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Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2016)

The initial step of photosynthesis occurs when light energy is absorbed by an array of pigments surrounding the photosynthetic reaction center. Chlorophyll and carotenoid molecules are coordinated by members of a family of intrinsic thylakoid membrane proteins known as Light-Harvesting Complex (LHC) proteins. LHCs are essential for stabilizing and tuning the spectroscopic characteristics of each individual pigment, as well as the pigment array, to allow for efficient energy transfer. Despite a high degree of sequence conservation within the protein family, each of the LHCs performs a different role in modulating the energy landscape of the thylakoid membrane. Two stress response LHC proteins, LHCSR and PSBS, are known to be essential for non-photochemical quenching in some photosynthetic eukaryotes. It has been shown that LHCSR is necessary for quenching in the green alga Chlamydomonas reinhardtii. Homologs of this stress-response protein are present in many photosynthetic eukaryotes, even those with chloroplasts that are of a red algal origin, but LHCSR is absent from terrestrial vascular plants. Likewise, PSBS is necessary for quenching in the plant Arabidopsis thaliana, but PSBS is only found in the green lineage of photosynthetic organisms. The molecular mechanism of both of these proteins is still unknown.

This work describes the relationship between protein structure and quenching function in the stress response protein LHCSR. I show the distinct contributions to non-photochemical quenching from each of the two LHCSR protein isoforms, LHCSR1 and LHCSR3, in C. reinhardtii. I also contributed to the identification of three lumen-exposed residues in LHCSR3 that are required for quenching function. By using directed mutagenesis and expression in heterologous and native organisms, this shows that while LHCSR is necessary for nearly all non-photochemical quenching in its native cellular environment within C. reinhardtii, it is not sufficient to activate quenching in plants when expressed heterlogously. By altering some of the pigment-binding sites within LHCSR3, however, the heterologously expressed mutant LHCSR3 can activate additional quenching in a plant system.

This also explores possible roles of the PSBS protein from C. reinhardtii, which has not previously been shown to have a function. Here, I present data showing that the algal PSBS protein can enhance non-photochemical quenching when expressed heterologously in plants. Since we do not see the same behavior in the alga, if PSBS is expressed in C. reinhardtii it most likely plays a different role or is not sufficient to induce quenching alone. Heterologous co-expression of both LHCSR and PSBS from C. reinhardtii results in enhanced quenching in the tobacco, Nicotiana benthamiana. The enhanced quenching phenotype is dependent upon PSBS being pH-sensitive. While no evidence has yet been obtained showing an interaction between PSBS and LHCSR in C. reinhardtii, this motivates further exploration.

In order to better understand the evolution of photoprotective mechanisms employed by photosynthetic eukaryotes, I have also contributed to a genome annotation project aimed at establishing the marine heterokont alga, Nannochloropsis oceanica CCMP 1779, as a new model alga for studying photosynthesis. I identified three potential stress-response LHCs in the organism’s genome based on sequence homology, whose functionality and expression characteristics are being investigated by other researchers.

    Cover page: Exploring the Role of LHC Protein Structure and Function in the Evolution of NPQ Mechanisms in Eukaryotic Photosynthetic Organisms
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2016)
    Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non-photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor-intensive and time-consuming generation of stable transgenic lines and mutant populations - approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ-related genes from diverse organisms, we performed Agrobacterium tumefaciens-mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.
      Cover page: Transient expression in Nicotiana benthamiana for rapid functional analysis of genes involved in non‐photochemical quenching and carotenoid biosynthesis
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2016)
      Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green algaChlamydomonas reinhardtii Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesisin vivoandin vitrofor identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp(117), Glu(221), and Glu(224)were shown to be essential for LHCSR3-dependent NPQ induction inC. reinhardtii Analysis of recombinant proteins carrying the same mutations refoldedin vitrowith pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.
        Cover page: Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii *
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2017)
        Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.
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