RESUMO
Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family's neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela; Lemna; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.
Assuntos
Araceae , Etilenodiaminas/farmacologia , Flores , Filogenia , Sementes , Araceae/genética , Araceae/crescimento & desenvolvimento , Flores/genética , Flores/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
The only generally accepted treatment of coeliac disease (CD) is a lifelong gluten-free diet. Wheat gluten proteins include gliadins, low and high molecular weight glutenins. However, we have found significant structural variations within these protein families among different cultivars. To determine which structural motifs might be less toxic than others, we assessed five variants of α-gliadin immunodominant CD-toxic peptides synthesised as 16mers in CD T cell stimulation assays with gluten-sensitive T cell lines generated from duodenal biopsies from CD-affected individuals. The peptides harboured the overlapping T cell epitopes DQ 2.5-glia-α-2 and naturally occurring variants that differed in certain amino acids (AA). The results revealed that introduction of two selected AA substitutions in α-gliadin peptides reduced immunogenicity. A peptide with three AA substitutions involving two glutamic acids (E) and one glutamine residue (G) revealed the peptide was negative in 5:5 samples. We used CD small-intestinal organ culture to assess CD toxicity that revealed two peptides with selected substitution of both glutamic acid (E) and proline (P) residues abrogated evidence of CD toxicity.
Assuntos
Doença Celíaca/imunologia , Gliadina/imunologia , Glutens/imunologia , Peptídeos/imunologia , Triticum/química , Aminoácidos , Duodeno/imunologia , Ácido Glutâmico/imunologia , Glutamina/imunologia , Humanos , Fenômenos Imunogenéticos , Prolina/imunologia , Linfócitos T/imunologiaRESUMO
Teff (Eragrostis tef) is a cornerstone of food security in the Horn of Africa, where it is prized for stress resilience, grain nutrition, and market value. Here, we report a chromosome-scale assembly of allotetraploid teff (variety Dabbi) and patterns of subgenome dynamics. The teff genome contains two complete sets of homoeologous chromosomes, with most genes maintaining as syntenic gene pairs. TE analysis allows us to estimate that the teff polyploidy event occurred ~1.1 million years ago (mya) and that the two subgenomes diverged ~5.0 mya. Despite this divergence, we detect no large-scale structural rearrangements, homoeologous exchanges, or biased gene loss, in contrast to many other allopolyploids. The two teff subgenomes have partitioned their ancestral functions based on divergent expression across a diverse expression atlas. Together, these genomic resources will be useful for accelerating breeding of this underutilized grain crop and for fundamental insights into polyploid genome evolution.
Assuntos
Eragrostis/genética , Evolução Molecular , Genoma de Planta , África , Eragrostis/classificação , Filogenia , TetraploidiaRESUMO
Mutation of o2 doubles maize endosperm lysine content, but it causes an inferior kernel phenotype. Developing quality protein maize (QPM) by introgressing o2 modifiers (Mo2s) into the o2 mutant benefits millions of people in developing countries where maize is a primary protein source. Here, we report genome sequence and annotation of a South African QPM line K0326Y, which is assembled from single-molecule, real-time shotgun sequencing reads collinear with an optical map. We achieve a N50 contig length of 7.7 million bases (Mb) directly from long-read assembly, compared to those of 1.04 Mb for B73 and 1.48 Mb for Mo17. To characterize Mo2s, we map QTLs to chromosomes 1, 6, 7, and 9 using an F2 population derived from crossing K0326Y and W64Ao2. RNA-seq analysis of QPM and o2 endosperms reveals a group of differentially expressed genes that coincide with Mo2 QTLs, suggesting a potential role in vitreous endosperm formation.
Assuntos
Proteínas de Plantas/genética , Zea mays/genética , Mapeamento Cromossômico , Endosperma/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genômica , Locos de Características QuantitativasRESUMO
Gluten-free foods cannot substitute for products made from wheat flour. When wheat products are digested, the remaining peptides can trigger an autoimmune disease in 1% of the North American and European population, called coeliac disease. Because wheat proteins are encoded by a large gene family, it has been impossible to use conventional breeding to select wheat varieties that are coeliac-safe. However, one can test the properties of protein variants by expressing single genes in coeliac-safe cereals like maize. One source of protein that can be considered as coeliac-safe and has bread-making properties is teff (Eragrostis tef), a grain consumed in Ethiopia. Here, we show that teff α-globulin3 (Etglo3) forms storage vacuoles in maize that are morphologically similar to those of wheat. Using transmission electron microscopy, immunogold labelling shows that Etglo3 is almost exclusively deposited in the storage vacuole as electron-dense aggregates. Of maize seed storage proteins, 27-kDa γ-zein is co-deposited with Etglo3. Etglo3 polymerizes via intermolecular disulphide bonds in maize, similar to wheat HMW glutenins under non-reducing conditions. Crossing maize Etglo3 transgenic lines with α-, ß- and γ-zein RNA interference (RNAi) lines reveals that Etglo3 accumulation is only dramatically reduced in γ-zein RNAi background. This suggests that Etglo3 and 27-kDa γ-zein together cause storage vacuole formation and behave similar to the interactions of glutenins and gliadins in wheat. Therefore, expression of teff α-globulins in maize presents a major step in the development of a coeliac-safe grain with bread-making properties.
Assuntos
Pão , Eragrostis/química , Farinha , Glutens/química , Zea mays/química , alfa-Globulinas/genética , Plantas Geneticamente Modificadas/química , Proteínas de Armazenamento de Sementes/genética , Triticum , Zea mays/genéticaRESUMO
[This corrects the article DOI: 10.1186/s13068-019-1612-7.].
RESUMO
BACKGROUND: Sorghum bicolor (L.) is an important bioenergy source. The stems of sweet sorghum function as carbon sinks and accumulate large amounts of sugars and lignocellulosic biomass and considerable amounts of starch, therefore providing a model of carbon allocation and accumulation for other bioenergy crops. While omics data sets for sugar accumulation have been reported in different genotypes, the common features of primary metabolism in sweet genotypes remain unclear. To obtain a cohesive and comparative picture of carbohydrate metabolism between sorghum genotypes, we compared the phenotypes and transcriptome dynamics of sugar-accumulating internodes among three different sweet genotypes (Della, Rio, and SIL-05) and two non-sweet genotypes (BTx406 and R9188). RESULTS: Field experiments showed that Della and Rio had similar dynamics and internode patterns of sugar concentration, albeit distinct other phenotypes. Interestingly, cellulose synthases for primary cell wall and key genes in starch synthesis and degradation were coordinately upregulated in sweet genotypes. Sweet sorghums maintained active monolignol biosynthesis compared to the non-sweet genotypes. Comparative RNA-seq results support the role of candidate Tonoplast Sugar Transporter gene (TST), but not the Sugars Will Eventually be Exported Transporter genes (SWEETs) in the different sugar accumulations between sweet and non-sweet genotypes. CONCLUSIONS: Comparisons of the expression dynamics of carbon metabolic genes across the RNA-seq data sets identify several candidate genes with contrasting expression patterns between sweet and non-sweet sorghum lines, including genes required for cellulose and monolignol synthesis (CesA, PTAL, and CCR), starch metabolism (AGPase, SS, SBE, and G6P-translocator SbGPT2), and sucrose metabolism and transport (TPP and TST2). The common transcriptome features of primary metabolism identified here suggest the metabolic networks contributing to carbon sink strength in sorghum internodes, prioritize the candidate genes for manipulating carbon allocation with bioenergy purposes, and provide a comparative and cohesive picture of the complexity of carbon sink strength in sorghum stem.
RESUMO
Aquatic plants have to adapt to the environments distinct from where land plants grow. A critical aspect of adaptation is the dynamics of sequence repeats, not resolved in older sequencing platforms due to incomplete and fragmented genome assemblies from short reads. Therefore, we used PacBio long-read sequencing of the Spirodela polyrhiza genome, reaching a 44-fold increase of contiguity with an N50 (a median of contig lengths) of 831 kb and filling 95.4% of gaps left from the previous version. Reconstruction of repeat regions indicates that sequentially nested long terminal repeat (LTR) retrotranspositions occur early in monocot evolution, featured with both prokaryote-like gene-rich regions and eukaryotic repeat islands. Protein-coding genes are reduced to 18,708 gene models supported by 492,435 high-quality full-length PacBio complementary DNA (cDNA) sequences. Different from land plants, the primitive architecture of Spirodela's adventitious roots and lack of lateral roots and root hairs are consistent with dispensable functions of nutrient absorption. Disease-resistant genes encoding antimicrobial peptides and dirigent proteins are expanded by tandem duplications. Remarkably, disease-resistant genes are not only amplified, but also highly expressed, consistent with low levels of 24-nucleotide (nt) small interfering RNA (siRNA) that silence the immune system of land plants, thereby protecting Spirodela against a wide spectrum of pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, but also facilitates applications in bioenergy and phytoremediation.
