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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm

There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.

flux balance analysis↗
DOE OSTI2014

Expression, regulation and activity of a B2-type cyclin in mitotic and endoreduplicating maize endosperm

Cyclin-dependent kinases, the master regulators of the eukaryotic cell cycle, are complexes comprised of a catalytic serine/threonine protein kinase and an essential regulatory cyclin. The maize genome encodes over 50 cyclins grouped in different types, but they have been little investigated. We characterized a type B2 cyclin (CYCB2;2) during maize endosperm development, which comprises a cell proliferation phase based on the standard mitotic cell cycle, followed by an endoreduplication phase in which DNA replication is reiterated in the absence of mitosis or cytokinesis. CYCB2;2 RNA was present throughout the period of endosperm development studied, but its level declined as the endosperm transitioned from a mitotic to an endoreduplication cell cycle. However, the level of CYCB2;2 protein remained relatively constant during both stages of endosperm development. CYCB2;2 was recalcitrant to degradation by the 26S proteasome in endoreduplicating endosperm extracts, which could explain its sustained accumulation during endosperm development. In addition, although CYCB2;2 was generally localized to the nucleus of endosperm cells, a lower molecular weight form of the protein accumulated specifically in the cytosol of endoreduplicating endosperm cells. In dividing cells, CYCB2;2 appeared to be localized to the phragmoplast and may be involved in cytokinesis and cell wall formation. Kinase activity was associated with CYCB2;2 in mitotic endosperm, but was absent or greatly reduced in immature ear and endoreduplicating endosperm. CYCB2;2-associated kinase phosphorylated maize E2F1 and the “pocket” domains of RBR1 and RBR3. CYCB2;2 interacted with both maize CDKA;1 and CDKA;3 in insect cells. These results suggest CYCB2;2 functions primarily during the mitotic cell cycle, and they are discussed in the context of the roles of cyclins, CDKs and proteasome activity in the regulation of the cell cycle during endosperm development.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Maize Rough Endosperm6 (rgh6) Encodes A Predicted Dead-Box RNA Helicase and Affects Mirna Processing in Endosperm Development

Maize rough endosperm (rgh) mutants have defective kernels with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. Here, we report on the developmental and molecular function of the rgh6 locus. The rgh6 mutant was isolated from the UniformMu transposon tagging population. Mutant kernels have reduced endosperm size and defective embryos that develop in a more apical position than typical for defective embryos. TB translocation crosses revealed that rgh6 mutant endosperm inhibits normal embryo development. Positional cloning of the rgh6 locus found that it encodes a predicted DEAD-box RNA helicase. Consistent with a predicted function for RNA processing, transient expression of a RGH6-GFP fusion protein is localized to nucleolus and nuclear speckles in Nicotiana benthamiana leaves. Rgh6 transcripts are highly expressed in endosperm epidermal cell types such as the aleurone, basal endosperm cell layer, embryo surrounding region, and endosperm adjacent to scutellum. Markers of these cell types show increased levels in rgh6 mutant kernels. Mutant endosperm tissues have increased precursor microRNA (pre-miRNA) and decreased mature miRNA relative to normal sibling endosperm, indicating that rgh6 is required for miRNA processing. The transcript levels for most miRNA target genes accumulate to a higher level in rgh6 mutant tissue. These results suggest that miRNA processing and regulation of miRNA target genes are required for normal endosperm development.

Plant Sciences↗
NASA NTRS1988

Effect of salicylhydroxamic acid on endosperm strength and embryo growth of Lactuca sativa L. cv Waldmann's Green seeds

Salicylhydroxamic acid (SHAM) stimulated germination of photosensitive lettuce (Lactuca sativa L. cv Waldmann's Green) seeds in darkness. To determine whether SHAM acts on the embryo or the endosperm, we investigated separately effects of SHAM on growth potential of isolated embryos as well as on endosperm strength. Embryo growth potential was quantified by incubating decoated embryos in various concentrations of osmoticum and measuring subsequent radicle elongation. Growth potential of embryos isolated from seeds pretreated with 4 millimolar SHAM was equal to that of untreated controls. Rupture strength of endosperm tissue excised from seeds pretreated with SHAM was 33% less than that of controls in the micropylar region. To determine if the embryo must be in contact with the endosperm of SHAM to weaken the endosperm, some endosperms were incubated with SHAM only after dissection from seeds. Rupture strength of SHAM-treated, isolated endosperms in the micropylar region was 25% less than that of untreated controls. There was no difference in rupture strength in the cotyledonary region of endosperm isolated from seeds treated with SHAM in buffer or buffer alone. SHAM therefore stimulates germination not by enhancing embryo growth potential, but by weakening the micropylar region of the endosperm enclosing the embryo.

