Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes …

Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.

Seeds from the desert shrub Simmondsia chinensis (jojoba) are one of the only known natural plant sources to store a majority of its oil in the form of liquid wax esters (WE) instead of triacylglycerols (TAGs) and these oils account for ~55% of the seed weight. Jojoba oil is highly valued as cosmetic additives and mechanical lubricants, yet despite its value much is still unknown about its neutral lipid biosynthetic pathways and lipid droplet packaging machinery. Here, we have used a multi-"omics" approach to study how spatial differences in lipid metabolites, gene expression, and lipid droplet proteins influence the synthesis and storage of jojoba lipids. Through these studies mass spectrometry analyses revealed that WEs are compartmentalized primarily in the cotyledonary tissues, whereas TAGs are, surprisingly, localized to the embryonic axis tissues. To study the differences in gene expression between these two tissues, a de novo transcriptome was assembled from high throughput RNAseq data. Differential gene expression analysis revealed that the Jojoba Wax Synthase, which catalyzes the formation of wax esters, and the Diacylglycerol O-Acyltransferase1, which catalyzes the final acylation of triacylglycerol synthesis, were differentially expressed in the cotyledons and embryonic axis tissues, respectively. Furthermore, through proteomic analysis of lipid droplet …

In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD …