Proceedings of the National Academy of Sciences, 1997
Plants synthesize the osmoprotectant glycine betaine via the route choline → betaine aldehyde → g... more Plants synthesize the osmoprotectant glycine betaine via the route choline → betaine aldehyde → glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit ( M r 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type—i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two ...
Bioethanol production in the midwestern U.S. has largely focused on maize (Zea mays L.) grain for... more Bioethanol production in the midwestern U.S. has largely focused on maize (Zea mays L.) grain for starch-based ethanol production. There has been growing interest in lignocellulosic biomass as a feedstock for biofuels. Because maize adapted to the tropics does not initiate senescence as early as temperate-adapted maize, using a tropical germplasm could improve biomass yield. This study compares the suitability of temperate and tropical maize with differing relative maturities as feedstocks for bioethanol production. Field trials were established in central Iowa during the 2014 and 2015 growing seasons. Six hybrids of different relative maturities were grown at two levels of N fertilization and two row spacings to evaluate total biomass production and feedstock quality under midwestern U.S. conditions. Total biomass, height at the final leaf collar, stem diameter at one meter above ground, and lignocellulose concentration were measured at harvest. Tropical maize was taller and had greater non-grain and total biomass production (15% more than temperate maize), while temperate maize had greater grain yield and grain starch, as well as earlier maturation. Narrower row spacing had greater biomass and grain yield. Nitrogen fertilization rate affected grain and feedstock composition. Tropical maize had lower cellulose, lignin, and ash concentrations and higher nitrogen at harvest than that of temperate maize. Conversely, temperate maize had greater ash, cellulose, and lignin concentrations. Tropical maize planted at high densities has high potential as a feedstock for bioethanol production in the U.S. Midwest.
The main nutritional limitation of maize used for feed is the content of protein that is digestib... more The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine α-lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The α-lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine α-lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine α-lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine α-lactalbumin accumulation in transgenic maize kernels.
High activity levels of a transgene can be very useful, making a transgene easier to evaluate for... more High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27kDa gamma zein than the unselected con...
The discovery of the opaque2 (o2) mutation and o2 modifier genes in maize (Zea mays L.) has resul... more The discovery of the opaque2 (o2) mutation and o2 modifier genes in maize (Zea mays L.) has resulted in the development of Quality Protein Maize (QPM) lines with increased lysine and tryptophan content. The QPM lines BQPM9 (Reg. No.
Transgenic agricultural crops with increased nutritive value present prospects for contributing t... more Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron ho...
Methods in molecular biology (Clifton, N.J.), 2009
This method allows high-throughput qualitative screening to identify seeds containing a transgene... more This method allows high-throughput qualitative screening to identify seeds containing a transgene product in endosperm tissue. It is particularly useful for determining genetic segregation ratios or identifying seeds to be advanced in a breeding program. Tissue printing is used to avoid time-consuming extraction steps. Antibody-based detection of the transgene product makes this method suitable to any transgene product for which a specific antibody is available. It is possible to screen thousands of seeds per week using this method.
A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced po... more A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa c-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self-and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.
In order to meet the protein nutrition needs of the world population, greater reliance on plant p... more In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed
Brown midrib mutants in maize are known to be associated with reduced lignin content and increase... more Brown midrib mutants in maize are known to be associated with reduced lignin content and increased cell wall digestibility, which leads to better forage quality and higher efficiency of cellulosic biomass conversion into ethanol. Four well known brown midrib (bm) mutants, named bm1-4, were identified several decades ago. Additional recessive brown midrib mutants have been identified by allelism tests and designated as bm5 and bm6. In this study, we determined that bm6 increases cell wall digestibility and decreases plant height. bm6 was confirmed onto the short arm of chromosome 2 by a small mapping set with 181 plants from a F 2 segregating population, derived from crossing B73 and a bm6 mutant line. Subsequently, 960 brown midrib individuals were selected from the same but larger F 2 population for genetic and physical mapping. With newly developed markers in the target region, the bm6 gene was assigned to a 180 kb interval flanked by markers SSR_308337 and SSR_488638. In this region, ten gene models are predicted in the maize B73 sequence. Analysis of these ten genes as well as genes in the syntenic rice region revealed that four of them are promising candidate genes for bm6. Our study will facilitate isolation of the underlying gene of bm6 and advance our understanding of brown midrib gene functions. Communicated by M. Xu.
