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At least 613 records · Page 34

Changes in global land use and CO 2 emissions from us bioethanol production: what drives differences in estimates between corn and cellulosic ethanol?

Land use change (LUC) CO 2 emissions associated with bioenergy production depend on the amount of land required to produce bioenergy crops, the carbon stored in such crops (including in the leaves, stalk, roots and soil), and the carbon emitted when another land cover is directly or indirectly displaced as a result. In this study, we use a global integrated assessment model [the Global Change Analysis Model (GCAM)] to explore the differences in estimates of LUC CO 2 emissions for two crops (corn and switchgrass) used to produce ethanol in the United States under alternative assumptions about natural lands protection. Varying the latter assumptions for corn ethanol results in net LUC CO 2 emissions between 7 and 41 gCO 2 per MJ of ethanol, whereas varying the same assumptions for switchgrass ethanol results in net emissions between -26 and 14 gCO 2 per MJ of ethanol. The low-end estimate for each occurs when natural lands are assumed to be fully protected everywhere, which leads to significant cropland intensification. The high-end estimate for each occurs when natural lands are assumed to be unprotected everywhere, leading to greater cropland expansion and associated conversion of unmanaged forest and pasture. Results from this study could be used to inform scenarios of future energy system change or life cycle assessment of biofuels for which LUC emissions would be an input.

54 ENVIRONMENTAL SCIENCES↗

CA-ResidueRetentionAssumptionsBT16-BaseCase

As part of the Billion Ton resource assessment projections created in 2016 (see https://www.energy.gov/sites/prod/files/2016/12/f34/2016_billion_ton_report_12.2.16_0.pdf, DOI: 10.2172/1435342) -henceforth "BT16", this dataset was used as an assumption to limit the availability of residues under a "base-case" scenario (BC1). Crop residues that had this assumption applied include corn stover, cereal (wheat, oats, and barley) straws, and sorghum stubble. What is the purpose of the data set? Why were the data collected? Per request for use in subsequent research, we have summarized assumptions for California only and selected years (2020, 2030, 2040) that were used in the BT16's base-case projections for agricultural residues and have provided details by tillage class that limited residue availability for harvest (dry tons of residues that must remain). Note: '10' is a high number that assures that no residue harvested could occur. Note: Tillage classification assumptions are also of importance: a low flexibility was applied, allowing a moderate deviation from a baseline situation (using historic CTIC data on tillage type used in counties for each crop). A moderate flexibility, allows farmers to put land into another tillage type (no till, conservation till, and reduced till) where a higher net present value was calculated. This dataset includes all allowable tillage types by crop, but each tillage type may not have occurred in BT16 simulations.

Davis, Maggie↗

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

Abstract Background Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. Results We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T 1 generation of any crop transformed via Agrobacterium . We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. Conclusion This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

Abstract Background The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase 1-2 ( DGAT 1-2) and Oleosin 1 ( OLE 1) in combination with RNAi suppression of SUGAR-DEPENDENT 1 ( SDP 1) and Trigalactosyl diacylglycerol 1 ( TGD 1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson’s correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT 1-2, followed by the level of RNAi suppression for SDP 1. Conclusions This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

1-2↗

Rapid quantification of whole seed fatty acid amount, composition, and shape phenotypes from diverse oilseed species with large differences in seed size

Seed oils are widely used in the food, biofuel, and industrial feedstock industries, with their utility and value determined by total oil content and fatty acid composition. Current high throughput seed oil analysis methods either lack accuracy in total fatty acid profiling or require extensive labor for lipid extraction prior to derivatization to fatty acid methyl esters (FAME) and quantification by gas chromatography (GC). Alternatively, direct whole seed FAME production methods have been developed for the very small seeds in the model species Arabidopsis thaliana but these have generally not been adapted to larger seeds of most oilseed crops. High-throughput direct whole seed FAME production methods were optimized for seeds up to 5 mg each utilizing acid-catalyzed esterification. For the oilseed species Camelina sativa, Thlaspi avernse (pennycress), Cuphea viscosissima, and Brassica napus (var. Canola), the total seed fatty acid content and composition from direct seed esterification to FAME matched that of lipid extract derivatization demonstrating the accuracy of the methods. In combination with seed phenotyping using GridFree, this approach enabled the development of a rapid pipeline for simultaneous seed weight, count, size/shape phenotyping, and oil analysis. For the larger and tougher seeds produced by Limnanthes alba (Meadowfoam) and Cannabis sativa L. (hemp) the whole seed acid-based method proved insufficient, and prior laborious homogenization of seeds was required. Therefore, a rapid one-tube bead homogenization and base catalyzed-esterification method was developed. Base-derived fatty acid esterification cannot derivatize free fatty acids leading to slightly lower total seed fatty acid than acid-catalyzed methods, however the seed oil content and fatty acid composition that is valuable for screening large numbers of samples in research populations was accurately measured. New rapid whole seed fatty acid esterification and phenotyping protocols were developed to accurately assess oilseed lipid content. These methods are particularly valuable in oilseed research, breeding, and engineering applications where efficient analysis of large numbers of samples and accurate oil fatty acid profiling is essential. While having been developed for current and emerging oilseed crops, these methods also provide a foundation from which protocols might be established for new and emerging crop species.

59 BASIC BIOLOGICAL SCIENCES↗

Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor

Abstract Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum ( Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p -coumarate ( p CA), a higher amount of p -coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of p CA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound p CA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall p CA remains unchanged indicating that most p CA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound p CA and lignification in cell walls.

09 BIOMASS FUELS↗

Data for Process Strategies for Recovery of Sugars, Lipids, and Lignin from Oilcane Bagasse Using Natural Deep Eutectic Solvents (NADES)

Sugarcane is being enhanced as a bioenergy crop by engineering it to accumulate and store lipids along with polymeric sugars in vegetative tissues. However, there is no existing process that allows for processing this new crop to recover both lipid and cellulosic sugars from the oilcane bagasse. Therefore, a comprehensive investigation of two pretreatment methods—natural deep eutectic solvents (NADES) and chemical-free hydrothermal pretreatment (HT) was conducted to judge their suitability for recovering fermentable sugars, lipids, and lignin from bagasse. Two NADES, i.e., choline chloride: lactic acid (ChCl:LA) and betaine: lactic acid (BT:LA) were prepared using a 1:2 M ratio and were evaluated for pretreatment of oilcane bagasse at 10, 20, and 50 % (w/w) solids, followed by enzymatic hydrolysis at 10 % (w/w) solids. Notably, ChCl:LA NADES treatment at 10 % (w/w) solids at 140 °C for 2 h, solubilized 78.8 % of lignin and 80.4 % of hemicellulose and allowed 82.7 % enzymatic conversion of glucans to glucose. In contrast, HT pretreatment removed approximately 87.6 % of the hemicellulose and provided an enzymatic glucose yield of 69.7 %. Furthermore, ChCl:LA operated at 50 % solids loading the enriched lipids 2.6-fold (9.2 wt%) in recovered solids compared to HT (6.4 %) and BT:LA (5.1 %) pretreatment processes. NMR-HSQC and GPC analysis showed that ChCl:LA also cleaved the most lignin β–O–4 linkages and demonstrated lower molecular weight compared to HT. This study demonstrates that NADES pretreatment is an effective green processing method for recovering lipids, sugars, and lignin from bioenergy crops at high solid loading (50 % w/w) within the context of an integrated biorefinery.

Conversion↗

Data for Climate vs Energy Security: Quantifying the Trade-offs of BECCS Deployment and Overcoming Opportunity Costs on Set-Aside Land

Bioenergy with carbon capture and storage (BECCS) sits at the nexus of the climate and energy security. We evaluated trade-offs between scenarios that support climate stabilization (negative emissions and net climate benefit) or energy security (ethanol production). Our spatially explicit model indicates that the foregone climate benefit from abandoned cropland (opportunity cost) increased carbon emissions per unit of energy produced by 14–36%, making geologic carbon capture and storage necessary to achieve negative emissions from any given energy crop. The toll of opportunity costs on the climate benefit of BECCS from set-aside land was offset through the spatial allocation of crops based on their individual biophysical constraints. Dedicated energy crops consistently outperformed mixed grasslands. We estimate that BECCS allocation to land enrolled in the Conservation Reserve Program (CRP) could capture up to 9 Tg C year–1 from the atmosphere, deliver up to 16 Tg CE year–1 in emissions savings, and meet up to 10% of the US energy statutory targets, but contributions varied substantially as the priority shifted from climate stabilization to energy provision. Our results indicate a significant potential to integrate energy security targets into sustainable pathways to climate stabilization but underpin the trade-offs of divergent policy-driven agendas.

