SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

The major phospholipids found in plant tissues are similar to those found in all eukaryotes, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG). The precursor molecule common to the de novo synthesis of all phospholipids in prokaryotes and eukaryotes is phosphatidic acid (PA). The enzyme cytidine diphosphate (CDP)-diacylglycerol synthase catalyzes the reaction between PA and CTP to yield CDP-1,2-diacylglycerol (DAG) and inorganic phosphate. In E. coli and yeast, PG is synthesized by a two-step reaction; CDP-diacylglycerol reacts with glycerol phosphate to yield phosphatidylglycerolphosphate and cytidine monophosphate (CMP), a reaction catalyzed by the enzyme glycerophosphate: CDP-diacylglycerol phosphatidyltransferase. In yeast, PS is synthesized from CDP-diacylglycerol and serine, a reaction catalyzed by the enzyme CDP-diacylglycerol:L-serine O-phosphatidyltransferase. The source of choline for the nucleotide pathway of PC biosynthesis is even less clear than the origin of free ethanolamine. Methylation of free ethanolamine to choline has not been conclusively demonstrated in any organism.

Designing crops to achieve specific goals for value-added traits involves a complex interaction of plant genetics with environment, economic forces, cultural needs and desires, projected uses, and available technology. This chapter considers the genetic potential inherent within plant genomes to achieve specific quantitative or qualitative traits that may add value to the harvested crop. In the USA, protein sources are relatively cheap, whereas in other locations, such as northern Europe, imported protein meal may be more expensive. The chapter also considers progress and prospects for increasing protein quantity or improving protein quality, primarily in the grain crops. It examines saltering fatty acid content in oil seed crops for edible and industrial oils. The chapter presents a different approach, capitalizing on the wealth of knowledge that has been obtained from the genetic and biochemical examination of com mutants to elucidate pathways of starch biosynthesis.

Although there is much knowledge of the enzymology (and genes coding the proteins) of lipid biosynthesis in higher plants, relatively little attention has been paid to regulation. We have demonstrated the important role for cholinephosphate cytidylyltransferase in the biosynthesis of the major extra-plastidic membrane lipid, phosphatidylcholine. We followed this work by applying control analysis to light-induced fatty acid synthesis. This was the first such application to lipid synthesis in any organism. The data showed that acetyl-CoA carboxylase was very important, exerting about half of the total control. We then applied metabolic control analysis to lipid accumulation in important oil crops — oilpalm, olive, and rapeseed. Recent data with soybean show that the block of fatty acid biosynthesis reactions exerts somewhat more control (63%) than lipid assembly although both are clearly very important. These results suggest that gene stacks, targeting both parts of the overall lipid synthesis pathway will be needed to increase significantly oil yields in soybean. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Oils and fats are an important source of energy for the human diet and also contribute significantly to the sensory characteristics of food.Many oils are also used for non-food applications, although industrial use currently accounts for only a small proportion of the world vegetable oil production, less than 5% of total production, mostly for biodiesel. About 80% of edible oils are derived from plant sources and temperate annual oil seeds (soy, rapeseed, sunflower and peanut) account for about 60% of this total. Soybean oil is by far the dominant oil in this category, accounting for over half of the world vegetable oil production. Improving the functional and nutritional qualities of vegetable oils has garnered much attention over the last 15 years or so. This chapter will describe some of the attempts to genetically improve plant seed oils, with special emphasis on soybean oil, for food and non-food uses.

For most of their 300 year domesticated history soybeans have been grown to feed humans and animals. In the last decade there has been increasing use of soybean oil (which constitutes about 20% of the seed) for energy, in the form of methyl ester biodiesel. Soybean biodiesel, while more expensive to produce than petroleum diesel, offers a number of advantages over all petrochemical fuels, including increased fuel performance, lower carbon emissions and biodegradability. Soybean biodiesel use is limited by the functional properties of soybean oil, cost and oil supply. The oxidative instability of soybean oil will compromise ignition performance. Saturated fatty acids reduce the cold flow properties of soybean biodiesel and limit its use in cold environments. High oleic, low palmitic acid soybean oil addresses these functional limitations. Efforts by plant breeders over a 30 year period has resulted in high oleic, low palmitic acid soybean lines but they have not been commercialized due to the breeding challenges associated with pyramiding multigenic factors, required for full penetration of the target trait and the observed instability of the phenotype across environments, as well as poor agronomic performance of the crop. On the other hand, using the tools of biotechnology, single locus, environmentally stable, high oleic, low palmitic acid soybean lines have been produced in high performing elite varieties. These varieties are being commercialized by major seed companies. The methyl ester biodiesel from these lines has improved functional properties including cold flow characteristics similar to petroleum diesel. Future biotechnology efforts will be directed towards improving the oil yield of the soybean crop.

Numerous clinical studies have demonstrated that the omega-3 fatty acids in fish oil significantly reduce the risk of cardiovascular disease in adults. This chapter discusses the approach to introduce the genes encoding an omega-3 fatty acid biosynthesis pathway into an oleaginous yeast that synthesizes and stores triglycerides as an energy reserve when starved for nitrogen in the presence of an excess carbon source, such as glucose. It explains the development of a clean and sustainable source of omega-3 fatty acids by fermentation, which uses a metabolically engineered strain of the oleaginous yeast Y. lipolytica. While certain strains of Y. lipolytica can accumulate oil up to 40% of the dry cell weight, the only Polyunsaturated Fatty Acid (PUFA) normally synthesized by the organism is Linoleic Acid (LA). Coordinate expression of desaturase genes and elongase genes comprising a "delta6 pathway" was sufficient to demonstrate the synthesis of Eicosapentaenoic Acid (EPA). However, only an integrated strategy, based on the use of strong promoters, an increase in gene copy numbers, the push and pull of carbon into the engineered pathway, and the use of oleaginous condition, resulted in the generation of a high EPA production strain.

This chapter will take a broad look at non-acyl and acyl lipids found in seed. Initially it will focus on certain non-acyl lipids, such as sterols, carotenoids and tocochromanols. It will provide a brief overview of the function of these molecules in plants with a focus on seeds, review information on biosynthetic pathways and attempts at altering quality and quantity of these molecules through metabolic engineering. Our subsequent review of acyl lipid biosynthesis will mainly focus on recent findings related to modification of the pathways providing energy, reductant and carbon for fatty acid biosynthesis that allows for high levels of acyl lipid accumulation during seed filling. This section will have a specific focus on the interaction of primary metabolism and fatty acid biosynthesis. We will then briefly review fatty acid biosynthesis in seed plastids and cytosolic assembly of seed storage and membrane lipids in seed. Finally developmental regulation of seed storage lipid accumulation and transgenic approaches to increase the total oil content of seeds will be summarized.

Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom.