Detection and immunolocalization of glycoproteins of the plasma membrane of maize sperm cells

Summary After purifying plasma membranes from isolated maize sperm cells by aqueous polymer two-phase partition, peripheral and integral proteins were solubilized from the plasma membrane with Triton X-114 and separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining revealed 10 bands (19–68 kDa) of peripheral membrane proteins and about 40 bands (12–120 kDa) of integral proteins. Peroxidase-conjugated Con A was used to detect the surface glycopeptides. It was found that Con A particularly stained 8 peripheral polypeptide bands, including 68, 66, 55, 51,48, 44, 36, and 32 kDa, and 6 integral polypeptide bands, 68, 51, 48, 44, 38, and 34 kDa. These bands differed from those of somatic samples. Staining specificity was demonstrated by the control in the presence of competing inhibitory sugar. The above result indicates the existence of mannosyl and glucosyl residues in the surface glycoproteins of maize sperm cells. The prominent peripheral 68 kDa polypeptide was further separated into 4 spots by isoelectric focusing and sodium dodecyl sulfate two-dimensional (IEF-SDS 2-D) electrophoresis, showing pI values from 5.5 to 5.8. Three prominent glycopeptides (68, 48, and 32 kDa) were localized on the plasma membrane of maize sperm cells via the fluorescein isothiocyanate (FITC) technique. About 25% of sperm cells showed an intense positive reaction in each immunological labelling. The results agree with our previous labelling of the surface of isolated viable maize sperm cells with Con A-FITC.Abbreviations FITC fluorescein isothiocyanate - Con A Canavalia ensiformis agglutinin - HRP horseradish peroxidase - RCA Ricinus communis agglutinin - WGA Triticum vulgaris agglutinin     

Accumulation,assembly, and digestibility of amarantin expressed in transgenic tropical maize

An amaranth (Amaranthus hypochondriacus) 11S globulin cDNA, encoding one of the most important storage proteins (amarantin) of the seed, with a high content of essential amino acids, was used in the transformation of CIMMYT tropical maize genotype. Constructs contained the amarantin cDNA under the control of a tissue-specific promoter from rice glutelin-1 (osGT1) or a constitutive (CaMV 35S) promoter with and without the first maize alcohol dehydrogenase intron (AdH). Southern-blot analysis confirmed the integration of the amarantin cDNA, and copy number ranged from one to more than ten copies per maize genome. Western-blot and ultracentrifugation analyses of transgenic maize indicate that the expressed recombinant amarantin precursors were processed into the mature form, and accumulated stably in maize endosperm. Total protein and some essential amino acids of the best expressing maize augmented 32% and 8–44%, respectively, compared to non-transformed samples. The soluble expressed proteins were susceptible to digestion by simulated gastric and intestinal fluids, and it is suggested that they show no allergenic activity. These findings demonstrate the feasibility of using genetic engineering to improve the amino acid composition of grain crops.Communicated by J.W. Snape     

Polypeptides of the Maize Amyloplast Stroma : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.     

Patterns of polypeptide synthesis in various maize organs under anaerobiosis

The pattern of protein synthesis was compared in several organs of maize (Zea mays L.) under aerobic and anaerobic conditions. Protein synthesis was measured by [³⁵S]methionine incorporation and analysis by two-dimensional native-SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis and fluorography. The aerobic protein-synthesis profiles were very different for root, endosperm, scutellum and anther wall. However, except for some characteristic qualitative and quantitative differences, the patterns of protein synthesis during anaerobiosis were remarkably similar for these diverse organs and also for mesocotyl and coleoptile. The proteins synthesized were the anaerobic polypeptides (ANPs) which have been previously described in anaerobic roots of seedlings. Leaves exhibited no detectable protein synthesis under anaerobic conditions, and died after a short anaerobic treatment. Evidence is presented that the ANPs are not a generalized response to stress. This indicates that the ANPs are synthesized as a specific response to anaerobic conditions such as flooding.Abbreviations ADH alcohol dehydrogenase - ANP anaerobic polypeptide - SDS sodium lauryl sulfate     