Assuntos
Adaptação Fisiológica/genética , Araceae/genética , Genoma de Planta/genética , Organismos Aquáticos/genética , Organismos Aquáticos/fisiologia , Araceae/anatomia & histologia , Araceae/fisiologia , DNA de Plantas/genética , Resistência à Doença/genética , Evolução Molecular , Perfilação da Expressão Gênica , Proteínas de Plantas/genética , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Análise de Sequência de DNA , Sequências de Repetição em TandemRESUMO
Grain starch and protein are synthesized during endosperm development, prompting the question of what regulatory mechanism underlies the synchronization of the accumulation of secondary and primary gene products. We found that two endosperm-specific NAC transcription factors, ZmNAC128 and ZmNAC130, have such a regulatory function. Knockdown of expression of ZmNAC128 and ZmNAC130 with RNA interference (RNAi) caused a shrunken kernel phenotype with significant reduction of starch and protein. We could show that ZmNAC128 and ZmNAC130 regulate the transcription of Bt2 and then reduce its protein level, a rate-limiting step in starch synthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130 also reduced accumulation of zeins and nonzeins by 18% and 24% compared with nontransgenic siblings, respectively. Although ZmNAC128 and ZmNAC130 affected expression of zein genes in general, they specifically activated transcription of the 16-kDa γ-zein gene. The two transcription factors did not dimerize with each other but exemplified redundancy, whereas individual discovery of their function was not amenable to conventional genetics but illustrated the power of RNAi. Given that both the Bt2 and the 16-kDa γ-zein genes were activated by ZmNAC128 or ZmNAC130, we could identify a core binding site ACGCAA contained within their target promoter regions by combining Dual-Luciferase Reporter and Electrophoretic Mobility Shift assays. Consistent with these properties, transcriptomic profiling uncovered that lack of ZmNAC128 and ZmNAC130 had a pleiotropic effect on the utilization of carbohydrates and amino acids.
Assuntos
Proteínas de Plantas/genética , Sementes/genética , Amido/genética , Fatores de Transcrição/genética , Zea mays/genética , Sítios de Ligação/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Endosperma/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , Ativação Transcricional/genética , Zeína/genéticaRESUMO
The Lemnaceae family comprises aquatic plants of angiosperms gaining attention due to their utility in wastewater treatment, and rapid production of biomass that can be used as feed, fuel, or food. Moreover, it can serve as a model species for neotenous growth and environmental adaptation. The latter properties are subject to post-transcriptional regulation of gene expression, meriting investigation of how miRNAs in Spirodela polyrhiza, the most basal and most thoroughly sequenced member of the family, are expressed under different growth conditions. To further scientific understanding of its capacity to adapt to environmental cues, we measured miRNA expression and processing of their target sequences under different temperatures, and in the presence of abscisic acid, copper, kinetin, nitrate, and sucrose. Using two small RNA sequencing experiments and one degradome sequencing experiment, we provide evidence for 108 miRNAs. Sequencing cleaved mRNAs validated 42 conserved miRNAs with 83 targets and 24 novel miRNAs regulating 66 targets and created a list of 575 predicted and verified targets. These analyses revealed condition-induced changes in miRNA expression and cleavage activity, and resulted in the addition of stringently reviewed miRNAs to miRBase. This combination of small RNA and degradome sequencing provided not only high confidence predictions of conserved and novel miRNAs and targets, but also a view of the post-transcriptional regulation of adaptations. A unique aspect is the role of miR156 and miR172 expression and activity in its clonal propagation and neoteny. Additionally, low levels of 24 nt sRNAs were observed, despite the lack of recent retrotransposition.
