Source: MICHIGAN STATE UNIV submitted to
REGULATION OF LIPID METABOLISM IN PLANTS AND ALGAE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1004127
Grant No.
(N/A)
Project No.
MICL02357
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Nov 1, 2014
Project End Date
Oct 31, 2019
Grant Year
(N/A)
Project Director
Benning, C.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Biochemistry & Molecular Biology
Non Technical Summary
Seed oils provide food for human and animal consumption but also feedstocks for high energy transportation biofuels such as biodiesel or even jet fuels. However, the oil yield of currently grown seed oil crops such as canola, soybean, corn or sunflower is limited. Therefore, biofuels derived from temperate seed oil crops can only replace a small fraction of current transportation fuel needs without competing with the production of feed and food. Engineered plants that produce oils in tissues other than developing embryos have the potential to increase the oil yield per available land. Moreover engineering oil in vegetative tissues such as leaves, stems or roots enhances the energy density of potential fuel crops. The nature of the portfolio of biofuel crops or photosynthetic organisms that could be utilized for biofuel production is not yet defined. However, it is clear that potentially viable options aside from dedicated biofuel crops with novel properties include microalgal biomass enriched in oils (triacylglycerols). Unicellular microalgae species are known that have the propensity to produce and accumulate oil up to 60% of their cell mass. Knowledge and understanding of biological model systems coupled with recent advances in genetic and genomic technologies have opened a wealth of new possibilities. These new advances promise rapid progress towards the development of algal systems that could potentially produce oil on a scale that might soon supplement and possibly one day replace fossil high energy density liquid fuels. Algae have the potential advantage over conventional crops that they can be grown on marginal land not suitable for conventional agriculture. Thus, microalgal production systems do not compete with the production of food and feed using conventional crops. Moreover, algal culture systems are explored that can take advantage of flue gases emitted by coal-firing plants and waste streams of sewage treatment facilities. Because of the currently uncertain nature of biofuel feedstocks, it seems prudent to explore the potential of land based biofuel crops and microalgae in parallel. Both have the potential to transform agriculture as we currently know it and it seems likely that local solutions will require different feedstocks. The long-term goal of the proposed research is to gain a fundamental understanding of the regulation of oil biosynthesis in plants and algae. It is expected that this basic knowledge can be explored to engineer new biofuel crops and enhanced microalgal production strains in support of building a sustainable biofuel economy.
Animal Health Component
0%
Research Effort Categories
Basic
35%
Applied
35%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20324991000100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
OBJECTIVES The project is broadly organized into three objectives with subobjectives:1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress.1.1 We will continue to analyze the function of TGD proteins to gain mechanistic and structural insights and to identify the lipid substrates.1.2 The function of SFR2 and its mechanism of activation will be explored in plants with different sensitivity to abiotic stress. Based on these findings novel strategies for the engineering abiotic stress tolerance will be explored.1.3 The function of PGD1 like proteins in plants will be determined and their possible physiological roles in abiotic stress tolerance will be determined. Depending on the findings novel strategies for the engineering abiotic stress tolerance will be explored.2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae.2.1 We will continue explore the link between cell metabolic status and cell division initially by determining the function of the CHT7 protein and its interacting factors. Knowledge gained here will allow us to overcome the inverse relationship between growth and TAG accumulation hampering algal biofuel feedstock production.2.2 We will determine the role of TAG accumulation following nutrient stress in microalgae by detailed analysis of low TAG mutants. We expect to gain new insights into photosynthetic carbon partitioning.2.3 Lipid metabolism in Nannochloropsis will be explored using forward and reverse genetics. New target genes for the engineering of TAG biosynthesis in algae and plants will be identified. A sustained effort to build the molecular tool box for Nannochloropsis will ideally position this organism for further biotechnological applications.3. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues.3.1 Differences in lipid metabolism between Arabidopsis and Brachypodium as a grass model will be explored to identify possible limitations in the engineering of TAGs in vegetative tissues. Efforts will initially focus on the exclusive use of the ER pathway for thylakoid biosynthesis in grasses, differences in the function of TGD proteins and the role of chloroplast phosphatidic acid phosphatase in the production of diacylglycerol in grasses.3.2 New strategies for the engineering of TAG metabolism in grasses using algal and other genes will be developed in ways to allow better control for tissue specific accumulation of TAGs in stems and leaves.
Project Methods
In the following the main approaches used to accomplish the individual objectives will be summarized in order of the subobjectives listed above:1.1 Function of TGD proteins. We will use two strategies to define the lipid substrate transported by the TGD proteins: 1. In vivo tracking of ER-derived lipids. To directly test the lipid conduit model, we will produce transgenic plants that replace α-linolenic acid (ALA, 18:3 Δ9,12,15 (18 carbons : 3 double bonds at carbons 9, 12 and 15 from the carboxyl end)) with γ-linolenic acid (GLA, 18:3 Δ6,9,12) in ER lipids such as PtdCho. These lines will be crossed with individual tgd mutants to assess the function of the respective protein in acyl group distribution in the respective lines. 2. In vitro reconstitution of lipid import into chloroplasts. We will reconstitute a lipid transfer system with isolated mutant and wild-type chloroplasts to which mutant or wild-type ER microsomes or synthetic liposomes spiked with labeled lipids are added as lipid donors.1.2 Mechanism and engineering of SFR2. We will conduct a detailed structure/function study of SFR2. We have developed a set of highly specific antibodies against the N-terminal and C-terminal portions of the protein that will allow us to determine the topology of SFR2 relative to the membrane and the composition of the native protein complex. We will test whether Mg2+ plays a catalytic role. Other possible SFR2 activity-modulating factors will be systematically explored to determine the subcellular signals that tie SFR2 activation with its physiological role. Taking cues from the in vitro studies, we will test SFR2 activity in isolated chloroplasts, leaves, and plants to determine how SFR2 may be activated in response to freezing injury or other stresses. We will begin by comparing Arabidopsis and pea, because pea unlike Arabidopsis uses lipid precursors imported from the ER for galactolipid biosynthesis, it is freezing intolerant, and pea chloroplasts are easily isolated.2.1 Function of CHT7 and associated proteins. Identified in a novel mutant screen, "Compromised Hydrolysis of Triacylglycerols 7" (CHT7) is a protein of Chlamydomonas with predicted CXC DNA-binding domains. CHT7 is a component of a multiprotein complex that appears to adjust specific transcriptional subnetworks at quiescence entry and exit. We predict that CHT7 will bind regulatory DNA sequences either directly or indirectly. Therefore, a ChIP-Seq experiment (using either a CHT7 antibody, or transgenic lines expressing CHT7 tagged versions) will be conducted to identify possible target sequences. Targets will be tested in vitro for direct binding by recombinant CHT7 to distinguish whether CHT7 is a transcription factor or a corepressor/coactivator. The composition of the CHT7-containing complex will be determined using CoIP experiments. Proteins will be immuno-detected on gels where possible, or identified by mass spectrometry in case of novel components.2.2 Role of TAG accumulation in microalgae. The disruption in the Chlamydomonas pgd1 mutant specifically reduced TAG accumulation by 50% following nutrient deprivation, but not the major membrane lipids. The pgd1 mutant also exhibited a chlorotic phenotype along with an increase in reactive oxygen species following nutrient deprivation, which was rescued by blocking of photosynthetic electron transport at photosystem II. These observations support the current hypothesis of TAG accumulation in nutrient-stressed algae and make the pgd1 mutant an excellent tool to study the physiological role of TAG accumulation following nutrient deprivation and