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.

Carotenoids are isoprenoid pigment molecules with a long hydrocarbon skeleton distinguished by its system of conjugated double bonds. They are essential to photosynthetic organisms because of their ability to prevent photo-oxidative damage, and they also harvest light energy for photosynthesis. Chlamydomonas reinhardtii carotenoid-deficient mutants were generated using UV and DNA insertional mutagenesis and screened for pigment accumulation. Mutants that accumulate phytoene, zeta-carotene, or prolycopene were isolated and found to be genetically linked to the predicted phytoene desaturase (PDS1), zeta-carotene desaturase (ZDS1), and carotene isomerase (CRTISO1) genes, respectively.

The first C. reinhardtii pds1 mutant isolated, pds1-1, is a weak allele for phytoene desaturation; it is light-green and accumulates phytoene and downstream colored carotenoids. Two white mutants that appear to be null alleles of PDS1, pds1-2 and pds1-3, were identified by their phytoene accumulation and complete lack of colored carotenoids. Compared to white psy mutants, which also lack all colored carotenoids, phytoene-accumulating mutants exhibited slower growth rates in the dark and reduced plating efficiencies, indicating that phytoene accumulation may be deleterious. Three classes of pds1-2 suppressor mutants were identified that synthesized elevated levels of colored carotenoids and were less light sensitive than their parental strain. The pds1-2 suppressor mutants had mutations affecting amino acids at positions 64, 90, and 143 in the predicted PDS protein: pds1-4 (L64P), pds1-5 (L64F and E143K), and pds1-6 (L64P, K90M, E143K). Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 64 and 90 help maintain the structural stability and/or activity of the PDS enzyme.

Dark-grown C. reinhardtii zds1 and crtiso1 mutants are yellow in the dark and accumulate zeta-carotenes or prolycopene, respectively, but with gradual illumination they can turn green and accumulate higher levels of xanthophylls. In the case of crtiso1, dark-accumulated prolycopene can be photoisomerized and efficiently converted to downstream photoprotective carotenoids in the light. The presence of xanthophylls in dark-grown zds1 and crtiso1 null mutants provides evidence for an alternative carotene desaturase in C. reinhardtii. A suppressor of the zds1 mutant, zSup63, with a dominant very-green-in-low-light phenotype was isolated. Transcript levels for a putative protein (protein ID 516552) with homology to plant and algal CRTISO proteins were elevated at least ten-fold in light-grown zSup63 cells compared to the parental zds1 strain. A low light-sensitive, dark-green DNA insertional mutant (CAL028.02.09) with a disruption in the gene encoding protein 516552 has been identified and analysis of double mutants will determine if this protein is responsible for xanthophyll synthesis in dark-grown zds1 and crtiso1mutants.

The collection of early carotenoid biosynthetic mutants reported here, which accumulate cis-configured carotenes, conclusively demonstrate that carotenoids in C. reinhardtii are primarily synthesized by a plant-type poly-cis carotene desaturation pathway, making C. reinhardtii ideal for investigating carotenoid biosynthesis and regulation (and the processes that intersect with them).

