Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: A potential oral therapeutic to treat phenylketonuria

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (∼50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions.     

Effect of 1.10-phenanthroline, a photodynamic herbicide on the development and structure of maize chloroplasts

Effect of low (5 mmol·dm⁻³) and high (10 or 20 mmol·dm⁻³) doses of 1.10-phenanthroline (Phe), a photodynamic herbicide, on the development of chloroplasts in etiolated and subsequently illuminated maize seedlings and on the structure of already developed chloroplasts of green maize seedlings was examined. Etiolated and then irradiated plants were resistant to 5 mmol·dm⁻³ of Phe with respect to morphology, however Phe caused inhibition of greening and of grana formation. Higher Phe concentrations followed by exposure to light caused not only total inhibition of greening but also dilation of thylakoids, swelling of chloroplasts, and finally total destruction of chloroplast structure. Application of Phe in the same concentrations to green plants revealed that they were resistant to low dose of Phe with respect to morphology and structure of chloroplasts, however 10 and 20 mmol·dm⁻³ Phe and illumination caused the loss of turgor of treated plants and other photooxidative damages seen at the ultrastructural level. We concluded that maize, as representant of monocotyledonous plants, is resistant to low (5 mmol·dm⁻³) Phe concentration. Higher (10 or 20 mmol·dm⁻³) concentrations, used to determine the site of damage and mode of action of Phe on the level of cell revealed that action of photodynamic herbicides is based on standard photoinhibition mechanism and also probably on their chelating properties.     

Changes on the structure, consistency, physicochemical and viscoelastic properties of corn (Zea mays sp.) under different nixtamalization conditions

Aqueous corn (Zea mays sp.) dispersions with 1% (w/w) of calcium hydroxide/weight of corn were cooked at 90 °C (nixtamalization) for different times (10, 30, 50, 70 and 90 min), making five treatments. A control lot (nine treatments), was cooked for 10, 30, 50, 60, 70, 90, 120, 150 and 180 min. Then, processed corn samples were washed and milled to obtain masa (dough). Scanning electron microscopy, calorimetry, viscoamilography and rheological analysis were used to characterize the corn samples. The corn micrographs showed that the nixtamalization modified the shape of the starch granules and the protein bodies. Starch granules from nixtamalized samples, were round shaped, while control samples, showed polygonal shape. Proteins from nixtamalized samples usually exhibited two transition endotherms, while in control samples, only one transition was seen, suggesting some relationship with gelatinized starch. The nixtamalization shortened the corn cooking time to develop a proper texture in masa to obtain good quality tortillas, as seen in their consistencies. All corn masa samples showed weak gel-like viscoelastic behavior with the elastic modulus (G′) higher than the loss modulus (G″), over all strain and frequency domains.     

Aflatoxin accumulation in preharvest maize kernels: Interaction of three fungal species,european corn borer and two hybrids

Summary The interaction was studied among: 1) developing maize kernels (Zea mays L.); 2) European Corn Borer (ECB) (Ostrinia nubilalis Hubner); 3) and three fungal species,Aspergillus flavus Lk. ex Fr.,Penicillium oxalcium Currie and Thom, andFusarium moniliforme Sheld. Two hybrids with varying degrees of resistance to ECB stalk damage were grown in Iowa, Georgia, and Missouri in 1980. One-half of the plots were hand-infested with ECB egg masses. Fungal spores of individual isolates or combinations of the three species were introduced into the silk channels of developing ears in designated plots. ECB larvae were subsequently collected from developing ears. A higher incidence ofA. flavus group isolates was observed in ECB larvae collected from ears that had been inoculated withA. flavus than from insects collected from control ears. Although the resistant hybrid exhibited reduced ECB stalk damage compared with the susceptible variety, no consistent pattern of hybrid effect on the association betweenA. flavus and ECB was observed at all three locations. Differences in aflatoxin B₁ levels in mature kernels from the three locations ranged from 8 ppb in Iowa samples to 419 ppb in Missouri kernels. Conditions during crop development at the Missouri location were particularly conducive to elevated presence ofA. flavus propagules in ECB larvae, increased ECB-mediated stalk damage, and greater toxin concentration in mature kernels.     

The action of divalen Zn,Cd, Hg,Cu and Pb ions on the ATPase activity of a plasma membrane fraction isolated from roots ofZea mays

The effects of the divalent metal ions Zn, Cd, Hg, Cu and Pb on the ATPase activity of a plasma membrane fraction isolated from roots ofZea mays have been investigated. When Mg-ions (3 mM), with or without K-ions (50mM) are included in the reaction medium, inhibition of ATPase activity was found in all cases, the relative order of the inhibitors over the concentration range 10 to 100M, being Hg>>CuCd>ZnPb. Below 1.0M only Hg caused substantial inhibition. In the absence of Mg ions, Zn and to a lesser extent Cd, activated the enzyme up to a concentration of 1 mM, activity being further stimulated in the presence of K-ions (50mM). No activation of ATPase activity was found for Hg, Cu or Pb. It was concluded that Zn-ATP and Cd-ATP are both alternative substrates for the enzyme. Further experiments showed that both K_m and V_max for the substrates Zn-ATP and Cd-ATP are very much lower than for the usual substrate Mg-ATP.These present results are discussed in relation to the known actions of these divalent cations on the trans-root potential and H-ion efflux in excised maize roots (Kennedy and Gonsalves, 1987).     

Analysis and regulation of legume inoculants in Canada: The need for an increase in standards

Herbicide residues may affect seedlings during early stages of their development. We studied this possibility by the use of light and electron microscopy after incubation of germinating seeds ofPisum sativum L. andZea mays L. with different concentrations of chlorsulfuron and metsulfuron-methyl. By in vitro experiments, we have shown that both herbicides caused growth reduction of the very young roots, and severe ultrastructural alterations and injuries of the root caps of both species. Chlorsulfuron caused increase of electron-dense material in the vacuoles, cytoplasmic degeneration even in the inner secretory cell layers of the cap, and disruption of the amyloplast envelopes with release of the statolithic starch grains. In the initial cell complex of the root cap, the herbicides caused the formation of large concentric aggregates of the rough ER and wall disformations in the cells adjacent to this complex. Scanning electron microscopic observations revealed a decrease of the slime layer ensheathing the root cap and the subapical root surface.We conclude that even in early stages of seed germination, both herbicides seriously affect the gravity perception centre (consisting of the statocytes), and the secretory tissue of the root caps, thus probably disturbing the processes of gravitropism and the protective slime secretion of the roots.     

Soil fertility management as a factor in weed control: the effect of crimson clover residue,synthetic nitrogen fertilizer,and their interaction on emergence and early growth of lambsquarters and sweet corn

Previous experiments have shown that crimson clover (Trifolium incarnatum L.) used as a green manure may supply weed control benefits as well as nitrogen (N) to a subsequent crop of corn (Zea mays L.). In contrast to use of synthetic N fertilizer, use of fresh, incorporated crimson clover residue as an N source has been found to suppress lambsquarters (Chenopodium album L.) aboveground drymatter accumulation but to only temporarily reduce that of sweet corn. One possible cause of the clover's suppressive effect is the initial low availability of N that may occur after residue incorporation in the soil. A factorial treatment combination of +/– crimson clover residue and four rates of N fertilizer was used in two field experiments to further document the clover's influence on early plant growth and development and to test the hypothesis that low initial N availability is responsible for the clover's previously observed suppressive effects. The presence of crimson clover residue was found to reduce total emergence of lambsquarters by 27%, while application of N fertilizer increased lambsquarters emergence by almost 75%. Lambsquarters emergence was also delayed by the residue treatment. Addition of N did not alleviate the clover's suppressive effect on total emergence or emergence rate of lambsquarters. Sweet corn emergence and emergence rate differed by less than 5% in 0 N/+residue and 0 N/–residue treatments. Applications of N to residue plots suppressed rather than enanced sweet corn emergence. Lambsquarters aboveground biomass accumulation was 46% tower in the residue than nonresidue treatments at 23 days after planting (DAP) and remained 26% lower at 53 DAP. Addition of N did not alleviate the suppressive effect of the clover residue on lambsquarters aboveground drymatter accumulation. Sweet corn aboveground biomass accumulation was not affected by the presence of the clover residue. The results of the experiments indicate that the suppressive effect of crimson clover residue on lambsquarters emergence and growth is not attributable to initial low availability of N. However, given the stimulatory effect of N fertilizer on lambsquarters development, use of crimson clover as an N source would appear to provide weed control benefits both as a direct suppressant of weed emergence and growth and as a substitute for fertilizer N.     