to gain novel insights into the regulation of the photosynthetic electron transport chain and photosynthetic carbon partitioning.2.3 Nannochloropsis lipid metabolism. Studies will be undertaken to functionally validate key enzymes in lipid metabolism of Nannochloropsis. We will develop and apply gene inactivation strategies for Nannochloropsis, but initially we will rely on the expression of these genes in heterologous hosts for functional characterization. These studies will involve complementation of null phenotypes in yeast or E. coli mutants. For example, a TAG-deficient S. cerevisiae strain will be used to systematically characterize annotated Nannochloropsis DGAT and PDAT genes for their ability to restore TAG biosynthesis. Similarly, yeast and E. coli fatty acid synthase (FAS) mutants will be used as hosts to test the candidate Type II or type I FAS from Nannochloropsis for the ability to catalyze fatty acid biosynthetic reactions by expression of the complete polypeptide or individual domains using procedures described previously. Candidate fatty acid desaturase genes will be tested for their capacity to generate novel unsaturated fatty acids upon expression in yeast or other microbial host, as reported for the characterization of the Chlamydomonas ?12 desaturase. Because Nannochloropsis lipid metabolism is highly efficient, its genes identified as encoding bona fide enzymes or other proteins for energy dense compound synthesis and storage will be used for metabolic engineering studies in Brachypodium for enhanced production of triacylglycerols or for producing novel fatty acid structures.3.1 Brachypodium lipid metabolism. Basic lipid assembly pathways in grasses differ from those of Arabidopsis, as the latter utilizes both the ER and plastid pathways while grasses primarily assemble lipids through the ER pathway. We have mined the data base to identify orthologs of known or predicted Arabidopsis genes in the Brachypodium genome. A limited but significant number of genes appear to be absent in Brachypodium, which include desaturase and acetyl-CoA carboxylase genes. It has also been proposed that plastid phosphatidic acid phosphatase activity is missing in grasses and can explain the differences in the utilization of the ER and plastid pathways in monocots and dicots. We will test this hypothesis by introducing an Arabidopsis plastid phosphatidic acid phosphatase gene into Brachypodium. In addition, we will generate TGD1 KO lines of Brachypodium to determine verify the function of the TGD transporter in lipid trafficking in grasses. We will also introduce the Brachypodium TGD1 gene into the respective Arabidopsis tgd1 mutant to test for its functionality in the heterologous system. Since Brachypodium relies exclusively on the import of lipid precursors into the chloroplast for galactolipid biosynthesis, it seems possible that the TGD proteins from Brachypodium are more efficient than those from Arabidopsis to cope with the increased flux. We believe understanding the role of TGD is important for our understanding of lipid metabolism in grass leaves as we attempt to engineer vegetative oil content.3.2 Enhancing energy density of grasses. A series of new constructs will be produced for introduction and testing in Brachypodium. The Great Lakes Bioenergy Research Center has set up a Brachypodium transformation facility that we are using for all our needs to generate transgenic Brachypodium lines. A limited number of transgenic WRI1 lines are already available, but need to be combined with AGPAse RNAi. Focus will be on the expression of algal DGATs and a monocot DGAT alone and in combination with an algal (Nannochloropsis) lipid droplet protein known to work in plants as recently demonstrated. The latter presents a new strategy in preventing the turnover of lipid droplets. Recent reports by others suggest that coexpression of DGAT and oleosin from plants will enhance oil accumulation in dicot leaves (Winichayakul et al., 2013). Lipid droplets may be protected from lipases by the presence of lipid droplet proteins, especially if a heterologous lipid droplet protein is used.

Progress 11/01/14 to 10/31/19

Outputs
Target Audience:Students of plant sciences, scientists in academia with focus on plant sciences, scientists in biotech industries with focus on renewable energy and on industrial compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Altogether, 2 postdoc, 4 graduate students, 5 undergraduate students, one technician, and one emeritus professor participated in the described projects during the past year. One postdoc and one graduate student developed with the PI a course based undergraduate research experience (CURE) module for BioSci171, in which the students participate in the suppressor mutant screen in the PLIP3 overexpression line described above. How have the results been disseminated to communities of interest?Asides from the publications reported, Benning gave the following public lectures 09/30/18 A role for lipases in lipid droplet formation in plants and algae. 14th GERLI Lipidomics Meeting: Biogenesis and Fate Of Lipid Droplets. St. Maximin la Saint-Baume, France 10/04/18 Engineering Triacylglycerol Accumulation in Vegetative Tissues and Algal Cells. CEA Cadarache, France 10/17/18 Synthetic consortium between an alga and a fungus. The Portable Organelle Project. Norwich, UK. 10/27/18 Chloroplast lipases involved in seed oil biosynthesis and stress protection. USDA NC1200, D. Danforth Plant Science center, St. Louis, MO 11/26/18 Chloroplast lipases involved in membrane lipid remodeling and stress protection in plants and algae. RIKEN Center for Sustainable Resource Science, Yokohama, Japan. 01/31/19 The Essential Role of Metabolism and Transport of Phosphatidic Acid in Plastids. GRC Plant Lipids, Galveston, TX 05/29/19 The Chloroplast Envelope Membranes as Hub for Lipid Biosynthesis. CPLAS Summer School, Wermelskirchen, Germany 08/4/19 Regulation of cell division in response to the metabolic status of cells in Chlamydomonas. ASPB Cell Biology I, San Jose , CA What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress. 1.1 We continue to explore the function of chloroplast lipases of PLIP1, 2 and 3 in Arabidopsis. Overexpression of the respective cDNAs in Arabidopsis caused severe stunting of the plants, because these plants accumulate oxylipins, which leads to the induction of biotic defense pathways. We are conducting a physiological analysis of individual, double and triple mutants lacking combinations of the three isoforms PLIP1, 2, and 3. The seeds of some combinations showed an increased sensitivity to ABA, and we are investigating whether these mutants are also compromised in abiotic stress responses. Because the activity of these lipases leads to oxylipin formation and activation of biotic defense responses, we continue to investigate the interplay of ABA and oxylipin signaling in the mutants and the role of these lipases in this process. For example, we recently found that ABA hypersensitivity of seed germination of plip2 mutants can be reversed by treatment of methyl-jasmonate. 1.2 We are taking advantage of the strong growth phenotype of plants overproducing PLIP3 to look for suppressor mutants. We expect to identify novel factors involved in oxylipin biosynthesis and export from the chloroplast as well as signaling. Mutants have been isolated and we are currently in the process of identifying the responsible genes. 1.3 We continued the analysis of the reaction mechanism, activation, and function of the unusual FAD4 desaturase of Arabidopsis. FAD4 is responsible for the formation of a phosphatidylglycerol species in chloroplasts that contains a 16:1 delta 3 trans fatty acid at its sn-2 position. We identified a new protein cofactor, a specific peroxiredoxin, required for the activity of this protein in vivo and in vitro. We were able to reconstitute the desaturase and its cofactor in yeast microsomes. Specific cysteine residues in FAD4 and peroxiredoxing were identified that are essential for activation and activity of FAD4 activity. Based on these results, we hypothesize that the redox state of the chloroplast is linked with the activity of the FAD4 desaturase as a photoprotective mechanism under certain abiotic stress conditions. A manuscript describing these findings is under review. 1.4 We advanced the analysis of a novel rhomboid protease of Arabidopsis located in the inner chloroplast envelope membrane. We published the biochemical analysis of the respective mutant, in which the supply of lipid precursors, i.e. phosphatidic acid, from the inside of the chloroplast to the enzyme that assembles the galactolipid monogalactosyldiacylglycerol is disrupted, while the precursor supply from the ER compensates. We have made progress in determining that the respective rhomboid protease has catalytic activity, as we can demonstrate that it autocatalytically cleaves off its C-terminus, but we do not know at this time the relevance of this cleavage. The protein is associated with a large complex of the inner chloroplast envelope membrane and we are investigating its composition. We are raising antibodies against this protein to aide in this process. 