Gierz, Sarah Louise (2017) Thermal acclimation and light-harvesting complex expression in Symbiodinium. PhD thesis, James Cook University.

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Abstract

Endosymbioses observed between photosynthetic dinoflagellates of the genus Symbiodinium and reef-building (Scleractinian) corals are crucial to the success of diverse reef ecosystems. Dysfunction of this symbiotic relationship can occur under a number of stressors (including elevated sea surface temperatures and ocean acidification), resulting in the expulsion of Symbiodinium from host cells or loss of photosynthetic pigments, a process known as coral bleaching. While ocean temperatures fluctuate on a daily basis, the mean ocean temperature is predicted to rise approximately 1 – 2 °C over the next century and is expected to lead to more mass coral bleaching events.

Within coral bleaching experiments, elucidation of sites of thermal sensitivity within Symbiodinium has focused on potential points where damage may originate. One of these potential sites are the integral light-harvesting protein complexes (LHCs), which bind chlorophylls and accessory pigment molecules with roles in lightharvesting by receiving and transferring light energy to photosystems, and photoprotection by dissipating excess energy under stress conditions. Little is known about the response of the diversified integral LHC gene family (acpPCs) in Symbiodinium to thermal stress, as only short term (24 h), light stress and dissociation experiments have been reported. Additionally, few studies have examined the broad transcriptional response of Symbiodinium to thermal stress conditions.

Therefore, the aims of this research were to examine the effect of extended thermal stress on Symbiodinium to determine variations in gene expression and morphology both in vitro and in hospite and to link this to observed physiological parameters. To achieve these aims thermal stress experiments were performed on cultured Symbiodinium sp. (clade F), and in hospite utilising Acropora aspera harbouring Symbiodinium clade C3. A targeted quantitative PCR approach was utilised to determine the expression of five integral LHC genes within Symbiodinium in hospite and a transcriptome approach was utilised to identify differentially expressed transcripts within Symbiodinium sp. (clade F) in vitro. Variations in Symbiodinium morphology were characterized following exposure of A. aspera to thermal stress using confocal laser scanning microscopy.

Exposure of Symbiodinium sp. (clade F) cultures to a twenty-eight day thermal stress regime (~31 ϒC) elicited a stress response measured as reduced cell growth from day four onwards (p < 0.01) and decreased dark-adapted yield on days fourteen (p < 0.05), nineteen (p < 0.001) and twenty-eight (p < 0.001). Whole transcriptome sequencing of Symbiodinium cells on days four, nineteen and twenty-eight identified 23,654 unique genes (FDR < 0.05), though 92.49% differentially expressed genes displayed ≤ 2-fold change in expression. The transcriptional response included differential expression of genes encoding photosynthetic machinery subunits, integral LHCs, fatty - acid desaturases, metabolic enzymes, and components of stress response pathways. The results indicate a shift in metabolism, from carbon fixation to fatty acid catabolism under thermal stress, supported by upregulation of β- oxidation, glyoxylate cycle and gluconeogenic enzymes and has not previously been quantified in Symbiodinium.

Exposure of A. aspera to a sixteen-day thermal stress regime elicited a bleaching response measured as reduced Symbiodinium density (day sixteen, p < 0.001) and significantly decreased dark-adapted yield (day sixteen, p < 0.001). The expression of five integral LHC genes in Symbiodinium in hospite were measured using quantitative PCR employing previously established reference genes. Of the five integral LHC genes quantified, three acpPC genes exhibited upregulated expression when corals were exposed to temperatures above 31.5 ϒC (acpPCSym_1:1, day sixteen (1.74-fold, p < 0.001); acpPCSym_15, day twelve (1.33-fold, p < 0.05); and acpPCSym_18, day ten (2.44-fold, p < 0.05) and day sixteen (2.08-fold, p < 0.05)). In contrast, acpPCSym_5:1 and acpPCSym_10:1 exhibited constitutive expression throughout the experiment. Interestingly, the three acpPC genes with increased expression cluster together in a phylogenetic analysis of light-harvesting complexes.

Variation in an assemblage of cellular and photophysiological variables in individual and populations of Symbiodinium sp. (clade C3) cells within A. aspera were characterized following exposure to a sixteen day thermal stress (approximately +0.7 ϒC per d, maximum ~34 ϒC). Coral branches were maintained across four aquaria, with two tanks per condition, and were sampled on days zero, eight, ten, twelve and sixteen. Specific physiological parameters such as Symbiodinium density, dark-adapted yield, effective quantum yield, chlorophyll pigment content, cellular morphology and chlorophyll a fluorescence intensity were measured to assess the cytological response to extended exposure at elevated temperatures below and above the bleaching threshold of A. aspera. A variety of responses among the Symbiodinium populations both within and between coral branches were identified in the parameters assayed. Further demonstrating that broad, multifaceted approaches are required when assessing coral bleaching cellular responses to ensure an accurate representation of holobiont health.

The results of this thesis provide insights into the molecular response of Symbiodinium exposed to thermal stress, below the bleaching thresholds. As in previous gene expression analyses, relatively small transcriptional changes were detected in vitro and in hospite, further supporting the hypothesis that other mechanisms of regulation (post-transcriptional or translational regulation) are critical in Symbiodinium stress responses. Quantification of multiple integral LHCs in vitro and in hospite identified genes with constitutive and inducible expression within the highly expanded family, providing potential insights into the functional purpose for LHC diversification in Symbiodinium. The implications for altered Symbiodinium gene expression and metabolism under thermal stress and the effect this may have on host - symbiont metabolite transfer is unknown, although, the results presented here provide preliminary data for studies investigating the molecular response of Symbiodinium under future temperature conditions.

Item ID:	51805
Item Type:	Thesis (PhD)
Keywords:	Acropora aspera, bleaching, coral, dinoflagellate, gene expression, light harvesting complexes, quantitative PCR, RNA-Seq, Symbiodinium, thermal stress, transcriptome
Related URLs:	https://researchonline.jcu.edu.au/47474/ https://researchonline.jcu.edu.au/45220/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 2: Gierz, Sarah, Forêt, Sylvain, and Leggat, William (2017) Transcriptomic analysis of thermally stressed Symbiodinium reveals differential expression of stress and metabolism genes. Frontiers in Plant Science, 8. pp. 1-20. Chapter 3: Gierz, Sarah L., Gordon, Benjamin R., and Leggat, William (2016) Integral light-harvesting complex expression in Symbiodinium within the coral Acropora aspera under thermal stress. Scientific Reports, 6. Data deposition for Chapter 2: Data included in Appendices E,G, G and two additional tables relevant to the analysis are available on the James Cook University Tropical Data Hub. Illumina sequences read data reported in the chapter/article have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under accession number SRA467551, which is associated with BioProject number PRJNA342240. Data may be accessed from the Research Data field.
Research Data:	http://dx.doi.org/10.4225/28/598a96f71288a, https://www.ncbi.nlm.nih.gov/bioproject/342240
Date Deposited:	19 Dec 2017 03:38
FoR Codes:	06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060199 Biochemistry and Cell Biology not elsewhere classified @ 50% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060405 Gene Expression (incl Microarray and other genome-wide approaches) @ 50%
SEO Codes:	96 ENVIRONMENT > 9603 Climate and Climate Change > 960399 Climate and Climate Change not elsewhere classified @ 40% 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 40% 96 ENVIRONMENT > 9608 Flora, Fauna and Biodiversity > 960808 Marine Flora, Fauna and Biodiversity @ 20%
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