Integral light-harvesting proteins in the dinoflagellate, symbiodinium sp.

Reef building (Scleractinian) corals are the major component of coral reefs and form an intimate symbiotic relationship with a single celled dinoflagellate (Symbiodinium sp.). It is a relationship that is highly sensitive to environmental changes, particularly when thermal stress is combined with a non-optimal light level. The success of the symbiosis is driven by the dinoflagellates' ability to harvest the energy of the sun through the process of photosynthesis and translocate reduced organic carbon to the coral host for calcification and growth. The process of photosynthesis is a complex and hazardous process involving a balance between solar energy harvesting and energy utilization, or dissipation. Our current understanding of eukaryotic photosynthetic gene expression is predominately derived from plant and green unicellular algae and observations at the level of gene transcription indicate distinct differences in how unicellular algae and higher plants acclimate their photosystems to environmental changes. Therefore the question arises; how do unicellular symbiotic dinoflagellates, such as Symbiodinium, respond to distinct environmental cues, do they mimic the response of other unicellular algae or that of higher plants. To determine whether Symbiodinium mimic the response of other unicellular algae or that of higher plants, a better understanding of the major light-harvesting protein complex (LHCs) utilized by Symbiodinium is required. In addition, knowledge of the LHC genes possessed by Symbiodinium, the expression of these genes under a variety of environmental cues, and an understanding of how translation and degradation combine to restructure the photosynthetic apparatus, is necessary. Therefore, the aims of this research were to use expressed sequence tag data for Symbiodinium sp. sub-clade C3 to: sequence the chlorophyll a-chlorophyll c₂-peridinin protein complexes (acpPC); to compare C3 acpPC with LHCs from other photosynthetic organisms; to determine whether C3 acpPCs were present in cultured Symbiodinium of varying clades and sub-clades; and to investigate acpPC gene expression patterns in response to varying light conditions. To achieve these aims an extensive sequencing project was performed and once complete sequences were obtained bioinformatics analysis, including analysis of conserved amino acid residues and homology of translated protein sequences with known light harvesting genes from symbiotic and free-living dinoflagellates, algae and higher plants, was performed. Phylogenetic comparison of translated protein sequences with light harvesting protein complexes from a variety of organisms was undertaken and the diversity of acpPCs within Symbiodinium from divergent lineages was investigated. Transcript sizes were determined using northern blots and Southern blots were used to determine acpPC gene copy numbers. A polyclonal antibody generated in rabbits to a synthesized peptide fragment based upon acpPC cDNA sequences was obtained and western blots used to investigate protein expression. Real- Time PCR, a relatively new tool to the study of Symbiodinium, was used for gene expression work following validation of normalization genes. Phylogenetic analysis of 11 LHC sequences suggests the acpPC subfamily forms at least three clades within the Chl a/c-binding LHC family. The first clade includes rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences; the second peridinin binding dinoflagellates only; while the third contains heterokonts, fucoxanthin and peridinin binding dinoflagellate sequences. Symbiodinium sp. C3 acpPC sequences generally contain three transmembrane helices and at least two proteins with LHC membrane-spanning helix duplication, deletion, and / or degeneration are evident. In addition, Symbiodinium encode transcripts containing one, two or three LHC polypeptides and the genome encodes single copy and as well as high copy acpPC genes. Analysis of Symbiodinium from divergent lineages demonstrates that C3 acpPC sequence information can be utilized to investigate closely related Symbiodinium at the transcript and protein level, in particular Symbiodinium C1, and findings suggest a number of C3 acpPC genes are common to Symbiodinium clades. β-actin and proliferating cell nuclear antigen (PCNA) can be used in controlled light experiments to investigate acpPC expression having been validated as normalization genes. The use of a polyclonal antibody for acpPC suggests protein expression varies between Symbiodinium subclades. Nevertheless, changes at the transcript level are relatively small when Symbiodinium are exposed to different light environments suggesting acpPC genes are possibly subject to translational control mechanism or post-translational controls. With Symbiodinium genome information becoming publicly available insight into the complex genetic composition of these unique dinoflagellates will be possible. Until readily available, the information resulting from this research provides: a basis for future investigations into the major light-harvesting protein complexes utilized by Symbiodinium, specifically clade C Symbiodinium; a means to compare whether the acpPC subfamily differs between stress tolerant and susceptible Symbiodinium species; a methodology to measure how environmental factors impact the major LHCs used by these ecologically important dinoflagellates; and a potentially significant breakthrough in our understanding of the diverse and complex acpPC subfamily