1.5 The analysis of the rhomboid protease described above raised new questions about phosphatidic acid movement and metabolism in the inner envelope membrane. For example, which proteins participate in the conversion of phosphatidic acid to diacylglycerol, the precursor of galactolipid biosynthesis, remains unclear. We are currently studying three candidates encoding phosphatidic acid phosphatases and their respective loss-of-function mutants alone and in combinations and we identified a double mutant that shows strong growth reduction, which is further studied. The proteins have been previously shown to be in the chloroplast, but their exact membrane association and topology is critical for our understanding of phosphatidic acid metabolism and is currently under investigation. 2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae. 2.1 The analysis of the Chlamydomonas CHT7 protein in the regulation of the cell cycle and N deprivation-induced quiescence continues. Detailed phenotypic analysis of the cht7 mutant in a cell-walled strain has shown that viability is compromised during N deprivation. In addition we completed a meta-analysis of genes misregulated in the cht7 mutant and mapped these onto the Chlamydomonas transcriptional network. Most prominently were nodes of misregulated genes involved in different aspects of cell division. In fact, abnormal cell division has been observed in the cht7 mutant that can be linked to the misregulation of cell cycle genes during N deprivation-induced quiescence in the mutant. We completed the dissection of the functional domains of the CHT7 protein and corroborated that the presumed DNA binding, CXC domain is not required for function using complementation assays. However, several predicted protein-binding domains towards the C-terminus of CHT7 are essential for tis function. A paper describing these results is currently under review. 2.2 A next step to understand how CHT7 affects cell cycle gene expression involves the analysis of a large 275 kDa CHT7 complex, especially determining its composition to identify the presence of DNA binding proteins that we suspect to be present. Consistent with the fact that CHT7 does not seem to bind DNA directly through its CXC domain, we were unsuccessful in identifying CHT7 DNA binding sites during an extensive ChIP-Seq experiment. 2.3 We completed an RNAseq study on synchronized cultures of the cht7 mutant, and complemented lines in photobioreactors using different nutrient conditions. We expanded this analysis to other mutants such as mat3 and dp1 affecting cell cycle regulation in Chlamydomonas to begin to understand the possible interaction of these factors with CHT7 in the regulation of the cell cycle and N deprivation-induced regulation of cellular quiescence. Using CoIP, we have observed that CHT7 and MAT3 can occur in the same complex. 2.4 Co-cultivating the alga Nannochloropsis and an oleogenic fungus, Mortierella, we observed synergistic enhancement of oil production. In addition, we discovered that the algal cells become internalized by the fungal hyphae and we showed that this process resembles a first step in a mutualistic endosymbiosis. These findings were published. 2.5 We completed a transcriptomic analysis of the oleogenic alga Nannochloropsis oceanica under different conditions including N deprivation. A manuscript describing these results has been reviewed and will be resubmitted shortly.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Warakanont J, Li-Beisson Y, Benning C. 2019. LIP4 is involved in triacylglycerol degradation in Chlamydomonas reinhardtii. Plant Cell Phys. 60:1250-1259 doi: 10.1093/pcp/pcz037
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sadre R, Kuo P, Chen J, Yang Y, Banerjee A, Benning C., Hamberger, B. 2019. Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves. Nature Com. 10, Article number: 853, doi: 10.1038/s41467-019-08515-4
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lavell A, Froehlich JE, Baylis O, Rotondo A, Benning C. 2019. A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana. Plant J. 99:978-987 doi: 10.1111/tpj.14377
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Li L, Lavell A, Meng X, Berkowitz O, Selinski J, van de Meene A, Carrie C, Benning C, Whelan J, De Clercq I, Wang Y. (2019). Arabidopsis DGD1 SUPPRESSOR 1 is a subunit of the mitochondrial contact site and cristae organizing system and affects mitochondrial biogenesis. Plant Cell 31:1856-1878 doi: 10.1105/tpc.18.00885
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Chen B, Zhang G, Li P, Yang J, Guo L, Benning C, Wang X, Zhao J. 2019. Multiple GmWRI1s are redundantly involved in seed filling and nodulation by regulating plastidic glycolysis, lipid biosynthesis, and hormone signaling in soybean (Glycine max). Plant Biotechnol J. doi: 10.1111/pbi.13183
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Du ZY, Zienkiewicz K, Vande Pol N, Ostrom NE, Benning C, Bonito GM. 2019. Algal-fungal symbiosis leads to photosynthetic mycelium. Elife. 2019 Jul 16;8. pii: e47815. http://doi.org/10.7554/eLife.47815.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lavell AA, Benning C. 2019. Cellular Organization and Regulation of Plant Glycerolipid Metabolism. Plant Cell Physiol. 60:1176-1183 doi: 10.1093/pcp/pcz016.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang K, Durrett TP, Benning C. 2019. Functional diversity of glycerolipid acylhydrolases in plant metabolism and physiology. Prog. Lipid Res. doi: 10.1016/j.plipres.2019.100987


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Students of plant sciences, scientists in academia with focus on plant sciences, scientists in biotech industries with focus on renewable energy and on industrial compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Altogether, five postdocs, seven graduate students, six undergraduate students, and one technician participated in the described projects during the past year. Of the graduate students, three graduated with Anna Hurlock now working at BioFire Diagnostics, LLC, Eric Poliner working as postdoc in the Kramer Lab at the MSU-DOE plant Research Laboratory and Kun Wang joining the Farese and Walther lab at Harvard Medical School. Postdocs Patrick Horn took an assistant professor position at Eastern Carolina University, Yang Yang joined Wiley Science China in Beijing as science editor, and Asdrubal Jesus Birgos Gutierrez returned to Universidad de Guadalajara in Mexico as lecturer. How have the results been disseminated to communities of interest?Asides from the publications reported, Benning gave the following public lectures: 11/11/17 Mechanisms of lipid remodeling and transfer in the chloroplast. USDA NC1200, Arlington VA 11/30/17 Lipid Assembly, Remodeling, and Transport to Build and Protect the Photosynthetic Membrane. ASPL2017, Taipei, ROC\ 05/16/18 Membrane lipid remodeling for oil production and stress protection. Max-Planck-Institut für Pflanzenzüchtungsforschung, Cologne, Germany 05/23/18 Membrane lipid remodeling for oil production and stress protection. iGrad HHU-Duesseldorf, Germany. 07/09/18 Regulation of lipid movement in the chloroplast envelope membranes. The 23rd International Symposium on Plant Lipids 2018, Yokohama, Japan What do you plan to do during the next reporting period to accomplish the goals? Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress: We will continue to explore the biotechnological application of PLIP1-based engineering of seed oil content and quality in Camelina. We are conducting a genetic suppressor screen in the PLIP3 cDNA over expression line to identify new components involved in the coordination of abiotic and biotic stress responses in plants.The reaction mechanism of FAD4 and the role of cofactors in this reaction will be completed and published. A first publication of the role of a rhomboid protease located in the chloroplast envelope membranes on phosphatidic acid biosynthesis will be completed. Inspired by our results on the rhomboid protease, we will investigate the identity and location of chloroplast phosphatidic acid phosphatases to come to a better understanding of phosphatidic acid metabolism in the chloroplast envelope membranes. Phosphatidic acid is a central metabolite in lipid biosynthesis yet its metabolism and transfer within the chloroplast membranes remains to be determined. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae: A major emphasis will continue to be on the identification of direct target genes of the CHT7 complex and the composition of the CHT7 complex in synchronized cultures of Chlamydomonas. We will continue to explore the interaction of CHT7 with the cell cycle regulatory protein MAT3 in Chlamydomonas in dependence on the nutrient status of the cell. The findings should lead to engineering strategies towards the optimization of algal oil yields. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues: We will continue to explore fundamental difference in lipid metabolism between 16:3 plants such as Arabidopsis and 18:3 plants including grasses such as Brachypodium or Camelina. We will test a new hypothesis postulating that specific details in phosphatidic acid metabolism and transport within the chloroplast envelope membranes differ between these plant types.