Proceedings of the National Academy of Sciences, 2003
The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with ... more The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with potential for use as a vaccine, a carrier molecule to deliver antigens to gut-associated lymphoid tissues, and possibly an adjuvant to make coadministered vaccines more effective. LT-B produced in plants was shown to be functional and immunogenic in animals and humans. In this work, we show that maize-derived LT-B is strongly associated with starch granules in endosperm. Using immunogold labeling/electron microscopy, cell fractionation, and protein analysis techniques, we observed that LT-B protein could be detected both internally and externally in starch granules. This strong association confers an effective copurification of the antigen with the starch fraction of maize kernels, thermostability desirable in maize processing, and resistance to peptic degradation in simulated gastric fluid digests, an important attribute for an orally delivered antigen.
Cereal grains are an important nutritional source of amino acids for humans and livestock worldwi... more Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of c-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires c-zeins for its proper subcellular organization in maize.
Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polyme... more Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native...
We have developed a method for detecting a transgene and its protein product in maize endosperm t... more We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This technique could be highly useful for several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and testing for gene silencing in kernels.
The Germplasm Enhancement of Maize (GEM) Project is a cooperative effort between the USDA-ARS, pr... more The Germplasm Enhancement of Maize (GEM) Project is a cooperative effort between the USDA-ARS, private industry and public researchers to broaden and enhance the germplasm base of maize. In this program, selected accessions from the Latin American Maize Project, and seven tropical hybrids donated by DeKalb to the GEM Project, were crossed to elite proprietary inbred lines contributed by commercial plant breeding programs. In most cases, the resulting hybrids were crossed to a second commercial inbred line and the resulting 25% exotic hybrids were used as breeding populations for further development. To identify GEM germplasm with value to protein quality breeding programs, we developed a process for evaluating the content of the essential amino acids methionine, lysine and tryptophan in the grain of GEM germplasm that balances the need for multiple-year evaluations with the constantly changing entry list of this germplasm screening program. This process involves annual field trials ...
Proceedings of the National Academy of Sciences, 1997
Plants synthesize the osmoprotectant glycine betaine via the route choline → betaine aldehyde → g... more Plants synthesize the osmoprotectant glycine betaine via the route choline → betaine aldehyde → glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit ( M r 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type—i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two ...
Bioethanol production in the midwestern U.S. has largely focused on maize (Zea mays L.) grain for... more Bioethanol production in the midwestern U.S. has largely focused on maize (Zea mays L.) grain for starch-based ethanol production. There has been growing interest in lignocellulosic biomass as a feedstock for biofuels. Because maize adapted to the tropics does not initiate senescence as early as temperate-adapted maize, using a tropical germplasm could improve biomass yield. This study compares the suitability of temperate and tropical maize with differing relative maturities as feedstocks for bioethanol production. Field trials were established in central Iowa during the 2014 and 2015 growing seasons. Six hybrids of different relative maturities were grown at two levels of N fertilization and two row spacings to evaluate total biomass production and feedstock quality under midwestern U.S. conditions. Total biomass, height at the final leaf collar, stem diameter at one meter above ground, and lignocellulose concentration were measured at harvest. Tropical maize was taller and had greater non-grain and total biomass production (15% more than temperate maize), while temperate maize had greater grain yield and grain starch, as well as earlier maturation. Narrower row spacing had greater biomass and grain yield. Nitrogen fertilization rate affected grain and feedstock composition. Tropical maize had lower cellulose, lignin, and ash concentrations and higher nitrogen at harvest than that of temperate maize. Conversely, temperate maize had greater ash, cellulose, and lignin concentrations. Tropical maize planted at high densities has high potential as a feedstock for bioethanol production in the U.S. Midwest.
The main nutritional limitation of maize used for feed is the content of protein that is digestib... more The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine α-lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The α-lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine α-lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine α-lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine α-lactalbumin accumulation in transgenic maize kernels.
High activity levels of a transgene can be very useful, making a transgene easier to evaluate for... more High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27kDa gamma zein than the unselected con...