Assuntos
Adaptação Fisiológica/genética , Araceae/fisiologia , MicroRNAs/genética , Reguladores de Crescimento de Plantas/metabolismo , Organismos Aquáticos , Araceae/genética , Regulação da Expressão Gênica de Plantas , RNA de Plantas/genética , Estresse FisiológicoRESUMO
Sweet sorghum accumulates large amounts of soluble sugar in its stem. However, a system-based understanding of this carbohydrate allocation process is lacking. Here, we compared the dynamic transcriptome and metabolome between the conversion line R9188 and its two parents, sweet sorghum RIO and grain sorghum BTx406 that have contrasting sugar-accumulating phenotypes. We identified two features of sucrose metabolism, stable concentrations of sugar phosphates in RIO and opposite trend of trehalose-6-phosphate (T6P) between RIO vs R9188/BTx406. Integration of transcriptome and metabolome revealed R9188 is partially active in starch metabolism together with medium sucrose level, whereas sweet sorghum had the highest sucrose concentration and remained highly active in sucrose, starch, and cell wall metabolism post-anthesis. Similar expression pattern of genes involved in sucrose degradation decreased the pool of sugar phosphates for precursors of starch and cell wall synthesis in R9188 and BTx406. Differential T6P signal between RIO vs R9188/BTx406 is associated with introgression of T6P regulators from BTx406 into R9188, including C-group bZIP and trehalose 6-phosphate phosphatase (TPP). The inverted T6P signalling in R9188 appears to down-regulate sucrose and starch metabolism partly through transcriptome reprogramming, whereas introgressed metabolic genes could be related to reduced cell wall metabolism. Our results show that coordinated primary metabolic pathways lead to high sucrose demand and accumulation in sweet sorghum, providing us with targets for genetic improvements of carbohydrate allocation in bioenergy crops.
Assuntos
Carbono/metabolismo , Metaboloma , Sorghum/genética , Açúcares/metabolismo , Transcriptoma , Parede Celular/metabolismo , Genótipo , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sorghum/metabolismo , Especificidade da Espécie , Amido/metabolismo , Sacarose/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Trealose/metabolismoRESUMO
Bulked segregant analysis (BSA) is used to identify existing or induced variants that are linked to phenotypes. Although it is widely used in Arabidopsis and rice, it remains challenging for crops with large genomes, such as maize. Moreover, analysis of huge data sets can present a bottleneck linking phenotypes to their molecular basis, especially for geneticists without programming experience. Here, we identified two genes of maize defective kernel mutants with newly developed analysis pipelines that require no programing skills and should be applicable to any large genome. In the 1970s, Neuffer and Sheridan generated a chemically induced defective kernel (dek) mutant collection with the potential to uncover critical genes for seed development. To locate such mutations, the dek phenotypes were introgressed into two inbred lines to take advantage of maize haplotype variations and their sequenced genomes. We generated two pipelines that take fastq files derived from next-generation (nextGen) paired-end DNA and cDNA sequencing as input, call on several well established and freely available genomic analysis tools to call SNPs and INDELs, and generate lists of the most likely causal mutations together with variant index plots to locate the mutation to a specific sequence position on a chromosome. The pipelines were validated with a known strawberry mutation before cloning the dek mutants, thereby enabling phenotypic analysis of large genomes by next-generation sequencing.
Assuntos
Genoma de Planta/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fenótipo , Análise de Sequência de DNA/métodos , Zea mays/genéticaRESUMO
MAIN CONCLUSIONS: A high-throughput method combining liquid handling system and 96-well microplate pipetting format was developed for total sugar determination. With this new method, we characterized diverse sugar accumulation in sorghum varieties. Sweet sorghum accumulates large amounts of sucrose in its stalk and, therefore, has emerged as one important bioenergy crop. The commonly used sugar measurement, Brix, limits the characterization of internode variation of the sugar concentrations due to its low throughput. Here we developed a low-cost, high-throughput method to determine profiles of total sugars in sorghum internodes with a liquid handling system-based sample preparation and a phenol-sulfuric acid assay in 96-well microplate format. The present method generates results highly correlated with commonly used Brix measurements (r = 0.922). The inter-assay coefficient of variation ranged from 4.8 to 7.6%. The present method can reliably estimate mixed sugars composed of 80% sucrose. We characterized the profiles of 35 sorghum accessions and identified 21 accessions with significantly different sugar concentrations between internodes either due to dried-up internodes or concentration differences. As a high-throughput alternative to Brix measurements, the new method makes it possible to phenotype total sugars from large numbers of internode samples and, therefore, will be useful for genetic and breeding purposes.
