Mapping of zein polypeptides after isoelectric focusing on agarose gels

Isoelectric focusing of zein in agarose gels gives sharp separations of at least 25 bands noted among 25 corn-belt inbreds. Six inbreds provided standard bands which were used to construct a pattern map. A method is provided for comparing bands, identified by distance from the cathode, which differ only slightly in position. The 25 inbreds were separated into five groups on the basis of pattern similarity. Some groups contained inbreds derived from widely different sources. Zein isoelectric focusing in agarose should be useful for genotype identification and for determination of varietal purity.     

Linkages among zein genes determined by isoelectric focusing

Summary Genetic control of the major zein polypeptides in maize (Zea mays L.) was studied by isoelectric focusing (IEF) in agarose. Linkage relationships were determined by making a number of crosses, then determining the expression of zein polypeptides in backcross seeds. Chromosome linkages were determined by using the markers sugary-1 (for chromosome 4), yellow-8, and a waxy 7–9 translocation (for chromosome 7). Nine zeins were in one linkage group on chromosome 4, six in another linkage group on chromosome 4, and four zeins were in one linkage group on chromosome 7. Some IEF single bands consisted of at least two polypeptides, which were detected by subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis, by aberrant ratios in backcrosses, or by differing recombination percentages. One zein occurred only in homozygous sugary-1 seeds. Three sets of closely-linked zeins were noted that occurred together almost exclusively in certain inbreds.Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the Illinois Agricultural Experiment Station, Department of Agronomy, University of Illinois, Urbana, USAMention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned     

Inheritance of RAPDs in F1 hybrids of corn

Summary Random amplified polymorphic DNA (RAPD) markers were analyzed in materials from a partial diallel, including 16 corn F₁ hybrids (with five reciprocals) and their five parental inbreds. Using 21 primers, we scored a total of 140 different fragments for their presence/absence and intensity variation, where appropriate. When all 21 genotypes were taken into consideration, 20.7% of these fragments were nonpolymorphic, 37.1% were unambiguously polymorphic, and 42.1% were quantitatively polymorphic. Unambiguous polymorphisms were distinguished by the simple presence or absence of a specific fragment in the inbred genotypes, whereas quantitative polymorphisms exhibited a variation in the intensity of a fragment. Of the F₁ patterns, 95.2% of the unambiguously polymorphic situations could be interpreted genetically by assuming complete dominance of the presence of the parental fragment, while 3.2% of the F₁ patterns exhibited a fragment intensity that was intermediate between the two parental patterns (partial dominance). For quantitative polymorphisms, values of 88.1% for complete dominance and 5.0% for partial dominance were obtained. The results suggest that specific types of errors can be detected in RAPD analysis, that uniparental inheritance is not common, and that RAPD analysis might be more prudently used for some applications than for others.     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

In vitro pollination fertilization of maize: influence of explant factors on kernel development

The influence of donor plant genotype, ear maturity, explant size, and the ratio of ovule-to-cob tissue on kernel development from in vitro pollinated ovules was examined. All genotypes evaluated in this study were capable of in vitro pollination/fertilization, however, significant differences were observed for the responses measured. Genotype means for complete kernel formation ranged from 1.5% to 25.4% with B73 exhibiting the highest response. Averaged over all genotypes, ear maturity effects were not significant, however, the genotype x ear maturity mean square was significant for swelling percentage. Explant size had a profound effect on in vitro kernel development. Averaged over all genotypes and ear maturities, 30-ovule explants resulted in more than twice as many ovules classified as complete kernels when compared to 10-ovule explants. Ovule-to-cob tissue ratio was also found to have highly significant effects on all three variables measured. An ovule-to-cob tissue ratio of 4:24 resulted in the highest percentages of swelling, embryos with incomplete embryos, and complete kernels.     