NASA Discipline Plant Biology↗
DOE OSTI2018

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

Highlights: • Pectin methyl esterification of cell wall endosperm is involved in seed development. • Seed and embryo morphology are altered in bdx-1 mutant. • Low-esterified pectins are related to embryo bending in WT and bdx-1 seeds. The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana

Background: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. Results: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. Conclusions: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1987

Isolation and characterization of esters of indole-3-acetic acid from the liquid endosperm of the horse chestnut (Aesculus species)

Esters of indole-3-acetic acid were extracted and purified from the liquid endosperm of immature fruits of various species of the horse chestnut (Aesculus parviflora, A. baumanni, A. pavia rubra, and A. pavia humulis). The liquid endosperm contained, at least 12 chromatographically distinct esters. One of these compounds was purified and characterized as an ester of indole-3-acetic acid and myo-inositol. A second compound was found to be an ester of indole-3-acetic acid and the disaccharide rutinose (glucosyl-rhamnose). A third compound was partially characterized as an ester of indole-3-acetic acid and a desoxyaminohexose.

NASA Discipline Number 40-10↗
DOE OSTI2019

Tissue-specific differences in metabolites and transcripts contribute to the heterogeneity of ricinoleic acid accumulation in Ricinus communis L. (castor) seeds

Castor ( Ricinus communis L.) seeds are valued for their production of oils which can comprise up to 90% hydroxy-fatty acids (ricinoleic acid). Castor oil contains mono-, di- and tri- ricinoleic acid containing triacylglycerols (TAGs). Although the enzymatic synthesis of ricinoleic acid is well described, the differential compartmentalization of these TAG molecular species has remained undefined. To examine the distribution of hydroxy fatty acid accumulation within the endosperm and embryo tissues of castor seeds. Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to map the distribution of triacylglycerols in tissue sections of castor seeds. In addition, the endosperm and embryo (cotyledons and embryonic axis) tissues were dissected and extracted for quantitative lipidomics analysis and Illumina-based RNA deep sequencing. Results: This study revealed an unexpected heterogeneous tissue distribution of mono-, di- and tri- hydroxy-triacylglycerols in the embryo and endosperm tissues of castor seeds. Pathway analysis based on transcript abundance suggested that distinct embryo- and endosperm-specific mechanisms may exist for the shuttling of ricinoleic acid away from phosphatidylcholine (PC) and into hydroxy TAG production. The embryo-biased mechanism appears to favor removal of ricinoleic acid from PC through phophatidylcholine: diacylglycerol acylytransferase (PDAT) while the endosperm pathway appears to remove ricinoleic acid from the PC pool by preferences of phospholipase A (PLA 2 α) and/or phosphatidylcholine: diacylglycerol cholinepshphotransferase PDCT. In conclusion, collectively, a combination of lipidomics and transcriptomics analyses revealed previously undefined spatial aspects of hydroxy fatty acid metabolism in castor seeds. These studies underscore a need for tissue-specific studies as a means to better understand the regulation of triacylglycerol accumulation in oilseeds.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1988

Translocation of radiolabeled indole-3-acetic acid and indole-3-acetyl-myo-inositol from kernel to shoot of Zea mays L

Either 5-[3H]indole-3-acetic acid (IAA) or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[3H]indole-3-acetic acid or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.

Non-NASA Center↗
NASA NTRSDate not supplied

Engineering 6-Phosphogluconate Improves Grain Yield in Heat-Stressed Maize

Endosperm starch synthesis is a primary determinant of grain yield and is sensitive to high-temperature stress. The maize chloroplast-localized 6-phosphogluconate dehydrogenase (6PGDH), PGD3, is critical for endosperm starch accumulation. Maize also has two cytosolic isozymes, PGD1 and PGD2, that are not required for kernel development. We found that cytosolic PGD1 and PGD2 isozymes have heat-stable activity, while amyloplast-localized PGD3 activity is labile under heat stress conditions. We targeted heat-stable 6PGDH to endosperm amyloplasts by fusing the Waxy1 chloroplast targeting the peptide coding sequence to the Pgd1 and Pgd2 open reading frames (ORFs). These WPGD1 and WPGD2 fusion proteins import into isolated chloroplasts, demonstrating a functional targeting sequence. Transgenic maize plants expressing WPGD1 and WPGD2 with an endosperm-specific promoter increased 6PGDH activity with enhanced heat stability in vitro. WPGD1 and WPGD2 transgenes complement the pgd3-defective kernel phenotype, indicating the fusion proteins are targeted to the amyloplast. In the field, the WPGD1 and WPGD2 transgenes can mitigate grain yield losses in high-nighttime-temperature conditions by increasing kernel number. These results provide insight into the subcellular distribution of metabolic activities in the endosperm and suggest the amyloplast pentose phosphate pathway is a heat-sensitive step in maize kernel metabolism that contributes to yield loss during heat stress.

