A Novel Soybean Diacylglycerol Acyltransferase 1b Variant with Three Amino Acid Substitutions Increases Seed Oil Content.

Improving soybean (Glycine max) seed composition by increasing the protein and oil components will add significant value to the crop and enhance environmental sustainability. Diacylglycerol acyltransferase (DGAT) catalyzes the final rate-limiting step in triacylglycerol (TAG) biosynthesis and has a major impact on seed oil accumulation. We previously identified a soybean DGAT1b variant with 14 amino acid substitutions (GmDGAT1b-MOD) that increases total oil content by 3 percentage points when overexpressed in soybean seeds. In the present study, additional GmDGAT1b variants were generated to further increase oil with a reduced number of substitutions. Variants with one to four amino acid substitutions were screened in the model systems S. cerevisiae and transient N. benthamiana leaf. Promising GmDGAT1b variants resulting in high oil accumulation in the model systems were selected for over-expression in soybeans. One GmDGAT1b variant with three novel amino acid substitutions (GmDGAT1b-3aa) increased total soybean oil to levels near the previously discovered GmDGAT1b-MOD variant. In a multiple location field trial, GmDGAT1b-3aa transgenic events had significantly increased oil and protein by up to 2.3 and 0.6 percentage points, respectively. Modeling of the GmDGAT1b-3aa protein structure provided insights into the potential function of the three substitutions. These findings will guide efforts to improve soybean oil content and overall seed composition by CRISPR editing.

Loss-of-function of triacylglycerol lipases are associated with low flour rancidity in pearl millet [Pennisetum glaucum (L.) R. Br.].

Pearl millet is an important cereal crop of semi-arid regions since it is highly nutritious and climate resilient. However, pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids post-milling. We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions. The breakdown of storage lipids (triacylglycerols; TAG) was accompanied by free fatty acid accumulation over the time course for all lines. The high rancidity lines had the highest amount of FFA by day 21, suggesting that TAG lipases may be the cause of rancidity. Additionally, the high rancidity lines manifested substantial amounts of volatile aldehyde compounds, which are characteristic products of lipid oxidation. Lipases with expression in seed post-milling were sequenced from low and high rancidity lines. Polymorphisms were identified in two TAG lipase genes (PgTAGLip1 and PgTAGLip2) from the low rancidity line. Expression in a yeast model system confirmed these mutants were non-functional. We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity. These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life.

Plant and algal lysophosphatidic acid acyltransferases increase docosahexaenoic acid accumulation at the sn-2 position of triacylglycerol in transgenic Arabidopsis seed oil.

Although docosahexaenoic acid (DHA), an important dietary omega-3 polyunsaturated fatty acid (PUFA), is at present primarily sourced from marine fish, bioengineered crops producing DHA may offer a more sustainable and cost-effective source. DHA has been produced in transgenic oilseed crops, however, DHA in seed oil primarily occupies the sn-1/3 positions of triacylglycerol (TAG) with relatively low amounts of DHA in the sn-2 position. To increase the amount of DHA in the sn-2 position of TAG and in seed oil, putative lysophosphatidic acid acyltransferases (LPAATs) were identified and characterized from the DHA-producing alga Schizochytrium sp. and from soybean (Glycine max). The affinity-purified proteins were confirmed to have LPAAT activity. Expression of the Schizochytrium or soybean LPAATs in DHA-producing Arabidopsis expressing the Schizochytrium PUFA synthase system significantly increased the total amount of DHA in seed oil. A novel sensitive band-selective heteronuclear single quantum coherence (HSQC) NMR method was developed to quantify DHA at the sn-2 position of glycerolipids. More than two-fold increases in sn-2 DHA were observed for Arabidopsis lines expressing Schizochytrium or soybean LPAATs, with one Schizochytrium LPAAT driving DHA accumulation in the sn-2 position to 61% of the total DHA. Furthermore, expression of a soybean LPAAT led to a redistribution of DHA-containing TAG species, with two new TAG species identified. Our results demonstrate that transgenic expression of Schizochytrium or soybean LPAATs can increase the proportion of DHA at the sn-2 position of TAG and the total amount of DHA in the seed oil of a DHA-accumulating oilseed plant. Additionally, the band-selective HSQC NMR method that we developed provides a sensitive and robust method for determining the regiochemistry of DHA in glycerolipids. These findings will benefit the advancement of sustainable sources of DHA via transgenic crops such as canola and soybean.

Transgenic and Genome Editing Approaches for Modifying Plant Oils.

Vegetable oils are important for human and animal nutrition and as renewable resources for chemical feedstocks. We provide an overview of transgenic and genome editing approaches for modifying plant oils, describing useful model and crop systems and different strategies for transgenic modifications. We also describe new genome editing approaches that are beginning to be applied to oilseed plants and crops. These approaches are illustrated with examples for modifying the nutritional quality of vegetable oils by altering fatty acid desaturation, producing non-native fatty acids in oilseeds, and enhancing the overall accumulation of oil in seeds and leaves.

Cytochrome b5 reductase encoded by CBR1 is essential for a functional male gametophyte in Arabidopsis.

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 heterozygotes segregated 1:1 heterozygous:wild type, indicating a gametophyte defect. Homozygous cbr1-2 seeds were deformed and required Suc for successful germination and seedling establishment. Vegetative growth of cbr1-2 plants was relatively normal, and they produced abundant flowers, but very few seeds. The pollen produced in cbr1-2 anthers was viable, but when germinated on cbr1-2 or wild-type stigmas, most of the resulting pollen tubes did not extend into the transmitting tract, resulting in a very low frequency of fertilization. These results indicate that cytochrome b5 reductase is not essential during vegetative growth but is required for correct pollen function and seed maturation.

Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana.

Ricinoleic acid, a hydroxylated fatty acid (HFA) present in castor ( Ricinus communis) seeds, is an important industrial commodity used in products ranging from inks and paints to polymers and fuels. However, due to the deadly toxin ricin and allergens also present in castor, it would be advantageous to produce ricinoleic acid in a different agricultural crop. Unfortunately, repeated efforts at heterologous expression of the castor fatty acid hydroxylase (RcFAH12) in the model plant Arabidopsis thaliana have produced only 17-19% HFA in the seed triacylglycerols (TAG), whereas castor seeds accumulate up to 90% ricinoleic acid in the endosperm TAG. RcFAH12 requires an electron supply from NADH:cytochrome b5 reductase (CBR1) and cytochrome b5 (Cb5) to synthesize ricinoleic acid. Previously, our laboratory found a mutation in the Arabidopsis CBR1 gene, cbr1-1, that caused an 85% decrease in HFA levels in the RcFAH12 Arabidopsis line. These results raise the possibility that electron supply to the heterologous RcFAH12 may limit the production of HFA. Therefore, we hypothesized that by heterologously expressing RcCb5, the reductant supply to RcFAH12 would be improved and lead to increased HFA accumulation in Arabidopsis seeds. Contrary to this proposal, heterologous expression of the top three RcCb5 candidates did not increase HFA accumulation. Furthermore, coexpression of RcCBR1 and RcCb5 in RcFAH12 Arabidopsis also did not increase in HFA levels compared to the parental lines. These results demonstrate that the Arabidopsis electron transfer system is supplying sufficient reductant to RcFAH12 and that there must be other bottlenecks limiting the accumulation of HFA.