Photosynthesis of isolated chloroplasts and protoplasts under osmotic stress

1. Isolated intact spinach chloroplasts respond to changes of the sorbitol concentration of the suspending medium as near-perfect osmometers within a large range of osmotic potentials. Under isotonic conditions (=9–10 bar), their average osmotic volume is 24 m³ and the total volume 36 m³. The osmotic volume can be increased to 63 m³ by lowering the sorbitol concentration until a critical osmotic potential of =4 bar is reached. Below that value chloroplasts rupture. Between 10 bar and 4 bar, volume changes are reversible. 2. Increasing the chloroplast volume above 24 m³ causes inhibition of photosynthesis, with 50% inhibition occurring at an osmotic potential of =5–6 bar. This corresponds to an osmotic volume of 45–55 m³. Depending on the duration of hypotonic treatment, inhibition of photosynthesis is more or less reversible. 3. Between 4 and 10 bar, the chloroplast envelope exhibits a very low permeability for ferricyanide, many metabolites, and soluble stroma proteins. 4. Electron transport is not inhibited by swelling of chloroplasts. Also, the ATP/ADP-ratio remains unchanged. 5. The solute concentration in the chloroplasts appears to be optimal for photosynthesis at 10 bar. Increasing the chloroplast volume causes inhibition of photosynthesis by dilution effects.     

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl₂ 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.     

Transformation of protoplast-derived cell colonies and suspension cultures by Agrobacterium tumefaciens

In this paper we describe procedures for transforming micro colonies derived from mesophyll protoplasts of Petunia hybrida with Agrobacterium tumefaciens. The method is efficient, up to 70% of the colonies were transformed, and we used a similar method to transform cells from a suspension culture of haploid Nicotiana plumbaginifolia.Abbreviations RH Relative humidity - MES 2 (N - Morpholino) ethane sulphonic acid - NAA 1 - Naphthylacetic acid - BAP 6 - Benzyl amino purine - B5 Gambourg's B5 medium (Gambourg 1970) - MS Murashige and Skoog Medium (1962) - PZ saline 0.9% NaCl pH 7.0 - 2, 4-D 2,4, Dichlorophenoxyacetic acid - Ty tryptone yeast     

Effect of fusicoccin on plant cell cultures and protoplasts

We have assayed the capacity of the fungal toxin fusicoccin to induce some of its characteristic effects (acidification of the medium, stimulation of K⁺, and of 3-O-methyl-D-glucose uptake) in cell suspensions of Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L. and Oryza sativa L., and in protoplast suspensions prepared from leaves of Nicotiana tabacum L. and Spinacia oleracea L. or from cultures of P. tricuspidata. Evidence is presented showing that all tested biological materials respond to the addition of fusicoccin. The observation that the toxin is also active on protoplasts indicates that the cell wall is not involved in the mechanism of action of fusicoccin.Abbreviations FC Fusicoccin - 3-O-MG 3-O-Methyl-D-glucose     

Differential effects of calcium ion concentration on cell fusion,cell division and creatine kinase activity in muscle cell cultures

Cell fusion, cell number, soluble cell protein and creatine kinase activity have been measured simultaneously in chick muscle cell cultures exposed to various calcium ion concentrations for various periods of time, by adding either extra calcium chloride or the calcium-chelating agent, EGTA. Up to 0.75 mM EGTA cell fusion is not inhibited, but the specific activity of creatine kinase is reduced by 20–50%. Between 0.75 and 1.7 mM EGTA, cell fusion is gradually abolished and the increase in cell number prevented, but enzyme specific activity actually increases again and returns to control values. Adding extra Ca²⁺ produces small increases in cell fusion and soluble cell protein, but much greater increases in creatine kinase activity. EGTA stimulates thymidine incorporation into DNA at low concentrations and then inhibits again as its concentration is increased further. These effects of EGTA on cell division may be related to its effects on creatine kinase. The implications of these results are discussed in terms of current ideas about the inter-relationships between cell fusion, cell division and the accumulation of muscle proteins during differentiation. In particular they show that cell fusion is not essential for the attainment of normal levels of creatine kinase.     

