Kimberly Glassman

A phenylalanine in DGAT is a key determinant of oil content and composition in maize

Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.

Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds

Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (Pi) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.

Expression of ZmLEC1 and ZmWRI1 Increases Seed Oil Production in Maize

Increasing seed oil production is a major goal for global agriculture to meet the strong demand for oil consumption by humans and for biodiesel production. Previous studies to increase oil synthesis in plants have focused mainly on manipulation of oil pathway genes. As an alternative to single-enzyme approaches, transcription factors provide an attractive solution for altering complex traits, with the caveat that transcription factors may face the challenge of undesirable pleiotropic effects. Here, we report that overexpression of maize (Zea mays) LEAFY COTYLEDON1 (ZmLEC1) increases seed oil by as much as 48% but reduces seed germination and leaf growth in maize. To uncouple oil increase from the undesirable agronomic traits, we identified a LEC1 downstream transcription factor, maize WRINKLED1 (ZmWRI1). Overexpression of ZmWRI1 results in an oil increase similar to overexpression of ZmLEC1 without affecting germination, seedling growth, or grain yield. These results emphasize the importance of field testing for developing a commercial high-oil product and highlight ZmWRI1 as a promising target for increasing oil production in crops.

Nutritionally improved transgenic sorghum.

Sorghum(Sorghum bicolor L.)is the sixth most planted crop in the world, grown on over 100 million acres /year worldwide and currently produces ~60 million metric tons of grain per year. Sorghum is the dietary staple food to over half a billion people in developing countries. However, sorghum grain is low in protein quality due to its low content of essential amino acids, such as lysine. The reliance on sorghum as an important food in regions of Africa and Asia can result in problems associated with malnutrition, especially of children.

Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum.

Biofortified sorghum (Sorghum bicolor (L.) Moench) lines are being developed to target vitamin A deficiency in Sub-Saharan Africa, but the delivery of provitamin A carotenoids from such diverse germplasms has not been evaluated. The purpose of this study was to screen vectors and independent transgenic events for the bioaccessibility of provitamin A carotenoids using an in vitro digestion model. The germplasm background and transgenic sorghum contained 1.0-1.5 and 3.3-14.0 μg/g β-carotene equivalents on a dry weight basis (DW), respectively. Test porridges made from milled transgenic sorghum contained up to 250 μg of β-carotene equivalents per 100 g of porridge on a fresh weight basis (FW). Micellarization efficiency of all-trans-β-carotene was lower (p < 0.05) from transgenic sorghum (1-5%) than from null/nontransgenic sorghum (6-11%) but not different between vector constructs. Carotenoid bioaccessibility was significantly improved (p < 0.05) by increasing the amount of coformulated lipid in test porridges from 5% w/w to 10% w/w. Transgenic sorghum event Homo188-A contained the greatest bioaccessible β-carotene content, with a 4-8-fold increase from null/nontransgenic sorghum. While the bioavailability and bioconversion of provitamin A carotenoids from these grains must be confirmed in vivo, these data support the notion that biofortification of sorghum can enhance total and bioaccessible provitamin A carotenoid levels.

Elevated vitamin E content improves all-trans β-carotene accumulation and stability in biofortified sorghum

Significance Studies on the importance of vitamin A for human health continue to draw significant worldwide attention. However, the instability of provitamin A in crops resulted in a significant reduction of the potential nutrition values of these food crops. Our work demonstrates that provitamin A can be stabilized in sorghum by the coexpression of vitamin E through ectopic expression of homogentisate geranylgeranyltransferase (HGGT) and that vitamin E can enhance the stability of provitamin A in planta. This research has the potential to impact directly the lives of the millions of people who suffer from vitamin A deficiency, and we believe that these results will be applicable to enhancing provitamin A stability in many food crops. Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (β-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased β-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent β-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing β-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate β-carotene oxidative degradation, resulting in increased β-carotene accumulation and stability. A kinetic study of β-carotene degradation showed that the half-life of β-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.

A single point mutation in Ms44 results in dominant male sterility and improves nitrogen use efficiency in maize

Summary Application of nitrogen fertilizer in the past 50 years has resulted in significant increases in crop yields. However, loss of nitrogen from crop fields has been associated with negative impacts on the environment. Developing maize hybrids with improved nitrogen use efficiency is a cost‐effective strategy for increasing yield sustainably. We report that a dominant male‐sterile mutant Ms44 encodes a lipid transfer protein which is expressed specifically in the tapetum. A single amino acid change from alanine to threonine at the signal peptide cleavage site of the Ms44 protein abolished protein processing and impeded the secretion of protein from tapetal cells into the locule, resulting in dominant male sterility. While the total nitrogen (N) content in plants was not changed, Ms44 male‐sterile plants reduced tassel growth and improved ear growth by partitioning more nitrogen to the ear, resulting in a 9.6% increase in kernel number. Hybrids carrying the Ms44 allele demonstrated a 4%–8.5% yield advantage when N is limiting, 1.7% yield advantage under drought and 0.9% yield advantage under optimal growth conditions relative to the yield of wild type. Furthermore, we have developed an Ms44 maintainer line for fertility restoration, male‐sterile inbred seed increase and hybrid seed production. This study reveals that protein secretion from the tapetum into the locule is critical for pollen development and demonstrates that a reduction in competition between tassel and ear by male sterility improves grain yield under low‐nitrogen conditions in maize.

High level accumulation of α-glucan in maize kernels by expressing the gtfD gene from Streptococcus mutans

Glucosyltransferases (GTFs, EC.2.4.1.5) are bacterial enzymes that catalyze the polymerization of glucose residues from sucrose, leading to the production of high molecular weight glucan with α-1,3 /α-1,6 linkages. Such glucans, with many potential food and industrial applications, do not normally exist in higher plants. We fused a mutant form of the gtfD gene from Sreptococcus mutans with the maize (Zea mays L.) chloroplastic Brittle 1 transit peptide for amyloplast targeting. This construct, driven by the ubiquitin promoter, was introduced into maize by Agrobacterium-mediated transformation. We developed a novel HPLC-based method that enabled us differentially to distinguish transgene glucan from other endogenous polysaccharides in maize kernels. Using this method, we screened over 100 transgenic plants for the presence of GTF-produced glucan whose content varied between 0.8 and 14% of dry weight in the mature transgenic seeds. The mature transgenic plants were indistinguishable from wildtype plants in growth rate and morphology. Furthermore, starch granule size in the transgenic maize kernel was unaffected by the accumulation of the foreign polysaccharide. Mutation in Sh2, which encodes a subunit of ADP-glucose pyrophosphorylase, had no effect on glucan accumulation caused by gtfD expression. Our results indicated that high levels of novel carbohydrate polymer can be accumulated in crop plants through transgene technology.

Maize LEC1 Improves Transformation in Both Maize and Wheat

Since ectopic expression of LEC1 in Arabidopsis can lead to adventive formation of embryo-like structures (Lotan et al., 1998), it was hypothesized that ectopic expression of the maize LEC1 (Lowe et al., 2001) gene could be used to induce somatic embryogenesis in maize and wheat leading to an improved culture response and higher transformation frequencies.