Generating Pennycress (Thlaspi arvense) Seed Triacylglycerols and Acetyl-Triacylglycerols Containing Medium-Chain Fatty Acids

Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of seeds rich in triacylglycerols and protein, making it an attractive target for domestication into an offseason oilseed cash cover crop. Pennycress is easily genetically transformed, enabling synthetic biology approaches to tailor oil properties for specific biofuel and industrial applications. To test the feasibility in pennycress of producing TAGs and acetyl-TAGs rich in medium-chain fatty acids (MCFAs; C6–C14) for industrial, biojet fuel and improved biodiesel applications, we generated transgenic lines with seed-specific expression of unique acyltransferase (LPAT and diacylglycerol acyltransferase) genes and thioesterase (FatB) genes isolated from Cuphea viscosissima, Cuphea avigera var. pulcherrima, Cuphea hookeriana, Coco nucifera, and Umbellularia californica. Wild-type pennycress seed TAGs accumulate no fatty acids shorter than 16C and less than 5 mol percent C16 as palmitic acid (16:0). Co-expressing UcFatB and CnLPAT produced up to 17 mol% accumulation of lauric acid (12:0) in seed TAGs, whereas CvFatB1 CvLPAT2 CpDGAT1 combinatorial expression produced up to 27 mol% medium chain FAs Medium Chain Fatty Acids mostly in the form of capric acid (10:0). CpFatB2 ChFatB2 combinatorial expression predominantly produced, in equal parts, up to 28 mol% myristic acid (14:0) and palmitic acid. Genetically crossing the combinatorial constructs into a fatty acid elongation1 (fae1) mutant that produced no 22:1 erucic acid, and with an Euonymus alatus diacylglycerol acetyltransferase (EaDAcT)-expressing line that produced 60 mol% acetyl-TAGs, had no or relatively minor effects on MCFAs accumulation, suggesting fluxes to MCFAs were largely unaltered. Seed germination assays revealed no or minor delays in seed germination for most lines, the exception being CpFatB2 ChFatB2-expressing lines, which had substantially slower seed germination rates. Taken together, these data show that pennycress can be engineered to produce seeds accumulating modest amounts of MCFAs of varying carbon-chain length in TAGs and acetyl-TAGs, with rates of seed germination being delayed in only some cases. We hypothesize that increasing MCFAs further may require functional reductions to endogenous transferases and/or other FA elongases

Molecular tools enabling pennycress (\u3ci\u3eThlaspi arvense\u3c/i\u3e) as a model plant and oilseed cash cover crop

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyltriacylglycerol- containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress’ rapid domestication to meet the growing sustainable food and fuel demands

A Plant DJ-1 Homolog Is Essential for Arabidopsis thaliana Chloroplast Development

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development

Identification of a Consensus DNA-Binding Site for the \u3ci\u3eArabidopsis thaliana\u3c/i\u3e SBP Domain Transcription Factor, AtSPL14, and Binding Kinetics by Surface Plasmon Resonance

Proteins with a conserved Cys- and His-rich SQUAMOSA promoter binding protein (SBP) domain are transcription factors restricted to photosynthetic organisms that possess a novel two Zn-finger structure DNA-binding domain. Despite the fact that altered expression of some SBP-encoding genes has profound effects on organism growth and development, little is known about SBP domain protein target genes. Misexpression of the Arabidopsis thaliana AtSPL14 SBP domain gene confers resistance to programmed cell death and modifies plant architecture. A consensus DNA-binding motif for AtSPL14 was identified by systematic evolution of ligands by exponential enrichment (SELEX) or random binding site selection (RBSS). DNA recognized by AtSPL14 contained the core binding motif (GTAC) found for other SBP domain proteins, but mutational analyses indicated that at least one additional flanking nucleotide is necessary for effective AtSPL14−DNA interaction. Comparison of several SBP domain amino acid sequences allows us to hypothesize which specific amino acids might participate in this sequence-specific DNA recognition. Electrophoretic mobility shift assays (EMSA) with mutant AtSPL14 DNA-binding domain proteins indicated that not all of the Zn2+ ion coordinating ligands in the second Zn structure are strictly required for DNA binding. Surface plasmon resonance (SPR) was used to evaluate AtSPL14 in vitro binding kinetics for comparison of equilibrium binding constants with other SBP domain proteins. These data provide a strong basis for further experiments aimed at defining and distinguishing the sets of genes regulated by the closely related SBP domain family members. Includes Supporting Information

Ultrastructural Alterations in Arabidopsis thaliana dj1c Null Mutant Cotyledons

Increasing evidence suggests that deficits in mitochondrial function, oxidative and nitrosative stress, accumulation of aberrant or misfolded proteins, and ubiquitin-proteasome system dysfunction may represent the principal molecular pathways that commonly underlie the pathogenesis of neurodegenerative Parkinson’s disease (PD). Mutations in human DJ-1 lead to early onset PD. The subcellular distribution of DJ-1 (PARK7) is primarily cytoplasmic with smaller quantities found associated with mitochondria. Postulated functions include roles in the oxidative stress response, either as a redox sensor protein that can prevent the aggregation of alpha-synuclein or as an antioxidant. Homologs of DJ-1 are found in all kingdoms of life. To understand the functions of plant DJ-1 homologs we identified null mutants in the model plant Arabidopsis thaliana. One of the 3 Arabidopsis genes encoding DJ1 homologs (DJ1C) is essential for viability, and null knockout mutants are seedling lethal

A Plant DJ-1 Homolog Is Essential for \u3ci\u3eArabidopsis thaliana\u3c/i\u3e Chloroplast Development

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development

Molecular tools enabling pennycress (\u3ci\u3eThlaspi arvense\u3c/i\u3e) as a model plant and oilseed cash cover crop

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyltriacylglycerol- containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress’ rapid domestication to meet the growing sustainable food and fuel demands

Role of the Protein Corona Derived from Human Plasma in Cellular Interactions between Nanoporous Human Serum Albumin Particles and Endothelial Cells

The presence of a protein corona on various synthetic nanomaterials has been shown to strongly influence how they interact with cells. However, it is unclear if the protein corona also exists on protein particles, and if so, its role in particle-cell interactions. In this study, pure human serum albumin (HSA) particles were fabricated via mesoporous silica particle templating. Our data reveal that various serum proteins adsorbed on the particles, when exposed to human blood plasma, forming a corona. In human umbilical vein endothelial cells (HUVECs), the corona was shown to decrease particle binding to the cell membrane, increase the residence time of particles in early endosomes, and reduce the amount of internalized particles within the first hours of exposure to particles. These findings reveal important information regarding the mechanisms used by vascular endothelial cells to internalize protein-based particulate materials exposed to blood plasma. The ability to control the cellular recognition of these organic particles is expected to aid the advancement of HSA-based materials for intravenous drug delivery