Localization of a phytohormone using immunocytochemistry.

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.

Nuclear magnetic resonance measurement of oil "unsaturation" in single viable corn kernels.

High-resolution nuclear magnetic resonance spectroscopy has been used to demonstrate the feasibility of determining iodine value and average molecular weight of oil in individual corn kernels. The procedure is rapid and nondestructive. Depending on heritability of individual fatty acids, this technique may greatly increase selection efficiency in breeding programs to alter the fatty acid composition of corn oil.

Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii.

Chromatin has been prepared from nuclei isolated from the dinoflagellate alga Gyrodinium cohnii. This chromatin contains RNA, acid-insoluble proteins, and acid-soluble proteins; the respective ratios to amount of DNA are about 0.09, 0.48, 0.08 (by weight). Not only is the amount of acid-soluble protein associated with the DNA much less than it is in the typical eukaryote, but polyacrylamide gel electrophoresis in urea at pH 3.2 produces a banding pattern different from that of typical histones. There is one predominant band that migrates about as fast as does histone IV from corn. These findings are of interest, because the nuclear organization in the dinoflagellates appears to be intermediate between the prokaryotes and the eukaryotes.

Air containing nitrogen-15 ammonia: foliar absorption by corn seedlings.

Thirty-day-old corn seedlings, grown in the greenhouse with different concentrations of supplemental nitrate nitrogen, were moved to a constant-temperature growth chamber and sealed in a 560-liter tent made of polyvinyl chloride. The plants were exposed to air containing ammonia labeled with nitrogen-15 (1, 10, and 20 parts per million) for 24 hours and then harvested. The nitrogen-15 content of the tops and roots showed that at 1 part per million 43 percent of the ammonia was absorbed, whereas at 10 and 20 parts per million, 30 percent of the ammonia was absorbed. The results demonstrate that growing plants may be a natural sink for atmospheric ammonia.

Dihydroxyphenylalanine in rat food containing wheat and oats.

Dopa has been identified in rat food by three different fluorimetric assays and paper chromatography. Incubation of the rat food with proteolytic enzymes dramatically increased the measurable free dopa. Analysis of samples of six individual protein-containing constituents of rat food revealed that both wheat and oats contain dopa.

The helical content of zein, a water insoluble protein, in non-aqueous solvents.

The conformation of zein, a seed protein from corn, was determined by optical rotary dispersion measurements in a wide variety of nonaqueous solvents. Over a wide range of dielectric constants, in pure and mixed solvent systems, the helical content was independent of dielectric constant. Determination of the helical content of insulin and ribonuclease in several of these solvents indicated a variation in secondary structure comparable of that of zein. Though virtually insoluble in water, zein not only is a globular protein in nonaqueous solutions, but has conformational properites characteristic of more conventionally behaving globular proteins.

'Cap' structures in maize poly(A)-containing RNA.

Poly(A)-containing RNA was isolated from maize embryos by chromatography on columns of oligo(dT)-cellulose and exhaustively digested with ribonucleases T2, T1, and A. Fractionation of the digests by two-dimensional electrophoresis revealed the presence of three 7-methylguanosine-terminated 'cap structures' of the type m7GpppNp.

Diol lipids. Acylated ethyleneglycol glycosides. A new type of natural glycolipid.

A new type of glycolipid has been detected in ripening corn seeds. The presence of ethyleneglycol, galactose, glucose and fatty acids was shown by degradation studies. The products of alkaline deacylation were identified as galactosyl- and glycosylethyleneglycol by thin-layer chromatography and combined gas-liquid chromatography-mass spectrometry. The native ethyleneglycol galactolipid was isolated by distribution of the total lipids between heptane and 95% methanol, following silica gel column chromatography of the methanol soluble fraction. Analysis of the alkaline deacylation products of the isolated ethyleneglycol lipid as well as examination of the mass-spectrum of its tetraacetate showed the new lipid to have the structure of 1-acyl-2-(O-beta-D-galacto-pyranosyl)ethyleneglycol with palmitic, oleic and stearic acids as the main fatty acid components. The fragmentation patterns under electron impact of trimethylsilyl ethers of synthetic ethyleneglycol glycopyranosides and of 1-palmitoyl-2-(O-beta-D-tetraacetylgalactopyranosyl)ethyleneglycol are described.

[Independence of special forms of chloeophyll a and chlorophyll holochromes]. / Indépendance des formes spectrales de chlorophylle a et des holochromes chlorophylliens

Zea mays L. seedlings were cultivated for 10 days with submission to 4 s illumination periods interspersed with dark periods varying in length from 30 min to 6 h depending on the lot analyzed. The results show that, for the case in which the dark periods were shorter than 1 h, the relative proportions of different spectroscopic chlorophyll forms (maxima at 662, 670, 677.5, and 684 nm) were constant. For longer durations of darkness between illuminations, the relative proportion of the form Da670 increases, while that of Ca684 diminishes with the length of darkness; to a lesser extent, the relative proportion of Ca662 increases and a form Ca692 disappears. A scheme is proposed to explain the evolution of the relative proportions of the different spectral forms. The different chlorophyll holochromes present in the chloroplasts were also analysed. If the dark period was longer than 1 h, chlorophyll was associated with peptide chains of molecular weights 21 000 and 29 000. If the dark period was shorter than 1 h chlorophyll was associated with four peptide chains of molecular weights 21 000, 25 000, 29 000 and 70 000. The results taken together demonstrate that a given spectral chlorophyll a form cannot be associated with a definite chlorophyll holochrome.

Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments.

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.

Molecular weights of maize mitochondrial and cytoplasmic ribosomal RNAs under denaturing conditions.

The molecular weights of maize cytoplasmic and mitochondrial rRNAs were determined by gel acrylamide electrophoresis under non-denaturing and denaturing conditions. The molecular weights of mitochondrial rRNAs (0.76-10-6 and 1.25-10-6) exceeded those of cytoplasmic rRNAs (0.67-10-6 and 1.19-10-6) when electrophoresed in 8 M urea at 60 degrees C. Electrophoresis in 1.1 M formaldehyde resulted in similar values except for heavy mitochondrial rRNA, which exhibited a higher molecular weight than observed in 8 M urea. The observed values for cytoplasmic rRNAs, especially the heavy component, represent a decrease from estimates obtained under non-denaturing conditions. This is the first report of the electrophoretic examination of higher plant rRNAs under denaturing conditions.

Lipid molecular species composition of thylakoid membranes.

Lipid molecular species compositions of chloroplast thylakoid membranes of mesophyll cells from Spinacia oleracea, Glycine max, Oryza sativa and Zea mays and of bundle sheath cells from Zea mays have been quantitatively determined. No significant difference in the lipid molecular species composition was found among the five membrane sources. The predominant molecular species of monogalactosyldiacylglycerol was the 1-linolenoyl parallel to 2-linolenoyl species. The 1-linolenoyl parallel to 2-linoenoyl and 1-palmitoyl parallel to 2-linolenoyl species were the major molecular species of digalactosyldiacylglycerol. 6-Sulfoquinovosyldiacylglycerol was mainly composed of the palmitoyl parallel to linolenoyl and palmitoyl parallel to lineolyl species. Almost all of the C-2 position of phosphatidylglycerol were esterified with the palmitoyl or delta 3-trans-hexadecenoyl residue. The molecular species compositions of phosphatidylcholine and phosphatidylinositol were basically similar to those of membranes in non-photosynthetic tissues.

A chemical and physical method for determining the complete base composition of plant DNA.

Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.

Fiber of the maize tortilla.

Fiber concentrations were measured in maize tortillas and in masa, the lime-treated dough from which they are made, with the aid of Van Soest's methods. Neutral detergent fiber of tortillas was 6.60 +/- 0.98% (mean and SD) and acid detergent fiber 3.75 +/- 0.83% of the dry weight corrected for ash. The hemicellulose fraction was 2.89 +/- 0.94%. Both neutral and acid detergent fiber were significantly higher in tortilla than in masa. The gain in fiber by tortillas during baking is attributed to the formation of browning reaction (Maillard) products resistant to the detergent-containing fiber reagents.

Protein quality of two varieties of high-lysine maize fed alone and with black beans or milk to normal and depleted weanling rats.

Opaque-2 corn, modified high-lysine corn with vitreous endosperm, and common corn were compared in rat growth studies. Corn alone supplied 8.6% protein; and at both 8.6 and 11.4% protein, corn furnished 60% of the protein and either milk or black beans supplied 40%. The modified corn, which also was high in both lysine and tryptophan, did not differ significantly from Opaque-2 corn in its ability to stimulate growth and nitrogen deposition in both well-nourished and partially protein-depleted rats; and both were significantly superior to common corn when fed alone. Milk was a more effective supplement for corn than black beans, especially when high-lysine varieties were fed, whereas beans exerted a similar effect on all varieties of corn. The depleted rats deposited more nitrogen as a result of eating most diets than did well nourished rats. Implications of these findings for preschool children are discussed.

[Zein of maize grain. Preparation and characterization]. / La zéine du grain de maïs. Préparation et caractérisation.