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

Suitability of pollen sources for the development and reproduction of Coleomegilla maculata (Coleoptera: Coccinellidae) under simulated drought conditions

Laboratory experiments compared the nutritive value of various pollen sources for the development of Coleomegilla maculata DeGeer under conditions of continuous water availability and simulated drought. When water was continuously available, larval survival was not different from 100% on diets of frozen eggs of Ephestia kuehniella Zeller, corn pollen, sorghum pollen, or pulverized bee pollen, whereas survival of larvae was significantly reduced on the latter three diets in the simulated drought treatment. Pollen of cultivated sunflower, Helianthus annus L., proved fatal to both larvae and adults; its surface structure caused clumping and accumulation on the insect cuticle that led to death from exhaustion/desiccation in petri dishes. The Ephestia egg diet yielded shorter developmental times and heavier adult weights than any pollen diet in both treatments. The drought treatment increased developmental time on all diets with a significant treatment–diet interaction. Drought reduced the adult weight of females on the sorghum pollen diet, and that of both sexes on the bee pollen diet, again with a significant treatment–diet interaction. Initial water content was highest in corn pollen (36.8%), followed by Ephestia eggs (29.2%), sorghum pollen (25.3%), sunflower pollen (8.7%), and bee pollen (4.6%), but did not appear correlated with C. maculata larval survival on pollen sources under drought conditions. Reproductive adult females that received corn or sorghum pollen as a supplement to Ephestia eggs did not differ in fecundity or fertility from those fed only Ephestia eggs.     

The quiescent center in roots of maize: initiation,maintenance and role in organization of the root apical meristem

Summary Using roots of maize, we tested the hypothesis that the origin and maintenance of the quiescent center (QC) are a consequence of polar auxin supply. Exposing roots to the polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), or to low temperature (4 °C, with subsequent return to 24 °C), enhances mitotic frequency within the QC. In both treatments, the QC most typically is activated at its distal face, and the protoderm/dermatogen undergoes several periclinal divisions. As a result, the root body penetrates and ruptures the root cap junction and the characteristic closed apical organization changes to open. A QC persists during these changes in apical organization, but it is diminished in size. The data from the TIBA-treated roots suggest a role for auxin in the origin and maintenance of the QC, and further, that alterations in QC dimensions are a consequence of polar auxin supply. We hypothesize that the root cap, and specifically the root cap initials, are important in regulating polar auxin movements towards the root apex, and hence are important in determining the status of the QC.Abbreviations QC quiescent center - TIBA 2,3,5-triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Immunodetection of four mitotic cyclins and the Cdc2a protein kinase in the maize root: Their distribution in cell development and dedifferentiation

Summary Cyclin proteins and cyclin-dependent kinases play a key role in the regulation of cell division. We have therefore studied the relationship of the level of four mitotic cyclin proteins and the Cdc2a kinase protein to cell division in maize root tissue with respect to cessation of division as cells leave the primary meristem region, resumption of division in formation of lateral-root primordia, and induced division following wounding. All four mitotic cyclins and Cdc2a were most abundant in dividing cells. The only examined cell cycle protein which was restricted to dividing tissue was cyclin ZmCycB1;2 (previously ZmIb) and may thus be a limiting factor for cell division. All other cyclin proteins, i.e., ZmCycB1;1 (previously ZmIa), ZmCycA1;1 (previously ZmII), and ZmCycB2;1 (previously ZmIII), and the Cdc2a kinase declined shortly after cells had ceased division. The distance from the root tip at which cells ceased division was tissue-specific and reflected the distance at which decrease of cell cycle proteins was detected. Whereas cyclin ZmCycB1;2 rapidly declined to a hardly detectable level in either nucleus or cytoplasm, in the nuclei of nondividing cells there was persistence of Cdc2a and of cyclins ZmCycB1;1, ZmCycCA1;1, and ZmCycB2;1, indicating that there are plant cyclins which are tightly linked to cell division and others that persist, especially in the nuclei, in nondividing cells. The transition from division to differentiation may thus partly be triggered and enforced by the decrease of the cell cycle proteins and especially the decline of cyclins in the cytoplasm. In the resumption of cell division, both in lateral-root formation and in wound response, high nuclear and low cytoplasmic accumulation of cyclin ZmCycB2;1 was the first visible sign of cell dedifferentiation, implying a role for cyclin ZmCycB2;1 in the G₀–G₁ phase transition. Next, cytoplasmic accumulation of cyclin ZmCycA1;1, followed by a rearrangement of cortical microtubules, was observed and since both the cyclins ZmCycA1;1 and ZmCycB2;1 were found at places of high tubulin concentration, they may function in the microtubule rearrangement for cell division. When the nuclei of dedifferentiating cells had visibly enlarged, all cyclins and Cdc2a accumulated there, possibly contributing to DNA replication and preparation for mitosis. Later, presumably during G₂ phase, cytoplasmic accumulation was observed for Cdc2a at low levels, as observed in G₂ phase cells of the primary meristem, and for cyclins ZmCycB1;1 and ZmCycB1;2 accumulation was observed above the levels found in undisturbed meristems, suggesting special contributions to late dedifferentiation processes in both wound-induced and lateral meristems.Abbreviations CDK cyclin-dependent kinase - LRP lateral-root primordium - Mt microtubule - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