Field Data↗

UAV remote sensing imagery - Miscanthus trials 2020 - Energy Farm - UIUC

Aerial imagery utilized as input in the manuscript "Deep convolutional neural networks exploit high spatial and temporal resolution aerial imagery to predict key traits in miscanthus" . Data was collected over M. Sacchariflorus and Sinensis breeding trials at the Energy Farm, UIUC in 2020. Flights were performed using a DJI M600 mounted with a Micasense Rededge multispectral sensor at 20 m altitude around solar noon. Imagery is available as tif file by field trial and date (10). The post-processing of raw images into orthophoto was performed in Agisoft Metashape software. Each crop surface model and multispectral orthophoto was stacked into an unique raster stack by date and uploaded here. Each raster stack includes 6 layers in the following order: Layer 1 = crop surface model, Layer 2 = Blue, Layer 3 = Green, Layer 4 = Red, Layer 5 = Rededge, and Layer 6 = NIR multispectral bands. Msa raster stacks were resampled to 1.67 cm spatial resolution and Msi raster stacks were resampled to 1.41 cm spatial resolution to ease their integration into further analysis. 'MMDDYYYY' is the date of data collection, 'MSA' is M. Sacchariflorus trial, 'MSI' is Miscanthus Sinensis trial, 'CSM' is crop surface model layer, and 'MULTSP' are the five multispectral bands.

bioenergy↗

AmeriFlux FLUXNET-1F US-RC3 WSU Lind Dryland Research Station

This is the AmeriFlux Management Project (AMP) created FLUXNET-1F version of the carbon flux data for the site US-RC3 WSU Lind Dryland Research Station. This is the FLUXNET version of the carbon flux data for the site US-RC3 WSU Lind Dryland Research Station produced by applying the standard ONEFlux (1F) software. Site Description - RC3 operated from 2011-2016 at the Washington State University Lind Dryland Experimental Station, as part of a cluster of 5 towers (RC1 to RC5) operated for the Regional Approaches to Climate Change (REACCH) USDA-supported research project. Lind is in the low precipitation region of the Columbia Plateau dryland cropping region. The area is characterized as a stable crop-fallow agroecological zone, with crops typically grown every second year to allow soil water to accumulate during intervening fallow years. Winter wheat was grown August 2012-August 2013, and August 2014-July 2015. The growing seasons of 2012 and 2014 were fallow years. Soils at Lind are predominantly silt loam texture Mollisols in the Shano and Ritzville series. Site topography is flat.

Chi, Jinshu [The Hong Kong University of Science a↗

AmeriFlux US-NYo NYSM - Penn Yan

This is the AmeriFlux version of the carbon flux data for the site US-NYo NYSM - Penn Yan. Site Description - Located in a crop field setting. It is very flat and open, at the edge of a crop field. Obstructions within 100m include high crops. The soil type is Honeoye silt loam. This site is a part of the New York State Mesonet Flux Network, one of multiple subnetworks (e.g. Profiler, Snow) operated by the New York State Mesonet. It also operates as a Standard meteorological site.

Miller, Scott [University at Albany]↗

U.S. Department of Energy Free-Air CO 2 Enrichment Experiments: FACE Results, Lessons, and Legacy

