Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : IV. Characteristics of the Perisperm during Seed Development

We previously reported that an apparent water potential disequilibrium is maintained late in muskmelon (Cucumis melo L.) seed development between the embryo and the surrounding fruit tissue (mesocarp). To further investigate the basis of this phenomenon, the permeability characteristics of the tissues surrounding muskmelon embryos (the mucilaginous endocarp, the testa, a 2- to 4-cell-layered perisperm and a single cell layer of endosperm) were examined from 20 to 65 days after anthesis (DAA). Water passes readily through the perisperm envelope (endosperm + perisperm), testa, and endocarp at all stages of development. Electrolyte leakage (conductivity of imbibition solutions) of individual intact seeds, decoated seeds (testa removed), and embryos (testa and perisperm envelope removed) was measured during imbibition of freshly harvested seeds. The testa accounted for up to 80% of the total electrolyte leakage. Leakage from decoated seeds fell by 8- to 10-fold between 25 and 45 DAA. Presence of the perisperm envelope prior to 40 DAA had little effect on leakage, while in more mature seeds, it reduced leakage by 2- to 3-fold. In mature seeds, freezing, soaking in methanol, autoclaving, accelerated aging, and other treatments which killed the embryos had little effect on leakage of intact or decoated seeds, but caused osmotic swelling of the perisperm envelope due to the leakage of solutes from the embryo into the space between the embryo and perisperm. The semipermeability of the perisperm envelope of mature seeds did not depend upon cellular viability or lipid membrane integrity. After maximum seed dry weight is attained (35-40 DAA), the perisperm envelope prevents the diffusion of solutes, but not of water, between the embryo and the surrounding testa, endocarp, and mesocarp tissue.     

Leakage of solutes from leaf discs of alfalfa plants susceptible and resistant to bacterial wilt caused byCorynebacterium michiganense pv.insidiosum

Seven day- and six week-old alfalfa (Medicago sativa L.) plants, susceptible and resistant to bacterial wilt, were inoculated withCorynebacterium michiganense pv.insidiosum (McCulloch) Dye & Kemp. Leakage of solutes absorbing UV light from leaf discs into distilled water was investigated. The bacterial infection did not affect solute leakage rate from unifoliate and trifoliate leaves of either susceptible or resistant plants at an early stage of the disease. This may indicate that cell membrane integrity in alfalfa leaf tissues was not impaired.     

On the mechanism of aging in soybean seeds

Changes in seeds of soybeans (Glycine max [L.] Merr. var. Wayne) which occur during accelerated aging (41 C, 100% relative humidity) showed subsequent loss of vigor, a decline in early respiratory activity, increased leakage of electrolytes, losses of as much as 10% dry weight from imbibing cotyledons, and a decrease in the swelling response of the imbibing system (seed plus H₂O). Each of these changes with aging is interpreted as resulting from deteriorative changes in membranes.     

Solute Leakage from the Isolated Parenchyma of Allium cepa and Kalanchoe daigremontiana

Efflux of different solutes from leaf slices of Kalanchoëdaigremontiana and from slices of the onion bulb scale was reinvestigatedwith respect to (1) dependence on turgor, (2) selectivity, (3)integrity of protoplasts and cellular changes. In both materials efflux of solutes (electrolytes or sugars)is non-selective and strongly dependent on turgor. Treatmentof tissue slices with hypotonic solutions (below a criticalosmotic pressure) resulted in high leakage rates, an increasein free space and an increased number of damaged cells. Lowconcentrations of calcium did not prevent this loss of retentionand cell stability. Part of the surviving cells were found to have a strongly decreasedosmotic pressure of cell sap. Leakage did not occur simultaneouslyat all cells of the tissue slice. It can be concluded that effluxfrom parenchyma cells in hypotonic solutions results from irreversibleosmotic breakdown and reversible membrane defects both favouredby high turgor. Key words: Parenchyma cells, Allium cepa, Kalanchoé daigremontiana, Solute efflux, Viability, Permeability, Plasmoptysis     

Yeast killer factor: ATP leakage and coordinate inhibition of macromolecular synthesis in sensitive cells

The action of the yeast killer factor proteins on sensitive yeast cells has been examined. The killer factor caused a coordinate inhibition of protein synthesis, nucleic acid synthesis and d-[⁴C]glucose incorporation into macromolecules in growing sensitive cells. During the inhibition period ATP became detectable in the growth medium and the cellular ATP pool level fell to exhaustion. ATP synthesis continued over this period as extracellular ATP accumulated to levels 4–20-fold those found in the cellular pools of control cultures. Leakage studies on other cellular components over the ATP leakage period indicated little loss of macromolecules, but an increased efflux of pools of leucine and glucose. The results are consistent with a killer-induced alteration in the yeast cell membrane.     