Hydrolysis of organic phosphates by corn and soybean roots

Because of the importance of organic phosphates as sources of P for plants, this work was performed to study the hydrolysis of nine organic phosphates by sterile, intact corn (Zea mays L.) and soybean (Glycine max L.) roots. Results showed that the rates of hydrolysis ofp-nitrophenyl phosphate (PNP) in buffered solutions by roots of three varieties of corn and three varieties of soybean ranged from 13 to 22 μmol PO₄−P g⁻¹ root h⁻¹ and from 2.1 to 2.2 μmol PO₄−P 0.1 g⁻¹ root h⁻¹, respectively. The average rate of hydrolysis of PNP in nonbuffered solutions was 2- to 3-fold lower for corn roots and 6- to 10-fold lower for soybean roots as compared with those obtained with buffered solutions. The orthophosphate released from hydrolysis of organic P compounds in buffered solutions during a 48-h incubation of corn roots showed that the maximum rate of hydrolysis of PNP was 4 to 6 times greater than the commonly used substrates: α- and β-glycerophosphates, phenolphthalein diphosphate, and glucose-6-phosphate. The rates of hydrolysis of glucose-6-phosphate and glucose-1-phosphate were similar and about 6- to 12-fold lower than that of PNP. Phosphoethanolamine and phosphocholine were hydrolyzed slightly, ando-carboxyphenyl phosphate was not hydrolyzed. The rates of hydrolysis of organic P compounds in nonbuffered solutions by corn and soybean roots were 1 to 3 and 1 to 10 times lower than those in buffered solutions, respectively. The trends in rates of hydrolysis by soybean roots of buffered organic P substrates were similar to those observed with corn roots, with the exception of glucose-1-phosphate and phosphoethanolamine.     

A comparison of results obtained with two methods for assessing yield stability

Summary A major objective of the CIMMYT Maize Program is to develop open-pollinated varieties of maize (Zea mays L.) that are well adapted to a wide range of environments. To achieve this breeding goal, it is essential that the program use a stability technique that will identify high-yielding, stable genotypes accurately in international trials conducted under different environmental conditions. The objective of this study was to compare a spatial method with a modified conventional regression analysis method to determine the yield stability of 27 CIMMYT maize varieties evaluated at 37 locations. The methods also were compared on the basis of their consistency in assessing the stability of varieties when certain locations were omitted, and when subsets of varieties were analyzed. The varieties found to be stable by the spatial method with all sites included in the analysis were also stable (1) when the lowest and highest yielding sites were excluded from the analyses, and (2) when the varieties were considered, along with others, as a subset of the original group of materials. Stability parameters determined by regression analysis, however, varied for some varieties when (1) extreme sites were excluded, and (2) a subset of entries was considered in isolation. Because the spatial method was more consistent in identifying high-yielding stable varieties, it was considered the more useful of the two methods.     

Depletion of macro-nutrients from rhizosphere soil solution by juvenile corn,cottonwood, and switchgrass plants

In situ sampling of rhizosphere solution chemistry is an important step in improving our understanding of soil solution nutrient dynamics. Improved understanding will enhance our ability to model nutrient dynamics and on a broader scale, to develop effective buffers to minimize nutrient movement to surface waters. However, only limited attention has been focused on the spatial heterogeneity and temporal dynamics of rhizosphere solution, and still less is known about how rhizosphere solution chemistry varies among plant species. Nutrients in rhizosphere soil solution and changes in root morphology of juvenile corn (Zea mays L. cv. Stine 2250), cottonwood (Populus deltoids L.), and switchgrass (Panicum virgatum L.) were monitored using mini-rhizotron technology. Plants were grown for 10 days in a fine-silty, mixed, superactive, mesic Cumulic Hapludoll (Kennebec series). Micro-samples (100–200 μL) of rhizosphere and bulk soil solution were collected at 24-h intervals at a tension of −100 kPa and analyzed for P, K, Ca, and Mg concentration using Capillary Electrophoresis techniques. Plants were harvested at the end of the 10-day period, and tissue digests analyzed for nutrient content by Inductively Coupled Plasma Spectroscopy. Corn plants produced roots that were 1.3 times longer than those of cottonwood, and 11.7 times longer than those of switchgrass. Similar trends were observed in number of root tips and root surface area. At the end of 10 days, rhizosphere solution P and K concentrations in the immediate vicinity of the roots (<1 mm) decreased by approximating 24 and 8% for corn, and 15 and 5% for cottonwood. A rhizosphere effect was not found for switchgrass. After correction for initial plant nutrient content, corn shoot P, K, and Mg were respectively 385, 132, and 163% higher than cottonwood and 66, 37, and 10% higher than switchgrass. Cottonwood shoot Ca concentration, however, was 68 to 133% higher than that of corn or switchgrass. There was no difference in root P concentration among the three species. Nutrient accumulation efficiency (μg nutrient mm⁻¹ root length) of cottonwood was 26 to 242% higher for P, 25 to 325% higher for Ca, and 41 to 253% higher for Mg than those of corn and switchgrass. However, K accumulation efficiency of corn was four to five times higher than that of the cottonwood and switchgrass. Nutrient utilization efficiency (mg of dry weight produced per mg nutrient uptake) of P, K, and Mg was higher in cottonwood than in corn and switchgrass. These differences are element-specific and depend on root production and morphology as well as plant nutrient status. From a practical perspective, the results of this study indicate that potentially significant differences in rhizosphere solution chemistry can develop quickly. Results also indicate that cottonwood would be an effective species to slow the loss of nutrients in buffer settings. An erratum to this article can be found at     

