Living on the Edge: Protective Mechanisms Underlying Thermal Tolerance in High Latitude Symbiodinium spp.

<p>The association between symbiotic dinoflagellates (Symbiodinium spp.) and corals extends to subtropical and temperate regions, where sea surface temperatures (SSTs) are generally lower than in the tropics and can vary substantially over the course of the year due to seasonal changes. These high latitude coral-dinoflagellate symbioses might be better able to withstand thermal variability and might be particularly well equipped to cope with lower SSTs compared to their tropical relatives. The aim of this thesis was to analyze the cellular mechanisms that underlie heat and/or cold tolerance in a range of reef-building corals (Acropora yongei, Acropora solitariensis, Isopora palifera, Pocillopora damicornis, Porites heronensis and Stylophora sp.), as well as the symbiotic sea anemone Entacmaea quadricolor. In particular, the study focussed on protective mechanisms in their dinoflagellate symbionts as a potential determinant of thermal sensitivity (i.e. bleaching) or resistance of the intact symbiosis. High latitude reef-building corals were analyzed at the world’s southernmost coral reef at Lord Howe Island, while E. quadricolor was sampled at the subtropical coral community at North Solitary Island; both sites are located in New South Wales, Australia. The specific objectives were to assess the roles of: (1) xanthophyll deepoxidation; (2) thylakoid fatty acid composition; (3) Symbiodinium superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity; and (4) D1 repair on the photophysiology, bleaching susceptibility and survivorship of a range of high-latitude coral-Symbiodinium associations from Lord Howe Island when exposed to elevated or decreased temperature. Furthermore, I aimed to: (5) characterise Symbiodinium diversity in the anemone E. quadricolor on the west coast of Australia; and (6) measure the dynamics of Symbiodinium ITS2 populations and SOD activity in two E. quadricolor phenotypes (green and pink colour phenotypes) in response to elevated temperature. I showed that thermal responses in high latitude corals and their dinoflagellate symbionts are highly variable, depending on host species (or phenotype) and Symbiodinium genotype, and that the activation of protective mechanisms in Symbiodinium was not necessarily correlated with sub-lethal bleaching susceptibility or survivorship of their coral hosts. More specifically: (1) In response to short-term heat stress and cold stress, xanthophyll de-epoxidation increased in some but not all bleaching susceptible (e.g. P. damicornis) and bleaching tolerant (P. heronensis) corals; (2) overall unsaturated thylakoid fatty acids increased in symbionts of a bleaching tolerant coral association, yet was not correlated with PSII photochemical efficiency; and (3) SOD and APX activity remained unchanged in the majority of Symbiodinium types regardless of bleaching susceptibility of the coral host, but decreased in bleaching susceptible Pocillopora damicornis when exposed to short-term heat stress. Elevated temperatures resulted in enhanced D1 turnover in two warm-water bleaching susceptible Symbiodinium-host combinations; however a direct link between increased dependence on D1 turnover and bleaching susceptibility was not demonstrated. From the results obtained it seems unlikely that the specific cellular adaptations in Symbiodinium alone determine the tolerance of Lord Howe corals to thermal variations. In contrast, the results highlight the significance of the particular host-symbiont combination and it appears that the host is important in determining, at least in part, the thermal response of the coral. Additionally, this study revealed a high diversity of Symbiodinium ITS2 (internal transcribed spacer 2) types in E. quadricolor from five locations on the west coast of Australia. E. quadricolor predominantly associated with six types of clade C (four of which were novel) and most anemones harboured multiple types simultaneously. At North Solitary Island, anemones simultaneously harboured Symbiodinium C25 and C3.25 (a novel variant of C3). Experimentally, I showed that anemones shuffled the relative proportions of C25 and C3.25 in response to elevated temperature, but not in both anemone colour phenotypes analyzed. Furthermore, baseline photobiological characteristics were distinct in the two different anemone colour morphs but were not correlated with the ratio of Symbiodinium C25 to C3.25, suggesting that host mechanisms such as pigmentation were involved in regulating light utilization by the symbionts. My hypothesis that symbiont shuffling was related to SOD activity, as such that those symbionts with enhanced SOD activity and increased capability to scavenge superoxide anion would increase in relative abundance in response to short-term heat stress, could not be proved. In summary, this thesis provides detailed information on some key cellular mechanisms that could underpin thermal sensitivity and resistance in high latitude Symbiodinium, and most importantly highlights the significance of the host-symbiont combination in determining the response to thermal stress. The various mechanistic findings described here further our understanding of the coral bleaching process in general and particularly give insight into physiological and cellular responses to coldwater stress in reef-building corals at high-latitude sites. The results of this thesis indicate that in light of ongoing climate change, as episodes of cold-water and warm-water anomalies will become more frequent, branching corals such as Acropora yongei or Pocillopora damicornis and their symbionts will experience physiological stress more frequently than massive species such as Porites heronensis. This might have profound impacts on the long-term stability and species composition of high latitude coral reefs.</p>

Living on the Edge: Protective Mechanisms Underlying Thermal Tolerance in High Latitude Symbiodinium spp.