Quantitative changes in maize cytoplasmic proteins induced by aluminium

Three-day-old maize seedlings were subjected to 100 μM AlCl3 for 24 h. Cytoplasmic proteins were isolated from root tips, root base and from coleoptiles. After fractionation of cytoplasmic proteins on anion chromatography column Bio-Scale Q2 sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to monitor Al-induced changes in polypeptide composition of particular fractions. Four (root) and 7 (coleoptile) fractions were eluted from the column with linear 0 - 1.0 M NaCl gradient. In fraction 1 of cytoplasmic proteins from root tips Al induced accumulation of polypeptide with molecular mass of 16 kD and simultaneous reduction of two polypeptides (67.5 and 60 kD). In fraction 1 isolated from mature zone of maize roots Al-induced accumulation of 22 kD polypeptide and reduction of 67.5, 60, and 14 kD polypeptides. Most pronounced changes were revealed in coleoptile. In three protein fractions increased accumulation of polypeptides with molecular mass of 14, 17.5, 20, 24.5, 28, 30, and 37.5 kD were observed. In the remaining three root or four coleoptile fractions of cytoplasmic proteins, no differences were found between Al-treated and control maize seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans-isomerase that interacts with calmodulin and a 36-kDa cytoplasmic protein

A member of a eukaryotic gene superfamily, encoding a peptidylproline cis-trans-isomerase (rotamase) has been isolated from a maize (Zea mays L. A69Y+) endosperm cDNA library. The maize sequence (mzFKBP-66) encodes a 66-kDa polypeptide most closely related to the subclass of rotamases which bind an immunosuppressive drug, FK506, (termed FK506-binding proteins, FKBPs), and possesses four tandem copies of the FKBP-like binding domain. The sequence mzFKBP-66 is expressed ubiquitously in the maize plant, and the protein encoded is present in both cytosolic and nuclear compartments within the cell. Both the native mzFKBP-66 and a recombinant protein overexpressed in Escherichia coli showed peptidylproline␣cis-trans-isomerase (PPIase) activity at rates comparable to those reported for mammalian immunophilins. This activity was also sensitive to inhibition by FK506. Immunoaffinity chromatography using anti-mzFKBP66 demonstrated an association of the protein with an unknown 36-kDa polypeptide, and affinity chromatography of mzFKBP-66 on calmodulin-agarose beads indicated the presence of a calmodulin-binding site. The existence of mzFKBP-66-associated proteins suggests that plant immunophilins may act as part of multicomponent complexes, as has been shown for other representatives of this class of enzyme. Received: 9 June 1997 / Accepted: 19 August 1997     

urf13-T of T cytoplasm maize mitochondria encodes a 13 kD polypeptide

Polyspecific antibody to a 17 amino acid synthetic peptide from the maize T-cytoplasm urf13-T mitochondrial open reading frame immunoprecipitated a 13 kD polypeptide from ³⁵S-methionine incorporations of T cytoplasm maize. Male-fertile, toxin-insensitive mutants in which the urf13-T sequence is deleted do not synthesize the 13 kD polypeptide. A mutant designated T-4, which carries a 5 bp insertion and a premature stop codon, synthesizes a truncated polypeptide, corresponding to an open reading frame of 8.3 kD. Thus the 13 kD polypeptide is trunctated or absent in mutants expressing male fertility and toxin insensitivity in T-cytoplasm maize.USDA-ARS     

Isolation and characterization of male flower cDNAs from maize

Differential screening of two libraries made from whole, immature maize tassels was used to isolate six cDNAs which show enhanced levels of expression in male flowers. MFS1, MFS2, MFS4, MFS10 and MFS18, which were isolated from a 5 cm tassel library, are expressed throughout tassel growth up until mature pollen is produced in the anthers. MFS14, which was isolated from a 10–12 cm tassel library, has a narrower window of expression associated with microsporogenesis and declines as mature pollen is produced. MFS18 mRNA accumulates in the glumes and in anther walls, paleas and lemmas of mature florets. MFS18 mRNA is particularly associated with the vascular bundle in the glumes and encodes a polypeptide of 12 kDa, rich in glycine, proline and serine that has similarities with other plant structural proteins. In contrast, MFS14 mRNA accumulates in the tapetum and encodes a polypeptide of 13 kDa that is rich in alanine. The MFS14 and MFS18 proteins are basic (isolectric points of 11.56 and 9.54, respectively) and both have hydrophobic N-termini which display all the characteristics of signal peptides, indicating that these proteins may be secreted.     

