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 Effect of the WRKY76 transcription factor on starch biosynthesis and plant growth(Montana State University - Bozeman, College of Agriculture, 2016) Estabrooks, Hannah Margaret; Chairperson, Graduate Committee: Jack MartinStarch is an important contributor to plant growth as excess photosynthate is stored in leaves as starch during the day to be mobilized at night and re-synthesized in sink tissues. Starch is also the principal constituent of cereal seeds and its variation greatly influence crop yields. The starch pathway is complex and its regulation is not fully understood. Transcription Factors (TFs) are known to act as master regulators of whole biosynthetic pathways and the work presented here was aimed at gaining a better understanding of starch production in leaves by identifying a TF which specifically regulates the leaf starch biosynthetic pathway. Leaf starch levels are regulated in part by ADP-glucose pyrophosphorylase (AGPase), the rate limiting step of starch biosynthesis. Transgenic rice event (NR16+) with increased leaf AGPase activity (due to overexpression of the AGPase large and small subunit transgenes Sh2r6hs and Bt2) was subjected to RNA-sequencing. The results indicated that the leaf specific AGP transgene, which increased leaf starch, also had upregulation of the WRKY76 TF. Another regulatory protein, F-Box, was chosen as a candidate due to the F-box family's involvement in plant development. The current study examines the potential of these gene products for increasing starch biosynthesis in leaves via leaf specific overexpression. Results indicate that overexpression of WRKY76 increases leaf starch an average of 39% at both the one month and anthesis growth stages in comparison to the Varietal Control Nipponbare. WRKY76 transgenic lines have enhanced phenotype with an improved harvest index due to biomass and immature panicles trending down by 4% and 21% respectively, while seed weight trended 12% higher. Events overexpressing WRKY76 were also found to upregulate important starch biosynthetic and carbon metabolism genes including AGPL1, AGPS2, SSIIIb, GBSII, Rbcs, PRK, and GS2 as well as leading to a general upregulation of leaf tissue carbohydrates. Events 1, 2, and 12 additionally had on average 13% increased photosynthetic rate at the one month growth stage. The findings of this study support WRKY76 as a dynamic regulator of the starch biosynthetic pathway conferring more efficient carbon assimilation leading to an increased harvest index.Item Modification of a starch biosynthetic enzyme : potential for increased seed yield in wheat (Triticum aestivum)(Montana State University - Bozeman, College of Agriculture, 2002) Meyer, Fletcher DamienItem Biosynthesis of bromegrass mosaic virus ribonucleic acid(Montana State University - Bozeman, College of Agriculture, 1967) Branson, Dean RussellItem 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.