Maize Endosperm Tissue Research Articles

Characterization of Endosperm Carbohydrates in isa2–339 Maize and Interactions with su1‐ref

Selection for genetic modifiers of starch and sugar accumulation in sugary1‐ref (su1‐ref) maize (Zea mays L.) has led to the improvement of flavor characteristics in su1 sweet corn hybrids. The Su1 gene encodes a starch debranching enzyme that when defective causes large quantities of water‐soluble polysaccharides (WSP) to be produced in maize endosperm tissue. Another gene in maize, Isoamylase2 (Isa2), produces a starch debranching enzyme known to interact with the wild‐type SU1 protein in developing maize endosperm. Experiments were conducted to determine if a mutant of the Isa2 gene significantly modifies carbohydrate composition in su1‐ref inbreds and therefore is potentially useful for modifying flavor characteristics of su1 sweet corn. Using isa2–339, a mutant at the Isa2 locus, a population was created in which the su1‐ref and isa2–339 alleles were segregating. Lines developed from this population were used for single kernel carbohydrate analysis in 2012 and carbohydrate analysis of replicated ears in 2013. In 2012 and 2013, individual kernels and replicated ears fixed for the su1‐ref and isa2–339 alleles contained less starch and more total sugar compared with the su1‐ref, Isa2 parent, which was the source of the su1‐ref allele in this population. When the su1‐ref, isa2–339 mutant lines were reciprocally crossed with the Su1, isa2–339 parent, starch and WSP contents were significantly different among the Su1, isa2–339 parent and the hybrids. Kernels of both types of reciprocal hybrids had more WSP and less starch than did the Su1, isa2–339 parent. Kernels with two doses of the su1‐ref allele have more WSP and less starch than kernels with a single dose of the su1‐ref allele. In this population, starch content is further reduced in su1‐ref, isa2–339 double mutant genotypes compared with the su1‐ref parent. The su1‐ref/su1‐ref, isa2–339/isa2–339 lines developed in this experiment are the first documented double mutants of su1‐ref with another isoamylase starch debranching enzyme mutant.

Comprehensive analysis of imprinted genes in maize reveals allelic variation for imprinting and limited conservation with other species.

In plants, a subset of genes exhibit imprinting in endosperm tissue such that expression is primarily from the maternal or paternal allele. Imprinting may arise as a consequence of mechanisms for silencing of transposons during reproduction, and in some cases imprinted expression of particular genes may provide a selective advantage such that it is conserved across species. Separate mechanisms for the origin of imprinted expression patterns and maintenance of these patterns may result in substantial variation in the targets of imprinting in different species. Here we present deep sequencing of RNAs isolated from reciprocal crosses of four diverse maize genotypes, providing a comprehensive analysis that allows evaluation of imprinting at more than 95% of endosperm-expressed genes. We find that over 500 genes exhibit statistically significant parent-of-origin effects in maize endosperm tissue, but focused our analyses on a subset of these genes that had >90% expression from the maternal allele (69 genes) or from the paternal allele (108 genes) in at least one reciprocal cross. Over 10% of imprinted genes show evidence of allelic variation for imprinting. A comparison of imprinting in maize and rice reveals that 13% of genes with syntenic orthologs in both species exhibit conserved imprinting. Genes that exhibit conserved imprinting between maize and rice have elevated nonsynonymous to synonymous substitution ratios compared with other imprinted genes, suggesting a history of more rapid evolution. Together, these data suggest that imprinting only has functional relevance at a subset of loci that currently exhibit imprinting in maize.

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain. Utilization of maize in bioethanol industry in the United States reached ±13.3billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85°C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2±2.63% (14.7±0.90g ethanol per 100g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5% (33.1±0.66g ethanol per 100g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.

Binding of ABI4 to a CACCG motif mediates the ABA-induced expression of the ZmSSI gene in maize (Zea mays L.) endosperm

Starch synthase I (SSI) contributes the majority of the starch synthase activity in developing maize endosperm. In this work, the effects of various plant hormones and sugars on the expression of the starch synthase I gene (ZmSSI) in developing maize endosperms were examined. The accumulation of ZmSSI mRNA was induced using abscisic acid (ABA) but not with glucose, sucrose, or gibberellin treatment. To investigate the molecular mechanism underlying this effect, the ZmSSI promoter region (-1537 to +51) was isolated and analysed. A transient expression assay in maize endosperm tissue showed that the full-length ZmSSI promoter is activated by ABA. The results of deletion and mutation assays demonstrated that a CACCG motif in the ZmSSI promoter is responsible for the ABA inducibility. The results of binding shift assays indicated that this CACCG motif interacts with the maize ABI4 protein in vitro. The overexpression of ABI4 in endosperm tissue enhanced the activity of a promoter containing the CACCG motif in the absence of ABA treatment. Expression pattern analysis indicated that the transcription pattern of ABI4 in the developing maize endosperm was induced by ABA treatment but was only slightly affected by glucose or sucrose treatment. Taken together, these data indicate that ABI4 binds to the CACCG motif in the ZmSSI promoter and mediates its ABA inducibility.