Photosynthetic organisms must balance light absorption and light utilization. Excess absorbed light leads to the production of reactive oxygen species that can cause photo-oxidative damage to the cell and even cell death. To counteract the excess absorbed light, plants and algae turn on the rapid component of non-photochemical quenching (NPQ) known as qE to dissipate the extra energy as heat. From studies of higher plants, it is known that three factors are needed for the activation of qE: 1) de-epoxidized xanthophylls synthesized by a xanthophyll cycle, 2) a high proton gradient across the thylakoid membrane, and 3) the PsbS protein, a member of the light-harvesting complex (LHC) protein superfamily. Although the first two components are also involved in qE in the green alga Chlamydomonas reinhardtii, PsbS does not appear to play a role in quenching. The role of a different protein, called light-harvesting complex stress-related (LHCSR), was demonstrated by molecular genetic analysis of the npq4 mutant of Chlamydomonas. This mutant is deficient in qE and lacks two closely linked LHCSR genes (LHCSR3.1 and LHCSR3.2) that encode identical LHCSR3 proteins. A third gene, LHCSR1, encodes a second LHCSR isoform that is also involved in qE. The LHCSR1 protein is overexpressed in a suppressor of npq4 that partially restores the qE capacity of the mutant. A loss-of-function lhcsr1 mutation was isolated using reverse genetics, and an lhcsr1 npq4 double mutant exhibited no qE. Thus, all qE in Chlamydomonas is dependent on LHCSR proteins. Based on biochemical and biophysical analysis of a reconstituted LHCSR3 pigment-protein complex and site-directed mutagenesis of LHCSR3, LHCSRs have a dual function in Chlamydomonas, acting as sensors of lumen pH and as the sites of quenching, unlike in higher plants where the role of high light sensing is attributed to PsbS and the sites of quenching are in the minor and/or major antenna complexes. These findings have interesting implications for the evolution of qE in the green lineage.

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.

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.

This 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.

Chapter 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.

In 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.

Chapter 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.

Over 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.

Plants are able to dissipate excess absorbed energy as heat through several mechanisms collectively termed non-photochemical quenching (NPQ). Because these pathways can compete with photochemistry, they must be tightly regulated in order to optimize photosynthesis. In high light, the primary component of NPQ is feedback de-excitation (qE), which is known to require the protein PsbS. In order to investigate additional NPQ pathways and to identify novel proteins involved, I performed a suppressor screen in PsbS-deficient (npq4) mutants of Arabidopsis thaliana. One of the mutants identified, called suppressor of quenching 1 (soq1), displayed considerably higher NPQ relative to npq4 plants after illumination with high light.

The characteristics of the NPQ demonstrated by the soq1 mutant point to a new component of NPQ that has not been previously described. The NPQ seen in soq1 plants had slower induction and relaxation kinetics relative to qE, relaxing over the course of several hours. Additionally, fluorescence measurements of soq1 single mutants in an otherwise wild-type background demonstrated an additive capacity for quenching, and this quenching was dependent on the light intensity. The additional quenching formed independently of both changes in the pH gradient and zeaxanthin formation based on treatments with nigericin and dithiothreitol, respectively. The additional NPQ in soq1 was also still observed in xanthophyll mutants, and the pigment composition was identical in wild type and soq1 leaves before and after illumination. State transitions are also not involved in soq1 quenching, as quenching occurred in the absence of the STN7 kinase and did not cause changes in phosphorylation of thylakoid proteins or the 77 K fluorescence spectra when compared to wild type. Increased damage to reaction centers is unlikely to be the cause of quenching, as determined by treatment with lincomycin, light curves, and a seemingly wild-type growth phenotype in high light. Using time-resolved fluorescence, it was shown that quenching in soq1 mutants was the result of a decrease in average lifetime of excited chlorophyll.

The soq1 mutation was mapped to an uncharacterized chloroplast protein containing three domains: a haloacid dehalogenase-like hydrolase (HAD), a thioredoxin-like (Trx-like) fold and a beta-propeller. The NPQ phenotype of T-DNA lines and complementation with the wild-type SOQ1 gene confirmed that this locus was responsible for the soq1 phenotype, and that the soq1 mutants obtained by mutagenesis were loss-of-function alleles. SOQ1 is found in all green plants with the exception of prasinophytes and certain species of algae and monocots that have unique domain architecture. Trx-like and beta-propeller domains are also found together in diverse groups of organisms including animals, bacteria and archaea. I demonstrated that SOQ1 is a thylakoid membrane protein with both Trx-like and beta-propeller domains located in the lumen and the HAD domain in the stroma. Successful complementation with a truncated SOQ1 protein lacking the HAD domain indicated that it did not contribute to the formation of the NPQ seen in soq1 plants.

