Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

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.     

Wide hybridization: pollination of Zea mays L. by Sorghum bicolor (L.) Moench

Summary Foreign pollen tubes in the stigma of Zea mays can be prevented from reaching the ovary cavity by the unusual length of the pollen tube pathway. A simple and rapid procedure is described for overcoming this difficulty by pollinating the basal parts of the stigmas without removing the ensheathing bracts (husks). The method maintains high humidity in the vicinity of the ovaries, and by conserving photosynthetic tissues probably also ensures a more normal O₂ /CO₂ balance in the neighbourhood of the stigmas than do bagging procedures. It is shown that Sorghum pollen tubes readily reach the ovary after pollination by the method. Their presence induces some of the characteristic post-pollination effects caused by Zea pollen tubes, but they frequently also stimulate premature enlargement of the nucellus and lysis of nucellar cells. Although Sorghum tubes have been traced across the inner ovary wall, they have not been seen to enter the micropyle, and hybrid embryos have not yet been obtained.     

Fertilization inNicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cell with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed coronas. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.Abbreviations ES embryo sac - FA filiform apparatus - Mf microfilament - Mt microtubule - PT pollen tube - RF-FS rapid-freeze freeze-substitution - TEM transmission electron microscopy We thank Gregory W. Strout for technical assistance in the use of the RF-FS technique and Dr. Hongshi Yu for providing Fig. 1. This research was supported by U.S. Department of Agriculture grants 88-37261-3761 and 91-37304-6471. We gratefully acknowledge use of the Samuel Robert Noble Electron Microscopy Laboratory of the University of Oklahoma.     

Callose in the walls of mature embryo sac ofTorenia fournieri

Summary During the course of a fluorescence microscopic investigation on the extra-ovular micropylar portion of the embryo sacs ofTorenia fournieri Lind. (Scrophulariaceae) a callosic wall was found which surrounded it almost completely until the time of anthesis. In addition, the walls of young synergids and the filiform apparatus also showed callosic fluorescence. Treatments with PAS reaction revealed a PAS-positive substance filling up the locular cavity. Our attempts to induce fluorochromasia by employing fluorescein diacetate failed, indicating the low permeability of the callosic wall around the embryo sac. It is assumed that the callose wall around the embryo sac isolates the latter from the contents of the locular cavity whereas the callose in the synergid walls may represent an intermediate stage in the maturation of these walls; the filiform apparatus is mainly composed of callose.     

In vitro pollination as a model for studying fertilization in maize (Zea mays L.)

Summary In vitro pollination was conducted using excised segments of maize female spikelets to determine the effects of age and silk length on fertilization efficiency and developmental pattern. Ovary development after 15 days resulted in: (1) normal kernels, (2) abnormal kernels and (3) enlarged ovaries; the percentages of each class varied with age. Evidence of double fertilization was observed in both normal and abnormal kernels. In vitro fertilization was traced using silk excision and autoradiography with ³²P-radiolabelled pollen and occurred between 4 and 7 h after the pollination of 4.5-cm-long silks. This study supports the validity of the in vitro pollination method for studying fertilization and emphasizes the importance of using a developmentally sensitive index (silk length) for establishing female developmental stage.     

Biochemical selection of immature,haploid embryos of Zea mays L.

Summary A method was devised for the biochemical selection of immature, haploid Zea mays embryos using Adh1 ^– and either the Stock 6 or indeterminate gametophyte (ig in W23) high haploid-inducing systems. Haploid (Adh1 ^–) embryos survived exposure to levels of allyl alcohol which killed diploid (Adh1 ⁺/Adh1 ^–) embryos. Of the total surviving embryos which were examined cytologically 15% (using ig) and 22% (using Stock 6) were haploid. In two experiments with Stock 6, 100% of the surviving embryos were haploid. To obtain maximum effectiveness of Stock 6 and ig, Adh1 ^– was transferred to stock 6 and W23 backgrounds. Immature, haploid embryos are being used to develop haploid, morphogenic tissue cultures of Zea mays.     

Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag

Summary The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene tag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.     

The duration of DNA synthetic (S) period in Zea mays: A genetic control

Summary The nuclear cycle among several diverse genetic stocks of Zea mays root meristem cells was compared and it was found that there were no significant differences among the nuclear cycle durations and its component phases. The durations of various periods of their mitotic cycles were studied by autoradiography of cells pulse-labelled with tritiated thymidine (3H-TdR). The total nuclear cycle was 10 to 11.5 hours and mitosis was 0.81 to 1.34 hours at 25°C. The S period is the longest interval (50% of the total time) of the nuclear cycle; of the rest of the cycle, G₂ is longer than G₁ or mitosis among all stocks. The constancy of the nuclear cycle among several stocks was adduced as evidence for strict genetic control of the cycle. Furthermore, it is demonstrated the DNA synthesis period is not dependent upon the amount of DNA present.This study is based on a portion of the dissertation presented by the senior author to the Graduate School, The University of Western Ontario, London, Canada, in partial fulfillment of the requirement for the Ph. D. degree     

Deleterious effect of minimal enzymatic treatments on the development of isolated maize embryo sacs in culture

The long-term viability of isolated embryo sacs was studied in maize. Fertilised embryo sacs were digested in order to remove most of the nucellus cells present on their surfaces and then transferred to culture. Experiments on 161 embryo sacs showed that isolation treatments using even minimal enzymatic digestion affected the further development of the embryo sacs. Few embryo sacs survived in culture and those produced only abnormal embryos; they produced no plants. We concluded that embryo sacs isolated through enzymatic digestion may offer limited prospects for long-term studies where normal embryogenic development is required. Alternative strategies are discussed for maize.     

Isolation of the lily embryo sac

Summary A 5%–8% yield of isolated embryo sacs of Lilium longiflorum was obtained using an enzymatic isolation procedure. The best results were obtained with a maceration mixture containing mannitol, pectinase, pectolyase, cellulase, hemicellulase, CaCl₂ and NaOH. All developmental stages of the female gametophyte can be isolated in the living condition, although fewer than expected mature stages were observed. Moreover, only some of the more mature embryo sacs showed a positive fluorochromatic reaction (FCR) at the time of liberation. When the embryo sacs were stored in the enzyme or sugar solution after isolation, the positive reaction rapidly diminished for all stages. The isolated embryo sac and its nuclei were similar in size, shape, and position to the in situ embryo sac. Light microscopical observations of sectioned material revealed an intact cellular structure. However, the deleterious effects of the enzyme solution were sometimes observed in the form of lipid-like accumulations inside the isolated embryo sac.In collaboration with: Reconnaissance Cellulaire et Amélioration des Plantes, Université Cl. Bernard-Lyon I; INRA 879, 43, Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France     

Occurrence of spermine in chromatin of Zea mays

Chromatin prepared from maize shoot tips using as extraction medium including quinacrine as an inhibitor of polyamine oxidase, contained 1.6 pmol spermidine g DNA^-1 and 14.8 pmol spermine g DNA^-1, respectively. This represented 0.1% spermidine and 3.7% spermine as compared with the content of those amines in the whole tissue. No putrescine was detectable in the chromatin preparation. When contamination of polyamines in the preparation was determined by the addition of labeled polyamines to the extraction medium, the ratio of the polyamines in the preparation to those in the extraction medium was 0.1% spermidine and 0.7% spermine, respectively. Spermine in the chromatin preparation was almost fully solubilized by a DNase-treatment, but spermidine was less easily solubilized. Most of the spermine associated with the chromation is chromatin-specific.     

Sterols of male and female compound inflorescences of Zea mays L.

A comparison has been made between the sterols of male and female inflorescences and of pollen from Zea mays. The female inflorescence was shown to contain cholesterol, 24-methylcholesterol, 24-ethyl-5,22-cholestadien-3-ol, 24-ethylcholesterol and (28Z)-24-ethylidenecholesterol. Themale inflorescence contained the same five compounds together with 24-methylenecholesterol. Pollen contained 24-methylenecholesterol as its main sterol together with lesser amounts of cholesterol, 24-ethylcholesterol, (28Z)-24-ethylidenecholesterol, 24-methylene-5-cholest-7-en-3-ol and 4-methyl-24-methylene-5-cholest-7-en-3-ol.     

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.     

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.     

Stress Responses of Zea mays to Cadmium and Mercury

A hydroponic experiment was carried out to characterize the oxidative stress responses of maize seedlings (Zea mays L. cv. Dekalb DK604) to cadmium (Cd) and mercury (Hg). Plants were grown hydroponically for 7 days in a nutrient solution supplemented with several concentrations of Cd and Hg: 0.0 (control), 6 or 30 μM. Growth was inhibited by both metals. The effect was more severe in plants exposed to Hg. Oxidative stress was caused by the exposure to the metals, as quantified by malondialdehyde and carbonyl accumulation, by-products of lipid peroxidation and protein oxidation, respectively. The activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD), enzymes involved in the scavenging of reactive oxygen species, were measured upon metal treatment. We found an activation of a cytosolic APX isoform, as identified by using a specific polyclonal antiserum. However, there were negligible changes in SOD activity. Analysis of thiol-peptides revealed that at 6 μM Cd a remarkable increase in root reduced glutathione (GSH) content occurred, and little effect on the relative content of oxidised glutathione (GSSG) was observed. However, at 30 μM Cd and in plants exposed to 6 and 30 μM of Hg, GSH root content either remained stable or decreased significantly, while the proportion of GSSG increased. Moreover, only Cd was able to induce accumulation of phytochelatins at both assayed concentrations. Apparently, Hg was more toxic than Cd, as inferred from the magnitude of the changes found in the physiological parameters tested.     

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

Summary Sperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [³H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-³H]uridine, about 7 times. Very low incorporation of [6-³H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.Supported by funds from a Strategic Grant (D.D.C.) and an Operating Grant (D.J.G.) from the Natural Sciences and Engineering Research Council of Canada     

The effect of vesicular-arbuscular mycorrhizae and chilling on two hybrids of Zea mays L.

In order to investigate the effect of vesicular-arbuscular mycorrhizae on the chilling resistance of Zea mays, seeds of two hybrids (Pioneer 3902 and Pride 5) were grown in soil inoculated with Glomus mosseae. Germination tests at 10° C and 25° C showed that Pride 5 was more resistant to chilling than Pioneer 3902. Plants grown at 25° C for 6 weeks were given a 1-week chilling treatment at 10° C and the responses of mycorrhizal and nonmycorrhizal plants of the two hybrids were compared. At 10° C, the mycorrhizal plants had greater biomass, carbohydrate, and protein content than the nonmycorrhizal plants.     

Some aspects of organization and histochemistry of the embryo sac ofScilla sibirica sato

Summary The present investigation deals with some of the organizational and histochemical aspects of the embryo sac ofScilla sibirica. Both the synergids and egg cell are invested by PAS-positive complete walls. The filiform apparatus comprises an elaborate system of fibrillar projections, showing extensive ramifications. The micropylar region of the embryo sac wall from where the filiform apparatus originates is composed of three distinct layers. On a histochemical basis it may be surmised that, unlike the egg cell, the synergids are metabolically very active. Two kinds of wall ingrowths (i) massive and highly branched very much akin to the filiform apparatus, and (ii) small tuberculate wall projections, are unique to the antipodal cells of S.sibirica. Small tuberculate projections have also been observed along the wall of the central cell adjacent to the nutrient-rich nucellar cells. The antipodals and the central cell show the presence of starch grains and abundant total proteins. All the cell types in the embryo sac ofS. sibirica are structurally so organized as to meet the requirements of its nutrition during pre- and postfertilization development. The presence of abundant PAS-positive granular substance in the cells of nucellar epidermis probably establishes a gradient which assists in the pollen tube growth.