Impacts
What was accomplished under these goals? 1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress. 1. 1 To utilize the PLIP1 pathway for the engineering of seed oil content in a potential crop plant, we expressed the PLIP1 cDNA under the control of a seed-specific promoter in Camelina. A large number of transgenic plants was obtained that we are now studying in the T3 generation. The growth of these plants seems normal and preliminary results suggest that oil content per seed may be slightly increased, although seed number per plant may be slightly reduced. More importantly, overexpression of the PLIP1 cDNA caused a change in seed oil fatty acid content towards fatty acids with shorter chain length and fewer double bonds. In essence, PLIP1 cDNA overexpression in a seed-specific manner led to Camelina oil similar to that of Canola. 1.2. We have completed and published the analysis of two paralogs of PLIP1 in Arabidopsis, PLIP2 and 3. These are also chloroplast lipases targeting specific thylakoid membrane lipids. We were able to show their activity in vivo and in vitro. The expression of the two respective genes is induced by the plant abiotic stress hormone ABA. Overexpression of the respective cDNAs in Arabidopsis caused severe stunting of the plants especially in case of PLIP2. As a cause, we determined that these plants accumulate oxylipins, which leads to the induction of biotic defense pathways. Introducing a mutation that causes the loss of the JA-Ile receptor COI1 in Arabidopsis reversed the growth defect in the PLIP cDNA overexpression lines. We also constructed triple mutants lacking the three isoforms PLIP1, 2, and 3. The seeds and seedlings showed an increased sensitivity to ABA. Based on our findings on PLIP2 and 3, we proposed a new model that places these two plastid lipases at a key junction between abiotic and biotic stress responses. 1.3. We advanced the analysis of the reaction mechanism of the unusual FAD4 desaturase of Arabidopsis and are currently preparing a publication on this topic. FAD4 is responsible for the formation of a phosphatidylglycerol species in chloroplasts that contains a 16:1 delta 3 trans fatty acid at its sn-2 position, which is the substrate for PLIP1 mentioned above. Loss of this lipid in the fad4 mutant reduces seed oil content in Arabidopsis. We identified a new protein cofactor, a specific peroxiredoxin, required for the activity of this protein in vivo and in vitro. We were able to reconstitute the desaturase and its cofactor in yeast microsomes. Specific cysteine residues in FAD4 were identified that are essential for its activity. Based on these results, we are proposing a hypothesis linking the redox state of the chloroplast to the activity of the FAD4 desaturase. 1. 4. We are studying a novel rhomboid protease of Arabidopsis located in the inner chloroplast envelope membrane. The biochemical analysis of the mutant indicates that the supply of lipid precursors from the inside of the chloroplast to the enzyme that assembles the galactolipid monogalactosyldiacylglycerol is disrupted, while the precursor supply from the ER compensates. Label experiments with isolated chloroplasts show a buildup of phosphatidic acid and a decrease of label in the galactolipid. However, neither phosphatidic acid phosphatase in the chloroplast nor the galactolipid synthase are decreased in their activity in the mutant. Based on these results we are proposing a new hypothesis that takes into account the topology of the enzymes and we postulate that phosphatidic acid transfer from the inside of the inner chloroplast envelope membrane to its intermembrane face is disrupted. How the rhomboid protease affects this process is currently being investigated and one clue will come from the analysis of proteins associated with it in a large complex of the inner chloroplast envelope membrane. 1.5. We published a study of transgenic Arabidopsis plants expressing the cDNA encoding an ER delta 6 desaturase to tag ER lipids and monitor their import in to plastids. Key findings from this study were that a fraction of phosphatidylglycerol in the chloroplast is derived from imported lipid precursors and that acyl editing on chloroplast lipids is extensive. 2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae. 2.1. The analysis of the Chlamydomonas CHT7 protein in the regulation of the cell cycle and N deprivation-induced quiescence continues. Detailed phenotypic analysis of the cht7 mutant in a cell-walled strain has shown that viability is compromised during N deprivation. Abnormal cell division has been observed that can be linked to the misregulation of cell cycle genes during N deprivation-induced quiescence in the mutant. We have also begun the dissection of the functional domains of the CHT7 protein and discovered that the presumed DNA binding, CXC, domain is not required for function using complementation assays. However, several predicted protein-binding domains towards the N-terminus of the protein are essential. A paper describing these results will be resubmitted for publication soon. 2.2 We are focusing now on the analysis of a large 275 kDa CHT7 complex determining its composition and phosphorylation state during different stages of the cell cycle. We hypothesize based on the result described above that this complex contains (a) DNA-binding protein(s) other than CHT7. 2.3. We completed and published an RNAseq study of the original cht7 mutant with a particular focus on N refeeding. We were able to discern distinct transcriptional programs during quiescence exit compared to quiescence entry. 2.4. We are in the final stages of a new RNAseq study on synchronized cultures of the cht7 mutant, and complemented lines in photobioreactors using different nutrient conditions. Because all RNAseq data suggest that the CHT7 complex affects the transcriptional regulation of cell cycle genes depending on the nutrient status of the cell, we are conducting a ChIPseq experiment to determine directly what promoter sequences and genes this complex binds. We are also in the process to expand this analysis to other mutants such as mat3 and dp1 affecting cell cycle regulation in Chlamydomonas to begin to understand the possible interaction of these factors with CHT7 in the regulation of the cell cycle and N deprivation-induced regulation of cellular quiescence. Using CoIP, we have observed that CHT7 and MAT3 can occur in the same complex. 