<p>The association between symbiotic dinoflagellates (Symbiodinium spp.) and corals extends to subtropical and temperate regions, where sea surface temperatures (SSTs) are generally lower than in the tropics and can vary substantially over the course of the year due to seasonal changes. These high latitude coral-dinoflagellate symbioses might be better able to withstand thermal variability and might be particularly well equipped to cope with lower SSTs compared to their tropical relatives. The aim of this thesis was to analyze the cellular mechanisms that underlie heat and/or cold tolerance in a range of reef-building corals (Acropora yongei, Acropora solitariensis, Isopora palifera, Pocillopora damicornis, Porites heronensis and Stylophora sp.), as well as the symbiotic sea anemone Entacmaea quadricolor. In particular, the study focussed on protective mechanisms in their dinoflagellate symbionts as a potential determinant of thermal sensitivity (i.e. bleaching) or resistance of the intact symbiosis. High latitude reef-building corals were analyzed at the world’s southernmost coral reef at Lord Howe Island, while E. quadricolor was sampled at the subtropical coral community at North Solitary Island; both sites are located in New South Wales, Australia. The specific objectives were to assess the roles of: (1) xanthophyll deepoxidation; (2) thylakoid fatty acid composition; (3) Symbiodinium superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity; and (4) D1 repair on the photophysiology, bleaching susceptibility and survivorship of a range of high-latitude coral-Symbiodinium associations from Lord Howe Island when exposed to elevated or decreased temperature. Furthermore, I aimed to: (5) characterise Symbiodinium diversity in the anemone E. quadricolor on the west coast of Australia; and (6) measure the dynamics of Symbiodinium ITS2 populations and SOD activity in two E. quadricolor phenotypes (green and pink colour phenotypes) in response to elevated temperature. I showed that thermal responses in high latitude corals and their dinoflagellate symbionts are highly variable, depending on host species (or phenotype) and Symbiodinium genotype, and that the activation of protective mechanisms in Symbiodinium was not necessarily correlated with sub-lethal bleaching susceptibility or survivorship of their coral hosts. More specifically: (1) In response to short-term heat stress and cold stress, xanthophyll de-epoxidation increased in some but not all bleaching susceptible (e.g. P. damicornis) and bleaching tolerant (P. heronensis) corals; (2) overall unsaturated thylakoid fatty acids increased in symbionts of a bleaching tolerant coral association, yet was not correlated with PSII photochemical efficiency; and (3) SOD and APX activity remained unchanged in the majority of Symbiodinium types regardless of bleaching susceptibility of the coral host, but decreased in bleaching susceptible Pocillopora damicornis when exposed to short-term heat stress. Elevated temperatures resulted in enhanced D1 turnover in two warm-water bleaching susceptible Symbiodinium-host combinations; however a direct link between increased dependence on D1 turnover and bleaching susceptibility was not demonstrated. From the results obtained it seems unlikely that the specific cellular adaptations in Symbiodinium alone determine the tolerance of Lord Howe corals to thermal variations. In contrast, the results highlight the significance of the particular host-symbiont combination and it appears that the host is important in determining, at least in part, the thermal response of the coral. Additionally, this study revealed a high diversity of Symbiodinium ITS2 (internal transcribed spacer 2) types in E. quadricolor from five locations on the west coast of Australia. E. quadricolor predominantly associated with six types of clade C (four of which were novel) and most anemones harboured multiple types simultaneously. At North Solitary Island, anemones simultaneously harboured Symbiodinium C25 and C3.25 (a novel variant of C3). Experimentally, I showed that anemones shuffled the relative proportions of C25 and C3.25 in response to elevated temperature, but not in both anemone colour phenotypes analyzed. Furthermore, baseline photobiological characteristics were distinct in the two different anemone colour morphs but were not correlated with the ratio of Symbiodinium C25 to C3.25, suggesting that host mechanisms such as pigmentation were involved in regulating light utilization by the symbionts. My hypothesis that symbiont shuffling was related to SOD activity, as such that those symbionts with enhanced SOD activity and increased capability to scavenge superoxide anion would increase in relative abundance in response to short-term heat stress, could not be proved. In summary, this thesis provides detailed information on some key cellular mechanisms that could underpin thermal sensitivity and resistance in high latitude Symbiodinium, and most importantly highlights the significance of the host-symbiont combination in determining the response to thermal stress. The various mechanistic findings described here further our understanding of the coral bleaching process in general and particularly give insight into physiological and cellular responses to coldwater stress in reef-building corals at high-latitude sites. The results of this thesis indicate that in light of ongoing climate change, as episodes of cold-water and warm-water anomalies will become more frequent, branching corals such as Acropora yongei or Pocillopora damicornis and their symbionts will experience physiological stress more frequently than massive species such as Porites heronensis. This might have profound impacts on the long-term stability and species composition of high latitude coral reefs.</p>

Involvement of the HtrA family of proteases in the protection of the cyanobacterium Synechocystis PCC 6803 from light stress and in the repair of photosystem II

Photosystem II (PSII) is prone to irreversible light–induced damage, with the D1 polypeptide a major target. Repair processes operate in the cell to replace a damaged D1 subunit within the complex with a newly synthesized copy. As yet, the molecular details of PSII repair are relatively obscure despite the critical importance of this process for maintaining PSII activity and cell viability. We are using the cyanobacterium Synechocystis sp. PCC 6803 to identify the various proteases and chaperones involved in D1 turnover in vivo . Two families of proteases are being studied: the FtsH family (four members) of Zn 2+ –activated nucleotide–dependent proteases; and the HtrA (or DegP) family (three members) of serine–type proteases. In this paper, we report the results of our studies on a triple mutant in which all three copies of the htrA gene family have been inactivated. Growth of the mutant on agar plates was inhibited at high light intensities, especially in the presence of glucose. Oxygen evolution measurements indicated that, under conditions of high light, the rate of synthesis of functional PSII was less in the mutant than in the wild–type. Immunoblotting experiments conducted on cells blocked in protein synthesis further indicated that degradation of D1 was slowed in the mutant. Overall, our observations indicate that the HtrA family of proteases are involved in the resistance of Synechocystis 6803 to light stress and play a part, either directly or indirectly, in the repair of PSII in vivo .