To determine the role of SOQ1 in the thylakoid and how it prevents NPQ, biochemical and high-resolution imaging techniques were used to explore differences between soq1 and wild-type plants. SOQ1 was shown to bind thylakoid proteins in plants treated with high light, however these interactions did not affect the composition of supercomplexes. To identify these interacting proteins, co-immunoprecipitation and yeast two-hybrid analysis were performed and indicated that SOQ1 might interact with HCF136 and At2G26340. Fluorescent labeling of reduced thiols using monobromobimane could lead to additional targets of SOQ1. Electron microscopy (EM) showed that soq1 plants did not appear to have significantly different chloroplast or thylakoid structures, except for a possible accumulation of plastoglobules in the soq1 background. When investigated using atomic force microscopy (AFM), the organization of supercomplexes within the grana was unique in soq1 mutants in both dark-adapted and high light-treated plants.

The role of the HAD domain of SOQ1 has also been investigated. I have shown that both the full-length protein and the HAD domain alone can act as phosphatases on small molecules. Initially, I demonstrated that SOQ1 removed phosphate groups from a general phosphate substrate, p-nitrophenyl phosphate. Using the malachite green assay, I narrowed SOQ1's substrate specificity to a pentose sugar phosphate. The highest activity was seen using ribose-5-phosphate as the substrate, and this activity was redox regulated and had a pH optimum near 7. Initial characterization of the sugar content of wild type and soq1 mutants may improve our understanding of the role of the HAD domain in vivo.

Other aspects of NPQ that I have investigated include the dynamic mechanical changes to the thylakoid that occur upon illumination. Using a modified AFM instrument, changes in the stiffness and height of thylakoid membranes were followed in real time. Upon illumination with photosystem II-specific light, an immediate increase in membrane stiffness was observed that was attributed to lumen expansion. Visco-elastic measurements suggest that some undetermined interaction between opposing lumen membranes was involved in this change. After a short delay, the change in stiffness was followed by an increase in the height of the membrane. Both the change in stiffness and height required formation of the pH gradient as determined by the addition of an uncoupler during the measurements. Using measurements on the stn7 mutant that is incapable of phosphorylating antenna complexes, the height change was shown to result from membrane unstacking.

The identification and characterization of a novel quenching pathway and the use of new AFM techniques to monitor dynamic changes in the thylakoid membrane will lead to a better understanding of how plants regulate photosynthesis.

Protein oxidation is ubiquitous throughout the tree of life and is important for all organisms to respond to both biotic and abiotic stress conditions. This is especially true in photosynthetic organisms, because they must contend with a daily onslaught of variable light conditions. Under optimal photon flux densities, a plant or alga can use the majority of the light for photochemistry. However, photosynthetic organisms are often inundated with excess light that cannot all be used photochemically due to sink constraints. This excess absorbed excitation energy must be dissipated or else the oxidative stress, caused by the formation of reactive oxygen species (ROS), would cause detrimental damage. These organisms have evolved a number of different mechanisms to dissipate excess energy, collectively known as non-photochemical quenching (NPQ). However, even with these safeguards, excess energy remains and ROS are still formed. Over the last few decades, research has shown that ROS may be more than just damaging to the cell. In fact, ROS can function in a second level of defense through protein oxidation-induced regulation and signaling. To investigate the effects of protein oxidation in photosynthetic organisms, I completed a proteomic analysis to identify cysteine oxidation sites in both Nannochloropsis oceanica cells and Arabidopsis thaliana chloroplasts, and I characterized cysteine-modified mutants that affect target proteins identified from the proteomics.