Small RNAs: How Seeds Remember To Obey Their Mother

The endosperm is one of two products from the double fertilization event that occurs during sexual reproduction in flowering plants. A series of recent reports highlights the unusual genetic regulatory mechanisms that occur in endosperm and suggests a role for transposon regulation in imprinting.

Nonadditive Expression and Parent-of-Origin Effects Identified by Microarray and Allele-Specific Expression Profiling of Maize Endosperm

Plant endosperm cells have a nuclear ratio of two maternal genomes to one paternal genome. This 2 to 1 dosage relationship provides a unique system for studying the additivity of gene expression levels in reciprocal hybrids. A combination of microarray profiling and allele-specific expression analysis was performed using RNA isolated from endosperm tissues of maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal hybrids at two developmental stages, 13 and 19 d after pollination. The majority of genes exhibited additive expression in reciprocal hybrids based on microarray analyses. However, a substantial number of genes exhibited nonadditive expression patterns, including maternal like, paternal like, high parent like, low parent like, and expression patterns outside the range of the parental inbreds. The frequency of hybrid expression patterns outside of the parental range in maize endosperm tissue is much higher than that observed for vegetative tissues. For a set of 90 genes, allele-specific expression assays were employed to monitor allelic bias and regulatory variation. Eight of these genes exhibited evidence for maternally or paternally biased expression at multiple stages of endosperm development and are potential examples of differential imprinting. Our data indicate that parental effects on gene expression are much stronger in endosperm than in vegetative tissues.

Molecular and Biochemical Analysis of the Plastidic ADP-glucose Transporter (ZmBT1) from Zea mays

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 mum, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988-2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1-2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.

O-Linked glycosylation in maize-expressed human IgA1

O-Linked glycans vary between eukaryotic cell types and play an important role in determining a glycoprotein's properties, including stability, target recognition, and potentially immunogenicity. We describe O-linked glycan structures of a recombinant human IgA1 (hIgA1) expressed in transgenic maize. Up to six proline/hydroxyproline conversions and variable amounts of arabinosylation (Pro/Hyp + Ara) were found in the hinge region of maize-expressed hIgA1 heavy chain (HC) by using a combination of matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), chromatography, and amino acid analysis. Approximately 90% of hIgA1 was modified in this way. An average molar ratio of six Ara units per molecule of hIgA1 was revealed. Substantial sequence similarity was identified between the HC hinge region of hIgA1 and regions of maize extensin-family of hydroxyproline-rich glycoproteins (HRGP). We propose that because of this sequence similarity, the HC hinge region of maize-expressed hIgA1 can become a substrate for posttranslational conversion of Pro to Hyp by maize prolyl-hydroxylase(s) with the subsequent arabinosylation of the Hyp residues by Hyp-glycosyltransferase(s) in the Golgi apparatus in maize endosperm tissue. The observation of up to six Pro/Hyp hydroxylations combined with extensive arabinosylation in the hIgA1 HC hinge region is well in agreement with the Pro/Hyp hydroxylation model and the Hyp contiguity hypothesis suggested earlier in literature for plant HRGP. For the first time, the extensin-like Hyp/Pro conversion and O-linked arabinosylation are described for a recombinant therapeutic protein expressed in transgenic plants. Our findings are of significance to the field of plant biotechnology and biopharmaceutical industry-developing transgenic plants as a platform for the production of recombinant therapeutic proteins.

Characterization of the structure and transcription of an ubiquitin fusion gene from maize.

We have identified a maize ubiquitin (Ubi) fusion gene (UBF9) by screening a maize W22 genomic phage lambda library with a short (16-nucleotide) oligodeoxyribonucleotide probe derived from the sequence for the extension sequence of a yeast UB13 fusion gene. UBF9 consists of an UB monomer sequence (228 bp long) joined to an extension sequence (237 bp long). The extension sequence encodes a protein of 79 amino acids which shares extensive identity with similar extension aa sequences found in yeast, humans, barley and Arabidopsis thaliana. UBF9 encodes a small-size class of Ubi mRNAs in the maize tissues investigated. The UBF9 transcript is present in high levels in maize endosperm tissues 22 days after pollination. Genomic Southern blots of maize inbred W22 DNA indicate that the fusion gene sequences are present in multiple copies in the maize genome. Primer extension experiments indicate that the transcription start point is located at 80 bp upstream from the translation start codon of UBF9. Two 37-bp tandem repeated A + T-rich sequences are found in the 5'-flanking region of UBF9. The A + T-rich sequences share the motif, AATATTTTATT, which is present in a diverse set of plant genes.

Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants.

Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.

Maize high mobility group proteins bind to CCAAT and TATA boxes of a zein gene promoter.

A 216-base pair promoter fragment of the 19-kDa zein storage protein gene pMS1, containing the CCAAT and TATA boxes, was analyzed for its in vitro interactions with purified high mobility group (HMG) proteins from maize endosperm tissue. It was found that the HMG proteins display a preferential binding of A/T-rich DNA regions like their animal counterparts, although the A/T content seems not to be the only determinant for binding specificity. Surprisingly, a specific binding of the HMG proteins occurs at the CCAAT and the TATA boxes as indicated by footprinting experiments.

A nuclear casein type II kinase from maize endosperm phosphorylating HMG proteins

A casein kinase of the type II was isolated and enriched from nuclear lysates of maize endosperm tissue. The kinase activity requires 10 mM Mg 2+ for maximal activity, can utilize either ATP or GTP as phosphate donors and is inhibited by polyamines, heparin and monovalent cations. A substrate specificity of the kinase activity towards specific nuclear proteins is indicated by its phosphorylation of high mobility group (HMG) proteins isolated from endosperm and its lack of accepting histones as protein substrates.

Ferredoxin and pyridine nucleotide-dependent glutamate synthase activities in maize endosperm tissue

NADH-dependent (EC 1.4.1.14) and ferredoxin-(Fd) dependent (EC 1.4.7.1) glutamate synthase (GOGAT) activities are present in extracts from developing corn endosperm. The estimated molecular weights were 171 000 for the Fd-GOGAT and 270 000 for the NADH-GOGAT. An activity with NADPH was shown to be artifact mediated by the conversion of NADPH to NADH by a phosphatase.

A Simplified Medium for the Growth of Maize (Zea mays) Endosperm Tissue in Suspension Culture

Abstract In vitro cultures of maize (Zea mays L.) endosperm derived from the dent inbred A636 have been maintained in liquid culture using Straus' medium for over six years. We have studied the growth of this tissue in four basic media and various modifications of the organic constituents of these media. Auxins and kinetin did not improve growth rate or degree of cell dispersion and thiamine (0.4 mg/l) was the only vitamin required by this tissue. Growth equal to that in the standard Straus medium and improved cell separation was obtained in a medium containing only inorganic salts, sucrose, and thiamine. Although asparagine was not required when high quantities of NH4NO3 and KNO3 were included, more rapid growth was obtained when 2 g/l of asparagine was added. The simplified medium reported in this paper should facilitate the use of maize endosperm tissue in various studies of metabolism, hormone biosynthesis, etc.

Growth of maize endosperm tissue in vitro I.—Nitrogen requirement of tissue grown on solid media

AbstractAmmonium salts can replace asparagine as the main nitrogen source for maize endosperm tissue culture. Although the acetate, oxalacetate and oxoglutarate are toxic, the succinate or citrate may be used.

Amino acid incorporation in a cell-free system from submerged tissue cultures of Zea mays L.

Summary An amino acid-incorporating system from maize endosperm tissue culture cells has been obtained and characterized. It exhibits the established requirements for protein synthesis and is sensitive to ribonuclease and puromycin but not to deoxyribonuclease or chloramphenicol. The system is dependent on a supernatant fraction from maize seedlings. The supernatant fraction from the tissue culture cells themselves is inhibitory to protein biosynthesis. A new method for the preparation of ribosomes from large amounts of starting material is described, which makes use of a Gaulin homogenizer for the homogenization of tissue and a Sephadex treatment to reduce the volume of the 30,000× g supernatant fraction.

Maize Endosperm Tissue Grown in Vitro. III. Development of a Synthetic Medium

Straus, Jacob. (U. Oregon, Eugene.) Maize endosperm tissue grown in vitro. III. Development of a synthetic medium. Amer. Jour. Bot. 47(8) : 641–647. Illus. 1960.—The development of a synthetic medium for the growth of endosperm tissue cultures derived from the maize variety, ‘Black Mexican Sweet,‘ is described. Previously, these tissues required yeast extract, casein hydrolyzate, or tomato juice in the medium in order to grow. The growth-supporting activity of these complexes could be attributed to their organic nitrogen content. The effect of juice extracted from fresh tomatoes is enhanced by autoclaving under acid conditions. Presumably this treatment increases the free amino acid content of the tomato juice. One-dimensional paper chromatograms of tomato juice autoclaved under acid conditions indicated the presence of a large amount of free amino acids. Addition of 1.5 × 10–2 M asparagine to the basal mineral-sugar-vitamin medium (White's medium plus Nitsch's trace-element solution) resulted in better growth than that supported by yeast extract, tomato juice, or casein hydrolyzate. Arginine was ineffective. Glutamine, glutamic acid and aspartic acid (all at 1.5 × 10–2 M) supported appreciable growth of the tissue but none of them were nearly so good as asparagine in this respect. Thus, a medium containing minerals, sugar, vitamins, and asparagine is capable of supporting excellent growth of maize endosperm tissue cultures.

