Identification of embryonal antigens of maize: Globulins as primary reserve proteins of the embryo

Normal and -irradiated caryopses of Zea mays L. were germinated, and the degradation of embryonal antigens (EA) was followed in the endosperms, scutella and embryonic axes of the seedlings, using double immunodiffusion, immunoelectrophoresis and quantitative immunoprecipitation. The predominant transient EA were presumed to be storage proteins related to the reserve globulins of dicotyledonous seeds. Therefore globulins were isolated from maize scutella, purified by (NH₄)₂SO₄ fractionation and isoelectric precipitation, and the molecular weights of the polypeptide units were estimated by discontinuous sodium-dodecyl-sulphate slab electrophoresis. The globulins were found to be identical with the predominant EA and amounted to about 40% of the protein nitrogen in the embryos of mature, non-germinated caryopses. The presumed reserve function of the globulins and the characteristic time course of their degradation in embryonic axes and scutella of maize seedlings are discussed in relation to the two-step pattern of mobilization of nitrogen reserves in germinating cereal caryopses.Abbreviations AG, AR and AS antisera to globulins and to root and scutellum proteins, respectively - CA common antigens - DAP days after pollination - EA embryonal antigens - ME 2-mercaptoethanol - SDS-PAGE discontinuous sodium-dodecylsulphate slab polyacrylamide-gel electrophoresis - TCA trichloroacetic acid     

Evidence for proteins specific for vascular elements in intact and cultured tissues and cells of maize

A group of antigenically distinct proteins characteristic for the tissue complex of the vascular cylinders was found in maize (Zea mays L.) seedlings using an immunofiltration technique. Specific stelar antigens present in the fully developed stele (vascular cylinder) of the primary root were also found in steles extracted from adventitious roots and from the mesocotyl but were absent, within the limits of sensitivity of the immunodiffusion tests employed, in root cortex and epidermis. Some of the stelar antigens were also evident in the meristem of the primary root and were present in traces in the scutellum, the mesocotyl node, and the primary leaves plus coleoptile. The specific stelar antigens could be traced in 13- and 15-day-old developing embryos and were definitely expressed by the 21 st day after pollination. Several stelar-specific antigens were found in embryo-derived callus tissues and in stem-derived cells maintained in serial suspension culture. Higher resolution of the stelar antigens by a modified technique of crossed immunoelectrophoresis was used to demonstrate several minor stelar antigens that were presumably characteristic exclusively of the completely differentiated stele. This technique along with sequential immunoprecipitation of labelled proteins provided a semiquantitative estimate of the specific stelar antigens in the meristem and the stele of the primary root, and in suspension-cultured cells which were devoid of noticeable signs of vascular differentiation.     

Identical embryonal proteins in intact and isolated tissues of maize (Zea mays L.)

Minor antigens characteristic of developing and mature embryos were not found in the shoot and root meristems of the seedlings. Some of these embryonal antigens (EA) were present, however, in callus and cell-suspension cultures, irrespective of their tissue origin, and were maintained throughout repeated subcultures, in some cases for more than 2 years. These EA were distinct both from the meristematic antigens found in the intact seedlings and in callus cultures, and from organ-specific antigens found only in intact plants. The EA of callus tissues derived from several maize genotypes were serologically identical. We therefore assume that these EA are proliferation proteins or early proteins expressed by cells that have not undergone any determination and lack any tissue or organ specificity.     

The embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.     

Is the wild oat embryo monocotylous?

The embryogeny of the wild oat (Avena fatua L.) was studied in detail. The pattern of embryo development was observed to be similar to the other investigated grass taxa, conforming to thePoa variation of the Asterad type. The embryogeny and anatomy of young seedlings showed that the embryo of the wild oat was not monocotylous, but dicotylous. The scutellum of the embryo, as reported for other grasses, was regarded as the first cotyledon, and the first leaf primordium, which developed later into a photosynthesizing leaf and situated opposite the scutellum, was interpreted as the second cotyledon. Observations indicated that the cotyledons of the embryo were placed lateral to the shoot apical meristem, which was terminal in position. The cotyledons were found to be dimorphic in structure and function. The scutellum, a modified cotyledon, functioned as a suctorial organ, transporting nutrients from the endosperm to the embryo axis. The second cotyledon or the first true leaf supplied nutrients directly to the embryo axis through the process of photosynthesis.     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.     

Characterization of Various Recombinant Antigens from <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> for Use in the Immunodiagnosis

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser²³–Glu¹⁷⁶ of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen B1 did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.     

Analysis of variants affecting the catalase developmental program in maize scutellum

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

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.     

Xylanase XYL1p from Scytalidium acidophilum: Site-directed mutagenesis and acidophilic adaptation

The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH’s and temperatures were determined. All mutations increased the pH optimum by 0.5–1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T_m maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.     

Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium. An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction. Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands. We observed that the physical presence of preincubated scutella is required for vir gene induction. Conditioned medium from which preincubated scutella were removed did not induce the vir genes. Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level. These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.     

The anatomy of secondary morphogenesis in cultured scutellum tissues ofSorghum bicolor

Summary Cells of the scutellum of immature embryos ofSorghum bicolor plated onto an agar medium containing 2,4-D give rise to shoots and embryo-like structures and to some callus. Some of the embryo-like structures later develop into typical sorghum embryos complete with scutellum, coleoptile and coleorhiza. The results of anatomical studies of the development of these secondary growth forms by light and scanning electron microscopy suggest that shoots and embryo-like structures can arise directly from cells of the primary scutellum without an intervening callus phase. In some cases it appears that the scutellum of the secondary embryos arises by folding of the scutellum of the sexual embryo and does not arisede novo. In other cases the structures arise from single cells. No evidence was found to indicate that organized structures arose from proliferating callus cells. The unorganized callus which arises initially is not capable of growth through continuous subculture; it produces a purple-black pigment and rapidly becomes necrotic. The significance of these observations is discussed in relation to present views on morphogenesis in cereal cell cultures and their implications forin vitro cell genetics.Abbreviations used in the text 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

桦褐孔菌提取物抗氧化活性研究

以乙酸乙酯和甲醇为提取剂,采用索氏提取法,剩余残渣采用热水浸提,最终得到桦褐孔菌不同极性提取物,对其DPPH自由基、羟基自由基及超氧阴离子自由基清除活性作用进行了研究,确定桦褐孔菌的抗氧化能力,为深入研究和开发桦褐孔菌功能性食品奠定理论基础。实验结果表明桦褐孔菌具有较好的抗氧化活性,其中乙酸乙酯提取物的DPPH自由基清除率、羟基自由基清除率和超氧阴离子自由基清除率均高于其他两组分及BHT,桦褐孔菌提取物有望成为功能性食品组分中合成抗氧化剂的天然替代品。     

Iron Mediated Nitrate Reductase Activity in Different Parts of Young Maize Seedlings

Incubation of 5-d-old maize seedlings in the half-strength Hoagland's nutrient solution containing 10 mM KNO₃ with FeCl₃ or FeSO₄ (0.5 or 2.0 mM) caused a significant increase in nitrate reductase (NR) activity and slightly increased total protein content in root, shoot and scutellum. In case of root, NADPH:NR activity was inhibited contrary to the NADH:NR activity. In spite of NR activity, nitrate uptake was inhibited from 13 to 37 % by the iron. The results presented demonstrate an isoform specific, organ specific, and to some extent salt specific responses of NR to iron.     

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) ⁺ RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.Abbreviations HRGP(s) hydroxyproline-richglycoprotein(s) - DAP days after pollination The present work was supported by grants from Plan Nacional de Investigation Cientifica y Técnica (grant BI088-0242) and European Communities (grant BAP-374). L.R.-A. is the recipient of a fellowship from the Plan Nacional de Formación de Personal Investigador.     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Expression of embryonic and MHC antigens in heterokaryons of murine embryonal carcinoma and human melanoma cells

Mouse embryonal carcinoma cells were fused with human melanoma cells or with cytoplasts of these cells. The expression of embryonic and major histocompatibility complex (MHC) antigens was studied in single heterokaryons and cybrids in the population after fusion. Recognition of heterokaryons by differential staining of mouse and human nuclei was combined with indirect immunofluorescent staining of specific membrane antigens. Complete suppression of embryonic antigen expression was found in heterokaryons within 2 days after fusion. Cybrids, formed by fusion of embryonal carcinoma cells with melanoma cytoplasts, showed a transient decrease in the expression of embryonic antigens. The expression of human MHC antigens, both class I (HLA-A, B, C) and class II (HLA-DR), was only slightly influenced in heterokaryons. No activation of mouse MHC antigens was found. The results indicate that melanoma cells contain trans-acting factors exerting a negative control on the expression of embryonic antigens. In contrast the continued expression of human MHC antigens in heterokaryons suggests that embryonal carcinoma cells either are devoid of or contain only a very limited amount of trans-acting factors controlling the expression of MHC antigens.     

Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) somatic embryogenesis from isolated scutellum: Days post anthesis,days of spike storage,and sucrose concentration affect efficiency

Summary To improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l⁻¹ casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.     

Synthesis of the peptide-transport carrier of barley scutellum during the early stages of germination

Development of peptide-transport activity in the scutella of isolated barley (Hordeum vulgare l. cv. Maris Otter, Winter) embryos is shown to increase rapidly after about 15 h of imbibition, with the bulk of the transport activity being expressed by 24 h. This development is prevented by treatment of 15 h embryos with cycloheximide. Protein synthesis is found to increase in a closely related manner and also to be abolished by cycloheximide. Measurement of the incorporation of bound [³⁵S]methionine by 15 to 21-h embryos indicates that de-novo protein synthesis during this period is greater in the scutellum than in the embryonic axis. Previous studies have shown that the peptide-transport system possesses essential dithiol groups, probably located at the substrate-binding site (Walker-Smith and Payne 1983 b, 1984b). Treatment of 15-h embryos with the non-penetrant thiol reagent p-chloromercuribenzene sulphonic acid did not affect development of peptide-transport activity during the following 6 h, whereas with 3-d embryos identical treatment inhibited uptake almost completely during a subsequent 6-h period. Radioautography revealed that amongst the proteins synthesised during this early phase of germination and labelled in vitro with [³⁵S]methionine some are found within the epithelial plasmalemmae of the scutellum, which is the location of the peptide-transport carrier identified previously by externally labelling with a radioactive thiol reagent. The results provide evidence that protein(s) of the peptide-transport system are synthesised and inserted into the scutellum during early germination, allowing the system to play a major role in the nitrogen nutrition of the embryo.Abbreviations Gly Glycine - Phe phenylalanine     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.