In the 1970s, growing concern about potential long-range consequences of carbon dioxide (CO 2 ) emissions from the combustion of fossil fuels prompted the U.S. Department of Energy (DOE) to begin developing a CO 2 research program. The goal was to obtain the scientific information needed to reduce uncertainty and develop a knowledgebase for decision-making. At its start in 1980, this program had three components: the global carbon cycle, climate effects, and vegetation effects. The vegetation effects component recognized that increased atmospheric CO 2 could be a potential benefit to vegetation and crops because CO 2 is an essential resource for plant growth. In addition to increasing crop yield, program developers and researchers recognized that this fertilizer effect could also result in greater storage of fossil fuel CO 2 by forests. Initial experiments were small in scale and scope and focused on the photosynthesis, physiology, and water use of crop plants, but the focus shifted toward responses of plants in natural systems, as needed for connections to the global carbon cycle. Program research investigated CO 2 interactions with other environmental variables, emphasizing field research whenever possible and recognizing that exposure to elevated CO 2 concentrations under realistic field conditions should lead to better predictions of plant response. Whole-ecosystem field experiments were instituted in low-stature ecosystems, but experiments to address forest responses necessarily relied on tree seedlings or saplings in open-top chambers (OTCs) and not within intact forest ecosystems. The need for larger-scale, longer-duration experiments in each of the world’s six major biomes (tundra, boreal forest, temperate forest, tropical forest, grassland, and desert) became clear, and for these experiments, free-air CO 2 exposure facilities were necessary. This report summarizes the objectives and main results of DOE-sponsored experiments employing such facilities, along with the outcomes from research on ecosystem responses using field chambers (see Fig. ES.1, p. x). Additionally, this report highlights the many successes of DOE CO 2 enrichment studies and considers the lessons learned that can be used to guide future initiatives.

54 ENVIRONMENTAL SCIENCES↗

Raman Spectroscopy to Detect and Measure NOW Pheromones. Annual Report [Jan 2021]

Insect sex pheromones are chemical compounds that insects release to attract their partners over distances of hundreds of meters or even kilometers, in complete darkness and without any audible signals. Use of synthetic forms of key compounds have in some cases become an essential component of monitoring and/or managing key pests of agricultural crops, including navel orangeworm (Amyelois transitella) (NOW) in California tree nuts. These pheromone-based strategies can include monitoring, mating disruption, mass trapping, attract-and-kill and push-pull. There are currently multiple commercially available mating disruption products available for NOW and recent studies have demonstrated that they can be effectively used to reduce crop damage by this pest. Just how mating disruption works is not fully established and likely varies across products and target species. For instance, the extent to which synthetic pheromones compete with natural pheromone is not well understood, or in the case of monitoring, how efficaciously the insect follows the diffusing plumes, especially across large blocks and at plot borders. Furthermore, there may be specific conditions under which poor or impeded diffusion of synthetic pheromone diminishes the disruption effect, which could result in some males effectively locating females for reproduction. At the same time, many commercially available synthetic lures are also available, and growers have been effectively using them to track population development – although the attractive range of these lures remains unclear. That is, while pheromone lures can attract many moths, the relationship between trap capture and local populations, much less crop damage, remains unclear.

47 OTHER INSTRUMENTATION↗

Advancing Enhanced Raman Spectroscopy to Detect and Measure NOW Pheromones (CPRB 2022 proposal)

Insect sex pheromones are chemical compounds that insects release to attract their partners over distances of hundreds of meters or even kilometers, in complete darkness and without any audible signals. Use of synthetic forms of key compounds have in some cases become an essential component of monitoring and/or managing key pests of agricultural crops, including navel orangeworm (Amyelois transitella) (NOW) in California tree nuts. These pheromone-based strategies can include monitoring, mating disruption, mass trapping, attract-and-kill and push-pull. There are currently multiple commercially available mating disruption products available for NOW and recent studies have demonstrated that they can be effectively used to reduce crop damage by this pest. Just how mating disruption works is not fully established and likely varies across products and target species. For instance, the extent to which synthetic pheromones compete with natural pheromone is not well understood, or in the case of monitoring, how efficaciously the insect follows the diffusing plumes, especially across large blocks and at plot borders. Furthermore, there may be specific conditions under which poor or impeded diffusion of synthetic pheromone diminishes the disruption effect, which could result in some males effectively locating females for reproduction. At the same time, many commercially available synthetic lures are also available, and growers have been effectively using them to track population development – although the attractive range of these lures remains unclear. That is, while pheromone lures can attract many moths, the relationship between trap capture and local populations, much less crop damage, remains unclear. Regardless of the emission source (aerosol puffers, meso emitters, lures etc.), we currently lack the ability to fully understand how these synthetic pheromone compounds diffuse away from their point-source of emission – and subsequently how this might affect the efficacy of mating disruption and/or the accuracy of monitoring efforts. Raman spectroscopy (RS) may offer a means to detect synthetic pheromone because of its analytical properties and recent technological advancements. In combination with nanostructured probes, RS has a demonstrated capability to detect volatiles at extremely low concentrations. While other methods for detecting pheromones have proven to be successful, such analytical methods like mass spectrometry (MS) gas chromatography (GC) require sampling in the fields and are not real-time. Other methods such as EAG/ EAD are also invasive (use antennas) but are of course valuable. Opto-electronic, micro and nano sensors just recently enticed interest. We fall in this latter category, proposing the use of optical spectroscopy (Raman) in a fiber aided by nanostructures to provide a chemical fingerprint without transductions and in real-time (i.e. no sampling). To date, we have been able to generate Raman signatures for the main chemicals in synthetic pheromones (a first for science, to the best of our knowledge), observe their trends at different concentrations, and also provide a clear distinction of such signatures in mixed samples via Principal Component Analysis (PCA) – all of which strengthens our conclusions on the ability of RS to detect emissions in more realistic conditions. We also established processes to enhance the detection sensitivity via preconcentration with Solid Phase Micro Extraction (SPME) fibers and Surface Enhanced Raman Spectroscopy (SERS) to increase the overall optical signals. Furthermore, we explored limitations and requirements for detecting from dispensers, lures, or live moths, including Dept. of Energy and CA State regulatory approvals. Finally, we setup some of our first “orchard in a box” experiments to measure pheromone diffusion along with developing appropriate models.

