UNDERSTANDING REGULATION OF PHOTOSYNTHESIS FOR OPTIMIZING PRODUCTION OF BIOFUELS AND AGRICULTURE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1000766
• Grant No.: 2013-67012-21272
• Project No.: CA-B-2013-MSR
• Proposal No.: 2013-03174
• Program Code: A7201
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2016
• Grant Year: 2013

Project Director
Roth, M. S.

Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720

Performing Department
Plant and Microbial Biology

Non Technical Summary
The growing human population creates an increasing demand for food and energy while exacerbating environmental problems including global climate change. The increased pressures on the environment and the global depletion of oil reserves, however, have created a less stable environment for producing food and a need for renewable energy resources. Microalgae biofuels have tremendous potential to become a major source of future energy, because microalgae utilize solar energy, are fast growing, and can be produced on non-arable land. Biofuels from microalgae feedstocks can currently be produced, but there are considerable limitations from high costs and low productivity. Because photosynthesis, both of biofuel feedstocks and agricultural crops, operates far below the theoretical yield, there may be approaches to optimize production. The subject of the proposed research- a green microalga- is unique in that it has the potential to be used for biofuels and as a food supplement (an antioxidant for human consumption). Using algae that are deficient in regulating photosynthesis, this project will elucidate the genes and mechanisms involved in photoprotection and biogenesis of the photosynthetic unit. Not only will understanding regulation of photosynthesis near the base of the extant green lineage provide insights into basic mechanisms of photosynthesis, but modifying photosynthetic regulatory mechanisms may be used to enhance biofuel and agricultural production through increasing yields and/or engineering algae and plants that are less susceptible to environmental stressors.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	4099	1103	25%
206	4099	1020	25%
206	4099	1040	25%
206	4099	1000	25%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1020 - Physiology; 1040 - Molecular biology; 1000 - Biochemistry and biophysics; 1103 - Other microbiology;

Keywords

rna-seq

algae

biofuel

biogenesis/breakdown of chlorophyll

photoprotection

photosynthesis

Goals / Objectives
The overall goal of the project is to elucidate the mechanisms of photosynthesis regulation. In particular the main objectives are 1) determine how high photoprotection capacity is achieved, and 2) determine the genes involved in biogenesis/breakdown of chlorophyll complexes. Specific Goals: Develop high quality RNA extraction protocol for M. zofingiensis Develop transformation protocol for M. zofingiensis Determine the genetic basis for npq mutants Conduct NPQ experiments and analyses Rescue npq mutants and conduct experiments Conduct TA/TCSPC measurements Conduct glc RNA-seq experiments and analyses Determine the genetic basis for glc mutants Rescue glc mutants and experiments