The effect of agricultural practices on the development of indigenous arbuscular mycorrhizal fungi. I. Field studies in an Indonesian ultisol

Two pre-established agricultural field trials were assessed for the abundance of arbuscular mycorrhizal fungi (AMF) in the soil (density of spores, species richness and lengths of extra-radical mycelium [ERM]) in association with one of three tropical plant species (Gliricidia sepium, Peltophorum dasyrachis and Zea mays). The trials were managed by one of three agricultural practices: soil disturbance in a monoculture system, a root barrier to prevent interactions between plants in an agroforestry system or the addition of organic matter (OM) in an agroforestry and a monoculture system. The lengths of ERM of AMF in the soil were greater in the agroforestry system than the monoculture system. These were greater when a root barrier was present, but decreased when OM was added. Soil disturbance reduced the density of spores, species richness and the lengths of ERM of AMF compared with the undisturbed soil. This work indicates that agricultural trials may provide a useful tool to monitor the abundance of AMF in the field. Clearly, there is potential to increase the abundance of AMF, from different genera, in the soil through the management of agricultural practices. The significance of the abundance of AMF for subsequent benefits to plant growth and development and ultimately the sustainability of tropical agro-ecosystems are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Fusarium</Emphasis> species of the <Emphasis Type="Italic">Gibberella fujikuroi</Emphasis> complex and fumonisin contamination of pearl millet and corn in Georgia,USA

This study was designed to identify and compare the Fusarium species of the Gibberella fujikuroi complex on pearl millet (Pennisetum glaucum (L.) R. Br) and corn (Zea mays L.) crops grown in southern Georgia, and to determine their influence on potential fumonisin production. Pearl millet and corn samples were collected in Georgia in 1996, 1997 and 1998. Three percent of the pearl millet seeds had fungi similar to the Fusarium species of the G. fujikuroi species complex. One hundred and nineteen representative isolates visually similar to the G. fujikuroi species complex from pearl millet were paired with mating population A (Fusarium verticillioides (Sacc.) Nirenberg), mating population D (F. proliferatum (Matsushima) Nirenberg) and mating population F (F. thapsinum (Klittich, Leslie, Nelson and Marasas) tester strains. Successful crosses were obtained with 50.4%, 10.1% and 0.0% of these isolates with the A, D and F tester strains, while 39.5 of the isolates did not form perithecia with any tester strains. Two of the typical infertile isolates were characterized by DNA sequence comparisons and were identified as Fusarium pseudonygamai (Nirenberg and ODonnell), which is the first known isolation of this species in the United States. Based on the pattern of cross-compatibility, conidiogenesis, colony characteristics and media pigmentation, a majority of the infertile isolates belong to this species. Fumonisins FB₁ and FB₂ were not detected in any of the 81 pearl millet samples analyzed. The species of the G. fujikuroi species complex were dominant in corn and were isolated from 84%, 74% and 65% of the seed in 1996, 1997 and 1998, respectively. Representative species of the G. fujikuroi species complex were isolated from 1996 to 1998 Georgia corn survey (162, 104 and 111 isolates, respectively) and tested for mating compatibility. The incidence of isolates belonging to mating population A (F. verticillioides) ranged from 70.2% to 89.5%. Corn survey samples were assayed for fumonisins, and 63% to 91% of the 1996, 1997 and 1998 samples were contaminated. The total amount of fumonisins in the corn samples ranged from 0.6 to 33.3 g/g.     