MAIZE ENDOSPERM TISSUE GROWN IN VITRO III. DEVELOPMENT OF A SYNTHETIC MEDIUM

Straus, Jacob. (U. Oregon, Eugene.) Maize endosperm tissue grown in vitro. III. Development of a synthetic medium. Amer. Jour. Bot. 47(8) : 641–647. Illus. 1960.—The development of a synthetic medium for the growth of endosperm tissue cultures derived from the maize variety, ‘Black Mexican Sweet,‘ is described. Previously, these tissues required yeast extract, casein hydrolyzate, or tomato juice in the medium in order to grow. The growth‐supporting activity of these complexes could be attributed to their organic nitrogen content. The effect of juice extracted from fresh tomatoes is enhanced by autoclaving under acid conditions. Presumably this treatment increases the free amino acid content of the tomato juice. One‐dimensional paper chromatograms of tomato juice autoclaved under acid conditions indicated the presence of a large amount of free amino acids. Addition of 1.5 × 10–2 M asparagine to the basal mineral‐sugar‐vitamin medium (White's medium plus Nitsch's trace‐element solution) resulted in better growth than that supported by yeast extract, tomato juice, or casein hydrolyzate. Arginine was ineffective. Glutamine, glutamic acid and aspartic acid (all at 1.5 × 10–2 M) supported appreciable growth of the tissue but none of them were nearly so good as asparagine in this respect. Thus, a medium containing minerals, sugar, vitamins, and asparagine is capable of supporting excellent growth of maize endosperm tissue cultures.

MAIZE ENDOSPERM TISSUE GROWN IN VITRO II. MORPHOLOGY AND CYTOLOGY

American Journal of BotanyVolume 41, Issue 10 p. 833-839 Article MAIZE ENDOSPERM TISSUE GROWN IN VITRO II. MORPHOLOGY AND CYTOLOGY Jacob Straus, Jacob Straus Biological Laboratories, Harvard University, Cambridge, MassachusettsAmerican Cancer Society Research Fellow.Search for more papers by this author Jacob Straus, Jacob Straus Biological Laboratories, Harvard University, Cambridge, MassachusettsAmerican Cancer Society Research Fellow.Search for more papers by this author First published: 01 December 1954 https://doi.org/10.1002/j.1537-2197.1954.tb14422.xCitations: 34 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume41, Issue10December 1954Pages 833-839 RelatedInformation

Maize Endosperm Tissue Grown In vitro. I. Culture Requirements

American Journal of BotanyVolume 41, Issue 8 p. 687-694 Article MAIZE ENDOSPERM TISSUE GROWN IN VITRO I. CULTURE REQUIREMENTS Jacob Straus, Jacob Straus Department of Botany, University of Michigan, Ann Arbor, MichiganAmerican Cancer Society Research Fellow. Present address: The Biological Laboratories, Harvard University, Cambridge, Mass. Some of the data in this paper were taken from a dissertation by Jacob Straus presented to the Graduate School of the University of Michigan in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Parts of the present work were supported by a research grant to C. D. LaRue by the Horace H. Rackham Research Fund of the University of Michigan and grateful acknowledgment for this assistance is made.Search for more papers by this authorCarl D. LaRue, Carl D. LaRue Department of Botany, University of Michigan, Ann Arbor, MichiganSearch for more papers by this author Jacob Straus, Jacob Straus Department of Botany, University of Michigan, Ann Arbor, MichiganAmerican Cancer Society Research Fellow. Present address: The Biological Laboratories, Harvard University, Cambridge, Mass. Some of the data in this paper were taken from a dissertation by Jacob Straus presented to the Graduate School of the University of Michigan in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Parts of the present work were supported by a research grant to C. D. LaRue by the Horace H. Rackham Research Fund of the University of Michigan and grateful acknowledgment for this assistance is made.Search for more papers by this authorCarl D. LaRue, Carl D. LaRue Department of Botany, University of Michigan, Ann Arbor, MichiganSearch for more papers by this author First published: 01 October 1954 https://doi.org/10.1002/j.1537-2197.1954.tb14396.xCitations: 35 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume41, Issue8October 1954Pages 687-694 RelatedInformation