Exploring new fronts in cell biology through a discovery-based study of a new model organism, Nannochloropsis oceanica (Eustigmatophyceae).

Photosynthesis drives, directly or indirectly, the metabolism of the vast majority of living things on earth, and thus it has a central role in supporting ecosystems and human societies As such, photosynthesis and the organisms that perform it have been the subject of extensive research, and these efforts have yielded impressive gains in our understanding of the fundamental concepts underlying this critical metabolism. However, most of this knowledge originates from studies of a select few model organisms from the green lineage (Viridiplantae), despite the much wider diversity that exists in photosynthetic life, including algae with secondary plastids that predominate in marine environments. With technological advances in high-throughput DNA sequencing, genome editing, molecular imaging, etc., the research community is poised to expand the knowledge gained from traditional models like Arabidopsis thaliana to new, under-studied taxa that may hold key insights into the diversity of photosynthetic life./nThis dissertation exemplifies such a mindset. Nannochloropsis oceanica (Eustigmatophyceae) is a unicellular, ochrophyte nanoplankton known for producing large quantities of lipids, including the valuable polyunsaturated omega-3 fatty acid, eicosapentaenoic acid. With a sequenced genome and a variety of genetic tools available, research into members of this genus has increased rapidly through the 2010s. While much of the published literature focus on optimizing the biosynthesis of lipids, studies such as the ones set forth here demonstrate how novel findings for basic research can be gleaned from studies of new model algae like Nannochloropsis./nChapter 2 details work related to the carbon-concentrating mechanism (CCM) in N. oceanica CCMP 1779. Knock-outs of CCM gene candidates were generated through homologous recombination, and the alpha-type carbonic anhydrase (CAH1) was found to have a severe high-CO2-requiring growth phenotype. Further characterization with a Venus fluorescent protein tag demonstrated a primarily ER localization for CAH1, and experiments with predicted catalytic null mutants strongly support its function as a carbonic anhydrase. The working model to emerge from these findings, in combination with published physiological studies, is a pyrenoid-less “pump-leak” CCM that transports bicarbonate into the ER lumen where CAH1 catalyzes its conversion to CO2, which may diffuse into the chloroplasts to Rubisco for fixation or back out to the environment. This type of CCM is a departure from the high-efficiency CO2 re-capture mechanisms proposed for the CCM in Chlamydomonas and for carboxysomes in cyanobacteria./nIn Chapter 3, I report on findings related to mutants generated in a predicted cellulose synthase gene, CESA2. Nannochloropsis possesses a thick inner, cellulose cell wall within a thinner outer layer made of the hydrophobic material, algaenan. No experimental evidence has been published regarding the possible function of the different CESA genes in this species. In this work, a ~150-bp deletion of the coding sequence of CESA2 led to reduced growth rates (perhaps particularly severe in diurnal conditions), abnormal cell shape, and loss of synchronous division in diurnal conditions. This preliminary work sets the stage for follow-up validation and elaboration, and it starts to provide functional insight that complements published diurnal and circadian RNA expression studies of this gene. This work also forms a starting point for future assessments of redundancy and interaction with the other putative cellulose synthases and studies of the interaction between the cellulose and algaenan cell wall layers./nChapter 4 presents a discovery-based series of experiments aimed at characterizing the red body of Nannochloropsis oceanica. This pigmented subcellular structure has been noted sporadically in the literature, and appears to be found in most eustigmatophyte algae, yet next to nothing is known about its biogenesis, composition, or function. Here, it was found to exhibit fluorescence, and this property was utilized to track its biogenesis from chloroplast-associated to apoplastic during the course of diurnal cell division. Membranes surrounding the structure were visible by electron microscopy. It was found to contain tocopherol(s) and carotenoids, including the ketocarotenoid canthaxanthin, and a ketocarotenoid-over-accumulating mutant was generated that produced abnormally large numbers and sizes of red bodies. FTIR analysis of isolated red bodies indicates that they have a similar chemical composition to that of shed outer walls made of algaenan. The red body of Nannochloropsis was observed to be shed with the autosporangial outer wall, a property that was shared with a species located on a nearby phylogenetic branch, Monodus unipapilla, but not others for which a red body was observed only intracellularly. Our current hypothesis is that the red body in Nannochloropsis is involved in the formation of the algaenan cell wall, perhaps by delivering precursor molecules to the apoplast for polymerization into mature algaenan. Possible implications for kerogen formation and global carbon cycling are discussed./nOver the course of completing this work, a few themes emerged. First, even in a relatively simple, unicellular organism like Nannochloropsis, the aspects of time, cell cycle, and development are critical to interpreting results and fully understanding most cellular processes. In all three chapters, the characteristic being measured varies with time of day and developmental stage of the cells. Second, cellular processes are, perhaps unsurprisingly, intimately connected with one another. Chapters 3 and 4 are particularly close in proximity, as they both are concerned with the cell wall. Lastly, each of these chapters has provided a novel, if sometimes subtle, addition to our current framework of thinking, from the alternative arrangement of CCM components, to rethinking cellulose synthase complex subunit combinations, to possibly adding another known step between sunlight and fossil fuels. In this way, Nannochloropsis, Eustigmatophyceae, and “new” model organisms in general, offer us fresh avenues of inquiry that may lead us into the coming decades where new mysteries and phenomenon await investigation.

Advisor: Niyogi, Krishna K