Quantitative Changes in Maize Membrane Proteins Induced by Aluminium

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for monitoring Al-induced changes in polypeptide composition of membrane proteins isolated from 3-d-old maize seedlings subjected to aluminium stress. Analysis of peripheral membrane proteins isolated from maize root showed an Al-induced increase in accumulation of 14 polypeptides with apparent molecular mass from 10 to 135 kDa. Qualitative differences were found between peripheral membrane proteins isolated from root tip (increased accumulation of 4 polypeptides with M_r 42 000 – 135 000) and from root base (increased accumulation of 10 polypeptides with M_r 10 000 – 59 000). On the other hand, no Al-induced changes were observed in peripheral membrane proteins isolated from maize coleoptile and integral membrane proteins isolated either from root or coleoptile. These results indicate that peripheral membrane proteins undergo considerable changes during 24-h Al treatment while integral membrane proteins pattern is stable.     

Effect of Ca<Superscript>2+</Superscript> and cAMP on protein phosphorylation in mitochondria of maize seedlings

The effect of common intracellular signals (Ca²⁺ and cAMP) on the activity of protein phosphorylation in mitochondria was investigated in coleoptiles of maize (Zea mays L.). Treatment of isolated mitochondria with 2 mM CaCl₂ brought about an increase in the level of phosphorylation of proteins with mol ws of 74, 60, and 33 kD but considerably reduced phosphorylation of the protein with a mol wt of 51.5 kD. In the presence of Ca²⁺, phosphorylation of polypeptides with mol wts of 59 and 66 kD was also detected. cAMP considerably reduced phosphorylation of essentially all the investigated proteins in isolated mitochondria, which could be explained by activation of their dephosphorylation. Phosphorylation of mitochondrial proteins involves a polypeptide of about 94 kD showing kinase activity, which may be proper protein kinase or one of the subunits of a compound structure. In maize mitochondria, PP1A phosphatases were found. A hypothesis was advanced that redox-dependent phosphorylation/dephosphorylation of mitochondrial proteins plays an important role in mitochondrial signaling in higher plants.     

Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP auxin-binding protein - DEAE diethylaminoethyl - Ig immunoglobulin - kDa kilodalton - NAA naphthalene-1-acetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

The roles of low temperature and light in accumulation of a 31-kDa polypeptide in the light-harvesting apparatus of maize leaves

Abstract Previously, accumulation of a 31-kDa polypeptide had been observed in the light-harvesting apparatus of thylakoids of maize leaves exposed to 5°C and high light (Hayden et al., 1986). The accumulation and disappearance of this 31-kDa polypeptide in thylakoids of maize leaves are examined as a function of photon flux density and temperature. The accumulation of large amounts of the polypeptide at 5°C was light-dependent during a 6-h chill period, with 50% of maximal accumulation occurring at a photon flux density of 60 μmol m^?2 s^?1.Some polypeptide accumulation did occur in leaves kept in the dark at 5°C for 6 h, i.e. ca. 18% of that accumulating at a photon flux density of 1500 μmol m^?2 s^?1. The temperature optimum for polypeptide accumulation was ca. 9°C with greater than 50% of maximal accumulation being achieved between 5 and 11°C. The breakdown of maximally accumulated polypeptide on returning leaves to 25°C was complete after 1 h, had a half-time of ca. 20 min and was independent of light. Breakdown of the polypeptide was also observed when thylakoids isolated from chilled leaves were incubated at 25°C. Reductions of thylakoid incubation temperature between 13 and 5°C markedly reduced the rate of polypeptide disappearance. The accumulation of the polypeptide is discussed in relation to temperature and light effects on the rate of the polypeptide synthesis and of peptidase activities. The results are also discussed in the context of accumulation of the polypeptide in maize leaves in the field and consideration is given to the possible physiological significance.     

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector

Summary The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein.     