Effect of fungal homogenate,enzyme inhibitors and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures

The addition of Aspergillus niger homogenate to Catharanthus roseus cell suspension cultures produced an increment of more than 60% in the alkaloid content of two different cell lines. The use of an inhibitor of phenylalanine ammonia lyase, i. e. cinnamic acid, along with the homogenate, resulted in an appearance of 90% of the alkaloids in the medium. Furthermore, even in the absence of fungal homogenate, there was a marked increase in the alkaloid content. The exposure of the cells to an osmotic stress produced a marked increment (320%) in the total alkaloid content. When both stress treatments were applied sequentially, an additive effect on alkaloid accumulation was observed. It was 300% higher than in cells cultured without treatment, the majority of the alkaloids found in the medium.Abbreviations BAP benzylaminopurine - CM chorismate mutase - DW dry weight - FW fresh weight - IAA indole-3-acetic acid - MS Murashige-Skoog - PAL phenylalanine ammonia lyase - PC Phillips-Collins     

Differential induction of enzyme in soybean cell cultures by elicitor or osmotic stress

Soybean cell cultures were challenged either by glucan elicitor from Phytophthora megasperma f.sp. glycinea or by osmotic stress (0.4 M glucose). Osmotic stress induced production of a microsomal NADPH-dependent flavone synthase (flavone synthase II) which catalyses conversion of (2S)-naringenin to apigenin. In one of our cell-lines this enzyme activity was not detected either in unchallenged cells or in cells treated with glucan elicitor. Inducibility of flavone synthase II by 0.4 M glucose was highest at the end of the linear growth phase. Changes in the activities of a number of other enzymes were determined after treatment of the cells with elicitor or 0.4 M glucose. The activities of phenylalanine ammonialyase, cinnamate 4-hydroxylase, chalcone synthase and dihydroxypterocarpan 6a-hydroxylase all increased with elicitor and with osmoticum, albeit to a different degree. The rise in enzyme activity occurred later with osmoticum than with elicitor. The prenyltransferase involved in glyceollin synthesis was induced strongly by elicitor but only very weakly by osmoticum, whereas isoflavone synthase and NADPH: cytochrome-c reductase were only induced by elicitor. The activity of glucose-6-phosphate dehydrogenase did not change with elicitor or with osmoticum. Different product patterns were also obtained: whereas with elicitor, glyceollin I was the major product, intermediates of the glyceollin pathway (7,4-dihydroxyflavanone, trihydroxypterocarpan) accumulated with osmoticum.     

Effects of osmotic stress on mast cell vesicles of the beige mouse

The large size of the vesicles of beige mouse peritoneal mast cells (4 microns in diameter) facilitated the direct observation of the individual osmotic behavior of vesicles. The vesicle diameter increased as much as 73% when intact cells were perfused with a 10 mM pH buffer solution; the swelling of the vesicle membranes exceeded that of the insoluble vesicle gel matrix, which resulted in the formation of a clear space between the optically dense gel matrix and the vesicle membrane. Hypertonic solutions shrank intact vesicles of lysed cells in a nonideal manner, suggesting a limit to the compressibility of the gel matrix. The nonideality at high osmotic strengths can be adequately explained as the consequence of an excluded volume and/or a three-dimensional gel-matrix spring. The observed osmotic activity of the vesicles implies that the great majority of the histamine known to be present is reversibly bound to the gel matrix. This binding allows vesicles to store a large quantity of transmitter without doing osmotic work. The large size of the vesicles also facilitated the measurement of the kinetics of release as a collection of individual fusion events. Capacitance measurements in beige mast cells revealed little difference in the kinetics of release in hypotonic, isotonic, and hypertonic solutions, thus eliminating certain classes of models based on the osmotic theory of exocytosis for mast cells.     

Effects of nitrogen source on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana L., betacyanin accumulation per cell increased with increasing total nitrogen concentration (initial NH⁺₄:NO⁻₃ ratio 1:2) in the range 0–40 m M and then remained almost constant in the range 40–80 m M . Increasing ammonium increased growth while betacyanin accumulation was reduced. On the other hand, betacyanin accumulation increased when nitrate was increased while growth was almost constant in the concentration range examined. A time-course study of ammonium and nitrate concentration changes in the medium showed that betacyanin accumulation was associated with nitrate uptake.     

Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca²⁺]_i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm² for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of Definity^TM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca²⁺]_i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca²⁺]_i compared to statically cultured cells. The maximum increases of PI uptake and [Ca²⁺]_i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca²⁺]_i waves in shear cultured HUVECs was reduced compared to the statically cultured cells.     

Effects of cotyledons and nitrate on the nitrogen assimilation ofPhaseolus vulgaris

Summary In an experiment performed under greenhouse conditions usingPhaseolus vulgaris cv. Carioca inoculated withRhizobium strain CO5, effects of cotyledons and mineral nitrogen on the initial process of nitrogen assimilation were evaluated. Plants were maintained intact or had either both or half of both cotyledons removed six days after planting. Levels of mineral nitrogen corresponded to the addition of 0 or 1.5 mg N/plant/day three days before each of the four harvests (8, 10, 12 and 14 days after planting). Cotyledon removal generally decreased nodule number and dry weight and total nitrogenase activity, although there was no effect on specific nodule efficiency, but the nitrate reductase activity was increased in both shoots and roots. Mineral nitrogen decreased nodulation and nitrogenase activity when applied 9 and 11 days after planting, but increased shoot and root nitrate reductase activity and total nitrogen incorporation, indicating that plants could be nitrogen limited during the initial period of growth.

