Brief Summary of Minutes of Annual Meeting

The meeting was held at the National Capital Region campus of Virginia Tech, in Arlington, VA. Glenda Gillaspy (VAES) began the meeting with introductions. The two federal agency program directors were introduced (Shing Kwok, USDA and Gerald Schoenknecht, NSF). Christoph Benning; MI-ABR, PRL talked about his work on the plant Lipidome. The DOE PRL mission is to work on energy capture, with a focus on the chloroplast, the main organelle containing photoysynthetic membranes. He detailed the identification of genes in the galactolipid pathway, and prospects for engineering an increase in oil production.

Benning also described the work of NC1200 member, David Kramer, who was not present this year. The Kramer lab is investigating photosynthesis by using imaging  of the dynamic environment (DEPI). DEPI has been used to screen various mutants, including Benning’s lipid mutants. This approach is revealing alterations in the mutants in response to changes in various photosynthesis parameters.

Discussion after this talk centered around how to bridge discovery in the lab before a company gets interested in investing. Discussion also centered around using CRISPR to avoid issues of being labelled as a GMO.  The difficulty of using CRISP in crop species with much redundancy was discussed. The idea that seed size is a constraint on total productivity was brought up by Rob Aiken, as well as  trade-offs when bigger, but fewer seed result from metabolic engineering. Potential strategies to address this were discussed: altering oil production after the embryo is formed. Krishna Jagadish brought up the issue of canola being an indeterminate plant, and the prospect that this would reduce the trade-off during seed set. He also mentioned that soy or peanuts might be good to engineer for this trait. Vara Prasad brought up the idea that similar strategies might be useful for engineering seed size or germination, and having a biomarker for something like oxylipins could be a powerful marker to streamline strategies. The issue of toxicity of fatty acids in plants was discussed and a remark made that Ruth Welti’s group at KSU can measure lipids for about $100/sample.

Anastasios Melis, CA-AES spoke about examining ways to engineer better efficiency of photosynthesis by lowering the size of the antenna within chloroplasts. Compelling data were presented that efficiency can be improved, at least under standard, non-stressful greenhouse conditions. Discussion centered around speculation as to what would happen in this system in the field, where stress would be a likely factor.

Helmut Kirchoff, WAES, spoke about his work on the biophysical properties of photosynthetic membranes and efficiency of photosynthesis. He addressed the issue of dynamic environments, pointing out that most are examining  static images of the system. He detailed his approach analyzing grana stacks within chloroplasts. Of note were results presented on specific lipid mutants which appear to have a faster NPQ, and associated changes in movement of molecules through photosynthetic membranes. In the discussion following, the issue of plastoglobuli was raised: whether these are a reservoir for lipids in chloroplasts, and whether these are involved in membrane remodeling.

Shing Kwok, NIFA, and our NIFA Rep presented information on NIFA funding programs, and pointed out that there are not enough translational photosynthesis research projects. Shing also provided advice and instructions on how to prepare NIFA reports, so as to highlight activities properly. Discussion after this presentation entered around federal funding rates, and the future of science.

Krishna Jagadish, KAES, spoke on season temperature and growth potential, highlighting that climatic challenges impact productivity in Sorghum. He described work on new sources of cold tolerance that were introgressed. The work utilizes field phenotyping with a College Precision Ag specialist, and aerial high throughput phenotyping. With metabolomics and transcriptomics on hybrids it was found that chilling stress and seed size are not associated. Beginning work on temperature stress in rice was presented.

Vara Prasad, KAES, spoke on sustainable farming. His work with USAID in Senegal was described, which focuses on the Agro-forestry system in West Africa. He also described research on sorghum, pointing out that this crop needs stress tolerance and recovery from stress pathways. He pointed out increases in global surface temperature by NASA show an upward trend. There is also a trend of high night time temperatures, will impact carbon balance and respiration. Hot weather, and extreme weather events are also important for agriculture. It is projected that 41% of land will be exposed to heat stress by 2050s. With major food crops exposed to heat, we need to understand the critical stage for heat stress.  He also spoke about using Technology kits at high schools in Cambodia and Senegal- to engage youth interested in agriculture. Lastly, he brought up the Womanization of agriculture, with legumes, chickens, and milk being grown by women, whereas grain crops are more grown by men.

Glenda Gillaspy, VAES, spoke about work on inositol phosphate signaling molecules and their relationship to phosphate sensing. It was pointed out that phosphate is a problem in certain areas of the US, while being not available to sue to stimulate growth in other parts of the world. Discussion centered around whether lipids or fatty acids were involved in some of the inositol phosphate signaling mutant phenotypes. Discussion on a science high school outreach project and critical thinking followed.

Business meeting. It was decided to ask Doug Allen to host the 2018 meeting at the Danforth Institute. Gillaspy agreed to ask Allen about this. It was agreed that Washington State University would be the back-up plan, and that 2019 meeting would be hosted by Melis in Berkeley, CA.

Activities: Progress from several groups was reported on our 2018 Milestone to engineer DNA plasmids expressing photosynthetically relevant genes and gene fusion constructs (1, 2, 3, 4; CA-AES, IL-ARS, MI-ABR, MS-AES, NE-AES, NV-AES, OH-AES, VA-AES, WA-AES). In 2017 several groups reported success with gene constructions that have either already been transferred into plants, or are ready for transfer. These include: three plastidial glucose-6-phsphate dehydrogenases (MI-ABR), RBP-P, an RNA binding protein required for glutelin RNA localization (WA-AES), fusions of genes hypothesized to be involved in thylakoid biogenesis fused to cutting edge fluorescent molecules (NE-AES), a truncated Rubisco activase (RCA) gene (IL-AES), one of three chloroplast located lipases, PLIP1, of Arabidopsis, and the Brachypodium TGD1 protein, which is part of a lipid transfer complex in the inner chloroplast envelope membrane (MI-ABR), and inositol pyrophosphate kinases (VA-AES).