42 ENGINEERING↗

The 5 Cs of Agrivoltaic Success Factors in the United States: Lessons from the InSPIRE Research Study

The concept of agrivoltaics (combining agriculture and solar photovoltaics technologies on the same land in novel configurations) has emerged as an approach to mitigate conflicts between solar and agricultural activities by providing mutual benefits and added values to each sector. The U.S. Department of Energy has supported agrivoltaics research since 2015 through its Innovative Solar Practices Integrated with Rural Economies and Ecosystems (InSPIRE) research project (National Renewable Energy Laboratory 2022). The InSPIRE project is the most comprehensive coordinated research effort on agrivoltaics in the United States and has examined opportunities and tradeoffs at over 25 sites across the country that span crop production, pollinator habitat, ecosystem services, animal husbandry, and d. Integrating research sites with active commercial agricultural operations can introduce unique challenges for conducting research. This synthesis aims to highlight the technical and non-technical insights from InSPIRE agrivoltaic field research sites from 2015-2021 to support i) appropriate deployment of agrivoltaic projects; ii) more successful research on agrivoltaics; and iii) more effective partnerships on agrivoltaic projects. The synthesized lessons discussed here are focused less on specific case study outcomes (i.e., the percent change in crop yield in an agrivoltaics configuration), and instead more on the elements that enable and facilitate agrivoltaics projects to be installed and operated along with research to be conducted at those sites. We find that there are some insights that are applicable across all types of agrivoltaic projects, while ecosystem service projects and crop production agrivoltaic projects can often have other unique considerations.

14 SOLAR ENERGY↗

BTRIC Technical Support for Appalachia: FY 2023 Summary Report on Net Negative Carbon Building Demonstration with AgPod

This study investigates the effects of various concentrations of carbon dioxide (CO 2 ) on the photosynthesis and biomass accumulation of crops growing in an Agricultural Pod (AgPod). The initial test crop was kale. It has been found that increasing the CO 2 concentration in the AgPod promotes higher photosynthetic rates in kale plants up to 1300 ppm (highest CO 2 concentration). An interesting finding was that net photosynthetic rates of kale leaves remain unsaturated until unusually high levels of photosynthetically active radiation (PAR, > 1200 µmolm-2s-1) and CO 2 concentration (> 1100 ppm) are reached. Such high levels of saturating PAR and CO 2 concentration values rarely occur under natural conditions, suggesting a promising potential for carbon capture via enriched greenhouse crop production. This task is one part of an overall plan to incorporate an AgPod to utilize carbon captured from the atmosphere to achieve net negative carbon emissions in a small neighborhood or community.

