Photosynthesis by plants and algae is the foundation for nearly all of Earth’s food webs. Maintaining photosynthesis is metabolically costly with a high demand for mineral nutrients. Most photosynthetic organisms live in conditions insufficient for optimal rates of photosynthesis. Consequently, diverse biochemical and genetic regulatory mechanisms have evolved to enable acclimation of photosynthesis to non-ideal conditions and thus organismal survival. Sugars are both primary products of photosynthetic fixation and signals to regulate photosynthesis gene expression in plants and algae. The emerging model green alga Chromochloris zofingiensis completely represses photosynthesis in the presence of glucose via a hexokinase 1 (HXK1) dependent pathway. Unlike other green algal models, C. zofingiensis upregulates total fatty acids and accumulates the valuable ketocarotenoid astaxanthin in either glucose-fed or nutrient stress conditions, making it a viable candidate for metabolic bioprospecting. In this dissertation, I reveal that iron deficiency, one the major limits on global photosynthesis, is required for C. zofingiensis’ photosynthetic switch off in addition to HXK1-dependent glucose signaling. Replete iron added at the same time as glucose creates a distinct mixotrophic state which concurrently maintains lipid accumulation with photosynthesis and supports the highest biomass growth rate of any other trophic state.

In Chapter 1, I briefly review sugars as signals in genetic regulation across organisms, which has been involved in some of the most foundational discoveries of molecular biology. Because the emergence of oxygenic photosynthesis drastically changed the carbon repertoire of Earth’s ecosystems, the impacts of carbon sink regulatory feedback and its interplay with mineral nutrient signaling are relevant to all experimental systems understanding sugar feedback. In Chapter 2, I employ physiology and combinatorial proteomics to distinguish the proteins that are distinctly regulated when photosynthesis is switched off from the states where lipid accumulation is activated. Incorporating orthology and cross-species multi-omics experiments reveals novel universal players in photosynthesis and lipid accumulation and reveals how glucose-fed C. zofingiensis has developed evolutionarily novel iron priorities. In Chapter 3, the implications of glucose interactions necessitated an experimental redesign of the medium, where incorporating ionomics with biomass of long-term glucose growth was used to create a medium that could maintain long-term glucose cultures in a replete nutrient state. In Chapter 4, a comprehensive multi-omics time course covering switch off and switch recovery of C. zofingiensis is analyzed to understand the regulatory cascades behind various metabolic transitions. Using time-ordered piece-wise linear models, I reveal temporally distinct molecular events that precede photosynthetic, lipid, iron, and biomass transitions. I incorporate these models to find enriched cis-regulatory elements, in particular, a domain architecture which will aid in engineering green algae to accumulate more biofuel precursors. Finally in Chapter 5, the evolutionary perspective of metabolism is also considered in a literature review with a specific focus on how two distinct algae, C. zofingiensis and Haematococcus pluvialis, accumulate astaxanthin. Phylogenomic and gene expression comparisons show a divergence in carotenoid accumulation and partitioning strategies in both organisms, which will also inform engineering of industrially useful ketocarotenoids.

As described in the chapters of this dissertation, investigating the synergistic impacts of iron and glucose with systems biology approaches provided an unprecedented ability to distinguish novel molecular players and strategies in regulating photosynthesis and lipid biochemistry. Secondly, incorporating bioinformatic predictions of orthologs across several plants and algae helped to reveal several universal players involved in photosynthetic development and divergent strategies of lipid and astaxanthin accumulation. These players will provide a framework for redesigning transcriptional networks for improved photosynthesis and metabolic engineering to enhance the environment and economy.

Carbon fixation by photosynthetic organisms provides the bulk of energy that most other life depends on. Many photosynthetic organisms can shift metabolism in order to metabolize other sources of energy when necessary. The unicellular, Chlorophycean alga, Chromochloris zofingiensis, is one example of a photosynthetic organism with metabolic flexibility. When supplemented with glucose, C. zofingiensis will utilize both photosynthesis and glucose as an energy source. In specific culture conditions, supplemental glucose causes the total loss of photosynthetic capacity. Additionally, this phenomenon is reversible, and C. zofingiensis is able to rapidly regenerate the photosynthetic apparatus if glucose is removed. This observation, that glucose can trigger a reversible collapse of photosynthesis, provides the opportunity to investigate fundamental metabolic and signaling processes and the genes required to rebuild photosynthetic capacity. Chapter 1 introduces C. zofingiensis, its scientific history, and places it in the context of current research.