Project Methods
Approach for elucidating high capacity NPQ mechanisms Using wild type, mutant, and rescued mutant algae in the following experiments will reveal the NPQ mechanism used to attain high capacity NPQ and associated photoprotection. My proposed research for this objective includes two main sets of experiments and uses ultrafast spectroscopy to measure biophysical interactions. Experiment 1 will test the photoacclimation capacity and biomass productivity of wild type, mutant, and rescued mutant strains of M. zofingiensis grown under low, medium, and high light conditions (respectively 40, 90, and 400 µmol quanta m-2 s-1) and measure NPQ capacity, photosynthetic efficiency, photosynthetic pigments, rates of photosynthesis, growth and survivorship. Results will be analyzed with student t-tests and analysis of variance (ANOVA) when appropriate. This experiment will demonstrate the differences between NPQ capacities and elucidate what mechanisms are used in high NPQ. Based on preliminary experiments, I expect medium light to have the highest NPQ because high light appears to turn on different photoprotective mechanisms including high abundances of carotenoids (data not shown). If the mutant has a defective LHCSR gene and the rescued mutant with the wild-type LHCSR gene has high NPQ capacity, then it would suggest that LHCSR is important for the NPQ mechanism. Experiment 2 will test the photoprotection capacity of wild type, mutant, and rescued mutant strains by acclimating them to low, medium and high light conditions and then exposing them to excess light (1,100 µmol quanta m-2 s-1) for at least 4 hours. Cells will be plated and placed under high light. After a week, I will measure NPQ capacity and survivorship. Control samples will not be exposed to excess light. A similar set of measurements and statistical analyses as used in experiment 1 will be employed in experiment 2. This experiment will reveal if more NPQ was used during light stress and whether higher NPQ capacity was correlated with survivorship, which would suggest that the NPQ photoprotection mechanism is essential for maximal growth and survivorship during dynamic light conditions. Preliminary experiments show that a npq mutant had reduced survivorship as compared to wild type. Recent experiments on Chlamydomonas reinhardtii in our laboratory have shown that over-expression of LHCSR was able to nearly double NPQ capacity in Chlamydomonas (Niyogi, unpublished data). These results suggest the hypothesis that M. zofingiensis may have extremely high expression of LHCSR. However even in over-expressed LHCSR strains of Chlamydomonas, NPQ capacity is 6-fold lower than in M. zofingiensis, which may suggest that an additional mechanism is utilized to achieve high NPQ capacity. In collaboration with Prof. Graham Fleming (UC Berkeley), we will determine how the energy is quenched and dissipated in high capacity qE. In plants, Prof. Fleming and co-workers, have found that the quenching occurs in coordination with the protein PsbS (Holt et al., 2005, Ahn et al., 2008). Ultrafast transient absorption (TA) and time-correlated single-photon counting (TCSPC) spectroscopy have emerged as powerful techniques to investigate the biophysical interactions between molecules involved in photosynthesis. We plan to use both types of spectroscopy to show the changes in excitation energy state of chlorophyll and carotenoid molecules, possibly bound to LHCSR, after they absorb light. Our laboratories have conducted TA and TCSPC on intact cells of Chlamydomonas, which provides feasibility for both methods on M. zofingiensis. In fact, it is expected to be easier with M. zofingiensis because the NPQ is much higher so the signal to noise ratio will be lower. However, if we are limited by signal, I will resolve this problem by using reconstituted LHCSR pigment-protein complex. Approach for elucidating genes involved in biogenesis/degradation of chlorophyll Using wild type, mutant, and rescued mutant algae in the following experiments will reveal the genes involved in turning off/on photosynthesis. Currently, 25 glc mutants have been isolated; however, it is possible that some of these mutants are defective in glucose uptake rather than regulation of chlorophyll complexes. To separate out the uptake mutants, I will grow the mutants on glucose plates in the dark, and those that survive will be the mutants of interest and used in the following experiments. Although there are many interesting experiments to pursue in the future, I will focus on the following two critical experiments. Experiment 1 will explore the turning off/on of photosynthesis by adding/removing glucose to wild type and glc mutant algae in light (90 and 400 µmol quanta m-2 s-1). I will conduct RNA-seq to reveal differences in gene expression and characterize pigments, proteins, photosystem I and II activity, and photosynthetic rates. Preliminary experiments have shown dramatic declines in photosystem II activity and chlorophyll content with the addition of glucose (Fig. 3). Photosystem I activity and oxygen evolution will indicate whether there is partial or complete shutting off of photosynthesis. Comparing the genes that change expression in wild type cells with the addition of glucose against the controls (both under 90 µmol quanta m-2 s-1) will reveal genes involved in the degradation of chlorophyll complexes. Comparisons of wild type algae in the control media in high light vs. medium light will account for the genes responsible for the up-regulation of carotenoids, which are likely the cause of the pink appearance (Fig 3b). RNA-seq data of glc mutants will confirm target genes revealed by wild type RNA-seq experiments. Comparing genes that change expression in wild type cells with the removal of glucose against controls remaining in glucose will define the genes involved in biogenesis of chlorophyll complexes. Experiment 2 will confirm which genes are involved in sensing glucose and turning off photosynthesis. I will add glucose to the media of the rescued glc mutants and anticipate the rapid decline of photosystem II efficiency. For each mutant, if the wild type gene restores the wild type phenotype, then that gene is responsible for shutting down photosynthesis. My plan for evaluating progress toward achieving my project objectives will be to ensure that I meet my milestones. My milestones include: submission of M. zofingiensis genome paper (10/14), determination of the genetic basis of npq mutants (3/14), completion of NPQ and analyses experiments (9/14), restoration of NPQ in npq mutants and experiments (2/15), completion of TA/TCSPC measurements with collaborator (3/15), completion of RNA-seq experiment (3/14), completion of RNA transcriptomics with collaborator (10/14), restoration of phenotype in glc mutants and experiments (3/15), completion of data analyses (7/15) and submission of NPQ and glc papers (9/15). Learning the skills and obtaining the different data sets by the established date will determine if the activity was successful. The findings will be communicated at scientific meetings and published in scientific journals.