Synthetic Biology↗
DOE OSTI2019

Comparison of Zn accumulation and speciation in kernels of sweetcorn and maize differing in maturity

Abstract Background and Aims Understanding the speciation of Zn in edible portions of crops helps identify the most effective biofortification strategies to increase the supply of nutrients for improving the health and nutrition of consumers. Methods Kernels of 12 sweetcorn and three maize (Zea mays) varieties were analysed for Zn concentration and content. The speciation of the Zn in the embryos, endosperms and whole kernels at 21, 28 and 56 days after pollination (DAP) was then examined for one maize and one sweetcorn variety using synchrotron-based X-ray absorption spectroscopy (XAS). Key Results Averaged across all sweetcorn and maize varieties at 21 DAP, the embryo contributed 27–29% of the whole kernel Zn whilst the endosperm contributed 71–73 %. While sweetcorn embryos contributed a lower proportion to the total kernel Zn than those of maize, the proportion of total Zn in the embryo increased as kernels aged for both varieties, reaching 33 % for sweetcorn and 49% for maize at 28 DAP. Using XAS, it was predicted that an average of 90 % of the Zn in the embryos was present as Zn-phytate, while in the endosperm the Zn was primarily complexed with an N-containing ligand such as histidine and to a lesser extent with phytate. However, in maize endosperms, it was also observed that the proportion of Zn present as Zn-phytate increased as the kernel matured, thereby also probably decreasing its bioavailability in these mature maize kernels. Conclusions The apparent low bioavailability of Zn supplied in maize at its consumption stage (i.e. mature kernels) probably undermines the effectiveness of biofortification of this crop. Conversely, successful biofortification of Zn in sweetcorn and green maize consumed as immature kernels could potentially provide a good source of bioavailable Zn in human diets.

Cheah, Zhong Xiang↗
NASA NTRSDate not supplied

The maize rough endosperm6 (rgh6) mutant encodes a predicted DEAD-box RNA helicase

Maize rough endosperm (rgh) mutants have defective seeds with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. We isolated the rgh6 locus from the UniformMu transposon tagging population. Mutant kernels have reduced grain-fill with defective embryos that fail to germinate. Self-pollination of rgh6 heterozygotes produces mutant seeds at frequency consistent with a single recessive mutation. Molecular mapping localized rgh6 to a 60 kbp interval on chromosome 5. PCR analysis of gene models within the fine-map interval identified a Mutator (Mu) transposon insertion within a predicted DEAD-box RNA helicase gene. A second allele of rgh6 was identified from UniformMu population. Reciprocal crosses between rgh6-umu1 and rgh6-umu2 heterozygotes produces mutant seeds at a frequency consistent with a 3:1 ratio indicating these mutants are allelic. Both rgh6-umu1 and rgh6-umu2 have Mu element insertions at the same insertion site within the DEAD-box RNA helicase gene. However, the terminal inverted repeat sequences are polymorphic between the alleles showing the alleles are independent mutations. We conclude that mutation of the DEAD-box RNA helicase causes the rgh6 phenotype. Further characterization of the rgh6 mutant phenotype and biochemical investigation of the RGH6 protein is expected to give further insights on the roles of RNA processing in endosperm development.

maize↗
NASA NTRSDate not supplied

Paternal Imprinting of Dosage-Effect Defective1 (Ded1) Contributes to Seed Weight Xenia in Maize

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinted dosage-effect defective1 (ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5-10% seed weight reduction when ded1 is transmitted through the male, while homozygous mutants are defective with a 70-90% seed weight reduction. Ded1 encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

Genomics↗
NASA NTRSDate not supplied

Paternal Imprinting of Dosage-Effect Defective1 Contributes to Seed Weight Xenia in Maize4

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinted dosage-effect defective1 (ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5-10% seed weight reduction when ded1 is transmitted through the male, while homozygous mutants are defective with a 70-90% seed weight reduction. Ded1 encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

Genomics↗
DOE OSTI2022

Altering the localization and toxicity of arsenic in rice grain

Previous work has shown that inorganic As localizes in rice bran whereas DMA localizes in the endosperm, but less is known about co-localization of As and S species and how they are affected by growing conditions. We used high-resolution synchrotron X-ray fluorescence imaging to image As and S species in rice grain from plants grown to maturity in soil (field and pot) and hydroponically (DMA or arsenite dosed) at field-relevant As concentrations. In hydroponics, arsenite was localized in the ovular vascular trace (OVT) and the bran while DMA permeated the endosperm and was absent from the OVT in all grains analyzed, and As species had no affect on S species. In pot studies, soil amended with Si-rich rice husk with higher DMA shifted grain As into the endosperm for both japonica and indica ecotypes. In field-grown rice from low-As soil, As localized in the OVT as arsenite glutathione, arsenite, and DMA. Results support a circumferential model of grain filling for arsenite and DMA and show Si-rich soil amendments alter grain As localization, potentially lessening risk to rice consumers.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1986

Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

NASA Discipline Number 40-30↗