Integrated Transcriptomic and Metabolomic Approaches to Identify Genes and Gene Networks Involved in Lipid Biosynthesis in Camelina sativa (L.) Crantz Seeds

Camelina sativa is an emerging oilseed crop dedicated for biofuel and biodiesel applications as well as a source for edible and general-purpose oils. Such valuable oilseed crop is subjected to plant breeding programs and is suggested for large-scale production of better seed and oil qualities. Due to the positive agronomic attributes and the oil qualities of Camelina, it is suggested for plant breeding programs as a biotechnological platform for industrial oils and for biodiesel applications. It is also proposed as an ideal “resilient” crop to be grown on marginal lands, where other plants cannot grow sufficiently. Hence, in an attempt to develop Camelina as a dedicated crop for industrial oils, we designed the current research project with multiple objectives aiming to i) increasing seed and oil yields to the upper possible limits via genetic engineering, ii) engineering Camelina for production of industrial oils, and iii) enhance Camelina tolerance to severe environmental stresses, including drought and salinity. To accomplish these research objectives, a better understanding of lipid metabolism at the molecular and biochemical levels in Camelina seeds is critical. In the current research, we integrated different ‘omics’ approaches, including transcriptomics, metabolomics, and lipidomics to identify and profile the gene(s) and metabolite(s) networks associated with triacylglycerol (TAG, the main lipid in seeds) biosynthesis. Further interests are to investigate how these two networks are interacting to determine the quantity and quality of Camelina oil during seed development, and finally to reveal the rate-limiting step(s) in TAG biosynthesis pathways for metabolic engineering. The whole seed-specific transcriptome revealed the identification of approximately 40,000 actively expressed transcripts, and of these, 7932 genes showed temporal and differential gene expression during the seed development. The differentially expressed genes (DEGs) were annotated and were found to encode key enzymes controlling alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. Further, we quantified the relative contents of over 240 metabolites by using GC/MS and LC/MS/MS platforms, and the results indicated that Camelina seed development is associated with temporally major metabolic switches. Furthermore, we were able to determine the potential limiting factor(s) in oil synthesis pathways, and accordingly, we selected several x candidate genes/enzymes for metabolic engineering of Camelina and overexpression of those genes has led to increased seed and oil yields in Camelina. Further, we proved that combining the overexpression of two enzymes involved in TAG biosynthetic pathway (DGAT1 and GPD1) has positively enhanced seed oil content (up to 13% increase), seed mass (up to 52% increase), and overall seed and oil yields in Camelina. This improvement in seed yield and oil contents was also associated with distinct molecular and biochemical consequences and therefore, identifying the metabolic bottlenecks for engineering Camelina for further increased seed oil yields is critical. Accordingly, we utilized metabolites profiling, in conjugation with transcriptome profiling during seed development in Camelina DGAT1 and GPD1 transgenics in comparison with their WT relatives. The whole seed-specific transcriptome of transgenic lines revealed the identification of approximately 1,566 and 2,102 transcripts were differentially regulated in Camelina transgenics and many of them were found to be involved in the alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. Further, the metabolome analysis indicated major switches in transgenic seeds, which are associated with significant changes in the levels of glycerolipids, phospholipids, most amino acids, sugars and organic acids, especially the ones involved in TCA cycle and glycolysis. Additionally, to introduce Camelina as a biotechnological platform for the production of industrial oils, we metabolically engineered Camelina seed oils for producing wax esters with desired physical and chemical properties for better fitting with industrial purposes. Two genes from jojoba encode the two enzymes; fatty acyl-CoA reductase (FAR) and wax synthase (WS), and a gene from Arabidopsis encode a wax synthase enzyme (WSD1) were overexpressed into Camelina under the control of seed-specific promoters to modify seed metabolism for wax ester synthesis in the transgenic lines. The overexpression of wax ester enzymes caused has allowed the transgenic lines to produce a substantial amount of wax esters higher than the amount produced naturally in WT seed oils. These obtained wax esters are currently subjected to subsequent purification and quantification processes, and their structural compositions will be resolved, and data will be available in the near future. xi Moreover, in order to develop Camelina to be grown under a wide range of climates, our research project was aimed to better understand the physiological roles of wax biosynthetic genes in response to environmental stresses. We targeted an Arabidopsis gene encode a cuticular wax synthase enzyme (namely, WSD1) and we investigated whether increasing the transcriptional activity of WSD1 would affect the amount of cuticular waxes, which can contribute to conferring tolerance to drought and/or salinity in Arabidopsis and also in Camelina. Transgenic Arabidopsis lines were tested for increased deposition of epicuticular waxes and the quantity and the structural compositions of surface waxes were determined using gas chromatographic (GC) techniques and scanning electron microscopy (SEM), and finally were evaluated in response to several abiotic stresses. The preliminary results indicated that WSD1 transgenic plants exhibited strong tolerance phenotype, as they were able to recover from drought and salinity better than the WT plants. This stress-resistant phenotype observed in WSD1 transgenics could be physiologically associated with the reduced cuticular transpirational rates and cuticle permeability, increased deposition of epicuticular wax crystals, and increased leaf and stem wax loading, in the transgenics as compared to WT plants. These promising data from Arabidopsis has encouraged us to investigate Camelina plants overexpressing WSD1 in response to drought and salinity which is still under development. Collectively, the integration of transcriptome and metabolome can be highly useful for understanding the regulation of TAG biosynthesis and identifying the bottlenecks in TAG pathways, providing a precise selection of candidate genes for generating Camelina varieties with improved seed and oil yields. Further, Camelina can serve as a biotechnological platform for the sustainable production of wax esters, and by using a similar approach, many oilseed crops could be metabolically engineered for the production of oil-based products of high value for the industry. Finally, manipulation of the cuticular waxes could be an effective strategy to confer abiotic stress tolerance in plants