Analysis of variants affecting the catalase developmental program in maize scutellum

Summary The catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6–67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6–67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Haplo-diploid gene expression and pollen selection for tolerance to acetochlor in maize

The objectives of this research were to determine if genes controlling the reaction to the herbicide acetochlor in maize (Zea mays L.) are active during both the haploid and the diploid phases of the life cycle and if pollen selection can be utilized for improving sporophytic resistance. Pollen of eight inbred lines, previously characterized through sporophytic analysis for the level of tolerance to acetochlor, showed a differential reaction to the herbicide forin vitro tube length; moreover, such pollen reactions proved to be significantly correlated (r =0.786^*,df=6) with those of the sporophytes producing the pollen. Pollen analysis of two inbred lines (i.e. Mo17, tolerant, and B79, susceptible) and their single cross showed that thein vitro pollen-tube length reaction of the hybrid was intermediate between those of two parents. An experiment on pollen selection was then performed by growing tassels of Mo17xB79 in the presence of the herbicide. Pollen obtained from treated tassels showed a greater tolerance to acetochlor, assessed asin vitro tube length reaction, than pollen obtained from control tassels. Moreover, the backcross [B79 (Mo17xB79)] sporophytic population obtained using pollen from the treated tassels was more tolerant (as indicated by the fresh weight of plants grown in the presence of the herbicide) than was the control backcross population. The two populations did not differ when grown without the herbicide. These findings indicate that genes controlling the reaction to acetochlor in maize have haplodiploid expression; consequently, pollen selection can be applied for improving plant tolerance.     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F₁ primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F₁ hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.     

鸡马立克氏病病毒B抗原片段在大肠杆菌中表达

鸡马立克氏病病毒ＢａｍＨＩ基因文库Ｉ３质粒中含有编码Ｂ抗原膜外Ｄｏｍａｉｎ的ＤＮＡ序列。经ＳｃａＩ和ＳｐｈＩ双酶酶解Ｉ３质粒,分离获得７６４ｂｐＤＮＡ片段,并克隆进Ｍ１３ｍｐ１９中。ＤＮＡ序列测定分析表明克隆片段为ＭＤＶ－Ｂ抗原基因的４９４－１２５８ｂｐ部分序列。进一步分离ＮｃｏＩ－ＨｉｎｄＩＩＩ部分基因片（５３０ｂｐ）,克隆于ＰＬＰｒｏｍｏｔｅｒ控制下的含有修饰型ｃＩｔｓ８５７基因的表达载体中,     

Efficient extracellular production of hybrid E. coli heat-labile enterotoxin B subunits in a marine vibrio

Abstract Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15–20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies.     

Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide

The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni²⁺-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni²⁺-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.     

Zinc deficiency and pollen fertility in maize (Zea mays)

Zinc deficiency decreased pollen viability in maize (Zea mays L. cv. G2) grown in sand culture. On restoring normal zinc supply to zinc-deficient plants before the pollen mother cell stage of anther development, the vegetative yield of plants and pollen fertility could be recovered to a large extent, but the recovery treatment was not effective when given after the release of microspores from the tetrads. If zinc deficiency was induced prior to microsporogenesis it did not significantly affect vegetative yield and ovule fertility, but decreased the fertility of pollen grains, even of those which visibly appeared normal. If the deficiency was induced after the release of microspores from the tetrads, not only vegetative yield and ovule fertility but pollen fertility also remained unaffected.     

Phytoliths as indicators of prehistoric maize (Zea mays subsp.mays,Poaceae) cultivation

Maize (Zea mays L. subsp.mays) has been identified in archaeological contexts by a high proportion of large cross-shaped phytoliths. Given the numerous races of maize, this study was undertaken to determine if differences below the species level could be noted. It was also designed to see if phytoliths differed in various plant parts at various stages of growth. Several races were grown under experimental conditions. No significant differences were found. Furthermore, few phytoliths alleged to be diagnostic of maize were discovered. Systemic studies of maize and analyses of prehistoric cultivation by means of phytoliths seem not to be as promising as some researchers have argued.     

Cleavage polyembryony in maize

Summary Two types of cleavage polyembryony are described in the inbred line VIR 17 of maize. Suspensorial embryony was observed to occur spontaneously. Typical cleavage of the zygotic proembryo occurred spontaneously, but could also be induced by treating the developing caryopses with 2,4-Dichlorophenoxyacetic acid (2,4-D) on the second day after pollination. 2,4-D was active as a decorelative factor also evoking the expression of totipotency in individual proembryonal cells.     

Effect of gibberellic acid on the ion ratios in a dwarf maize mutant (Zea mays L. d1)

The effect of gibberellic acid (GA₃) on ion ratios in a dwarf maize mutant (Zea mays L. d₁) exhibiting normal growth after hormone treatment has been investigated by electron microprobe analysis. Gibberellic-acid treatment increased the ion content in chloroplasts and vacuoles whereas no change of the ion content was found in the cytoplasm. The relation of these observations to the action of the hormone is discussed.Abbreviation GA₃ Gibberellic acid     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

Chlorophyll-deficient gene and morphological variations in Korean populations of maize (Zea mays)

We studied cultivated and naturalized Korean maize populations to determine the extent to which the chlorophylldeficient mutation and the phenotypic variations of two morphological characters (i.e., red coleoptiles and epicotyls, and the number of the first root hairs) are maintained. The frequency of the chlorophyll-deficient mutant gene (2.73% on average) was highly variable. Frequencies of red coleoptiles and epicotyls also were higher than expected from a mutation-selection balance. The average number of hairy phenotypes within populations was 1.8, ranging from 0.0 to 4.0. Naturalized populations were closely related to with cultivated communities. Most striking, however, was the more significant difference among populations than within populations with regard to both the frequency of chlorophyll-deficient mutant genes and the phenotypic variations of our two morphological characters. On a per-gene basis, the majority of the phenotypic variation (mean of 73.3%) resided among populations.     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Evaluation of in vitro selection regimes for a mitochondrial trait (methomyl resistance) in cms-T maize

Several factors affecting the success of selection in plant populations were examined for their relevance to in vitro selection. Three in vitro selection schemes and two growth assessment procedures were evaluated for effectiveness in selecting for a mitochondrial trait in maize: resistance to the insecticidal compound methomyl. Regenerable maize callus was derived from immature embryos of the three-way hybrid P39/IL766A2 x W182BN containing Texas male sterile cytoplasm (cms-T). Either low, gradually increasing, or high selection pressures were used to grow callus over a period of 3–5 months. There was no significant difference in recovery of resistant plants using these 3 methods. Growth of callus on medium containing methomyl was assessed by increase in fresh weight during the final month of selection or by increase in number of callus pieces over the course of selection. These quantitative measures of growth were unreliable indicators for gain in resistance within the callus population. A procedure for recovery of methomyl resistant and male-fertile cms-T plants is suggested.