Assuntos
Sucos de Frutas e Vegetais/análise , Ensaios de Triagem em Larga Escala/métodos , Sorghum/química , Sacarose/análise , Açúcares/análise , Biocombustíveis , Ensaios de Triagem em Larga Escala/instrumentação , Fenol , Fenótipo , Melhoramento Vegetal , Caules de Planta/química , Caules de Planta/genética , Caules de Planta/metabolismo , Sorghum/genética , Sorghum/metabolismo , Ácidos SulfúricosRESUMO
The domestication of maize has spanned a period of over 9000 years, during which time its wild relative teosinte underwent natural and artificial selection. We hypothesize that environmental conditions could have played a major role in this process. One factor of environmental variation is soil composition, which includes sulfur availability. Sulfur is reduced during photosynthesis and is used to synthesize cysteine and methionine, which drive the accumulation of δ10 (Zm00001d045937), δ18 (Zm00001d037436), ß15 (Zm00001d035760), γ16 (Zm00001d005793), γ27 (Zm00001d020592), and γ50 (Zm00001d020591) zeins, representing the zein2 fraction (z2) of storage proteins in maize seeds. In this study, polymorphisms and haplotypes were detected based on six z2 genes in 60 maize and teosintes lines. Haplotypes were unevenly distributed, and abundant genetic diversity was found in teosintes. Polymorphism was highest in z2δ18, whereas for z2ß15 single nucleotide polymorphism (SNP) density and insertion/deletion (indel) abundance were the lowest, indicating differential roles in seed evolution. Indels showed a clustered distribution, and most of these derived from teosintes. The indels not only led to tandem repeat polymorphisms, but also to frameshift mutations, which could also be used as null variants. In addition, neutral evolutionary tests, phylogenetic analyses, and population structures indicated that z2δ10 and z2γ50 had undergone natural selection. Indeed, a natural selection imprint could also be found with z2γ27 and z2γ16, whereas z2δ18 and z2ß15 tended to be under neutral evolution. These results suggested that genetic diversity and evolution of a subset of sulfur-rich zeins could be under environmental adaptation during maize domestication.
Assuntos
Domesticação , Enxofre/metabolismo , Zea mays/genética , Evolução Biológica , Sequência Conservada/genética , Genes de Plantas/genética , Variação Genética , Haplótipos/genética , Zea mays/metabolismoRESUMO
Maize kernels do not contain enough of the essential sulphur-amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)-amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S-assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S-assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell-specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12-fold higher SAT activity without negative impact on growth. S-assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10-kDa δ-zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40-fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high-Met zein accumulation. Moreover, SAT overcomes the shortage of S-amino acids that limits the expression and accumulation of high-Met zeins during kernel development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Galinhas/crescimento & desenvolvimento , Metionina/metabolismo , Serina O-Acetiltransferase/genética , Esterol O-Aciltransferase/metabolismo , Zea mays/genética , Zeína/metabolismo , Animais , Proteínas de Arabidopsis/genética , Expressão Gênica , Valor Nutritivo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Serina O-Acetiltransferase/metabolismo , Esterol O-Aciltransferase/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Zeína/químicaRESUMO
The TEL2, TTI1, and TTI2 proteins are co-chaperones for heat shock protein 90 (HSP90) to regulate the protein folding and maturation of phosphatidylinositol 3-kinase-related kinases (PIKKs). Referred to as the TTT complex, the genes that encode them are highly conserved from man to maize. TTT complex and PIKK genes exist mostly as single copy genes in organisms where they have been characterized. Members of this interacting protein network in maize were identified and synteny analyses were performed to study their evolution. Similar to other species, there is only one copy of each of these genes in maize which was due to a loss of the duplicated copy created by ancient allotetraploidy. Moreover, the retained copies of the TTT complex and the PIKK genes tolerated extensive retrotransposon insertion in their introns that resulted in increased gene lengths and gene body methylation, without apparent effect in normal gene expression and function. The results raise an interesting question on whether the reversion to single copy was due to selection against deleterious unbalanced gene duplications between members of the complex as predicted by the gene balance hypothesis, or due to neutral loss of extra copies. Uneven alteration of dosage either by adding extra copies or modulating gene expression of complex members is being proposed as a means to investigate whether the data supports the gene balance hypothesis or not.