2.5. We published the analysis of the low triacylglycerol pgd1 mutant of Chlamydomonas under different growth conditions during incubation in photobioreactors. The mutant shows increased abiotic stress sensitivity, changes in thylakoid stacking and carbon utilization. The overall conclusion is that the impaired ability to remodel the photosynthetic membrane during changes in conditions makes this mutant vulnerable to the toxic effects of reactive oxygen inside chloroplast. 2.6. Co-cultivating the alga Nannochloropsis and an oleogenic fungus, Mortierella, we observed synergistic enhancement of oil production and were able to develop an improved protocol maximizing the oil yield of Nannochloropsis cultures. A paper describing this new system has been published. 2.7. An episome-based approach to disrupt genes in Nannochloropsis suing CRISPR/Cas9 was developed. This system has the advantage that the modified strain can be cured of the episome leaving no traces of foreign DNA behind. Therefore, this approach allows genetic engineering of Nannochloropsis without necessarily generating a GMO. This work has been published. 3. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues. Much of this work has been completed in the Benning lab and further studies on the WRI1 transcription factor will be conducted in the lab at Nanyang technological University in Singapore of Prof Wei Ma, who is a former postdoc working on this project in the lab.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hurlock AK, Wang K, Takeuchi T, Horn PJ, Benning C. 2018. In vivo lipid "tag and track" approach shows acyl editing of plastid lipids and chloroplast import of phosphatidylglycerol precursors in Arabidopsis thaliana. Plant J. 2018 Jun 19. doi: 10.1111/tpj.13999
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Tsai CH, Uygun S, Roston R, Shiu SH, Benning C. 2018. Recovery from N deprivation is a transcriptionally and functionally distinct state in Chlamydomonas. Plant Physiol., 176:2007-2023 doi: 10.1104/pp.17.0154
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Du Z-Y, Alvaro J, Hyden B, Zienkiewicz K, Benning N, Zienkiewicz A, Bonito, G. Benning C. 2018. Enhancing oil production and harvest by combining the marine alga Nannochloropsis oceanica and the oleaginous fungus Mortierella elongata. Biotechnol. Biofuels 11:174, https://doi.org/10.1186/s13068-018-1172-2.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Du Z-Y, Lucker BF, Zienkiewicz K, Miller TE, Zienkiewicz A, Sears BB, Kramer DM, and C Benning. 2018. Galactoglycerolipid lipase PGD1 is involved in thylakoid membrane remodeling in response to adverse environmental conditions in Chlamydomonas. Plant Cell, 30:447-465. doi: 10.1105/tpc.17.00446.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Poliner E, Takeuchi T, Du ZY, Benning C, Farr� EM. 2018. Nontransgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779. ACS Synth Biol. 7:962-968 doi:10.1021/acssynbio.7b00362.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang K, Guo Q, Froehlich JE, Hersh HL, Zienkiewicz A, Howe GA, C Benning. 2018. Two abscisic acid responsive plastid lipase genes involved in jasmonic acid biosynthesis in Arabidopsis thaliana. Plant Cell, 30:1006-1022, doi: 10.1105/tpc.18.00250
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Severin GB, Ramliden MS, Hawver LA, Wang K, Pell ME, Kieninger A-K , Khataokar A, O'Hara BJ, Behrmann LV, Neiditch MB, Benning C, Waters CA, Ng W-L. 2018. Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholera. Proc. Natl Acad Sci. 115:E6048-E6055. doi: 10.1073/pnas.1801233115
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yang, Y., Benning., C. 2018. Functions of triacylglycerols during plant development and stress. Curr. Opin. Biotech. 49:191-198. https://doi.org/10.1016/j.copbio.2017.09.003
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Poliner E, Farr� EM, Benning C. 2018. Advanced genetic tools enable synthetic biology in the oleaginous microalgae Nannochloropsis sp. Plant Cell Rep. doi:10.1007/s00299-018-2270-0.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Students of plant sciences, scientists in academia with focus on plant sciences, scientists in biotech industries with focus on renewable energy and on industrial compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Altogether, 5 postdocs, 6 graduate students, 4 undergraduate students, and 1 technician participated in the described projects. Three graduate students are on track to graduate before the end of this year and two postdocs are currently applying for positions and will likely move on during the coming year to new positions. How have the results been disseminated to communities of interest?Asides from the publications reported Benning gave the following public lectures: 11/10/16 Regulation of Cellular Quiescence and Lipid Droplet Formation in Microalgae.Lipids and Metabolic Diseases II Inaugural Symposium of Big Ten Academic Alliance Lipids 11/18/16 Greasing photosynthesis: From membranes to lipid droplets. West Virginia State University, Institute, WV 01/31/17 Contribution of Chloroplast Phosphatidylglycerol Metabolism to Cellular Lipid Homeostasis. GRC on Plant Lipids: Structure, Metabolism & Function. Galveston, TX 05/24/17 From Membranes to lipid Droplets. The Plant Journal Editor's Symposium. Windsor, UK 09/04/17 Lipid Assembly, Remodeling, and Transport to Build and Protect the Photosynthetic Membrane. Institute for Basic Science, Republic of Korea 09/22/17 Lipid Assembly, Remodeling, and Transport to Build and Protect the Photosynthetic Membrane. Plant Biology, Cornell University, Ithaca, NY What do you plan to do during the next reporting period to accomplish the goals?1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress: PLIP1 based engineering of seed oil content will be explored in Camelina. The role of PLIP2 and 3 lipases in responses of plants towards abiotic stress will be further explored. The reaction mechanism of FAD4 and the role of cofactors in this reaction will be further investigated. The role of a rhomboid protease located in the chloroplast envelope membranes will be further investigated. 2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae: A major effort will continue to be on the identification of direct target genes of the CHT7 complex and the composition of the CHT7 complex in synchronized cultures of Chlamydomonas. The origin of ROS in the Chlamydomonas pgd1 mutant will be explored. 3. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues: The analysis of transgenic Brachypodium lines expressing different genes to enhance vegetative oil accumulation will be completed. We will complete the analysis of transcription factors interacting with WRI1.