The structure and prophylactic role of the angiosperm embryo sac and its associated tissues:Zea mays as a model

Summary Various developmental phases can be distinguished in the definition of the archesporium and the early life of the embryo, takingZea mays (maize) as a model within the family Gramineae, and other families where pertinent: (1) the isolation of the megasporocyte and the functional spore derived from it; (2) the maturation of the specialized walls of the embryo sac, and their reinforcement by ensheathments derived from the contiguous nucellar cells during a sequence of phased genetic ablation; (3) the differentiation of the synergids, the associated flange, and the filiform apparatuses; (4) the blocking of the pollen tube pathway by secondary secretions in the micropylar region and the coagulation of the pollen tube cytoplasm within the filiform apparatuses during the process of fertilization; and finally (5) the development of a compound cutinized envelope of four fused layers (six where the outer integument is also involved) after fertilization. For the nascent haploid generation, the period of maximum vulnerability in respect to both pathogen invasion and the transition from diplophase control occurs during these phases. It is concluded that many of the protective features form a prophylactic shield and are key components of the angiosperms in general, which may have contributed to their evolutionary success as a group. Other physiological or biochemical adaptations or barriers may also supplement the mainly structural features described here.     

The effects of nitrate:ammonium ratios and nitrapyrin on the nitrogen response ofZea mays L. under field conditions

Five nitrate:ammonium ratios at two N-levels were tested with and without nitrapyrin [2 chloro-6-(trichloromethyl) pyridine] for grain production on a sandy soil. Treatments were applied to field maize as nutrient solutions, in one application, six weeks after planting. Nytrapyrin resulted in an increase in grain yield at a nitrate:ammonium ratio of 1:3 but in a decrease at a 0:1 ratio. The optimum nitrate:ammonium ratio was close to 1:3 with nitrapyrin and close to 3:1 without nitrapyrin. Nitrapyrin had an effect on NH₄ ⁺-N in the topsoil and NO₃ ⁻-N in the subsoil at 70 days after application. Interactions of nitrate:ammonium ratios and N-levels were shown for leaf N concentration, soil mineral N and soil pH.     

Improved tissue culture response of an elite maize inbred through backcross breeding,and identification of chromosomal regions important for regeneration by RFLP analysis

Summary The frequency of initiation of friable, embryogenic callus from immature embryos of the elite maize inbred line B73 was increased dramatically by introgression of chromosomal segments from the inbred line A188 through classical backcross breeding. Less than 0.2% of the immature B73 embryos tested (5 of 3,710) formed embryogenic callus. The breeding scheme consisted of six generations of backcrossing to B73 with selection at each generation for high frequency initiation of embryogenic cultures. BC₆ individuals were selfed for four generations to select homozygous lines. The average embryogenic culture initiation frequency increased to 46% (256/561). Nearly all (91%) of the embryos from one BC₆ S₄ plant formed embryogenic cultures. RFLP analysis was used to determine the locations and effects of the introgressed A188 chromosomal segments. Five segments were retained through at least the fifth backcross generation. The hypothesis that one or more of these five regions contains genes controlling somatic embryogenesis in maize was tested using an F₂ population of the cross A188 X Mo17. A set of five DNA markers (three of them linked) explained 82% of the observed phenotypic variance for percentage of immature embryos forming embryognic callus. Four of the five markers were located in or near introgressed A188 chromosome segments.The region marked by probe c595 on the long arm of chromosome 9 was highly associated with several measures of in vitro culture response (percent embryogenic embryos, plants per embryo, and plants per embryogenic embryo). We propose that there is a major gene (or genes) in this region in A188 that promotes embryogenic callus initiation and plant regeneration in B73, Mo17, and probably many other recalcitrant inbred lines of maize.