A laboratory procedure for isolation and purification of zein from grains of 4 varieties of Maize was described. The preparations were characterized by their physicochemical properties. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS), native zein (from INRA 260 hybrid) was resolved into 2 major classes with average molecular weights of 45,000 and 22,000. After reduction with mercaptoethanol zein contained only two subunits of 22,000 and 24,000 daltons. Upon starch gel electrophoresis in 6 M urea at pH 3.5, native zein exhibited five major or medium intensity bands and several minor ones. The latter, under reducing conditions, disappeared to reinforce the major bands or to yield some new minor bands. Amino acid analysis revealed a very low content of lysine. The NH2-terminal amino acids were determined to be threonine and phenylalanine with a preponderance of the former. Zeins isolated from the varieties studied appeared tohave the same NH2-terminal residues and similar amino acid compositions with an arginine/histidine ratio ranging from 1.1 to 1.2. They differed in relative importance of components, detected by electrophoresis in the presence of SDS or urea. Changes in zein characteristics with the grain genotype allow one to conclude that the components of molecular weights of 22,000 and 24,000 consist of several subunits differing in charge and amino acid content.

Zein of maize grain: I--Isolation by gel filtration and characterization of monomeric and dimeric species.

Unreduced zein chromatographed on Sephadex G 200 in 8 M urea, on G 100 in 1.5 or 2.5% sodium dodecyl sulfate (SDS) and on hydroxypropylated G 100 in 70% ethanol was resolved into two minor fractions A and B and two major ones D and M irrespective of the medium. The quantitative importance of the fraction M was dependent on the isolation conditions of zein. It decreased from 53% of the proteins contained in ethanolic extract and chromatographed as they were extracted, to 40% of the purified zein. The molecular weight values obtained from SDS-polyacrylamide gel electrophoresis and amino acid compositional data indicated that fractions D and M, as isolated from purified zein in the presence of ethanol, represented respectively dimeric and monomeric forms of a mixture of Mr 22 000 and 24 000 polypeptides with threonine or phenylalanine as NH2-terminal residue. Electrophoretic analysis of selectively carbamylated fraction M on starch gel at pH 3.5 revealed that zein subunits comprised several polypeptides differing in the number and the nature of basic amino acids. At least one of these polypeptides contained one lysyl residue.

Zein of maize grain: II--The charge heterogeneity of free subunits.

The subunits present as monomers in unreduced zein and isolated as fraction M by gel filtration, were chromatographed on sulfoethyl-cellulose. Three major subfractions were detected and characterized. Each of them, submitted to electrophoresis at pH 3.5, migrated as a single band corresponding to each of the three major electrophoretic forms seen in fraction M at the same pH. The presence of lysine in some polypeptides, suggested by amino acid composition data, was confirmed by electrophoretic analysis of carbamylated subfractions at pH 3.5. At pH 8.9 each subfractions was further resolved into three cationic bands in starch gel and three (or more) anionic bands in polyacrylamide gel. The same fractionation was also obtained by submitting the major electroforms of fraction M, as isolated at pH 3.5, to isoelectric focusing. Based on these observations, the most probable distributions of basic amino acids in subunits detected by electrophoresis at pH 8.9 were specified and compared to those recently published for several zein clones. The presence per polypeptide chain of three carboxyl groups and occasionally of one lysine would be a feature of zein originating from maize hybrid Inra 260.

Inhibition of mitochondrial energy-linked reactions by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a hydroxamic acid from Gramineae.

DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) is the main hydroxamic acid isolated from maize extracts. It inhibited reversibly ATP synthesis, Pi-ATP exchange reaction and ATPase activity in submitochondrial particles from bovine heart. Half-maximal effects were obtained with 4, 2, and 6 mM DIMBOA respectively. At higher concentrations it also inhibited mitochondrial electron transport (I50 = 11 mM). Irreversible inactivation of mitochondrial electron transport, Pi-ATP exchange reaction and 8-anilino-1-naphthalene sulfonate energy-dependent fluorescence enhancement was also observed. These effects of DIMBOA on energy-linked mitochondrial reactions may explain the inhibitory action of DIMBOA on several aerobic organisms.

Microscopical comparison of cotton, corn, and soybean dusts.

Specialized analytical methods are required for identification of components of agricultural dusts such as those generated in harvesting, transportation, storage, and processing of cotton, corn, and soybeans. The larger particles and trash components of the dusts can often be identified visually or with the aid of an optical microscope (OM). The respirable portion of the dust, that which causes lung dysfunction, retains few structural features for identification. Electron microscopy and X-ray microanalysis, together with special optical microscopical techniques, can be used to characterize these microdusts. Combination studies with scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis of cotton dusts have shown the presence of mineralogical particles probably of a soil origin and materials that can be associated with plant parts. Even in screened and filtered cotton dusts, fibrillar fragments are usually present due to their ability to penetrate openings the size of their diameters. The corn and soybean dusts studied were different from the cotton dust in that the large fibrillar component of the cotton dust was absent in the screened grain dusts. However, these dusts consisted of structurally unrecognizable particles that appeared similar to those found in cotton dust. In addition they contained many spheroid particles identified as starch. Dusts from all three sources were found to agglomerate into larger particles, some of which were still less than 10 micron. This agglomeration could confuse the instrumental measurement of dust particle size.