Cooperation of epidermis and inner tissues in auxin-mediated growth of maize coleoptiles

The function of the epidermis in auxinmediated elongation growth of maize (Zea mays L.) coleoptile segments was investigated. The following results were obtained: i) In the intact organ, there is a strong tissue tension produced by the expanding force of the inner tissues which is balanced by the contracting force of the outer epidermal wall. The compression imposed by the stretched outer epidermal wall upon the inner tissues gives rise to a wall-pressure difference which can be transformed into a water-potential difference between inner tissues and external medium (water) by removal of the outer epidermal wall. ii) Peeled segments fail to respond to auxin with normal growth. The plastic extensibility of the inner-tissue cell walls (measured with a constant-load extensiometer using living segments) is not influenced by auxin (or abscisic acid) in peeled or nonpeeled segments. It is concluded that auxin induces (and abscisic acid inhibits) elongation of the intact segment by increasing (decreasing) the extensibility specifically in the outer epidermal wall. In addition, tissue tension (and therewith the pressure acting on the outer epidermal wall) is maintained at a constant level over several hours of auxin-mediated growth, indicating that the inner cells also contribute actively to organ elongation. However, this contribution does not involve an increase of cell-wall extensibility, but a continuous shifting of the potential extension threshold (i.e., the length to which the inner tissues would extend by water uptake after peeling) ahead of the actual segment length. Thus, steady growth involves the coordinated action of wall loosening in the epidermis and regeneration of tissue tension by the inner tissues. iii) Electron micrographs show the accumulation of striking osmiophilic material (particles of approx. 0.3 m diameter) specifically at the plasma membrane/cell-wall interface of the outer epidermal wall of auxin-treated segments. iv) Peeled segments fail to respond to auxin with proton excretion. This is in contrast to fusicoccin-induced proton excretion and growth which can also be readily demonstrated in the absence of the epidermis. However, peeled and nonpeeled segments show the same sensitivity to protons with regard to the induction of acid-mediated in-vivo elongation and cell-wall extensibility. The observed threshold at pH 4.5–5.0 is too low to be compatible with a second messenger function of protons also in the growth response of the inner tissues. Organ growth is described in terms of a physical model which takes into account tissue tension and extensibility of the outer epidermal wall as the decisive growth parameters. This model states that the wall pressure increment, produced by tissue tension in the outer epidermal wall, rather than the pressure acting on the inner-tissue walls, is the driving force of growth.Abbreviations and symbols E _el, E _pl elastic and plastic in-vitro cell-wall extensibility, respectively - E _tot E _el+E _pl - FC fusicoccin - IAA indole-3-acetic acid - IT inner tissue - ITW inner-tissue walls - OEW outer epidermal wall - osmotic pressure - P wall pressure - water potential     

Immunocytochemical evidence for methylation of the inactive X chromosome in human fetal oogonia

The state of DNA methylation of the X chromosomes of human interphase oogonia from a 46,XX and a 46,XX/47,XXX fetus at 17 weeks of gestation was tested immunocytochemically with an antibody to 5-methylcytosine (5MeC). Of 1637 oogonial nuclei from the 46,XX fetal ovary, 313 (19.1%) contained Barr bodies, of which 93.6% were positive for 5MeC. Of 1780 oogonia from the 46,XX/47,XXX fetus 327 (18.4%) contained Barr bodies; 175 oogonia had one Barr body and 152 had two. Of the single Barr bodies 145 (82.8%) had positive 5MeC reaction product. Of the 152 oogonia from the XXX line, 97 (63.8%) had positive 5MeC on both Barr bodies, 35 (23%) had one positive and one negative, and 20 (13.1%) had no product on either Barr body. This immunocytochemical evidence supports the hypothesis that the DNA of the inactive X-chromosome of the human 17-week gestation oogonium is methylated.     

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.     

Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations

Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl₂. On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl₂ the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K⁺ concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl₂ and probably played a key role in determining growth capacity. (b) Internal concentrations of Na⁺, Ca²⁺, or Cl⁻ could not be directly correlated with growth. (C) Internal Mg²⁺ concentration could be significant only in the presence of high external Ca²⁺ concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca²⁺)/(Na⁺)² concentration is crucial for growth. Ratios above 0.5 × 10⁻⁴ per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca²⁺)/(Na⁺)² ratio and the absolute external concentration of NaCl. However, a correlation between internal K⁺ concentration and growth capacity seemed independent of external NaCl concentration.     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

Modifications in repair and expression of potentially lethal damage (alpha-PLD) as measured by delayed plating or treatment with beta-araA in plateau-phase Ehrlich ascites tumor cells after exposure to charged particles of various specific energies

The ability of Ehrlich ascites tumor cells (EAT cells) to repair potentially lethal damage (alpha-PLD) as demonstrated by either an increase in survival after delayed plating or a decrease in survival after treatment with beta-arabinofuranosyladenine (beta-araA) was investigated after exposure to protons, deuterons, 3He, 4He, and heavy ions of various specific energies. A significant amount of repair or fixation was observed after delayed plating or treatment with beta-araA, respectively, in cells that were exposed to protons of 6-21 MeV energy, reflecting mainly variations in the survival curve shoulder width. Four-hour treatment with 80 microM/liter beta-araA resulted in an exponential survival curve for all proton energies tested. A decrease in particle energy increased killing and caused a reduction in Dq without a significant change in D0. The survival curve obtained after exposure of cells to 3.4 MeV protons had only a small shoulder and was only slightly modified by either delayed plating or treatment with beta-araA, suggesting a decrease in the induction rate of alpha-PLD. Similar results were also obtained after exposure to deuterons and 4He ions. The results are interpreted as indicating the importance of the specific particle energy and the delta-electron spectrum in the induction of alpha-PLD. When the results of delayed plating of cells exposed to protons, deuterons, or helium ions were pooled, an exponential relationship between Dq and penumbra radius was indicated. After exposure to 40Ar ions of 18 MeV specific energy, a shouldered survival curve was obtained, and beta-araA significantly enhanced killing by modifying Dq as well as D0, a result that also suggests induction of repairable damage by the delta particles produced and interaction of lesions induced within the core of the ion path with penumbra lesions. Based on these results a model is proposed assuming that alpha-PLD results from interaction, during the course of repair, of pairs of DNA lesions induced within a distance di. The model assumes the existence of a critical separation distance dic, with the property that pairs of lesions induced with separation distance shorter than dic (expressed as number of base pairs) will always be expressed as lethal, and the existence of a maximum separation distance dim, with the property that pairs of lesions induced with separation distance larger than dim will not interact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytoskeletal elements in cotton seed hair developmentin vitro: Their possible regulatory role in cell wall organization

Summary The localization and orientation of cytoskeletal elements in developing cotton fibres were studied by the indirect immunofluorescence and the dry cleaving technique. Microtubules are transversely arranged to the cell axis, most probably in a flat helix, in the cortex of expanding fibres. Since the innermost deposited cellulose microfibrils always show primarily the same orientation it is postulated that the microtubules control the transverse deposition of the cellulose fibrils. Little further cell expansion takes place during secondary wall formation and the microfibril pattern corresponds to that of the cortical microtubules,e.g., in the steepness of their helicoidal turns. Microtubules with a length of 7–20 m were observed, probably they are longer. The importance of microtubule length on microfibril deposition is discussed. The density of microtubule packing is in the range of 8–14 m^-1 as in other comparable cell types. In contrast to the microtubules, actin filaments are most likely longitudinally oriented during different phases of fibre development. The dry cleaving technique reveals numerous coated pits in the plasma membrane which are not crossed by microtubules. They seem to be linked to the latter by filamentous structures.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.