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Environmental effects on zein chromatograms of maize inbred lines revealed by reversed-phase high-performance liquid chromatography

Summary Alcohol soluble seed storage proteins (zeins and alcohol soluble glutelins) of maize (Zea mays L.) were separated by reversed-phase high-performance liquid chromatography (RP-HPLC). The objectives were to assess the reproducibility of chromatographic profiles using seed of inbred lines that had been produced in different locations and years. Reproducible differences between sources were seen but these were restricted to proteins that contributed 2% or less to an inbred profile. The majority of variation (93% for peak percent area; 99.8% for elution time) was between inbreds. RP-HPLC can therefore provide distinctive phenotypic profiles that are largely characteristic of genotype. Such qualitative and quantitative data will be valuable for studies of taxonomy, evolution, genetics, and germplasm identification.     

Calcium-dependent phospholipid-binding proteins in plants

There is evidence that Ca²⁺ can regulate vesicle-mediated secretion in plant cells, but the mechanism for this is not known. One possibility is that Ca²⁺ -dependent phospholipid-binding proteins (annexins) couple the Ca²⁺ stimulus to the exocytotic response. Using a protocol developed for the isolation of animal annexins we have identified proteins in maize (Zea mays L.) coleoptiles that have similar characteristics to annexins. The predominant polypeptide species run as a doublet of relative molecular mass (M_r) 33000–35000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); another less-abundant protein of M_r 23000 is also present. In the presence of Ca²⁺ these proteins bind to liposomes composed of acidic phospholipids. Calcium-sensitivity of binding differs for each protein and is also influenced by the pH of the buffer used for the liposome-binding assay. Antiserum raised to the 33 to 35-kDa doublet purified on SDS-PAGE recognises the doublet in crude extracts from maize and proteins of similar M_r in Tradescantia virginiana and tobacco Nicotiana tabacum L. The antiserum also recognises p68 (Annexin VI) from chicken gizzard extracts, indicating homology between animal annexins and the maize proteins. For the maize proteins to be involved in the regulation of exocytosis, binding to phospholipids would be expected to occur at physiological levels of Ca²⁺. The characteristics of the maize annexin-like proteins are described and attention drawn to the marked effect of pH in lowering the requirement for Ca²⁺ for phospholipid binding.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis (-aminoethyiether)-N,N,N,N-tetraacetic acid - kDa kilodalton(s) - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This work was funded by the Agricultural and Food Research Council. Our thanks also to Professor P. Lowry and Dr R. Woods, Department of Biochemistry, University of Reading for facilities and advice for antiserum production, and C. Boustead, Department of Biochemistry, University of Leeds for advice on immunoblotting and phospholipid-binding assays.     

Transfer ofE. coli gutD gene into maize and regeneration of salt-tolerant transgenic plants

GutD gene, encoding a key enzyme (glucitol-6-phosphate dehydrogenase) of sugar alcohol metabolic pathway inE. coli, was transferred into maize. Results of Southern and Western blotting analysis certified that this gene had integrated and been expressed in transgenic maize plants and their progeny. The synthesis and accumulation of sorbitol were detected in transgenic maize plants and a preliminary nutrient solution culture experiment showed thatgutD transgenic maize plants had an increased tolerance to salt stress compared with nontransgenic ones.     

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

Summary Plants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.Abbreviations kb kilobase pairs - ADH alcohol dehydrogenase     

Influence of GA3 and 4-PU-30 on Leaf Protein Composition, Photosynthetic Activity, and Growth of Maize Seedlings

The effects of gibberellic acid (GA3) and N1-(2-chloro-4-pyridyl)-N2 phenylurea (4-PU-30) on maize seedling growth, photosynthetic parameters, and leaf protein composition were investigated. The agents used alone or in combination increased leaf growth and photosynthetic rate of the seedlings. Chlorophyll and total nitrogen contents in leaves as well as the quantity of individual protein fractions increased simultaneously. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins (albumins and globulins) revealed quantitative differences between 4-PU-30-treated plants and the other experimental variants. They differed in polypeptide composition associated with changes in soluble proteins and amino acids. However, GA3 did not induce similar changes in polypeptide composition of soluble proteins. This revised version was published online in July 2006 with corrections to the Cover Date.