Dehydration effects on imbibitional leakage from desiccation-sensitive seeds

Changes in electrolyte leakage and viability in response to dehydration stress were examined in two species of seeds that do not survive desiccation. Leakage from silver maple (Acer saccharinum L.) seeds increased markedly as seed moisture contents decreased from 45 to 35% (fresh weight basis) and germination decreased from 97 to 5%, coincidentally. Time course curves of imbibitional leakage from areca palm (Chrysalido-carpus lutescens [Bory] Wendl.) embryos showed an increase in both initial leakage and steady-state leakage rates in response to dehydration from an original moisture content of 84 to as low as 53%. Absorbance at 530 nanometers of extracts from triphenyl tetrazolium chloride stained embryos of areca palm was used as a measure of viability. Absorbance decreased significantly in response to dehydration as embryo moisture content decreased from 80 to 30%. Collectively, the data suggest that membranes in the desiccation-sensitive seed tissues studied are damaged by dehydration below a critical moisture content, 40% in silver maple seed and 55% in areca palm embryos, and that the membrane damage contributes to loss of viability.     

Anaesthetic Enhancement of Echinochloa crus-galli (L.) Beauv. Seed Germination: Possible Membrane Involvement

Taylorson, R. B. 1988. Anaesthetic enhancement of Echinochloacrus-galli (L.) Beauv. seed germination: possible membrane involvement.—J.exp. Bot 39: 50–58. Dormant E. crus-galli seeds can be stimulated to germinate bycontact with solutions of several anaesthetic-like substances.The order of activity was n-pentanol > benzyl alcohol >n-butanol > n-propanol > ethanol. 2-propanol is nearlyinactive. The relative activity of the substances as germinationstimulants is closely related to their membrane-buffer partitioncoefficients in a manner similar to their effectiveness as anaestheticsin animals. Thus, n-pentanol is a more active stimulant of germinationthan ethanol and its increased effectiveness is correlated witha higher membrane-buffer partition coefficient Similarly, ananaesthetic that stimulated germination (n-propanol) causedgreater leakage of electrolytes, U.V. absorbing substances andamino acids from E. crus-galli seeds than a substance that wasnot active as a germination stimulant (2-propanol). In animals,action of anaesthetics can be prevented by application of externalpressure, an argument for membrane action. Action of n-propanolin stimulating germination can be similarly prevented by pressurein E. crus-galli seeds. The several lines of evidence supporta membrane action for anaesthetic-like substances in seeds and,accordingly, a membrane site as the locus of seed dormancy. Key words: Anaesthetics, germination, membranes     

DON抑制小麦种胚细胞周期启动的研究

小麦种子在０．５－４．０ｍｇ／Ｌ的脱氧雪腐镰刀菌烯醇（ＤＯＮ）中溶液中萌发,用流式细胞仪检测胚细胞的周期时相并观察黄化苗的生长。结果表明,对照组胚细胞在萌发１２ｈ开始启动细胞周期,此后Ｓ期细胞比率迅速增加并在２０ｈ达到最大值,较萌发１０ｈ的６．５％增加１．７８倍,约１４％的胚细胞在萌发的２２－２６ｈ间完成第一次分裂;ＤＯＮ抑制Ｇ１期胚细胞的启动,小麦胚中Ｓ期细胞比率随ＤＯＮ浓度增加呈指数下降,３．     

The diffusion of gibberellins into agar and water during early germination of Pharbitis nil Choisy

Agar diffusion of imbibed seeds yielded significant amounts of diffusible Gibberellin-like substances. An analysis of the extractable and diffusible gibberellin-like substance, including an analysis of the remaining imbibition water of the seeds, indicated that a significant part of these gibberellin-like substances could be attributed to a net biosynthesis of these substances in the imbibing seeds. At the same time it was found that water diffusion yielded considerably more gibberellin-like activities than comparable agar diffusions i.e. 10 to 12 fold in general.Agar as well as water diffusion showed a temperature effect with regard to the yield of gibberellin-like substances particularly during the first 6 h of diffusion. The yield of these substances is lower at 10°C, and remains lower as shown with consecutive diffusions, in comparison with the yields at 20°C or 30°C.With both agar and water diffusion the sum of activities obtained with consecutive diffusions is always higher, often considerably higher, than equal periods of continuous diffusion which is probably due to inactivation and/or interference of inhibitory substances with the bioassay responses. Finally, water diffusates of both seeds and seedlings of the normal growing cv. Violet of Japanese morning glory contained considerably more gibberellin-like activities than those of the dwarf cv. Kidachi which indicated that normals synthesize more gibberellins than dwarfs.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.     

