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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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

Possible effects of organelle charge and density on cell metabolism

A system of perception and transduction involving the gravity-induced asymmetric distribution of a plant growth hormone is studied. A theory is constructed which assumes that the perception of the gravitational stimulus involved a perturbation of the plant's bioelectric field and that the transduction of the stimulus involved voltage-gating of hormone movement from the plant's vascular tissue into the hormone responsive growing tissue. Particular attention is focused on the barriers to indole-3-acetic acid (IAA) transport from the seed to the mesocotyl cortex, the protoinhibition of IAA movement from the endosperm to the shoot, the effects of the gravitational stimulus on the movement of IAA from the kernel to the shoot, electrochemical gating as a target for the gravity stimulus, and the gravity sensing mechanism.

Bandurski, Robert S.↗

Measurement of the rates of oxindole-3-acetic acid turnover, and indole-3-acetic acid oxidation in Zea mays seedlings

Oxindole-3-acetic acid is the principal catabolite of indole-3-acetic acid in Zea mays seedlings. In this paper measurements of the turnover of oxindole-3-acetic acid are presented and used to calculate the rate of indole-3-acetic acid oxidation. [3H]Oxindole-3-acetic acid was applied to the endosperm of Zea mays seedlings and allowed to equilibrate for 24 h before the start of the experiment. The subsequent decrease in its specific activity was used to calculate the turnover rate. The average half-life of oxindole-3-acetic acid in the shoots was found to be 30 h while that in the kernels had an average half-life of 35h. Using previously published values of the pool sizes of oxindole-3-acetic acid in shoots and kernels from seedlings of the same age and variety, and grown under the same conditions, the rate of indole-3-acetic acid oxidation was calculated to be 1.1 pmol plant-1 h-1 in the shoots and 7.1 pmol plant-1 h-1 in the kernels.

NASA Discipline Plant Biology↗

Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

NASA Discipline Number 40-10↗

Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme

The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.

Non-NASA Center↗

Conserved Function of RNA Binding Motif Protein48 (RBM48) and Armadillo Repeat Containing 7 (ARMC7) in Maize and Human U12 Splicing

Splicing of pre-mRNA is fundamental for genes containing introns. Emerging data points to a deeply conserved role of this process in eukaryotic cell differentiation and proliferation. The vast majority of introns termed U2-type introns are spliced by a major spliceosome; however, there also exist rare and more conserved U12-type introns that are spliced by a minor spliceosome. Mutations that disrupt U12 splicing inhibit cell differentiation in both maize endosperm and human blood cells. However, the mechanism underlying this process is not well understood. The maize RNA Binding Motif Protein 48 (RBM48) plays an essential role as a U12 splicing factor and is required for proper maize kernel development. Using human cell lines, CRISPR-Cas9 knockdown of RBM48 demonstrated a conserved function in human U12 intron splicing. RBM48 and Armadillo Repeat Containing 7 (ARMC7) protein interact in both maize and human as part of the activated minor spliceosome. Here we show RBM48 and ARMC7 co-localization in the nucleus of human cell lines. Vertebrate ARMC7 is normally localized in the cytosol, whereas RBM48 is found in the nucleus. Our data suggests that the interaction plays a role in regulating ARMC7 localization or the efficiency of U12 intron splicing. We also performed comprehensive transcriptome profiling and identified a common subset of conserved Minor Intron Containing Genes (MIGs) impacted in both human and maize RBM48 knockout mutants. Of importance, the vast majority of these MIGs are associated with developmental defects in both plants and animals. This suggests that aberrant splicing of these targets has a high likelihood of mediating abnormal cell phenotypes. These data support evolutionarily conserved U12 splicing mechanisms between maize and humans with both RBM48 and ARMC7 having roles in the activated spliceosome.

maize↗