In N. oceanica, I identified several hundred proteins containing the primary oxidized state of cysteine, cysteine sulfenic acid (Cys-SOH), from three light conditions: dark, low light (LL), and high light (HL). Additionally, the proteomic analysis showed an increase in the number of Cys-SOH modifications with increases in light intensity. From this screen, several targets were selected for reverse genetics. CRISPR/Cas9 ribonucleoprotein-mediated homologous recombination was used to knockout (KO) these target genes. Four mutants showed an NPQ phenotype, with lhcx1 standing out above the rest. The lhcx1 KO exhibited a complete loss of the rapidly reversible, feedback de-excitation component of NPQ (qE), demonstrating that it is central to early NPQ induction. I transformed the mutant with the wild type LHCX1 gene (lhcx1+WT) as well as two modified versions of the gene: a cysteine to alanine mutant (lhcx1+C162A) to mimic the reduced state of the cysteine and a cysteine to serine (lhcx1+C162S) to mimic the Cys-SOH state. These modifications were chosen to determine what role the cysteine oxidation plays in the function of this protein.

Analysis of the cysteine-modified transgenics showed that LHCX1 is regulated by the oxidation state of its lone cysteine residue. In the lhcx1+WT and lhcx1+C162A lines, there was a recovery of qE back to the wild type state. In contrast, this recovery was not complete in the lhcx1+C162S line, which showed only ~60% of wild type total NPQ, and there was an additional sustained quenching phenotype: up to 50% of the total NPQ was slowly reversible compared to only 20% in lhcx1+WT and lhcx1+C162A. This sustained quenching was reminiscent of the qZ type of NPQ, which is slow to relax and dependent on zeaxanthin (Zea) formation. I examined Zea levels to determine if this higher qZ quenching was due to overaccumulation of Zea and found that there were no major differences. To determine if this sustained quenching would occur in the wild type, the period of actinic light was increased to allow for ROS build up. After 20 min, the level of qZ in the lhcx1+WT line had increased to ~40%, while qZ in lhcx1+C162A, which cannot be oxidized, remained low at 20%. These results strongly suggest that the oxidation of C162 to the Cys-SOH state modulates the quenching dynamics from the qE state to a qZ state. Based on protein modeling, it is possible that this switch is caused by a structural change in LHCX1, which directly alters the Zea binding affinity and causes the relocation of Zea away from the qE site(s) on LHCX1 toward qZ sites most likely on VCP type proteins.

In A. thaliana, I focused on the chloroplast proteome, as we are most interested in light-induced oxidation reactions. Hundreds of proteins with the Cys-SOH modification were identified from the three light conditions (dark, LL, and HL). Similar to N. oceanica, there was an increase in the number of Cys-SOH modified proteins with increases in light intensity, with the HL samples having twice as many modified proteins as the dark samples. The HL sample has an enrichment of proteins, based on GO term analysis, that are involved in translation, transport, and phosphorylation, which might be linked to downstream responses to oxidative stress. Based on the proteomics results, T-DNA lines were acquired in photosynthesis-associated targets for further characterization of possible Cys-SOH regulation. Three lines (lhca6, prxq, and atr2) had NPQ phenotypes, and one line (cyp38) had a strong growth phenotype. lhca6 had the strongest NPQ enhancement in both LL-grown and HL-treated conditions, so it was transformed with the wild-type LHCA6 gene (lhca6+WT) as well as the two cysteine-modified versions (lhca6+C58A and lhca6+C58S). The NPQ phenotype of lhca6+WT and lhca6+C58A both returned to the wild-type level, but the lhca6+C58S stayed at the level of the lhca6 T-DNA line. This is consistent with the possibility of Cys-SOH oxidation causing inactivation of LHCA6. However, this T-DNA line was only a knockdown with highly variable levels of mRNA, ranging from 20-65% of WT, so for complete confidence in the phenotype analysis of a complete KO is required.

NPQ dissipates excitation energy. If not tightly regulated this process could dissipate usable energy when light levels return to the optimal range. The oxidative regulation of proteins associated with NPQ could represent a second level of control, thereby preventing sustained NPQ quenching during short bursts of HL but allowing for greater overall quenching during longer periods of excess light. Additionally, the activation or inactivation of proteins by Cys-SOH formation could play a major role in many signaling and direct stress responses, beyond the light-mediated stress response examined here.