Chapter 2 presents results of a two-phase experiment, in which we driveC. zofingiensis into a state of photosynthetic repression using glucose in the first phase. In the second phase, we release that repression by removing glucose. We observe the severe impact on photosynthetic capacity, where oxygen evolution stops occurring and fluorescence signatures of a functional photosystem decrease to near zero. The proteins and lipids required for photosynthesis are greatly diminished. Cells grow larger in glucose, but thylakoid membranes in the chloroplast shrink, while storage molecules in the form of triacylglycerols and starch increase, as well as the keto-carotenoid, astaxanthin. In the second phase, when these cells are transferred into a glucose-free medium, photosynthetic repression is released, and the cells restart photosynthesizing within 12 hours and continue to regreen over the course of 48 hours. We analyze and observe gene expression during critical metabolic transitions and find a wholesale transcriptional reordering in glucose conditions that results in the differential expression of 41% of the genes in the genome. We find that glycolysis, lipid metabolism, chlorophyll biosynthesis, and the expression of photosynthetic proteins are tightly controlled by the presence or absence of glucose. This chapter presents C. zofingiensis as a model system to understand metabolic flexibility between photoautotrophic and heterotrophic modes, furthering our understanding of the regulation of algal metabolism. This work will facilitate engineering efforts to reroute metabolism towards beneficial bioproducts for energy, food, and potentially pharmaceuticals.

In Chapter 3, we seek to understand genetic determinants of trophic flexibility in C. zofingiensis. We utilize a genetic selection to identify mutants thatare unable to shift their trophic strategy when supplemented with glucose. UV- mutagenized cells were plated on medium containing the glucose analog, 2- deoxy-D-glucose, which induces photosynthetic repression, but is unable to be metabolized by C. zofingiensis. We identify potential causative mutations using whole-genome sequencing and find that all eight mutants generated and investigated have mutations in the critical glucose-phosphorylating enzyme, hexokinase (HXK1). We further characterize two of these independently generated mutants, which share the same hxk1 mutation. When treated with glucose, photosynthetic capacity, oxygen evolution, and proteins related to photosynthesis of hxk1 mutants are all much more similar to non-glucose-treated wild-type C. zofingiensis. The expression of genes representative of key pathways described in chapter 1, such as HXK1, a light-harvesting antenna protein (LHC16), A fatty acid de-esterase (FAD2), and the major lipid-droplet storage protein (MLDP1), is unresponsive to glucose. This provides the basis for our model of glucose-induced repression of photosynthesis, where the cellular response to glucose is mediated by HXK1.

Finally, in Chapter 4, we describe the potential of C. zofingiensis as an inducible model system of oxygenic photosynthesis to study thylakoid biogenesis. Utilizing a comparative genomics framework coupled with extant co- expression data for genes of the model plant Arabidopsis thaliana and data from a mutant library in model green alga Chlamydomonas reinhardtii, which contains many potential photosynthetic defects, we identify genes likely to be essential for thylakoid biosynthesis. By comparing sets of C. zofingiensis genes that are differentially expressed during the transition from glucose to non-glucose conditions to the A. thaliana and C. reinhardtii gene sets, we identify sets of genes, many of which are well-known components of thylakoid biogenesis, whereas others have roles that have yet to be described in thylakoid biogenesis. This work utilizes a recently developed bioinformatic tool called OrthoLang and presents a systematic approach to the integration of disparate data types, including RNA-seq data, a sequenced mutant library, and co-expression analysis.In summary, C. zofingiensis is emerging as a powerful model organism to study multiple aspects of photosynthetic metabolism. Signals that rewire metabolism in the presence of glucose pass through the HXK1 and result in global photosynthetic repression. Release of that repression allows C. zofingiensis to rapidly recover photosynthesis, providing an additional window into thylakoid biogenesis.

As atmospheric CO₂ rises, increases in photosynthesis and plant growth are routinely documented across ecosystems globally. Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO₂ fertilization responses by linking CO₂ assimilation with growth. However, little information is available on the in vivo activity of the plastidic phosphorylated serine pathway. Here, we show that the serine phosphate pathway is part of a ‘photosynthetic C₁ pathway’ and demonstrate its high activity in foliage of a C₃ tree where it rapidly integrates photosynthesis directly with C₁ metabolism contributing to new biomass via methyl transfer reactions and imparting a large natural ¹³C-depleted signature. Using ¹³CO₂-labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C₁ metabolism, unrelated to photorespiration, within minutes of light exposure. We speculate that the photosynthetic C₁ pathway and its key enzyme methionine synthase is highly conserved across the photosynthetic tree of life and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon assimilation with growth. Although the rise in atmospheric CO₂ inhibits major metabolic pathways like photorespiration in C₃ plants, our results suggest that the photosynthetic C₁ pathway may accelerate and represents a ‘missing link’ between enhanced photosynthesis and growth rates of modern plants and ecosystems during terrestrial CO₂ fertilization under a changing climate.