Approximately 50% of the photosynthesis on Earth occurs in the oceans, and diatoms are thought to be responsible for around half of this marine photosynthesis. The molecular machinery involved in the capture of light energy for photosynthesis is evolutionarily ancient and well conserved. However, the pathways for supplying the inorganic carbon that is fixed (i.e. incorporated into carbohydrates) during photosynthesis are quite diverse. These pathways, which evolved in response to a decrease in global carbon dioxide concentrations, elevate the concentration of carbon dioxide around the primary carbon-fixing enzyme RubisCO, which is both inefficient and located within the chloroplast. The goal of the proposed research is to identify and characterize the molecular components of the carbon dioxide concentrating mechanisms in Phaeodactylum tricornutum and Thalassiosira pseudonana, two marine diatoms for which whole genome sequences and genetic manipulation systems are available. A research focus will be bicarbonate transporters and carbonic anhydrases, which are known to be essential components of the carbon dioxide concentrating mechanisms. While these have been identified in the P. tricornutum and T. pseudonana genomes using computational approaches, there is little cytological or functional verification of the computational predictions. A variety of methods including microscopy, immunolocalization, over-expression of native protein-fluorescent protein fusions, RNA interference based expression knockdown, and membrane inlet mass spectrometry will be used to connect cytological and functional information to genomic entities. Such information will facilitate an extension to other diatoms as genomes or transcriptomes become available. The data will be used to test and refine conceptual models of the function of the carbon dioxide concentrating mechanisms.
Broader Impacts A graduate student and undergraduate students will be trained at the intersection of phytoplankton physiology and genetics. The wider community will be engaged through a collaboration with the Centers for Ocean Sciences Education Excellence-South East in which the investigators will collaborate with K-12 teachers and outreach specialists to develop materials on ocean acidification for use in K-12 classrooms.
Diatoms are heterokont phytoplankton responsible for upwards of 50% of marine primary production and are known for their ability to rapidly bloom with the delivery of nitrogen and iron to sunlit surface waters. Like most microalgae, diatoms elevate the concentration of CO2 around ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO), the enzyme that catalyzes carbon fixation in the Calvin-Benson cycle, to maximize its carbon fixation rate and reduce competitive fixation of O2. The system that achieves this CO2 increase is known as the CO2 concentrating mechanism (CCM). Diatoms require a CCM to overcome the low concentration and slow diffusion rate of CO2 in seawater. Bioinformatic analyses identified putative bicarbonate transporters in the existing diatom genomes and marine metagenomes. We produced a high quality phylogeny of putative bicarbonate transporters in marine algae (Fig. 1) and verified that these are related to the bicarbonate transporters found in metazoans, including humans, where they are involved in both bone growth and repair, as well as pH homeostasis. Over the course of the project, 10 putative bicarbonate transporters were cloned, overexpressed and localized in two marine diatoms. The different phylogenetic lineages of the transporters are located in different cellular membranes, including the outer membrane and the chloroplast membrane, providing a clear path for bicarbonate from outside the cell to RubisCO. Transgenic diatoms overexpressing the outer membrane transporters were examined to successfully verify these transporters do transport bicarbonate. The chloroplast transporters were targeted using RNA interference to reduce protein abundance, and this was shown to reduce growth rates at low CO2. We studied the role of carbonic anhydrases, enzymes that equilibrate CO2 and bicarbonate, in the CCMs of marine diatoms. Several carbonic anhydrases were localized in a centric diatom and compared with the localization of carbonic anhydrases in a more-well studied diatom. This comparison suggested that the CCMs of these two diatoms worked similarly except that the centric diatoms have a different strategy for concentrating bicarbonate in the chloroplast. Many diatoms have an extracellular carbonic anhydrase attached to their outermembrane, which is believed to supply CO2 for photosynthesis though other roles have been proposed. We developed a method to absolutely quantify the activity of this enzyme and studied its activity and regulation is several diatoms. This work showed that the enzyme does supply CO2 for photosynthesis and aids CO2 uptake by keeping the CO2 concentration gradient outside the diatom cell very small. To understand the expression of the these transporters and also possibly identify new CCM components, we examined gene and protein expression in cultures of diatoms grown at low, modern day, and Jurassic era CO2 concentrations. At the Jurassic era CO2 concentrations, the CCM component expression is repressed, suggesting they were not as necessary at the time when diatoms originally evolved. At lower CO2 concentrations, outer membrane transporter gene expression was upregulated, as was the protein abundance. The chloroplast localized transporter gene expression was relatively invariant, while the protein expression of one increased 100 fold at very low CO2 concentrations. This last result indicates that this vital transporter is regulated post-transcriptionally, but also suggests that the outer membrane transporter gene expression is the most responsive for interrogating wild populations of diatoms. A more global analysis of the RNA sequencing revealed a set of ~200 genes that are specifically responsive to changing CO2, which provides clear targets, including transcription factors, for future work. A comparison of the expression of all genes at night versus during the day showed a consistent diurnal cycle in gene expression that is modulated by the availability of CO2. That is, genes upregulated during the day are less so at low CO2 concentrations. Finally, many of the genes most upregulated by low CO2 are also responsive to Fe starvation. While these results are preliminary, they suggest that the CCM system is directly interacting with other cellular modules and metabolic pathways. In summary, several molecular components of the CCM in marine diatoms were identified and functionally characterized. These were integrated into a full cell model of the CCM that is fully consistent with current knowledge about the diatom CCM (Fig. 2). The model can be used to determine the energy requirements of a CCM and to predict the response of the CCM to environmental changes.
