Theses and Dissertations at Montana State University (MSU)
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Item The roles of Phospholipase C in oil biosynthesis in oilseeds(Montana State University - Bozeman, College of Agriculture, 2018) Aryal, Niranjan; Chairperson, Graduate Committee: Chaofu LuResearch on the biosynthesis pathways of storage oil (triacylglycerol, or TAG) in plants has gained momentum for the last couple of decades. Despite significant achievements, a complete understanding of such pathways is still lacking. Also, the production of industrially important unusual oils such as hydroxy fatty acids (HFA) at the commercial level is limited in both native and transgenic plants. In this study, I have examined the roles of Phospholipase C in the TAG biosynthesis. To test roles of non-specific PLCs (NPCs) in TAG synthesis, FA composition was analyzed in mature seeds of T-DNA lines of 6 Arabidopsis npc T-DNA mutants. Among them, npc1, npc4 and npc6 showed increases in oleic acid (18:1) and decreases in lineoleic acid (18:2) and linolenic acid (18:3). The 18:1 was further increased in the npc1/npc6 double mutant compared to npc1 and npc6. These changes in FA profile suggest the role of NPCs in TAG biosynthesis. Given the possible roles of NPCs in TAG synthesis in the seeds of Arabidopsis, I searched for suitable PLC from Castor (RcPLC) for further investigation and to transform to the 7-1 line of Camelina sativa expressing a fatty acid hydroxylase (RcFAH12). From previously published Castor transcriptome, RcPLCL1 was the phospholipase C-like gene that was primarily expressed in the endosperm. Exploration of the RcPLCL1 protein using bioinformatics tools placed it to the PLC-like phosphodiesterase family which has not been characterized yet. Based on the sequence analyses, the protein contains the X domain for the PLC activity on phosphatidylinositols (PI), but it lacks the Y and C2 domains. Enzyme assays using heterologous expression in yeast showed that the protein had both PC- and PI-PLC activities. Based on the genomic sequence analysis, 8 genes were found in Arabidopsis that were predicted to encode PLC-like phosphodiesterases. The seeds of 5 T-DNA knockout lines were analyzed for fatty acid composition, which showed decreased linolenic acid (18:3) and increased oleic acid (18:1). The FA composition change suggested the involvement of these proteins in oil biosynthesis in Arabidopsis seeds. Upon transformation of RcPLCL1 into the 7-1 line, HFA content in the seeds was increased to as high as 24% from 15%. Concurrently the amount of HFA decreased in the membrane lipid phosphatidylcholine (PC). However, the Arabidopsis homologue, AtPLCL1, did not increase the HFA in seeds of 7-1. These results suggest that RcPLCL1 is involved in HFA accumulation. The HFA-producing 7-1 plants were tested in the field. Results from two-year experiments indicated that both wild type and 7-1 demonstrated better performance for traits like oil content and yield. However, the HFA level in 7-1 was significantly decreased in field-grown plants than greenhouse plants.Item Modification of seed fatty acid composition by CRISPR/Cas9 targeting the fatty acid elongase1 in Camelina sativa(Montana State University - Bozeman, College of Agriculture, 2018) Ozseyhan, Mehmet Erkan; Chairperson, Graduate Committee: Chaofu LuThe low-input oilseed crop Camelina (Camelina sativa (L.) Crantz) is known for its high omega-3 (18:3) content, short growth season, and facile gene transformation. Camelina mostly contains unsaturated fatty acids, however its fatty acid composition needs optimization depending on the end uses, for example reduction of unsaturated fatty acid to use as biodiesels, or enhancing omega-3 fatty acid content to use as nutritional supplements. Very long chain fatty acid (VLCFAs, C20-C24), are undesirable for human consumption, and their accumulation in seed oil also needs to be diminished. VLCFAs are produced by the catalytic action of fatty acid elongase1 (FAE1), and Camelina contains three alleles of FAE1 genes (FAE1-A, FAE1-B, and FAE1-C) due to its allohexaploid nature. Recently, VLCFAs in camelina were decreased along with polyunsaturated fatty acids (PUFAs) using the RNA interference (RNAi) technology. A low VLCFA line was also isolated from ethyl methanesulfonate (EMS) induced mutants. Sequencing results indicated that FAE1-B gene was mutated and resulted in 60% reduction in VLCFAs, but other two FAE1 copies were presumably still active in the mutant. To address this multipleallele-knockout-at-once problem, here I investigated the effect of knocking out three alleles of FAE1 genes using CRISPR technology with egg cell-specific Cas9 expression. Due to the germline mutation, homozygous FAE1 knockout mutants were successfully created in a single generation. VLCFA accumulation was significantly decreased from 22% of total fatty acids in wild type to less than 2% in transgenic plants, and the C18 unsaturated fatty acids were improved since 18:1 substrates were diverted to desaturation pathway, rather than elongation. Analysis of the fatty acid composition of four transgenic generations indicated that the mutations that cause low VLCFA genotype were heritable. There was no significant difference observed in seed weight, plant height, total oil content, and seed germination in Cas9-induced mutants compared to the wild type. This study showed that polyploid Camelina can be modified rapidly and effectively through CRISPR/Cas9 to achieve desired fatty acid composition.Item The fatty acid elongase of Physaria fendleri increases hydroxy fatty acid accumulation in transgenic Camelina(Montana State University - Bozeman, College of Agriculture, 2013) Snapp, Anna Rose; Chairperson, Graduate Committee: Chaofu LuPlant oils containing hydroxy fatty acids (HFA) are desirable for a wide variety of applications including lubricants, plasticizers, surfactants, polyesters, paints, sealants, biodiesel, and more. Due to unfavorable agronomic attributes of natural accumulators such as castor and lesquerella, many efforts have been made to produce hydroxyl fatty acids in crop plants. The hydroxy fatty acid synthesis pathway has been extensively studied and key genes such as the castor fatty acid hydroxylase, RcFAH, have been discovered. However, insertion of the RcFAH gene into various Arabidopsis backgrounds under the control of seed specific promoters failed to result in high accumulation of the desired HFA products, highlighting a need for more research to uncover additional constraints and factors affecting the fluxes involved with the accumulation of these unusual fatty acids in seed oil. In this study I investigated the effect of co-expressing a fatty acid elongase gene, LfKCS3, from Lesquerella (Physaria) fendleri along with the castor hydroxylase gene, RcFAH, on accumulation of hydroxyl fatty acids in seed oil of the crop plant Camelina sativa. On its own, wild type camelina contains no hydroxy fatty acids but insertion of the RcFAH gene results in accumulation of around 15% HFA in transgenic camelina, however, addition of the LfKCS3 gene resulted in a significant increase in very long chain 20-carbon hydroxyl fatty acids from <2% to 8%; total hydroxyl fatty acids also increased from 15% to 22% in the highest accumulating lines. The presence of the LfKCS3 enzyme effectively increased total HFA levels at all stages of oil accumulation in developing seeds while also decreasing the amount of these fatty acids left on the phospholipid, phosphatidylcholine. This combination of increased 20- carbon and total hydroxyl fatty acid accumulation along with the decreased HFA levels in phosphatidylcholine indicates that the LfKCS3 gene helps to enhance the flux of HFA out of phosphatidylcholine for incorporation of HFA into triacylglycerol, aiding in relief of the metabolic bottleneck for engineering economically viable levels of these fatty acids in oilseed crops.