Progress 09/01/13 to 08/31/16

Outputs
Target Audience:In 2016, seminars and presentations were given at a variety of institutions including University of Rhode Island and Sonoma State University and meeting including the US National Academy of Sciences Indonesian-American Kavli Frontiers of Science Symposium in Malang, Indonesia and the 17th International Congress on Photosynthesis Research in the Netherlands. Changes/Problems:A significant amount of time was spent on generating a far higher quality genome and transcriptome than originally anticipated as well as on the high value carotenoid biosynthesis pathway. Because of this more time was spent on these aspects of the project and analyses of genomes etc and has delayed the photoprotection aspect of the research. However, this will result in a more useful resources for the community and provide greater impact to the community. What opportunities for training and professional development has the project provided?There were a variety of training acitivies and professional development from this project. Myself as well as 4 graduate students and 1 technician and 1 high school student have attained greater proficiency in a variety of techniques from molecular biology, microscopy, biochemisty, bioinformatics, next generation exquencing etc. Addiitionally, my professional development has continued from the already mentioned seminars and conferences as well as training in communicating science via workshops. How have the results been disseminated to communities of interest?As previously mentioned, the results have been disseminated to the communities of interest through presentations at meetings and seminars and will reach larger communities when the papers are published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The growing human population generates increasing demand for food and energy. Microalgae are a promising source of sustainable bioproducts whose production may not exacerbate worsening environmental problems. The green alga Chromochloris zofingiensis has potential as a biofuel feedstock and source of high-value pharmaceutical molecules, including the carotenoid astaxanthin. In collaboration with Matteo Pellegrini's and Sabeeha Merchant's labs at UCLA, we have generated a very high quality de novo genome and transcriptome of C. zofingiensis. Through our experiments and RNA-Seq analyses, we have identified novel candidate genes for biosynthesis of the high-value pharmaceutical molecule astaxanthin. Additionally, we have done extensive genome comparisons between C. zofingiensis, the model alga Chlamydomonas reinhardtii, the model plant Arabidopsis thaliana, and 3 other algae. We have continued to develop a transformation protocol for C. zofingiensis, which in combination with the genome and the published protocol, advance understanding of the green lineage and carotenoid production and enhance prospects for improving commercial production of C. zofingiensis. Additionally, we have identified the main gene involved in switching photosynthesis and metabolism and characterized these physiological and gene expression changes in response to environmental change. Greater understanding of the regulation of photosynthesis to its changing environment may improve yield for a variety of crops and algal feedstocks. Additionally, this research has led to a much larger direction in understanding sensing and signaling in algae. We are still working on determining how photoprotection capacity is achieved. The knowledge from studying the commercially valuable C. zofingiensis is valuable not only improving its own production, but also for basic understanding of photosynthesis in the green lineage as well as enhancing production of crops and other algal feedstocks. • Develop high quality RNA extraction protocol for M. zofingiensis 1. A high quality RNA extraction protocol has been generated and allowed for high quality RNA-Seq data collection. 2. This high quality RNA was use in RNA-Seq to build a transcriptome and conduct a variety of RNA-Seq physiological experiments. Additionally, this protocol has been used by a graduate student to collect high quality RNA for qPCR validation of the RNA-Seq data as well as generate data from experiments on a subset of genes. 3. These data were used in a variety of analyses including identifying significantly differentially expressed genes, investigating gene expression changes of specific pathways, and determining functional enrichment of gene ontology and protein domains. 4. The key outcomes from this goal has been a change in knowledge for myself and others including a graduate student under my training, my advisor, my collaborators etc and these data have been included in presentations at conferences and seminars and in publications in preparation. • Develop transformation protocol for M. zofingiensis 1. During this grant, another group has published a transformation protocol. The graduate student under my training has been refining this protocol. 2. We have generated new plasmids and conducted transformation, and we are currently awaiting results. 3. We are optimizing the protocol. 4. The key outcome would be a change in knowledge and to include these results in publication, but this research is still in progress. • Determine the genetic basis for npq mutants 1. NPQ mutants were generated using forward genetics and now the mutations must be identified. 2. First candidate genes were sequenced and the mutations were not identified. We have now planned out the future sequencing and a graduate student under my training will carry out the sequencing. 3. This work is still in progress and too early for results. 4. Once the mutation(s) have been identified, this will lead to change in knowledge and understanding photoprotection. • Conduct NPQ experiments and analyses/ Rescue npq mutants and conduct experiments/ Conduct TA/TCSPC measurements This research, experiments/data/results, are ongoing and not completed yet. • Conduct glc RNA-seq experiments and analyses 1. These experiments have been completed, the RNA has been extraction and the RNA-Seq has been conducted. 2. We have completed a preliminary set of analyses but need more analyses to be completed 3. These results show changes in gene expression with an environmental change and during a photosynthetic and metabolic switch. Further analyses including functional enrichment and significant differential gene expression are in progress. 4. These results will lead to a change in knowledge in understanding regulation of photosynthesis and metabolism during environmental change. • Determine the genetic basis for glc mutants 1. We have sequenced the genome of 9 glc mutant strains and conducted SNP analyses to determine the mutation. 2. We have found a mutation in a single gene in all 9 mutant strains, with mutations in three different locations in the gene. 3. We have conducted further experiments and analyses on these mutant strains. 4. The key outcome is a change in knowledge and these results will included in the publication in preparation. • Rescue glc mutants and experiments 1. The plasmid containing the resistance cassette and gene of interest has been generated. 2. A few attempts have been made to transform this plasmid into the mutant strains, but we are still working on optimizing the protocol. 3. We are still waiting for these results to confirm the phenotype by restoring the photosynthetic and metabolic switch. 4. The key outcome is a change in knowledge and these results will included in the publication in preparation. Additional goals that have been completed include: 1. High quality de novo genome including assembly into chromosomes 2. Building and assembling transcriptome from 14 diverse conditions 3. Genome comparison between 5 algae and 1 plant 4. Synteny analyses between the above 6 organisms 5. Identification of candidate genes involved in biosynthesis of high value pharmaceuticals 6. Generation/identification of mutants in producing the high value pharmaceuticals These have been completed and the key outcomes are changes in knowledge and are included in the manuscripts in preparation.