Impacts
What was accomplished under these goals? 1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress. 1.1 The analysis of one of three chloroplast located lipases, PLIP1, of Arabidopsis has been completed and published. We showed that PLIP1 releases acyl groups from a specific chloroplast phosphatidylglycerol species that contains a 16:1 delta 3 trans fatty acid at its sn-2 position. PLIP1 is primarily acting in developing embryos. We demonstrated that the PLIP1 based acyl export mechanism contributes approximately 10% of triacylglycerol found in Arabidopsis seeds. The findings provide a new avenue to engineer seed oil yield. The analysis of two additional isoforms (PLIP2, and PLIP3) continues. We were able to produce recombinant PLIP2 and assay its activity. We were unable to produced recombinant PLIP3. Individual and double and triple PLIP mutants have been produced and their analysis continues. In addition, we produced over expression line. These are impaired n growth which can be attributed to an over active defense response due to the accumulation of oxylipins. 1.2 We are studying the reaction mechanism of the unusual FAD4 desaturase of Arabidopsis responsible for the formation of a phosphatidylglycerol species in chloroplasts that contains a 16:1 delta 3 trans fatty acid at its sn-2 position, which is the substrate of PLIP1 mentioned. Loss of this lipid in the fad4 mutant reduces seed oil content. We identified a new protein cofactor required for the activity of this protein. 1.3 The Analysis of the Brachypodium TGD1 protein was published. We identified a specific loop of TGD1 proteins form Arabidopsis and Brachypodium that differs. Coevolution analysis showed that this loop provides an interaction domain with the TGD2. This study provides a detailed mechanistic insight into the different usage of plastid and ER-pathway of lipid assembly in monocotyledonous and dicotyledonous plant species. 1.4 We discovered and are studying a rhomboid protease located in the chloroplast envelope membranes of chloroplast. The phenotype of the respective mutant suggests that the protein affects lipid trafficking between the ER and the chloroplast. 1.5 We completed an analysis of transgenic Arabidopsis plants expressing an ER delta 6 desaturase to tag ER lipids and monitor their import in to plastids. Key findings from this study were that a fraction of phosphatidylglycerol in the chloroplast is derived from imported lipid precursors and that acyl editing on chloroplast lipids is extensive. A paper describing these results is under review. 2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae. 2.1 The cht7 mutation of Chlamydomonas was introduced into a new line with intact cell wall and complementation lines were generated in the same genetic back ground. A detailed phenotypic analysis of the cht7 mutant cells has shown that the cht7 mutation causes loss of viability during nutrient deprivation induced quiescence, a phenotype that could not be properly ascertained in the original cell wall compromised mutant background. Furthermore, meiotic viability was compromised in the cht7 mutant following mating. Cell cycle genes are misregulated during nutrient deprivation induced quiescence in the mutant. A manuscript describing these findings has been nearly completed. 2.2 We have established and studied synchronized cultures of the cht7 mutant, cell cycle mutants, double mutants and wild-types in photobioreactors. Using these conditions, we are in the process of generating RNAseq and ChIPseq data sets during different stages of the cell cycle. Furthermore, we have begun to analyze the composition and properties of the CHT7 complex during the cell cycle. 2.3 We have completed the analysis of the low triacylglycerol mutant of Chlamydomonas, pdgl1¸ under different growth conditions during incubation in photobioreactors. The mutant showed increased abiotic stress sensitivity, altered chlorophyll fluorescence and composition of the photosynthetic apparatus. A paper describing these results is under revision. 2.4 Cocultivating the alga Nannochloropsis and an oleogenic fungus Mortierella, we observed synergistic enhancement of oil production. Moreover, we discovered that the algal cells enter the fungal hyphae and are viable and dividing. In essence we were able to observe the initial steps of an endosymbiotic event reproducibly in the test tube. A paper describing this new system is under revision. 2.5 A toolkit for the expression of genes in the oleogenic algae was developed and used for the over expression of genes to enhance polyunsaturated fatty acid formation. This work has been completed and published. 3. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues. 3.1 The effects of WRI1 on root elongation was analyzed. It was shown that WR1 Affects auxin homeostasis in roots. A paper describing these results was published. 3.2 The interaction of WRI1 with other transcription factors is still investigated. The focus is currently on two transcription factors to determine their mode of interaction with WRI1.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zienkiewicz K, Zienkiewicz A, Poliner E, Du ZY, Vollheyde K, Herrfurth C, Marmon S, Farr� EM, Feussner I, Benning C. 2017. Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts. Biotechnol Biofuels. Online, doi: 10.1186/s13068-016-0686-8.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Yang Y, Zienkiewicz A, Lavell A, Benning C. 2017. Co-evolution of Domain Interactions in the Chloroplast TGD1, 2, 3 Lipid Transfer Complex Specific to Brassicaceae and Poaceae Plants. Plant Cell. 29:1500-1515, doi: 10.1105/tpc.17.00182.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Poliner, E., Pulman, J., Zienkiewicz, K., Childs, K., Benning, C., Farre, E. 2017. A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced fatty acid production Plant Biotech J. doi: 10.1111/pbi.12772
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, K, Froehlich, JE, Zienkiewicz, A, Hersh, LA, Benning C. 2017. A plastid phosphatidylglycerol lipase contributes to the export of acyl groups from plastids for seed oil biosynthesis. The Plant Cell 29:1678-1696, doi: https://doi.org/10.1105/tpc.17.00397
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 119. Kong Q, Ma W, Yang H, Ma G, Mantyla JJ, Benning C. 2017. The Arabidopsis WRINKLED1 transcription factor affects auxin homeostasis in roots. J. Exp. Bot. https://doi.org/10.1093/jxb/erx275.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Students of plant sciences, scientists in academia with focus on plant sciences, scientists in biotech industries with focus on renewable energy and on industrial compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Altogether, 7 postdocs, 6 graduate students, 6 undergraduate students, and 1 technician participated in the described projects. Of these one graduated finished and returned to a lecturer position at a university in Bangkok in her home country, Thailand. Two postdocs left to continue their work on algal lipid metabolism at the University of Goettingen in Germany, with one as part of his Marie-Curie fellowship from the EU. How have the results been disseminated to communities of interest?Asides from the publications reported DC. Benning gave the following public lectures: 10/08/15 Responsible Conduct of Research Presentation together with Cathy Martin. MSU BMB- Department 10/20/15 Greasing Photosynthesis; Regulation of Thylakoid Lipid Biosynthesis in Plants. DOE-BES PI meeting, Gaithersburg, MD. 11/07/15 Greasing Photosynthesis; Regulation of Thylakoid Lipid Biosynthesis in Plants. USDA NC1200 annual meeting. St Louis, IL. 02/10/16 Regulation of Cellular Quiescence and Lipid Droplet Formation in Microalgae. Inst. of Marine and Environ.l Tech., Baltimore MD. 03/07/16 Greasing Photosynthesis: From Membranes to Lipid Droplets. University of Hong Kong, Hong Kong 03/07/16 Lipid Remodeling to Build and Maintain the Photosynthetic Membrane. University of Hong Kong, Hong Kong. 03/10/16 Greasing photosynthesis: From Membranes to Lipid Droplets. Academia Sinica, Taipei, Taiwan. 05/18/16 Vegetable oils in photosynthetic tissues. GLLBRC annual Retreat, Lake Geneva, WI. 07/04/16 Biosynthesis and function of chloroplast 16:1t-phosphatidylglycerol. ISPL2016, Goettingen, Ger. What do you plan to do during the next reporting period to accomplish the goals?1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress: The analysis of TGD1 proteins in Brachypodium and Arabidopsis will be completed as will be the analysis of recombinant PGDL proteins. The role of PGDL lipases in seed oil biosynthesis will be further explored. Moreover, we are planning to gain a mechanistic understanding of growth inhibition in PGDL overproducing lines. We have first indications that these lipases release acyl groups from thylakoid membrane lipids and that these acyl groups serve as precursors for jasmonate biosynthesis. 2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae: A major effort will continue to be on the identification of direct target genes of the CHT7 complex in Chlamydomonas. The in-depth analysis of the cht7 mutant phenotype will be completed. The physiological analysis of the Chlamydomonas pgd1 mutant will be completed and published. We will complete the initial analysis of the newly discovered interaction between Nannochloropsis and Mortierella and publish a first paper on this new system. Subsequent studies will be pursued to explore whether we can gain mechanistic insights into the initial steps of an endosymbiotic event. 3. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues: The analysis of transgenic Brachypodium lines expressing different genes to enhance vegetative oil accumulation will be completed. We will complete the analysis of transcription factors interacting with WRI1.