54 ENVIRONMENTAL SCIENCES↗

Transforming our understanding of chloroplast-associated genes through comprehensive characterization of protein localizations and protein-protein interactions

Bioenergy crops are a renewable source of fuels and are a critical base for building a carbon-neutral economy. Rational engineering of bioenergy crops has the potential to enhance the yields. However, our ability to engineer plants is limited because the functions of most genes remain unknown. Systematic characterization of gene function in plants thus has the potential to greatly accelerate bioenergy research. Here, we focus on the chloroplast, an underexplored energy-producing organelle that is a hallmark of plants. The chloroplast is one of the promising targets of biofuel crop engineering efforts because of its central role in photosynthesis, metabolism, and intracellular signaling. However, the protein composition of the chloroplast and the functions of most of its proteins remain poorly characterized. At the core of this project, we sought to comprehensively determine the localization of chloroplast-associated proteins and generate a spatially defined protein-protein interaction network for chloroplast. For this purpose, we used the leading model alga Chlamydomonas reinhardtii, which greatly increased experimental speed and throughput. We illustrated the value of our findings to land plants by determining the localization of Arabidopsis thaliana land plant homologs of the Chlamydomonas proteins. Altogether, we were successful in determining the localization of 1,034 chloroplast-associated proteins in Chlamydomonas. The localizations provide numerous insights into the spatial organization of chloroplasts and how they function to support photosynthesis. The localization patterns of distinct proteins revealed new chloroplast structures and revealed new spatial organization inside the chloroplast. We also identified new components of known chloroplast structures, such as the chloroplast envelope, nucleoid, plastoglobuli, and pyrenoid. We identified these new components by investigating the interacting partners of known proteins. Many proteins localized in both the chloroplast and other cellular structures, thereby hinting at new functions and communication between cellular structures. We also applied machine learning on the atlas to generate predictions for the location of all of the proteins in Chlamydomonas. This enabled us to assign putative functions to many uncharacterized proteins based on their cellular location. Altogether, this research establishes a rich resource that opens new avenues of investigation and guides future work in deciphering and manipulating chloroplast function. Next, we developed an extensive protein-protein interaction network for the chloroplast by performing affinity purification-mass spectrometry on ~1,150 tagged chloroplast-associated proteins, the first such large-scale study in any photosynthetic organism. This dataset reveals 4,694 high-confidence protein-protein interactions, offering insights into the functions of thousands of conserved poorly-characterized chloroplast proteins. This systematic identification of protein-protein interactions in the chloroplast also provides multiple exciting new research directions and a detailed blueprint of the chloroplast's operation. This research lays the groundwork to decipher the inner workings of the chloroplast, the cell structure at the heart of photosynthesis. The spatial atlas and protein-protein interactions reveal chloroplast organizational features that would not have been accessible with traditional approaches. The localization mapping, insights into the function, and research materials generated further provide a rich resource for the research community to advance the understanding of how the chloroplast is organized to enable engineering of enhanced photosynthetic organisms.

59 BASIC BIOLOGICAL SCIENCES↗

A Systems Approach to Increasing Carbon Flux to Seed Oil for Biofuels and Bioproducts Production in Camelina sativa (Final Report)

To combat climate change and alleviate the dependency of the United States on fossil fuels, the transition to biofuel crops has long been proposed as a crucial part of the long-term solution. Camelina sativa has emerged as one of the leading commercially viable options for biofuel and bioproduct production for the U.S. Camelina has the advantages of low agronomic inputs and natural resistance to diverse biotic and abiotic stresses relative to other oilseed crops, and Camelina oil-based blends have been tested and approved as liquid transportation fuels. A major limitation in the widespread adoption of Camelina as a viable industrial oilseed crop is its modest oil yields. This project directly investigated possible paths towards increasing oil, by employing tissue-specific and whole plant systems approaches to identify major regulatory bottlenecks. We developed a new high-throughput method for the identification of multi-gene transformants in polyploid species like Camelina sativa and used this to determine that, contrary to what mathematical models suggested, introduction of the microbial Entner-Doudoroff (ED) pathway into Camelina sativa did not result in significant seed oil increases. We developed flux maps providing accurate and statistically robust models of central carbon metabolism in Camelina, including of cultured Camelina embryos. This knowledge was used in the capacitation of a number of junior researchers in gaining knowledge on approaches used for quantitative flux map analyses. We identified several novel Camelina transcription factor genes regulating fatty acid biosynthesis that showed variable effects on seed oil accumulation in transgenic plants. Among a number of community resources, we also developed a knowledge base web resource (CamRegBase) to integrate Camelina gene regulatory information. Together, research outcomes from this project have contributed to a much better understanding of the regulation and bottlenecks to engineer seed oil production in Camelina.

09 BIOMASS FUELS↗