A Physical Explanation for Solute Leakage from Dry Pea Embryos During Imbibition

The pattern of solute leakage from imbibing dead pea (Pisumsativum L.) embryos was the same as that from living embryos,with an initially high leakage declining to a low constant rateof leakage in the first 3 min of imbibition. The same patternof leakage occurred during each imbibition phase of repeatedimbibe/dry cycles of dead embryos. Living and dead seeds alsoshowed this pattern of leakage. These observations are usedto argue that leakage during imbibition of embryos and seedsis a physical diffusion phenomenon. Vital staining of livingembryos after imbibition revealed positive staining for dehydrogenaseenzymes in the cells on the outer surface of the cotyledonsonly when 0.5 mM sodium succinate solution was present duringimbibition and/or staining. This is discussed in relation tothe effect of rapid water uptake on these cells.     

Diurnal change in leakage of electrolytes from a long-day duckweed, Lemna gibba G3, under osmotic stress induced by water treatment

The leakage of electrolytes from Lemna gibba G3 placed on waterwas investigated in terms of the change in the electroconductivityof the ambient water. Amounts of the leaked electrolytes reacheda maximum 5–6 hr after transfer to water, followed bygradual decrease. The maximum amount of the leakage changeddiurnally, with the time of the water treatment, with a peakin the early subjective day and a trough in the early subjectivenight. The rhythm was reset by a light-on signal, persistedfor at least 3 days under continuous light, and its period wastemperature-compensated. The rhythm was maintained even in thepresence of 2,4-dinitrophenol or 3-(3,4-dichlorophenyl) 1,1-dimethylurea and under weak light, suggesting that a changein passive efflux of electrolytes was involved, and this wasconfirmed by kinetic studies which revealed that the leakageoccurred by simple diffusion. Strong illumination extinguishedthe rhythm, probably by promoting the active reabsorption ofthe leaked ions. Potassium ions were the major cations found,to the leakage of which was attributed half of the change inthe electroconductivity of the ambient water. (Received January 18, 1978; )     

Cp-S316菌株抗细菌活性物质对大肠杆菌的抑制作用及高产突变株选育

通过测定大肠杆菌K12 (Escherichia coli K12)菌悬液的OD260的变化, 研究了多粘类芽孢杆菌(Paenibacillus polymyxa)Cp-S316抗细菌活性物质对其细胞膜完整性的影响, 结果表明Cp-S316抗细菌活性物质可损伤大肠杆菌K12的细胞膜, 从而引起胞内RNA、DNA等大分子物质的泄漏。为获得抗细菌活性物质高产菌株, 以Cp-S316为出发菌株, 通过紫外诱变以及对自身产生的抗细菌活性物质的抗性筛选法进行预筛、摇瓶初筛和复筛, 获得突变株多粘类芽孢杆菌A17, 其发酵效价比出发菌株Cp-S316提高91%, 该突变株的高产遗传性状稳定。     

Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 μg/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺. Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca²⁺ and Na polygalacturonate.     

In vitro antibiofilm and anti‐adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic‐resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X‐ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram‐negative bacteria. Cell adherence assay indicated 25.3–49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time‐dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non‐toxic to HeLa cells at concentrations of 15–120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug‐resistant bacteria.

     

Prevention of imbibitional injury in low vigor soybean embryonic axes by osmotic control of water uptake

Deterioration of soybean [ Glycine max (L.) Merr. cv. Essex] seeds during accelerated aging at 41°C and 100% relative humidity predisposes the embryonic axis to injury during the initial period of imbibition. This injury was prevented or greatly reduced in severity when excised axes were imbibed on blotters containing 30% polyethylene glycol which slowed the rate of water uptake and when axes were pre-equilibrated to a high moisture level. Rates of water uptake by "high"(no treatment) and "low vigor"(accelerated aged) excised axes were identical. However, high vigor axes tolerated rapid water uptake during early imbition, whereas low vigor axes did not. Leakage of electrolytes during early imbibition was nearly six times greater in low than in high vigor axes. Polyethylene glycol significantly reduced the leakage of electrolytes from both low and high vigor axes. The data are in agreement with the hypothesis that seed deterioration in soybeans involves membrane changes which may predispose embryonic tissues to injury during imbibition. Reduction of the rate of water uptake during the initial period of imbibition would allow extra time for membrane repair or rearrangement, thus permitting the tissues to develop in a more orderly manner. The data indicate that deterioration in soybean seeds involves, at least in part, a decrease in ability of seed axes to tolerate rapid water uptake at the start of imbibition and that this weakness may be compensated by osmotic control of water uptake.     