Impacts
What was accomplished under these goals? 1. Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress. 1.1 Analysis of a Chlamydomonas mutant deficient in the TGD2 protein was completed and published. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant. 1.2 A study of the recombinant SFR2 protein of tomato has been completed. RNAi repression of SFR2 in tomato led to increased salt and drought sensitivity of tomato. In Arabidopsis, loss of SFR2 led to freezing sensitivity but not to sensitivity to salt or drought stress distinguishing the function of SFR2 in freezing sensitive and resistant plants. 1.3 The analysis of three chloroplast located lipases of Arabidopsis has been nearly completed. Overexpression of these proteins led to changes in thylakoid membrane lipid profiles that suggest that in vivo they prefer particular molecular species of phosphatidylglycerol or monogalactosyldiacylglycerol. Based on pulse chase experiments and lipid analysis of mutant and wild-type developing seeds of Arabidopsis, we hypothesize that these lipases are involved in acyl exchange and participate in the export of fatty acids from the chloroplast in developing embryos. 1.4 The Brachypodium TGD1 protein was found to only partially complement the Arabidopsis tgd1 mutant. Lipid trafficking was partially restored but activation of SFR2 was not reversed. To follow up on this observation, we generated chimera of the Arabidopsis and Brachypodium TGD1 protein and introduced them into the Arabidopsis tgd1 mutant for complementation. We identified a specific loop of TGD1 that is required for complementation. Protein modeling suggests that this loop interacts with another component of the TGD123 lipid transport complex. This hypothesis is currently tested. 2. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae 2.1 The detailed phenotypic analysis of the cht7 mutant cells of Chlamydomonas has been nearly completed. We showed that cell cycle genes are misregulated during N-deprivation. This is likely causal to other morphological cell phenotypes observed during N deprivation and N-refeeding. We have also begun to study the cht7 mutant and the wild type in photobioreactors. We established conditions to synchronize the growth of the cells, which will allow us to compare the transcriptome of mutant and wild type at different stages of the cell cycle. In addition, we developed new cell lines combining cell cycle mutations and the cht7 mutation into compatible background for further study. 2.2 The low triacylglycerol l mutant of Chlamydomonas, pdgl1¸ was characterized under different growth conditions during incubation in photobioreactors. The mutant showed altered chlorophyll fluorescence and composition of the photosynthetic apparatus as well as increased light sensitivity leading to reactive oxygen formation. 2.3 Cocultivating the alga Nannochloropsis and an oleogenic fungus Mortierella, we observed synergistic enhancement of oil production. Moreover, we discovered that the algal cells enter the fungal hyphae and are viable and dividing. In essence we were able to observe the initial steps of an endosymbiotic event reproducibly in the test tube. Because both organisms are experimentally tractable at the molecular level, this new experimental system provides an opportunity to gain mechanistic insights into the establishment of an endosymbiotic interaction between a photosynthetic alga and a heterotrophic fungus. 2.4 We collaborated in the analysis of a ferredoxin-5 mutant of Chlamydomonas leading to altered lipid metabolism and thylakoid disruption in the dark. 3. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues 3.1 The ectopic expression of WRI1 in Brachypodium led to increased lipid droplet formation in leaf blades but also induced localized cell death. To overcome this problem constructs were tested expressing WRI1 and other genes from algae under the control of stem-specific promoters. The analysis of these plants is ongoing. 3.2 The interaction of WRI1 with other transcription factors is investigated. The focus is currently on two specific transcription factors to determine their mode of interaction with WRI1. 3.3 An extensive analysis of diacylglycerol acyltransferase involved in triacylglycerol formation in Nannochloropsis was completed. A specific isoform has been explored for its efficacy in lipid droplet formation and enhancement of energy density of leaves in transgenic plants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang Y, Munz J, Cass C, Zienkiewicz A, Kong Q, Ma W, Sanjaya S, Sedbrook JC, Benning C. 2015. Ectopic expression of WRI1 affects fatty acid homeostasis in Brachypodium distachyon vegetative tissues. Plant Physiol. 169:1836-1847.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Warakanont J, Tsai CH, Michel EJ, Murphy GR 3rd, Hsueh PY, Roston RL, Sears BB, Benning C. 2015. Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue. Plant J. 84:1005-1020.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang, W., T. M. Wittkopp, X. Li, W. Warakanont, A. Dubini, C. Catalanotti, R. G. Kim, E. C. M. Nowack, L. C. M. Mackinder, M. Aksoy, M. D. Page, S. DAdamo, S. Saroussi, M. Heinnickel, X. Johnson, P. Richaud, J. Alric, M. Boehm, M. C. Jonikas, C. Benning, S. S. Merchant, M. C. Posewitz, and A. R. Grossman. 2015. Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism Proc. Natl. Acad. Sci. USA 112:14978-83.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Handee,W., Li, X., Hall, K.W., Deng, X., Li, P., Benning, C., Williams, B.L., Kuo, M.H. 2016. An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast. PLoS Genet. 12:e1005878. doi: 10.1371/journal.pgen.1005878.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Barnes, A.C., Benning, C., Roston, R. 2016. Chloroplast membrane remodeling during freezing stress is accompanied by cytoplasmic acidification activating SENSITIVE TO FREEZING2. Plant Physiol. 171:2140-9. doi: 10.1104/pp.16.00286.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ma, W., Kong, Q., Mantyla, J.J., Yang, Y., Ohlrogge, J.B., Benning, C. 2016. 14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1. Plant J. doi: 10.1111/tpj.13244. (Epub ahead of print).
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Wang, K., Hersh, H.L., Benning, C. 2016. SENSITIVE TO FREEZING2 aides in resilience to salt and drought in freezing-sensitive tomato. Plant Physiol. pii: pp.01183.2016 (Epub ahead of print).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kelly, A.A., Kalisch, B., H�lzl, G., Schulze, S., Thiele, J., Melzer, M., Roston, R.L., Benning, C., D�rmann, P. 2016. Proc. Natl. Acad. Sci U S A pii: 201609184. (Epub ahead of print).