---

Resumen En un experimento realizado en invernadero conPhaseolus vulgaris, cv. Carioca, inoculado conRhizobium (cepa CO5) fue evaluado los efectos de los cotiledones y del nitrógeno mineral en el proceso inicial de la assimilación de N. Las plantas fueron mantenidas intactas o con la mitad o todos los cotiledones retirados a los 6 dias despues de la siembra (DDS). El N mineral fue aplicado en la dosis de 0 ó 1.5 mg N/planta/dia, 3 dias antes de cada una de los 4 casechas realizadas (8, 10, 12 y 14 DDS). La retirada de los cotiledones generalmente disminuyó el número y el peso seco de los nódulos y la actividad de la nitrogenase, mas não hubo influencia en la eficiencia específica de los nódulos. La actividad de la nitrato reductasa aumentó tanto en la parte aérea como en las raíces. El nitrógeno mineral disminuyó la nodulación y la actividad de la nitrogenasa cuando la aplicación ocurrió a los 9 y 11 DDS, mas aumentó la actividad de la nitrato reductasa de las raíces y de la parte aérea, asi como el contenido de N en la planta, indicando que estas podrian estar limitadas por nitrógeno en la fase inicial de crescimento.

---

Résumé Dans une expérimentation réalisée dans les conditions de serre au moyen dePhaseolus vulgaris cv Carioca inoculée avec la souche CO5 deRhizobium, on a évalué les effets de cotylédons et de l'azote minéral sur le processus initial de l'assimilation azotée. Les plants ont été soit maintenus intacts soit amputés des deux ou de la moitié des deux cotylédons, 6 jours après la plantation. Les teneurs en azote minéral correspondaient à l'ajout de 0 ou 1.5 mg d'azote par plant et par jour, trois jours avant chacune des 4 récoltes (8, 10, 12 et 14 jours après la plantation). L'amputation de cotyledon diminue d'une manière générale le nombre de nodules, le poids sec et l'activité totale de nitrogénase, bien qu'il n'y ait aucun effet sur l'efficience spécifique de nodule mais l'activité de nitrate réductase était augmentée tant dans les pousses que dans les racines. L'azote minéral a déterminé la nodulation et l'activité de nitrogénase lorsque l'application avait lieu les 9ème et 11ème jours après la plantation, mais elle a augmenté l'activité de nitrate réductase chez les pousses et les racines ainsi que l'incorporation totale d'azote, indiquant par là que la plante pouvait être limitée en azote durant la période initiale de croissance.

     

The isolation, culture and division of protoplasts from citrus cotyledons

High yields (2.3 × 10⁵ to 1.3 × 10⁶ protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability.     

Effects of nitrogen on Pinus palustris foliar respiratory responses to elevated atmospheric CO2 concentration

Indirect effects of atmospheric CO₂ concentration [CO₂], onlongleaf pine (Pinus palustris Mill.) foliage respiration werestudied by growing trees in a factorial arrangement of low andhigh [CO₂] (369 and 729µmol CO₂ mol^–1) and low andhigh N (40 and 400 kg ha^–1 yr^–1). Direct effectsof [CO₂] on leaf respiration were tested by measuring respirationrates of foliage from all treatments at two CO₂ levels (360and 720µmol CO₂mol^–1) at the time of measurement.Elevated CO₂ did not directly or indirectly affect leaf respirationwhen expressed on a leaf area or mass basis, but a significantincrease in respiration per unit leaf N was observed in treesgrown in elevated [CO₂] (indirect response to elevated [CO₂]).The lack of a [CO₂] effect on respiration, when analysed onan area or mass basis, may have resulted from combined effectsof [CO₂] on factors that increase respiration (e.g. greateravailability of non-structural carbohydrates stimulating growthand carbon export from leaves) and on factors that decreaserespiration (e.g. lower N concentration leading to lower constructioncosts and maintenance requirements). Thus, [CO₂] affected factorsthat influence respiration, but in opposing ways. Key words: Pinus palustris, elevated CO₂, nitrogen, foliar, respiration     

Evaluation of parameters affecting the yield, viability and cell division of Pinus pinaster protoplasts