Effects of floury-2 locus on zein accumulation and RNA metabolism during maize endosperm development

Zein accumulation patterns during mutant and normal maize endosperm development were determined. Accompanying an increase in the number of floury-2 alleles present in the endosperm was a well-defined stepwise depression in zein accumulation. Analysis of the zein accumulated in endosperms containing zero, one, two, and three doses of the floury-2 allele by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a proportionate reduction in the two major zein components, Z1 and Z2. In contrast, the relative proportions of the minor zein bands were altered. Membrane-bound polysomes isolated from kernels of floury-2 and normal maize were predominantly large size classes. The presence of increasing numbers of the floury-2 allele in the endosperm decreased recovery of membrane-bound polysomal material in a stepwise fashion. However, major alterations in polysome size-class distributions were not observed. The reduction in membrane-bound polysome material correlated linearly with reductions in in vitro zein synthesis and in vivo zein accumulation.     

Interactions of Fusarium moniliforme,its metabolites and bacteria with corn

Fusarium moniliforme Sheldon is an economically important pathogen of corn (Zea mays L.) which causes stalk, root and ear rot. Several mycotoxins have also been isolated, identified and implicated in both animal and human toxicoses. The fungus can be disseminated in symptomless corn seed and can also survive in crop residues in the soil. Asymptomatic infection may be related to different corn cultivars, fungal strains, and environmental factors. Symptomatic expression of pathogenicity may vary, but usually the result of such infections is death of the plant. The greatest concern is the asymptomatic infection, since it is in this form that fungal toxins may surreptitiously enter animal and human food chains. F. moniliforme produces both fusaric acid, which is phytotoxic to corn and interferes with seed germination, and plant growth regulators that may affect pathogenicity of the fungus or be associated with the production of mycotoxins. Other metabolites, including fusarin C, moniliformin, and the fumonisins, may or may not be phytotoxic, but are associated with animal and human toxicoses. The control of F. moniliforme in corn is therefore quite important. One potential means to accomplish this reduction is biocontrol by the application of antagonistic rhizobacteria to corn kernels at planting. To be effective the bacteria must be able to colonize the corn root system and be able to prevent root infection by successful competing with F. moniliforme which may be accomplished by siderophore and or antibiotic activity.     

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.     