Publications

• Type: Journal Articles Status: Other Year Published: 2017 Citation: Roth, MS*, Cokus*, SC, Gallaher, SD, Lopez, D, Walter, A, Erickson, E, Endelman, B, Westcott, D, Larabell,C, Merchant, SS, Pellegrini, M, Niyogi, KK. Submitting to PNAS. Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production. *co-first authors
• Type: Journal Articles Status: Other Year Published: 2017 Citation: Roth, MS, Iwai, M, Muller, M, Walters, A, Gallaher, S, Erickson, E, Westcott, D, Merchant, SS, Larabell, C, Auer, M, Niyogi, KK. In prep. A molecular switch for photosynthesis in a unicellular green alga.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Roth, MS, Padilla-Gami�o, JL, Pochon, X, Bidigare, RR, Gates, RD, Smith, CM, Spalding, HL. 2015. Fluorescent proteins in dominant mesophotic reef-building corals. Marine Ecology Progress Series 521: 63-79.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Roth, MS. 2014. The engine of the reef: Photobiology of the coral-algal symbiosis. Frontiers in Microbiology 5, 422.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Melissa S Roth1, Masakazu Iwai1, Maria Mueller2, Andreas Walter3, Sean Gallaher4, Daniel Westcott1, Erika Erickson1, Winnie Wong1, Shawn Cokus5, David Lopez5, Matteo Pellegrini5, Sabeeha S Merchant4, Carolyn A Larabell2, 3, Manfred Auer2, Krishna K Niyogi1. A molecular switch for oxygenic photosynthesis and metabolism in a unicellular green alga. 17th International Congress on Photosynthesis Research. Maastricht, Netherlands
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Roth, MS. 2016. Coral reef conservation through insights in physiology and photosynthesis. US National Academy of Sciences Indonesian-American Kavli Frontiers of Science Symposium, Malang, Indonesia