Progress is reported on our (2018) Milestone to engineer the single RCA gene in rice which is required for redox regulation, using the CRISPR-Cas9 system (2.1, IL-ARS). Elimination of the carboxy-terminal portion of the RCA protein (by introduction of a stop codon) results in removal of redox regulation of RCA activity, which was postulated to enhance photosynthesis especially under non-steady state conditions.  Results of experiments in the greenhouse and growth chambers continue to suggest a positive impact on plant growth and seed yield per plant, but the mechanism is not yet clear.

Progress on our 2018 Milestone focused on the G6P shunt pathway (2.2, MI-ABR, WA-AES) is reported. Of the three plastidial glucose-6-phsphate dehydrogenases, one has been found to be redox sensitive with a midpoint potential of -378 mV. This positions it to be modulated by physiological changes in stromal redox potential. Phosphoglucoisomerase (PGI), one of the sources of G6P for the shunt is extremely sensitive to competitive inhibition by erythrose 4 phosphate. The Km for G6P is higher than for fructose 6-phosphate suggesting that flow is primarily from F6P to G6P.

Progress on our  (2019) Milestone focused on the biosynthesis and specific function of 16:1trans in phosphatidylglycerol and the role of oligogalactolipids in chloroplast membrane (1.1, NE-AES, MI-ABR) is reported: The analysis of one of three chloroplast located lipases, PLIP1, of Arabidopsis has been completed and published. It was shown 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. It was 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) is under way; 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 above. Loss of this lipid in the fad4 mutant reduces seed oil content. A new protein cofactor required for the activity of this protein has been identified (MI-ABR). NE-AES has discovered that SFR2, a chloroplast lipid modifying enzyme has a phosphorylation site when in its inactive state. They have also shown that triacylglycerol accumulates in response to cytoplasmic acidification, and chloroplast lipid changes due to sensing of freezing occur in warmer temperatures in less freezing tolerant species.

Progress on drought and heat stress-focused Milestones is reported. (Drought stress will be quantified by inferring canopy conductance from digital images of vegetative indices and thermal irradiance (4.1, KS-AES), and heat stress will be quantified by canopy temperature, gas exchange, leaf fluorescence, pollen viability, seed-set percentage, and harvest index (4.1, KS-AES). A novel method was developed to identify geographic regions which exhibit similar long-term climate dynamics. Simulation work demonstrated the capacity to relate wheat and sorghum grain productivity to water use. Carbon isotope discrimination can be used as an indicator of water stress avoidance, providing additional tools identifying genetic gain in winter wheat. NC1200 researchers also have quantified the impact of diurnal temperature change and night temperature on biomass accumulation, and showed that lipid composition of leaves under high temperatures influences physiological response which confers with tolerance or susceptibiliy of genotypes. It was determined that high temperature stress influences co-occurrence of certain lipid groups that are regulated by the same enzymes.

Progress on understanding the role of phosphorylation events in abiotic stress responses was reported, including the involvement of phospho-regulation of protein-protein interactions (4.2, IL-ARS, NV-AES, VA-AES). The cysteine residues targeted for glutathionylation in the receptor kinase Brassinosteroid Insensitive 1-Associated receptor-like Kinase 1 (BAK1) was examined using molecular dynamics simulations. It was found that Cys-408 greatly reduces the prevalence of an enzymatically competent conformation and has a significant effect of the conformations of distant residues identifying the major site responsible for oxidative modification of BAK1 kinase activity. It was also demonstrated that Tyr-610 of BAK1 is not essential for either brassinosteroid-mediated growth or innate immunity.  The possibility that phosphorylation of Tyr-610 is required for some other functional role remains a distinct possibility, and awaits further study (IL-AES). VA-AES reported specific phosphorylation sites within the inositol pyrophosphate kinases have been verified via Mass Spectrometry.

Milestones: Our description of activities addresses the milestones from our original proposal.

Outputs: See attached list of Publications.

PATENT APPLICATION FILED (Jul, 2017): PCT International Application No. PCT/US17/40859, Title: Mutated Rubisco Activase Applicant(s):         THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF AGRICULTURE, IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC. Refs: AG017316-PCT / D.N. 173.16  (David Marks); NC 1200 member Steve Huber

Plans for the Coming Year: The group will pursue three main lines of investigations. The first is a continued examination of basic processes that influence chloroplast membranes, carbon fixation, photoassimulate production and use. This work will involve investigating the role of PLIP2 and 3 lipases in responses of plants towards abiotic stress.  The reaction mechanism of FAD4 and the role of cofactors will be further investigated. The role of a rhomboid protease located in the chloroplast envelope membranes will be investigated, as will the role of protein kinase in chloroplast cold sensing. G6P shunt enzymes that could lead to significant flux through the shunt will be tested using transient expression of GPT2, plastidial starch phosphorylase, and other critical genes. The second area focuses on understanding carbon management and trade-offs in field grown crop species. We will continue to apply image analysis tools utilize simulation models to quantify crop water use and heat tolerance in a variety of crop species. The third area continues approaches to engineer plants with beneficial qualities. Examples include PLIP1 based engineering of seed oil content in Camelina, overexpression of AGPase in leaves as well as seeds for isoline development, collaborative studies with M. Spalding (ISU) to analyze existing transgenic rice lines with targeted editing (truncation) of the Rubisco activase gene to further test the hypothesis that maintaining Rubisco activation state at low light enhances photosynthesis and plant growth, and characterization of plant lines expressing novel AGPase isoforms.