Cadmium and zinc influx characteristics by intact corn (Zea mays L.) seedlings

Summary Cadmium and zinc uptake parameters were determined for intact corn (Zea mays L.) seedlings grown for 15 and 22 in nutrient solutions containing levels of Cd and Zn that were similar to those found in soil solutions. Uptake of both elements was assumed to follow Michaelis-Menten kinetics. Calculations were based on the concentrations of free ionic Cd (Cd²⁺) and Zn (Zn²⁺) rather than the total solution concentration. Rates of Zn uptake were measured by determining depletion of Zn for periods of up to 30 h from solutions containing initial concentrations of 1.5 and 10μmol Zn 1⁻¹. Depletion curves suggested that Zn uptake characteristics were similar at both levels of Zn in solution. The Imax for Zn uptake decreased from 550 to 400 pmol m⁻² root surface s⁻¹ between 16 and 22 d of growth while Km decreased from 2.2 to 1.5 μmol Zn²⁺ 1⁻¹. Cadmium uptake parameters were measured by controlling Cd²⁺ activities in nutrient solution betwen 6.3 to 164 nmol l⁻¹ by continuous circulation of nutrient solution through a mixed-resin system. Imax for Cd uptake was 400 pmol m⁻² root surface s⁻¹ at 15 and 22 d of growth. The magnitude of Km increased from 30 to 100 nmol Cd²⁺ 1⁻¹ during this time period. The Km value suggests that corn is efficient for Cd uptake. The results of these uptake studies are consistent with the observed uptake of Zn and Cd by corn seedlings in soils.     

Growth,yield, and yield components of ethephon-treated corn

Field studies were conducted during 1985 and 1986 to study the effect of stage and rate of ethephon application on growth, combine-harvested yield, and yield components of three corn (Zea mays L.) hybrids at two densities. Ethephon was applied at four rates from 0 to 560g ha^–1 at three growth stages: tassel elongation (TE 3mm), TE + 6d, and Ear elongation (EE 3mm). The greatest rate of ethephon decreased lodging by 85% in 1985 and 93% in 1986. Reduction in yield at the greatest rate of ethephon was 6% and 2% of the control in 1985 and 1986, respectively. Brace-root rating in 1986 was increased 20%, when comparing the greatest application rate with the control. In the same year, weight per seed was reduced 2%, which was equivalent to the percentage yield reduction. Plant and ear heights generally decreased in a linear fashion with increasing rate of ethephon. The growth stage at the time of ethephon application significantly altered all variables except grain moisture. Grain yield, seed weight, and lodging decreased as ethephon application was delayed. Decrease in seed weight probably caused the yield decrease with delayed application. Stages of application interacted with rate such that ear height was reduced less as ethephon application was delayed. The greatest rate of ethephon applied at the beginning of EE resulted in the best lodging control. However, reduction in lodging did not result in higher yield. Also, ethephon applied at the TE stage mainly affected elongation of internodes below the ear; at the EE stage, elongation of internodes above the ear was affected.     

Partitioning of reduced nitrogen derived from exogenous nitrate in maize roots: Initial priority for protein synthesis

Summary When roots of five day-old maize seedlings were exposed to¹⁵N-nitrate, a constant (25–29%) proportion of the reduced¹⁵N derived from the entering¹⁵N-nitrate accumulated as insoluble¹⁵N nitrogen. Constancy was established by two hours and lasted through 12 hours at ambient¹⁵N-nitrate concentrations of 0.05 mM to 20.0 mM. Even when little¹⁵N nitrate had been reduced (<2 moles), there was a linear relationship between accumulation of insoluble¹⁵N (but not accumulation or translocation of soluble reduced¹⁵N) and total reduced¹⁵N. It is proposed that protein synthesis from the entering nitrate occurs in close association with nitrate reduction.Paper No. 9764 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC, 27695-7619, USA. This research was supported by Grant No. PCM-8118661 from the National Science Foundation.Use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product's name or criticisms of similar ones not mentioned.     

Callus formation from cell culture protoplasts of corn (Zea mays L.)

Summary Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the quality of the culture used for protoplast isolation.     

Light-regulated protein and mRNA synthesis in root caps of maize

Illumination of maize roots initiates changes in mRNA levels and in the activities of proteins within the root cap. Using Northern analysis we showed a 5–6-fold increase in the levels of three specific mRNAs and a 14-fold increase in plastid mRNA. This increase is rapid, occurring within 30 minutes of illumination. With prolonged periods of darkness following illumination, messages return to levels observed in dark, control caps. For two species of mRNA illumination results in a reduction in message levels. Light-stimulated increases in the levels of specific mRNAs are proportionally greater than are increases in the activities of corresponding proteins. We suggest that the light-stimulated increase in protein activity in root caps may be preceded by and occur as a consequence of enhanced levels of mRNA. Our work suggests that photomorphogenesis in roots could involve changes in the levels of a wide variety of mRNAs within the root cap.     

Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.