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Du Z.-Y., Benning C. 2016. Triacylglycerol accumulation in photosynthetic cells in plants and algae. In: Lipids in Plant and Algae Development. Eds. Nakamura, Y., Li-Beisson, Y. Springer, Subcell. Biochem. 86:179-205.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zienkiewicz K, Du Z.-Y., Ma W., Vollheyde K., Benning C. (2016). Stress-induced neutral lipid biosynthesis in microalgae - Molecular, cellular and physiological insights. Biochim. Biophys. Acta. 1861:1269-81. doi: 10.1016/j.bbalip.2016.02.008.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Horn, P.J., Benning, C. 2016. The plant lipidome in human and environmental health. Science 353: 1228-32. doi: 10.1126/science.aaf6206


Progress 11/01/14 to 09/30/15

Outputs
Target Audience:Students of plant sciences, scientists in academia with focus on plant sciences, scientists in biotech industries with focus on renewable energy and on industrial compounds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for 7 postdoctoral researchers, 6 graduate students and approximately 5 undergraduate students during the reporting period. One graduate student, Chia-Hong Tsai graduated and is currently pursuing a career in the plant biotech industry. One undergraduate student, Elena Michel, graduated and entered graduate school. How have the results been disseminated to communities of interest?Aside from the reported publications, Christoph Benning gave the following presentations during the report period: Lipid transport and membrane remodeling to build and protect chloroplasts Chonnam National University, South Korea 10/10/14 Regulation of TAG biosynthesis in microalgae. POSTECH, IBB, Pohang, South Korea 11/11/14 Synthetic enzyme assemblies at the surface of lipid droplets. Synbio Conference, Berkeley, CA 11/15/14 Carbon partitioning into lipid droplets in microalgae. USDA NC1200 Annual Meeting, Kansas City, MO 12/17/14 Greasing photosynthesis: The influence of nutrient status on the lipidome. Washington State University, Pullman, WA 01/29/15 Learning from algae how to enhance the energy density of biofuel crops. Texas A & M, College Station, TX 02/01/15 Maximizing photosynthetic CO2 conversion into triacylglycerols in microalgae. GRC Plant Lipids, Galveston, TX 03/23/15 Greasing photosynthesis: The influence of nutrient status on the lipidome. Michigan State University-DOE-PRL. East Lansing MI. 03/31/15 Lipid Transport involving chloroplast envelope membranes in plants and algae. ASBMB, Plant Lipid Metabolism. Boston MA. 04/15/15 Greasing photosynthesis and making fuels. MSU Foundation Professorship Investiture. Michigan State University, East Lansing, MI 48823. 06/18/15 Understanding Photosynthetic CO2 Conversion into Triacylglycerols in Microalgae. Summer REU program in Plant Genomics at MSU. East Lansing, MI. 07/01/15 Regulation of Cellular Quiescence in Microalgae. iGRAD Plant Symposium. HHU Düsseldorf, Germany. What do you plan to do during the next reporting period to accomplish the goals? Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress: A focus will be on the refinement of in vitro transport assays to determine the lipid substrates for TGD proteins. The analysis of the role of SFR2 proteins in tomato will be completed and published. The analysis of recombinant PGDL proteins will be completed and published. The focus will shift to gain a better understanding of the function of the PGDL proteins in vivo through the detailed analysis of the respective mutants. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae: A major effort will be on the identification of direct target genes of the CHT7 complex in Chlamydomonas. A more in-depth analysis of the cht7 mutant phenotype will be published. The physiological analysis of the Chlamydomonas pgd1 mutant will be completed and published. The analysis of acyltransferases involved in triacylglycerol formation in Nannochloropsis will be completed and published. Their inactivation using CRISPR technology will developed. Selected lipid mutants of Nannochloropsis will be characterized to identify the mutated gene. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues: The analysis of Brachypodium lines partially deficient in the TGD1 protein will be completed. New transgenic Brachypodium lines expressing different genes potentially enhancing vegetative oil accumulation will be generated and characterized.

Impacts
What was accomplished under these goals? Deciphering the role of membrane lipid trafficking and remodeling in protection against abiotic stress. 1 Analysis of a Chlamydomonas mutant deficient in the TGD2 protein was completed. It was determined that Chlamydomonas uses ER-derived precursors for thylakoid lipid biosynthesis. 2 Recombinant SFR2 protein of tomato has been obtained in its active form. RNAi repression of SFR2 in tomato led to increased salt and drought sensitivity. 3 Three PGDL proteins of Arabidopsis have been produced in their recombinant form and their lipase activity has been characterized. The proteins were found to be associated with different chloroplast membranes. Overexpression of these proteins led to changes in thylakoid membrane lipid profiles. Identifying regulatory mechanisms controlling lipid biosynthesis in microalgae. Phenotypic analysis of the CHT7 mutant cells of Chlamydomonas has shown misregulation of cell cycle genes during N-deprivation. This is likely causal to other newly observed morphological cell phenotypes observed during N deprivation and N-refeeding. 1 The low triacylglycerol l mutant of Chlamydomonas, pdgl1¸ was characterized under different growth conditions during incubation in photobioreactors. The mutant showed altered chlorophyll fluorescence and composition of the photosynthetic apparatus as well as increased light sensitivity. 2 The mechanism of lipid droplet formation in Chlamydomonas under different growth conditions has been investigated. A role for the major lipid droplet protein of Chlamydomonas in the targeting of proteins to the lipid droplet was identified. 3 The major enzymes involved in triacylglycerol formation in Nannochloropsis have been identified and obtained in active recombinant form. A forward genetic mutant screen for lipid mutants of Nannochloropsis was conducted and led to the identification of promising mutants. Mapping of the affected genes is under way. Exploring lipid biosynthesis in grasses and enhancing the energy density in green tissues. 1 The Brachypodium TGD1 protein was found to only partially complement the Arabidopsis tgd1 mutant. Lipid trafficking was partially restored but activation of SFR2 was not reversed. Partial repression of TGD1 in Brachypodium lines does lead to mild lipid trafficking phenotypes but does not activate SFR2 as observed for Arabidopsis. 2 The ectopic expression of WRI1 in Brachypodium led to formation to increased lipid droplet formation in leaf blades but also induced localized cell death. It was determined that this phenotype was due to accumulation of free fatty acids which are toxic to cells. New construct were tested expressing WRI1 and other genes from algae under the control of stem-specific promoters. New, more stable variants of WRI1 were identified that led to higher accumulation of triacylglycerol in overexpressing tissues.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Panchy, N., Wu G., Newton, L., Tsai, C.-H., Chen, J., Benning, C. Farre, EM, Shiu, S.-H. 2014. Prevalence, evolution and cis-regulation of diel transcription in Chlamydomonas reinhardtii. G3, doi:10.1534/g3.114.015032
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tsai, CH, Zienkiewicz, K, Amstutz, CL, Brink, BG, Warakanont, J, Roston, R, Benning C. 2015. Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii. Plant J. doi: 10.1111/tpj.12917
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Tsai CH, Warakanont J, Takeuchi T., Sears BB, Moellering ER, Benning C. 2014.The protein COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas. Proc Natl Acad Sci U S A, doi: 10.1073/pnas.1414567111
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Ma, W., Kong, Q., Grix, M., Mantyla, J. J., Yang, Y., Benning, C., Ohlrogge, JB. 2015. Deletion of a C-terminal intrinsically disordered region of WRINKLED1 affects its stability and enhances oil accumulation in Arabidopsis. Plant J. DOI:10.1111/tpj.12933
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Poliner, E., Panchy, N., Newton, L., Wu, G., Lapinsky, A., Bullard, B., Zienkiewicz, A., Benning, C., Shiu, S.H., Farr�, E.M. 2015. Transcriptional coordination of physiological responses in Nannochloropsis oceanica CCMP1779 under light/dark cycles. Plant J. 2015 Jul 27. doi: 10.1111/tpj.12944.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Lipid trafficking in plant cells. 2014. Hurlock AK, Roston RL, Wang K, Benning C. (2014) Traffic. 15:915-32.