The discovery of the opaque2 (o2) mutation and o2 modifier genes in maize (Zea mays L.) has resul... more The discovery of the opaque2 (o2) mutation and o2 modifier genes in maize (Zea mays L.) has resulted in the development of Quality Protein Maize (QPM) lines with increased lysine and tryptophan content. The QPM lines BQPM9 (Reg. No.
Transgenic agricultural crops with increased nutritive value present prospects for contributing t... more Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron ho...
Methods in molecular biology (Clifton, N.J.), 2009
This method allows high-throughput qualitative screening to identify seeds containing a transgene... more This method allows high-throughput qualitative screening to identify seeds containing a transgene product in endosperm tissue. It is particularly useful for determining genetic segregation ratios or identifying seeds to be advanced in a breeding program. Tissue printing is used to avoid time-consuming extraction steps. Antibody-based detection of the transgene product makes this method suitable to any transgene product for which a specific antibody is available. It is possible to screen thousands of seeds per week using this method.
A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced po... more A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa c-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self-and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production.
In order to meet the protein nutrition needs of the world population, greater reliance on plant p... more In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed
Brown midrib mutants in maize are known to be associated with reduced lignin content and increase... more Brown midrib mutants in maize are known to be associated with reduced lignin content and increased cell wall digestibility, which leads to better forage quality and higher efficiency of cellulosic biomass conversion into ethanol. Four well known brown midrib (bm) mutants, named bm1-4, were identified several decades ago. Additional recessive brown midrib mutants have been identified by allelism tests and designated as bm5 and bm6. In this study, we determined that bm6 increases cell wall digestibility and decreases plant height. bm6 was confirmed onto the short arm of chromosome 2 by a small mapping set with 181 plants from a F 2 segregating population, derived from crossing B73 and a bm6 mutant line. Subsequently, 960 brown midrib individuals were selected from the same but larger F 2 population for genetic and physical mapping. With newly developed markers in the target region, the bm6 gene was assigned to a 180 kb interval flanked by markers SSR_308337 and SSR_488638. In this region, ten gene models are predicted in the maize B73 sequence. Analysis of these ten genes as well as genes in the syntenic rice region revealed that four of them are promising candidate genes for bm6. Our study will facilitate isolation of the underlying gene of bm6 and advance our understanding of brown midrib gene functions. Communicated by M. Xu.
Proceedings of the National Academy of Sciences, 2003
The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with ... more The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with potential for use as a vaccine, a carrier molecule to deliver antigens to gut-associated lymphoid tissues, and possibly an adjuvant to make coadministered vaccines more effective. LT-B produced in plants was shown to be functional and immunogenic in animals and humans. In this work, we show that maize-derived LT-B is strongly associated with starch granules in endosperm. Using immunogold labeling/electron microscopy, cell fractionation, and protein analysis techniques, we observed that LT-B protein could be detected both internally and externally in starch granules. This strong association confers an effective copurification of the antigen with the starch fraction of maize kernels, thermostability desirable in maize processing, and resistance to peptic degradation in simulated gastric fluid digests, an important attribute for an orally delivered antigen.
Cereal grains are an important nutritional source of amino acids for humans and livestock worldwi... more Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of c-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires c-zeins for its proper subcellular organization in maize.
Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polyme... more Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native...
We have developed a method for detecting a transgene and its protein product in maize endosperm t... more We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This technique could be highly useful for several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and testing for gene silencing in kernels.
The Germplasm Enhancement of Maize (GEM) Project is a cooperative effort between the USDA-ARS, pr... more The Germplasm Enhancement of Maize (GEM) Project is a cooperative effort between the USDA-ARS, private industry and public researchers to broaden and enhance the germplasm base of maize. In this program, selected accessions from the Latin American Maize Project, and seven tropical hybrids donated by DeKalb to the GEM Project, were crossed to elite proprietary inbred lines contributed by commercial plant breeding programs. In most cases, the resulting hybrids were crossed to a second commercial inbred line and the resulting 25% exotic hybrids were used as breeding populations for further development. To identify GEM germplasm with value to protein quality breeding programs, we developed a process for evaluating the content of the essential amino acids methionine, lysine and tryptophan in the grain of GEM germplasm that balances the need for multiple-year evaluations with the constantly changing entry list of this germplasm screening program. This process involves annual field trials ...
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