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 (CO 2 ) 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 CO 2 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 CO 2 assimilation. The Cysoleosins have applications in biofuels, animal feed, and human nutrition as well as in providing a tool for investigating fatty acid biosynthesis and catabolism.
Two TT2-type MYB transcription factors identified from tetraploid cotton are involved in regulating proanthocyanidin biosynthesis, providing new strategies for engineering condensed tannins in crops. Proanthocyanidins (PAs), also known as condensed tannins, are important secondary metabolites involved in stress resistance in plants, and are health supplements that help to reduce cholesterol levels. As one of the most widely grown crops in the world, cotton provides the majority of natural fabrics and is a supplemental food for ruminant animals. The previous studies have suggested that PAs present in cotton are a major contributor to fiber color. However, the biosynthesis of PAs in cotton still remains to be elucidated. AtTT2 (transparent testa 2) is a MYB family transcription factor from Arabidopsis that initiates the biosynthesis of PAs by inducing the expression of multiple genes in the pathway. In this study, we isolated two R2R3-type MYB transcription factors from Gossypium hirsutum that are homologous to AtTT2. Expression analysis showed that both genes were expressed at different levels in various cotton tissues, including leaf, seed coat, and fiber. Protoplast transactivation assays revealed that these two GhMYBs were able to activate promoters of genes encoding enzymes in the PA biosynthesis pathway, namely anthocyanidin reductase and leucoanthocyanidin reductase. Complementation experiments showed that both of the GhMYBs were able to recover the transparent testa seed coat phenotype of the Arabidopsis tt2 mutant by restoring PA biosynthesis. Ectopic expression of either of the two GhMYBs in Medicago truncatula hairy roots increased the contents of anthocyanins and PAs compared to control lines expressing the GUS gene, and expression levels of MtDFR, MtLAR, and MtANR were also elevated in lines expressing GhMYBs. Together, these data provide new insights into engineering condensed tannins in cotton.
Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75–93%) and procyanidins (PC, 17–25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6–2.4% of DM significantly reduced methane production by 19% (p ≤0.01) and ammonia production by 60% (p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.
SummaryWe have successfully created polyoleosins by joining multiple oleosin units in tandem head-to-tail fusions. Constructs encoding recombinant proteins of 1, 3 and 6 oleosin repeats were purposely expressed both in planta and in Escherichia coli. Recombinant polyoleosins accumulated in the seed oil bodies of transgenic plants and in the inclusion bodies of E. coli. Although polyoleosin was estimated to only accumulate to <2% of the total oil body protein in planta, their presence increased the freezing tolerance of imbibed seeds as well as emulsion stability and structural integrity of purified oil bodies; these increases were greater with increasing oleosin repeat number. Interestingly, the hexameric form of polyoleosin also led to an observable delay in germination which could be overcome with the addition of external sucrose. Prokaryotically produced polyoleosin was purified and used to generate artificial oil bodies and the increase in structural integrity of artificial oil bodies-containing polyoleosin was found to mimic those produced in planta. We describe here the construction of polyoleosins, their purification from E. coli, and properties imparted on seeds as well as native and artificial oil bodies. A putative mechanism to account for these properties is also proposed.
Our goal is to increase the metabolisable energy of forage species (such as Lolium perenne, perennial ryegrass) via accumulation of lipids in the leaves, and further improve upon this by the delivery of polyunsaturated fatty acids whilst maintaining a low input agricultural system. Keywords: lipid protection, triacylglyceride, DGAT, oleosin, biohydrogenation
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