Transcriptional activation of Brassica napus ß-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola ß-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.

The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis.

In higher plants, three subfamilies of sucrose nonfermenting-1 (Snf1)-related protein kinases have evolved. While the Snf1-related protein kinase 1 (SnRK1) subfamily has been shown to share pivotal roles with the orthologous yeast Snf1 and mammalian AMP-activated protein kinase in modulating energy and metabolic homeostasis, the functional significance of the two plant-specific subfamilies SnRK2 and SnRK3 in these critical processes is poorly understood. We show here that SnRK2.6, previously identified as crucial in the control of stomatal aperture by abscisic acid (ABA), has a broad expression pattern and participates in the regulation of plant primary metabolism. Inactivation of this gene reduced oil synthesis in Arabidopsis (Arabidopsis thaliana) seeds, whereas its overexpression increased Suc synthesis and fatty acid desaturation in the leaves. Notably, the metabolic alterations in the SnRK2.6 overexpressors were accompanied by amelioration of those physiological processes that require high levels of carbon and energy input, such as plant growth and seed production. However, the mechanisms underlying these functionalities could not be solely attributed to the role of SnRK2.6 as a positive regulator of ABA signaling, although we demonstrate that this kinase confers ABA hypersensitivity during seedling growth. Collectively, our results suggest that SnRK2.6 mediates hormonal and metabolic regulation of plant growth and development and that, besides the SnRK1 kinases, SnRK2.6 is also implicated in the regulation of metabolic homeostasis in plants.

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.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed.