Stimulation of Endomitotic DNA Synthesis and Cell Elongation by Gibberellic Acid in Epicotyls Grown from Gamma-irradiated Pea Seeds

Large doses of γ-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced.     

Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall.     

Gibberellic Acid Regulates Cell Wall Extensibility in Wheat (Triticum aestivum L.)

Mutations (Rht genes) blocking sensitivity to gibberellic acid (GA) were used to examine phytohormone mediated cell wall expansion in wheat (Triticum aestivum L.). Irreversible extensibility of immature leaf segments, as determined by stress/strain (instron) measurements, declined with Rht gene dose. Exogenous GA₃ significantly increased wall extensibility in the nonmutant controls but had no effect on the near-isogenic GA-insensitive genotypes. Furthermore, ancymidol, an inhibitor of gibberellin biosynthesis, diminished wall extensibility in the nonmutant control. Extensibility of immature segments was highly correlated with mature leaf sheath length (R = +0.95). The results indicate that wall yielding properties of expanding wheat leaves are associated with leaf cell expansion potential and that GA is involved in the determination of those properties.     

The Control of Elongation in Callitriche Shoots by Environment and Gibberellic Acid

Gibberellic acid (GA) was found to stimulate internode expansionin floating rosettes of the aquatic, Callitriche stagnalis.Treated shoots resembled those normally produced from submergedapices. The environmental factor which controls the growth ofuntreated floating rosettes was investigated, and found to beloss of water by transpiration. The morphological effects ofdifferent concentrations of GA are described. The similarityis pointed out between environmental dwarfing in Callitricheand genetic dwarfing in Pisum.     

The Effect of Indoleacetic Acid on Cell Wall Extensibility in Avena Coleoptiles

The extensibility of cell walls in segments of living Avenacoleoptiles has been determined after treatment with IAA bythe use of a microextensometer. It has been demonstrated thatIAA has an effect both on the plastic and elastic extensibility,which is more marked if the segments are allowed to grow, exceptat temperatures nearing 0° C. The auxin-induced increasein extensibility is therefore not a direct effect on the walland metabolic factors must be involved. The results are interpreted,along cunent lines of thought, as indicating effects on thelinkage between protopectin or hemicellulose chains themselvesand between these and the microfibrillar network of cellulose.The bearing of these interpretations on the normal growth processin cell walls is briefly discussed.     

小麦黄化胚芽鞘的细胞壁钙调素和钙调素结合蛋白

小麦黄化胚芽鞘经苯基琼脂糖亲和层析提取和纯化,其细胞壁CaM在有钙和缺钙时SDS电泳呈现不同的迁移率;依赖Ca~(2 )与苯基疏水结合;在紫外吸收光谱上具有五个特征峰;对PDE的激活剂量反应曲线和从非活性状态向活性状态转变时所需的Ca~(2 )浓度均和胞内CaM相同,说明细胞壁CaM和胞内CaM具有相同的基本理化特性。采用CaM琼脂糖亲和层析,发现在小麦细胞壁中存在CaM结合蛋白,其中以分子量为40.7 kD的 CaM结合多肽为主。细胞壁CaM结合蛋白不具有过氧化物酶、ATP酶或酸性磷酸酯酶的活性。     

Role of Polysaccharide Synthesis in Elongation Growth and Cell Wall Loosening in Intact Rice Coleoptiles

2,6-Dichlorobenzonitrile (DCB) inhibited only increases in levelsof the cellulosic polysac-charides while monensin and galactoseinhibited increases in levels of both the cellulosic and thematrix polysaccharides in intact rice coleoptiles that weresubmerged in water. Elongation growth of rice coleoptiles wassuppressed by DCB at 10^–6 M, by monensin at 10^–7M, and by galactose at 3 ? 10^–3 M and above. Thus, thesynthesis of both the cellulosic and the matrix polysaccharidesis essential for the elongation of intact rice coleoptiles.These inhibitors increased the minimum stress-relaxation timeand the relaxation rate and they decreased the mechanical extensibilityof the cell wall, indicating that they inhibited cell wall loosening.The concentrations of the inhibitors required for inhibitionof cell wall loosening were higher than those for suppressionof elongation. The data suggest that polysaccharides synthesisplays two roles in elongation. It keeps the cell wall in a "loosened"condition by producing new extensible cell walls, while itsother role is probably related to the fixation or extensionof polymers already present in the cell wall. (Received November 15, 1990; Accepted May 23, 1991)     

Effect of Gibberellic Acid and Metabolic Inhibitors of DNA and RNA Synthesis on Hypocotyl Elongation and Cell Wall Loosening in Dark-Grown Lettuce Seedlings

The effect of inhibitors of nucleic acid metabolism on cellwall loosening in hypocotyls of dark-grown lettuce seedlingswas studied in relation to the gibberellic acid (GA) actionthat stimulates hypocotyl elongation. 5-Fluorodeoxyuridine (FUDR),trifluorothymine deoxyribose, mitomycin C. aminopterin, 5-fluorouraciland cordycepin produced mechanical rigidity of the cell wallof the hypocotyl when they inhibited elongation in the presenceor absence of added GA. The effect of FUDR on the mechanicalproperty of the cell wall and on hypocotyl elongation was nullifiedby thymidine, but not by uridine. ¹Present address: Department of Tuberculosis, National Instituteof Health, Shinagawa-ku, Tokyo 141, Japan. (Received September 2, 1980; Accepted January 5, 1981)     

Abscisic Acid Suppresses the Increases in Cell Wall-Bound Ferulic and Diferulic Acid Levels in Dark-Grown Wheat (Triticum aestivum L.) Coleoptiles

Application of abscisic acid (ABA) to dark-grown wheat (Triticumaestivum L.) roots interfered the cell wall hardening of coleoptilesduring several days of the treatment. Although the amounts ofwall-bound diferulic (DFA) and ferulic (FA) acids in coleoptilesincreased as the coleoptiles grew, ABA substantially reducedtheir increases. When ABA was removed, however, these contentsincreased and reached levels near those of control coleoptiles.A close correlation was observed between the levels of DFA andFA and the mechanical properties of cell walls. The ratio ofthe amount of DFA to FA was almost constant irrespective ofgrowth conditions. The activities of phenylalanine- (PAL) andtyrosine-ammonia-Iyase (TAL) increased rapidly in the controlcoleoptiles. ABA greatly reduced the increases in these enzymeactivities. In response to ABA removal, the enzyme activitiesincreased rapidly. There was a close correlation between theincrease in FA level and the changes in enzyme activities. Theseresults suggest that ABA suppresses the increases in PAL andTAL activities in wheat coleoptiles, resulting in the reducedlevel of wall-bound FA, which, in turn, may cause the reducedDFA level and thereby maintain cell wall extensibility. (Received January 10, 1997; Accepted April 22, 1997)     

Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold-hardened Wheat Coleoptiles

Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of ¹⁴CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 μM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-¹⁴C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-¹⁴C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold-hardened and unhardened coleoptiles.     

Independence of Lateral and Differential Longitudinal Movement of Indoleactic Acid in Geotropically Stimulated Coleoptiles of Zea mays

The possible interdependence of the differential longitudinal, and the lateral movement of indoleacetic acid (IAA) in horizontal Zea coleoptile segments has been examined. The coleoptiles have been opened out into flat pieces of tissue and supplied apically with IAA-1-¹⁴C.     

Purification and Immunological Detection of Phytochrome from Wheat Coleoptiles

Phytochrome was partially purified from dark grown wheat coleoptiles by ammonium sulfate precipitation, hydroxyapatite column chromatography and polyethylene glycol precipitation, with a yield of about 27%. Phytochrome prepared by this method contains a dominant species having a molecular weight of 124 kD on SDS-PAGE. This 124 kD polypeptide is recognized by the antiserum raised against partially purified wheat phytochrome in rabbit. Oat anti-phytochrome polyclonal antibodies can also cross-react with phytochrome purified from wheat as assayed by ELISA and immunoblotting.     

Deoxyribonucleic Acid Distribution in Bacillus subtilis Independent of Cell Elongation

A temperature-sensitive DNA(-) mutant of Bacillus subtilis has been studied during the resumption of deoxyribonucleic acid (DNA) synthesis following a 45 to 30 C temperature shift. For several hours after return to 30 C, DNA synthesis proceeds although the cells fail to elongate appreciably. Autoradiographs of cell populations synthesizing DNA during the recovery period demonstrate that DNA can become distributed to previously unoccupied regions along the cell length. By varying the labeling regime, newly synthesized DNA as well as DNA present at the time of transfer from 45 to 30 C were followed independently. Measurements of the percent of cell length covered by grains ((3)H-thymine in DNA) demonstrate the progressive refilling of DNA-vacant cell regions by both newly synthesized and original DNA. These data indicate that cell surface growth is not an absolute requirement for segregation of bacterial DNA.     

Local Calcium Elevation and Cell Elongation Initiate Guided Motility in Electrically Stimulated Osteoblast-Like Cells

Background

Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration.

Methodology/Principal Findings

We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10–15 V/cm) and weak (≤5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation.

Conclusion/Significance

Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.     

Reversal of Ethionine-Induced Inhibition of Elongation of Avena Coleoptiles by Purines, Adenosine, and Glutamic Acid

Ethionine-induced inhibition of elongation of Avena coleoptile segments has been measured in water and in indol-3yl-acetic acid. In the presence of 10 mM L-ethionine the inhibition amounts to about 70 %. It has been shown previously that the addition of adenosine triphosphate effectively counteracts this inhibition; optimal ATP concentrations are between 0.25 and 0.5 mM. Adenine, adenine sulfate, adenosine, guanine, S-adenosylmethionine, and glutamic acid have now been shown to act similarly to ATP in reversing the ethionine-induced inhibition of elongation.     

Oxygen Dependence of Elongation Growth and Oxygen Uptake in Maize Coleoptiles

Auxin-mediated elongation growth of maize coleoptile segments is inhibited by reducing the O₂ concentration in the incubation medium to GT 100 μmol . 1^?1. The half-maximal elongation rate is reached at 40 μmol . 1^?1 O₂, i.e. about two orders of magnitude higher than with mitochondrial respiration. O₂ uptake of the segments measured under similar conditions with an O₂ electrode shows a very similar dependence on O₂ concentration. Auxin increases O₂ uptake by 5–10% when it induces growth. About 40% of the O₂ uptake is insensitive to inhibition by KCN. Auxin has no effect on O₂ uptake in the presence of KCN. The possibility that auxin-mediated elongation growth depends on a KCN-sensitive oxidative process, other than cytochrome c oxidase-catalyzed respiration, is discussed.