influenced by Both Photoperiod and Endosymbiotic Lifestyle

Abstract

Although the importance of anthozoan-dinoflagellate (genus Symbiodinium) endosymbioses in the establishment of coral reef ecosystems is evident, little is known about the molecular regulation of photosynthesis in the intra-gastrodermal symbiont communities, particularly with respect to the rate-limiting Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). In this study, we analyzed rubisco mRNA (rbcL) and protein (RBCL) concentrations over the diel cycle in both cultured and endosymbiotic Symbiodinium samples. In the former, rbcL expression increased upon illumination and decreased during the dark, a pattern that was upheld under continual dark incubation. A different trend in rbcL expression was observed in endosymbiotic Symbiodinium residing within sea anemone (Aiptasia pulchella) tissues, in which illumination gradually led to decreased rbcL mRNA expression. Unexpectedly, RBCL protein expression did not vary over time within anemone tissues, and in neither cultured nor endosymbiotic samples was a correlation between gene and protein expression documented. It appears, then, that photoperiod, lifestyle, and posttranscriptional regulation are all important drivers of RBCL expression in this ecologically important dinoflagellate.

Appendixes

Appendix 1 Primer Specificity Validation Study

Materials and Methods

Endosymbiotic sea anemones with an oral disc size of ~0.5 cm were collected and grown in 250 ml glass flasks containing 200 ml sterile filtered seawater (SFSW) for several months, and one specimen from each of three randomly selected clonal cultures was partially dissected to remove one tentacle for DNA extraction (described in Appendix 2), which was snap-frozen in a 1.5-ml microcentrifuge tube and stored at −80 °C. The partially dissected anemones were then visualized under a fluorescent microscope, snap-frozen in liquid nitrogen, and stored at −80 °C prior to extraction of their RNAs (described in the main article text) to serve as the healthy group (n = 3). Images were obtained with a DAPI filter set (UV/Violet; Ex: BP 365/12 nm; FT 395 nm; LP 397 nm) on a Zeiss dissecting microscope (SteREO Discovery V8, Carl Zeiss International, Germany) using a Zeiss Axiocam digital camera to detect Symbiodinium (red) chlorophyll fluorescence.

Six randomly selected anemone cultures were incubated at 4 °C for 2 h to induce bleaching (sensu Muscatine et al. 1991). After cold shock, one partially bleached anemone from each of the three randomly chosen cold-shocked cultures was dissected and visualized under the fluorescent microscope as described above, snap-frozen in liquid nitrogen, and stored at −80 °C to serve as the partially bleached group (n = 3). The remaining cold-shocked specimens were transferred to new flasks containing SFSW and then kept at 25–28 °C in total darkness for 8 months. The SFSW was replaced twice weekly, and, once Symbiodinium were no longer visible under the fluorescent microscope, one anemone from each of the three fully bleached clonal populations was imaged, dissected as above, snap-frozen in liquid nitrogen, and kept at −80 °C prior to extraction of RNAs to serve as the fully bleached group (n = 3). RNA extraction, cDNA synthesis, and SQ-RT-PCRs were conducted with the nine samples as described in the main article text.

Results

Symbiodinium density was diminished in partially bleached anemones, and in fully bleached specimens, no Symbiodinium were microscopically detected (Fig. s1a–c, right-most panels). act1 and rbcL cDNAs were amplified by PCR for either 27 or 40 cycles (Fig. s1d). As fewer Symbiodinium were found in partially bleached samples, decreased expression of rbcL and act1 was evident; PCR for 27 cycles did not amplify either gene at a detectable level (Fig. s1d). Detection of act1 expression in partially bleached A. pulchella specimens could only be achieved after 40 PCR cycles. Furthermore, neither rbcL nor act1 transcripts were detected in fully bleached anemones, even after 40 PCR cycles (Fig. s1d).

Appendix 2 RFLP Analysis of Cultured and Endosymbiotic Symbiodinium

To determine the genetic identity to clade level for both cultured and endosymbiotic Symbiodinium used in the study, DNAs were extracted from ~10⁶ cells from each of the 12 Symbiodinium cultures from which the experimental aliquots were removed, the individual tentacles dissected from each of the 9 anemones sampled for the primer specificity validation study (PSVS, Appendix 1), and the 24 A. pulchella (entire anemone) specimens sampled at 3-h intervals across one 12 L:12D cycle with the AxyPrep™ Multisource Genomic DNA Miniprep kit (Axygen Biosciences, CA, USA) according to the manufacturer’s recommendations after homogenizing the cells, tentacles, and anemones, respectively, in liquid nitrogen with a mortar and pestle. The 45 DNAs were PCR-amplified with the Symbiodinium 18 s rDNA primers (“ss3z-ss5z”), reagent concentrations, and thermocycling conditions of Rowan and Powers (1991), and the PCR products were excised from the gel with a razor blade and purified with the AxyPrep™ DNA gel extraction kit (Axygen Biosciences) according to the manufacturer’s recommendations.

The purified PCR amplicons were then digested with TaqI and Sau3 AI (New England Biolabs, MA, USA) in separate 20 μl reactions as in Yang (2001), and 10 μl of the digestion products were electrophoresed on 1.5 % tris-acetate-EDTA (TAE) agarose gels, stained in an ethidium bromide (EtBr) bath for 20 min, visualized at 610 nm on a Typhoon Trio™ Scanner (GE Healthcare, WI, USA), and the digestion patterns compared to those of Rowan and Powers (1991) and Yang (2001) to verify Symbiodinium identity in each of the 45 DNA samples. Subcladal diversity of Symbiodinium (sensu LaJeunesse 2002; LaJeunesse et al. 2010; Bellantuono et al. 2011) was not assessed, as the markers used to infer such diversity, particularly its2, are intragenomically variable (Stat et al. 2009, 2010) and hence unable to resolve subcladal genetic differences (Pochon et al. 2011).