RESUMO
Low levels of the essential amino acids lysine (Lys) and methionine (Met) in a maize-based diet are a major cost to feed and food. Lys deficiency is due to the abundance of Lys-poor proteins in maize kernels. Although a maize mutant, opaque-2 (o2), has sufficient levels of Lys, its soft kernel renders it unfit for storage and transportation. Breeders overcame this problem by selecting quantitative trait loci (QTL) restoring kernel hardness in the presence of o2, a variety called Quality Protein Maize (QPM). Although at least one QTL acts by enhancing the expression of the γ-zein proteins, we could surprisingly achieve rebalancing of the Lys content and a vitreous kernel phenotype by targeting suppression of γ-zeins without the o2 mutant. Reduced levels of γ-zeins were achieved with RNA interference (RNAi). Another transgenic event, PE5 expresses the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase involved in sulfate assimilation, specifically in leaves. The stacked transgenic events produce a vitreous endosperm, which has higher Lys level than the classical opaque W64Ao2 variant. Moreover, due to the increased sulfate reduction in the leaf, Met level is elevated in the seed. Such a combination of transgenes produces hybrid seeds superior to classical QPMs that would neither require a costly feed mix nor synthetic Met supplementation, potentially creating a novel and cost-effective means for improving maize nutritional quality.
Assuntos
Proteínas de Ligação a DNA/genética , Endosperma/genética , Proteínas de Plantas/genética , Locos de Características Quantitativas/genética , Fatores de Transcrição/genética , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Lisina/genética , Lisina/metabolismo , Metionina/genética , Metionina/metabolismo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Interferência de RNA , Sementes/genética , Sementes/crescimento & desenvolvimento , Zea mays/química , Zea mays/genéticaRESUMO
Sulfur assimilation may limit the pool of methionine and cysteine available for incorporation into zeins, the major seed storage proteins in maize. This hypothesis was tested by producing transgenic maize with deregulated sulfate reduction capacity achieved through leaf-specific expression of the Escherichia coli enzyme 3'-phosphoadenosine-5'-phosphosulfate reductase (EcPAPR) that resulted in higher methionine accumulation in seeds. The transgenic kernels have higher expression of the methionine-rich 10-kDa δ-zein and total protein sulfur without reduction of other zeins. This overall increase in the expression of the S-rich zeins describes a facet of regulation of these proteins under enhanced sulfur assimilation. Transgenic line PE5 accumulates 57.6% more kernel methionine than the high-methionine inbred line B101. In feeding trials with chicks, PE5 maize promotes significant weight gain compared with nontransgenic kernels. Therefore, increased source strength can improve the nutritional value of maize without apparent yield loss and may significantly reduce the cost of feed supplementation.
Assuntos
Sementes/genética , Enxofre/metabolismo , Zea mays/genética , Zeína/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Galinhas/fisiologia , Cisteína/química , Cisteína/metabolismo , Dieta/veterinária , Regulação da Expressão Gênica de Plantas , Metionina/química , Metionina/metabolismo , Plantas Geneticamente Modificadas , Sementes/fisiologia , Enxofre/química , Zea mays/fisiologia , Zeína/químicaRESUMO
We have used the newly engineered transposable element Dsg to tag a gene that gives rise to a defective kernel (dek) phenotype. Dsg requires the autonomous element Ac for transposition. Upon excision, it leaves a short DNA footprint that can create in-frame and frameshift insertions in coding sequences. Therefore, we could create alleles of the tagged gene that confirmed causation of the dek phenotype by the Dsg insertion. The mutation, designated dek38-Dsg, is embryonic lethal, has a defective basal endosperm transfer (BETL) layer, and results in a smaller seed with highly underdeveloped endosperm. The maize dek38 gene encodes a TTI2 (Tel2-interacting protein 2) molecular cochaperone. In yeast and mammals, TTI2 associates with two other cochaperones, TEL2 (Telomere maintenance 2) and TTI1 (Tel2-interacting protein 1), to form the triple T complex that regulates DNA damage response. Therefore, we cloned the maize Tel2 and Tti1 homologs and showed that TEL2 can interact with both TTI1 and TTI2 in yeast two-hybrid assays. The three proteins regulate the cellular levels of phosphatidylinositol 3-kinase-related kinases (PIKKs) and localize to the cytoplasm and the nucleus, consistent with known subcellular locations of PIKKs. dek38-Dsg displays reduced pollen transmission, indicating TTI2's importance in male reproductive cell development.