Location: Plant Physiology and Genetics Research
Project Number: 2020-21000-013-01-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2017
End Date: Aug 31, 2020
The goal of this collaborative research is to identify and characterize proteins involved in formation and function of lipid droplets in plant cells, with the end-goal of modifying lipid droplets to accumulate higher amounts of oil or increase crop tolerance to abiotic stress. Lipid droplets are small, spherical organelles that house neutral lipids (oil), which allows the oil to exist as an emulsion in the aqueous environment of the plant cell. Lipid droplets accumulate to high levels in the seeds of oilseed crops, where they “package” the high amounts of oil that are typical of these plant species. Lipid droplets are also present in the vegetative cells of plants, where they serve as transient depots for neutral lipids used in membrane remodeling, lipid signaling, and plant growth and development. Lipid droplets have also been shown recently to proliferate during biotic or abiotic stress responses, revealing previously unknown roles in plant stress responses. While the enzymes for the synthesis of oil are fairly well known, far less is known about the proteins involved in formation and function of lipid droplets. Our labs recently identified a new class of proteins called Lipid-Droplet Associated Proteins (LDAPs) that coat the surface of lipid droplets in non-seed organs of plants. There are three LDAP genes (LDAP1-3) in most plant species, and in Arabidopsis, they are similarly expressed throughout various organs and tissues, but during abiotic stress response, specific LDAP genes are induced. To identify additional proteins involved in lipid droplet formation and function in plants, ARS scientists used each of the three LDAP proteins as “bait” in a yeast 2-hybrid screen and identified approximately 15 interacting protein partners. To help narrow the field for further analysis, Arabidopsis mutants disrupted in each of the 15 genes were analyzed to determine the effects of gene loss on lipid droplet morphology and oil content and composition. Several plant lines were identified that showed pronounced changes in the number and size of lipid droplets, as well as increases in oil content in leaves and/or seeds. Two genes will be studied further in this collaborative effort. One encodes a protein of unknown function we termed “LDAP-Interacting Protein” (LDIP), and the second encodes a protein involved in autophagosome formation. Disruption of the former gene resulted in much larger lipid droplets in plant leaves and seeds, and a significant increase in oil content in both organs. Disruption of the latter gene is lethal, and thus has not been characterized extensively yet, but the identity of the encoded protein implies a role for vacuolar degradation in regulating lipid droplet abundance. The objectives of the work include: Analyze the lipidome of wild-type and LDIP-knockout plants to probe the mechanisms of LDIP function; disrupt the LDIP gene in camelina to determine whether loss of LDIP function increases seed oil content in an oilseed crop; characterize the physical interaction between LDAP and the autophagosomal protein and determine the importance of the vacuolar degradation pathway in regulating lipid droplets in plants.
Wild-type and LDIP mutant Arabidopsis plants will be grown under sterile conditions, then 15-day-old seedlings will be harvested and lipids extracted. Lipids will be analyzed using standard gas chromatograph/mass spectrometry techniques and complemented by high resolution, direct-infusion lipidomics analyses provided as a commercial service by the Kansas Lipidomics Research Center. Lipid droplets will also be purified from germinated Arabidopsis seedlings and lipids analyzed using similar approaches. Our working hypothesis is that LDIP is involved in formation of the phospholipid monolayer that surrounds the lipid droplet, and that comparison of the phospholipidome between wild-type and mutant plants should reveal the specific lipid molecular species associated with the high oil phenotype. The LDIP gene of camelina will be disrupted using CRISPR/Cas9 technology. Camelina is a hexaploid species, and as such, guide RNAs will be designed to knock out all three copies of the LDIP gene at once. Given that this technology is expected to generate a spectrum of plants containing mutation(s) in the LDIP gene(s), the plants will be genotyped to confirm gene knockouts. Complementary studies will be conducted to confirm that camelina LDIP, like the Arabidopsis homolog, is localized to lipid droplets in plant cells. The experiments will be conducted using fluorescently-tagged versions of the camelina LDIP protein transiently expressed in tobacco leaves, with visualization by confocal microscopy. The physical interaction of LDAP and the autophagosomal protein will be investigated using the yeast 2-hybrid system and complementary Bi-molecular Fluorescence Complementation (BiFC) imaging of the two proteins co-expressed in plant cells. Additional experiments to probe the role of vacuolar degradation will include the usage of drugs known to stimulate or block the autophagosomal process in plant leaves, and analyzing lipid droplet abundance by microscopic examination and quantifying lipid content using standard analytical techniques.