Nicholas John Roberts

In Vivo Packaging of Triacylglycerols Enhances Arabidopsis Leaf Biomass and Energy Density

The coexpression of a uniquely stabilized plant structural protein (Cys-oleosin) and diacylglycerol O-acyltransferase in Arabidopsis led to a 24% increase in the CO2 assimilation rate and a 50% increase in leaf biomass as well as oil accumulation in the leaves and roots. Our dependency on reduced carbon for energy has led to a rapid increase in the search for sustainable alternatives and a call to focus on energy densification and increasing biomass yields. In this study, we generated a uniquely stabilized plant structural protein (cysteine [Cys]-oleosin) that encapsulates triacylglycerol (TAG). When coexpressed with diacylglycerol O-acyltransferase (DGAT1) in Arabidopsis (Arabidopsis thaliana), we observed a 24% increase in the carbon dioxide (CO2) assimilation rate per unit of leaf area and a 50% increase in leaf biomass as well as approximately 2-, 3-, and 5-fold increases in the fatty acid content of the mature leaves, senescing leaves, and roots, respectively. We propose that the coexpression led to the formation of enduring lipid droplets that prevented the futile cycle of TAG biosynthesis/lipolysis and instead created a sustained demand for de novo lipid biosynthesis, which in turn elevated CO2 recycling in the chloroplast. Fatty acid profile analysis indicated that the formation of TAG involved acyl cycling in Arabidopsis leaves and roots. We also demonstrate that the combination of Cys-oleosin and DGAT1 resulted in the highest accumulation of fatty acids in the model single-cell eukaryote, Saccharomyces cerevisiae. Our results support the notion that the prevention of lipolysis is vital to enabling TAG accumulation in vegetative tissues and confirm the earlier speculation that elevating fatty acid biosynthesis in the leaf would lead to an increase in CO2 assimilation. The Cys-oleosins have applications in biofuels, animal feed, and human nutrition as well as in providing a tool for investigating fatty acid biosynthesis and catabolism.

Head-to-tail fusions of camelid antibodies can be expressed in planta and bind in rumen fluid

We have compared the accumulation of recombinant variable heavy‐chain portions [VHH (variable heavy‐chain antibody from camelids)] of camelid antibodies in a variety of subcellular compartments produced in planta. The VHH coding sequences were optimized for expression in thale cress (Arabidopsis thaliana) and placed individually or as fused tandem heterodimers in synthetic plant‐organelle‐targeting cassettes designed to target the protein to either the cytoplasm, ER (endoplasmic reticulum), protein storage vacuole or chloroplast. Accumulation of individual VHHs was only detected in plants transformed with the ER‐targeting cassette, whereas accumulation of the tandem VHHs was detected for all cassettes and was the highest with the ER cassette [0.1–0.7% (w/w) of total soluble proteins]. The ability of the plant‐produced tandem VHH to reduce TNFα (tumour necrosis factor α) cytotoxicity was found to be comparable with previously characterized recombinant VHHs. In vitro antigen binding and functional stability in rumen fluid were determined on both prokaryotically expressed and plant‐expressed tandem VHHs. The plant‐produced VHH did not appear to be any more stable in rumen fluid than other soluble plant proteins; however, it was able to bind equally well to the antigen in the presence or absence of rumen fluid.

Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion.

A variety of single-chain variable fragments (scFv) that had been previously developed to the surface epitopes of infective Trichostrongylus colubriformis L3 pathogenic gut nematodes of sheep were fused to a trimeric version of polyoleosin (three head-to-tail repeats of oleosin) and expressed in planta under the control of an Arabidopsis oleosin promoter. The fusion products were found to accumulate in oil bodies (OBs) at the range of 0.25-0.9% of the total seed protein which is comparable with the main 18 kDa isoform of Arabidopsis seed oleosin. Immunofluorescence microscopy and immuno-binding were used to demonstrate that it is possible to both purify the recombinant protein via enrichment for OBs as well as use the OBs emulsion to deliver functional recombinant scFv. This work presents a novel fusion strategy platform to boost the productivity and simplify the delivery of recombinant single chain antibodies and other like proteins.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an ‐apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co‐factor bound in the active site. A central hinge region creates a “butterfly‐like” motion of the domains that reduces the width of the inter‐domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.