Role of Endogenous Plant Growth Regulators in Seed Dormancy of Avena fatua: II. Gibberellins

Gibberellin A₁ (GA₁) was identified by combined gas chromatographymass spectrometry as the major biologically active gibberellin (GA) in seeds of wild oat (Avena fatua L.) regardless of the depth of dormany or stage of imbibition. Both unimbibed dormant and nondromant seeds contained similar amounts of GA₁ as estimated by the d5-maize bioassay. During imbibition, the level of GA₁ declined in both dormant and non-dormant seeds, although the decline was more rapid in dormant seeds. Only in imbibing nondormant seeds did the GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride (CCC), cause a reduction in the level of GA₁ from that observed in control seeds. These results are interpreted as an indication that while afterripening does not cause a direct change in the levels of GAs during dry storage, it does induce a greater capacity for GA biosynthesis during imbibition.

Nondormant seeds imbibed in the presence of 50 millimolar CCC germinated equally as well as untreated seeds. When wild oat plants were fed CCC throughout the entire life cycle, viable seeds were produced that lacked detectable GA-like substances. These seeds afterripened at a slightly slower rate than the controls. Moreover, completely afterripened (nondormant) seeds from plants fed CCC continuously contained no detectable GA-like substances, and when these seeds germinated, dwarf seedlings were produced, indicating GA biosynthesis was inhibited during and after germination. In total, these results suggest that the increased capacity for GA biosynthesis observed in imbibing nondormant seeds is not a necessary prerequisite for germination. It is therefore possible that GA biosynthesis in imbibing nondormant seeds is one of many coordinated biochemical events that occur during germination rather than an initiator of the processes leading to germination.

     

Decrease in sunflower (Helianthus annuus) seed viability caused by high temperature as related to energy metabolism, membrane damage and lipid composition

The aim of the present work was to investigate whether loss of germination ability and viability of sunflower (Helianthus annuus L.) seeds during incubation at a high temperature (45°C) was related to changes in energy metabolism, loss of membrane integrity, and/or changes in lipid composition. Pre‐treatment of seeds at 45°C progressively reduced subsequent germination at the optimal temperature (25°C). Seeds did not germinate at 45°C and almost all of them were dead after 72 h of soaking at this high temperature. This loss of seed viability was associated with a large increase in leakage of K⁺ and total electrolytes into the incubation medium, and with production of malondialdehyde in the embryonic axis and cotyledons, suggesting a loss of membrane integrity probably due to lipid peroxidation. ATP and ADP levels increased sharply during the first hours of imbibition at 45°C, remained high for about 24 h and then decreased. As a consequence, the energy charge followed a similar pattern. If the treatment at 45°C did not exceed 48 h, seeds recovered an apparently normal energy metabolism after transfer to 25°C, even though they lost their ability to germinate at this temperature. Therefore, energy metabolism at the whole embryo level cannot be considered as an indicator of germination ability. Incubation of seeds at 45°C resulted in an increase in triacylglycerols and diacylglycerols without a significant change in their fatty acid composition. It also induced a slight increase in phospholipid content with an increase in C_16:0, C_18:0 and C_18:1, but with no change in C_18:2. In phospholipids, the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios thus declined during treatment at 45°C. The results obtained suggest that deterioration of sunflower seeds during incubation at a high temperature is mainly related to membrane damage and alteration of energy metabolism, and that accumulation of malondialdehyde, which is an index of lipid peroxidation, does not correspond to a decrease in total lipids and phospholipids nor to a significant change in fatty acid composition, except in PL in which the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios slightly declined.     

The Effect of Temperature Change on Leakage from Pea Seeds

Intact seeds of P. sativum and P. elatius leaked electrolytes,sugars and proteins for as long as 48 h after the beginningof imbibition. Initial leakage was higher at 25 °C thanat 5 °C, but its extent was much greater at the lower temperature.Transfer of seed from 5 °C to 25 °C after 5 h imbibitionresulted in leakage for 48 h at the initial rate at 5 °C.The transfer from 25 °C to 5 °C gave leakage at a rateequivalent to the initial rate at 25 °C. The results arediscussed in relation to behaviour of membranes and to the sensingof the initial temperature. Key words: Pisum sativum, P. elatius, Leakage, Electrolytes, Sugars, Proteins, Temperature     

Effect of laser irradiation on microviscosity of aqueous medium in imbibing maize seeds as studied with a spin probe method

A spin probe method was used to study microviscosity of aqueous medium in embryos and endosperm of maize (Zea mays L.) seeds after their irradiation with a Lvov-1 Elektronika laser device. Based on parameters of electron spin resonance (ESR) spectra of nitroxyl radicals (probes) absorbed by imbibing seeds during water uptake, correlation times τ_C were determined for the rotational diffusion of probes in embryos and endosperm of seeds. The τ_C values were found smaller in embryos of irradiated seeds than in untreated seeds; the dependence of τ_C on the duration of seed imbibition was determined. It is concluded that laser irradiation of seeds decreases the microviscosity of aqueous medium in embryo cells and elevates the mobility of the probes. Effect of laser irradiation on τ_C in seed endosperm was less pronounced but also led to the increase in probe mobility.