Photoprotective and Photosynthetic Mechanisms of Eustigmatophyte Microalga, Nannochloropsis oceanica

Photosynthetic stramenopiles (stramenochromes) are a very diverse group of algae. They range from microscopic phytoplankton that serve as primary producers in oceanic environments to macroscopic seaweeds that provide food worldwide. Stramenochromes originated from secondary endosymbiosis involving a red-algal ancestor and exhibit unique features that make them different from well-studied green algae and plants. Because they occupy such a wide range of ecological niches, they have evolved different strategies in photosynthesis and photoprotection to regulate light harvesting under varying environmental conditions. Chapter 1 highlights these unique features and strategies among stramenochromes while encompassing classes beyond well-known groups like Bacillariophyceae and Eustigmatophyceae. Subsequent chapters focus on a particular Eustigmatophyte microalga that has emerged as a model organism, Nannochloropsis oceanica CCMP1779.Nannochloropsis oceanica is a stramenochrome that can produce high amounts of lipids including the valuable omega-3 fatty acid, eicosapentaenoic acid. This dissertation focuses on the mechanisms of photoprotection and photosynthesis in N. oceanica, holding potential applications in enhancing photosynthetic efficiency in mass cultures and algae farms. This begins with Chapter 2 which characterizes and investigates the mechanisms of a process known as non-photochemical quenching (NPQ), which dissipates excess absorbed light energy as heat. In this chapter we use chemical inhibitor treatments and mutant analysis to reveal that LHCX1 is crucial for qE induction, whereas LHCX2 and LHCX3 do not seem to play a role in NPQ in low-light grown cells. We also show that the vde mutant, deficient in the synthesis of the photoprotective pigment zeaxanthin, lacks both qE and qZ. These results show that photoprotection in N. oceanica is highly dependent on the formation of zeaxanthin and its collaboration with LHCX1. Chapter 3 focuses on resolving the molecular function of LHCX1 by testing the hypothesis that LHCX1 is involved in sensing a decrease in thylakoid lumen pH in excess light. Knock-in mutations targeting putative pH-sensing sites were generated in the native LHCX1 gene, although they revealed no discernable impact on qE capacity. This aligns with previous results in diatoms but is different from similar proteins in the green algae and plants, suggesting functional divergence in stramenochromes. Moreover, generation and analysis of the W143M mutant revealed that a mutation in this amino acid doesn’t impact a putative pigment binding site involved in qE. This contrasts with previous results in diatoms, suggesting species-specific divergence in N. oceanica. This work prompts a reconsideration of ΔpH-sensing mechanisms in photosynthetic organisms. While the overall function of LHCX1 in qE remains elusive, strides are made towards resolving its structure in Chapter 4. Chapter 4 focuses on developing methods towards unraveling the architecture and protein makeup of PSI-VCPI and PSII-VCPII supercomplexes that contain LHCX1. To do this, high-resolution single-particle cryogenic electron microscopy (EM) will be used. Developing the methods towards cryo-EM involves many steps, which were optimized in this chapter. Overall findings indicated that using maltose density gradients with gradient fixation (GraFix) improves sample quality and supercomplex isolation. These methods will be used for structural analysis to reveal the overall architecture, protein-protein interactions, and pigment locations in the photosynthetic supercomplexes, leading to inferences on energy-transfer and energy-dissipation pathways in N. oceanica and other diverse algae groups.

Advisor: Niyogi, Krishna K