Progress 09/01/13 to 08/31/14

Outputs
Target Audience:The overall goal of the project is to develop a molecular toolkit for the commercially alga, Chromochloris zofingiensis, and investigate its regulation of photosynthesis. This year much progress has been made. With my collaborators, we have been working on the de novo genome of the alga and have conducted a 4 tier hybrid approach including sequencing short reads (Illumina), long reads (Pacific Biosciences), optical mapping (Opgen), and transcriptome (RNAseq via Illumina). We are currently assembling the genome and at this point it is about 95% assembled. We are finishing the assembly, and then will rerun the genome annotation models, which were currently conducted with a draft genome. This research will establish a very high quality genome with gene ontology and models, which will be critical for future work. I have conducted the physiological experiments for the breakdown and biogenesis of the chloroplast and chlorophyll complexes. The RNAseq data have been collected and will need to be reanalyzed after completion of the genome. The breakdown and biogenesis experiments have also been completed measuring activity of photosystem I, activity of photosystem II, respiration and oxygen evolution, cell size, and confocal and super-resolution microscopy. However, these data still need to be analyzed. Additionally, samples were collected for analyses of lipid, proteins, pigments, and organelle structure and volume and I am currently processing these samples. I have added collaborations looking at the transmission electron microscopy and x-ray tomography to get a better understanding of the changes of the cell. The genetic glc mutants have now been determined and work is underway to rescue the glc mutants. I also have started doing live cell imaging of the cells under bright field and fluorescence conditions to image the cells over the course of the physiological experiment. These experiments will elucidate the genes controlling the breakdown of photosynthesis and characterize dynamic changes in cell metabolism. This alga produces the high value carotenoid astaxanthin, which is valued at about $2,500 per kilogram. I have bkt mutants that have mutations in the beta ketolase gene and no longer produce astaxanthin. These mutants confirm the function of this enzyme in this alga. I have conducted high light experiments and analyzed the pigments to show that they do not produce astaxanthin under conditions that wild type cell do. I have conducted a RNAseq transfer to high light experiment and after completion of the genome, will analyze the changes in gene expression. It appears there are about ~200 genes with different genetic expression under high light. With a graduate student in the lab, we are currently working on genetic transformation to rescue the bkt mutants. Changes/Problems:Because of the commercial importance of the carotenoid of this alga, I have added in additional work to characterize and identify the enzyme that produces this carotenoid. Additionally, we have done a higher level genome sequencing and analyses. This research has caused a delay in the NPQ work. What opportunities for training and professional development has the project provided?Training activities: I have greatly improved my molecular biology, biochemistry and microscopy skills. Additionally, I have been training a graduate student on many of these techniques as well. Professional development: I have presented at a variety of conferences including the International Photosynthetic Congress, the International Conference on the Cell and Molecular Biology of Chlamydomonas, and the NIFA PD Meeting. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The goals in the next reporting period: Finish and publish a paper with the following: De novo genome Transcriptome Gene models and annotation Compare genome with close relatives - chromosomal synteny Analysis of carotenoid pathways Analysis of TAG pathways Analysis of high light experiment Bkt mutants - identification of enzyme that produces astaxanthin Finish and publish a paper with the following: Analyze physiological results from breakdown and biogenesis experiments Finish processing lipid, protein and pigments samples from breakdown and biogenesis experiments Conduct TEM from breakdown and biogenesis experiments Analysis of x-ray tomography from breakdown and biogenesis experiments Transformation of glc mutants and conduct experiments Put together live cell time lapse video from breakdown and biogenesis experiments After completion of above two papers (which may extend beyond the next reporting period), continue with NPQ characterization: • Determine the genetic basis for npq mutants • Conduct NPQ experiments and analyses • Rescue npq mutants and conduct experiments

Impacts
What was accomplished under these goals? Accomplished: Develop high quality RNA extraction protocol for M. zofingiensis Develop transformation protocol for M. zofingiensis Conduct glc RNA-seq experiments and analyses Determine the genetic basis for glc mutants Additionally: Sequence de novo genome Sequence the transcriptome (using 14 different conditions) Determine genetic basis for bkt mutants (lack of carotenoid producing algae) Conduct highlight experiments Conduct x-ray tomography and TEM for glc experiments

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Roth, MS, Niyogi, KK. 2013. Elucidating high photoprotective capacity in a unicellular green alga. 16th International Congress on Photosynthesis Research. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Roth, MS, Niyogi, KK. 2013. Elucidating high photoprotective capacity in a unicellular green alga. Light Harvesting Satellite Meeting. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Roth, MS, Gallaher, S, Cokus, S, Lopez, D, Pellegrini, M, Merchant, SS, Niyogi, KK. 2014. An emerging unicellular green alga model system for studying regulation of photosynthesis. 16th International Conference on the Cell and Molecular Biology of Chlamydomonas.