Various factors affecting the yield and viability of Pinus pinaster Ait. cotyledon protoplasts and the mitotic activity of or regenerated cells are described. A study of the effect of sterilization procedures of the plant material showed that whereas the organs collected from disinfested seedlings allow for good yield and viability of isolated protoplasts, germination under non-sterile conditions favours a greater germinating capacity and stronger mitotic activity. Numerous clusters of from 10 to 15 cells were formed after 20 days of culture when a 5% aqueous solution of calcium hypochlorite was used as a sterilizing agent.
The effects of an additional purification of the enzymes showed that although yield and viability of the protoplasts are only slightly improved, the more highly purified enzymes on the other hand enhanced the mitotic activity markedly. Between the two total enzyme concentrations used (0.2 and 0.4%, and in which the relative ratio of each element was unchanged), only the lowest level supplied a debris-free protoplast suspension; mitotic activity occurred only in that case.
Comparison of the populations of cotyledon protoplasts collected from seedlings at two different growth stages (not fully-developed or fully-expanded cotyledons) did not reveal any appreciable difference in their size distribution. Neither was the extent of cellular viability affected by the degree of cell differentiation at the time of collecting. On the other hand, the yield of protoplasts and the mitotic activity of the regenerated cells were greater when partially-developed organs were used. Moreover a pretreatment of the elongating cotyledons with a mineral (half-strength MS macronutrients and full-strength micronutrients) and hormonal (15 μ M BAP, 0.5 μ M NAA) solution improved cell division frequency.     

Effects of silicon on enzyme activity and sodium,potassium and calcium concentration in barley under salt stress

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown in a hydroponics system containing 120 mol m^-3 NaCl only and 120 mol m^-3 NaCl with 1.0 mol m^-3 Si (as potassium silicate). Compared with the plants treated with salt alone, superoxide dismutase (SOD) activity in plant leaves and H⁺-ATPase activity in plant roots increased, and malondialdehyde (MDA) concentration in plant leaves decreased significantly for both cultivars when treated with salt and Si. The addition of Si was also found to reduce sodium but increase potassium concentrations in shoots and roots of salt-stressed barley. Sodium uptake and transport into shoots from roots was greatly inhibited by added Si under salt stress conditions. However, Si addition exhibited little effect on calcium concentrations in shoots of salt-stressed barley. Thus, Si-enhanced salt tolerance is attributed to selective uptake and transport of potassium and sodium by plants. The results of the present study suggest that Si is involved in the metabolic or physiological changes in plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction,maintenance, and differentiation of rice callus cultures on ammonium as sole nitrogen source

Rice callus was initiated from cultured anthers and was maintained on medium containing ammonium as sole nitrogen source and supplemented with either a buffer or an organic acid. Plant regeneration also was obtained on this medium, but the morphogenetic capacity of the callus culture declined as rapidly as on a medium containing a mixture of ammonium and nitrate. The results of the studies suggest that organic acids provide an additional function beyond that of buffering the medium during growth on ammonium.     

Effects of osmotic stress on rabbit corneal endothelium

The effects of osmotic stress on corneal endothelium were investigated by exposing rabbit corneas to anisosmotic conditions, and then perfusing the corneas with isosmotic glutathione bicarbonate Ringer solution for 4 hr at 35 degrees C. During the perfusion, endothelial function was assessed by measuring corneal thickness with a specular microscope. After perfusion, the corneas were prepared for scanning and transmission electron microscopy. Endothelial ultrastructure and function were well maintained after exposure to a wide range of osmolality (0.12-2.7 osmol/kg), but this tolerance of osmotic stress was dependent both on the duration and the temperature of exposure to the anisosmotic conditions. Exposure to an osmolality of 2.7 osmol/kg for 15 min at 23 or 37 degrees C resulted in gross damage to the endothelium when the hyperosmotic agent was sodium chloride. This damage was not the result of increased osmolality per se nor cellular shrinkage because the endothelium tolerated exposure to a sucrose solution of the same osmolality for 15 min at 37 degrees C. The detrimental effect of sodium chloride, however, was mitigated by reducing the temperature of exposure to 0 degrees C or reducing the duration of exposure to 5 min. These results suggest that endothelial cells become more tolerant of high electrolyte concentrations with reducing temperature, and this could be an important factor in the survival of the endothelium in corneal cryopreservation. The results also help to define the limits of osmotic shrinkage and swelling tolerated by endothelial cells. This will be of value in overcoming the detrimental osmotic effects associated with the addition and, in particular, the removal of cryoprotectants.     

The procyanidins of Douglas fir seedlings,callus and cell suspension cultures derived from cotyledons

While cotyledons of Douglas fir seedlings contain only 2–3% of their dry weight as procyanidins (mainly in an insoluble form), callus cultures and cell suspension cultures derived from them contain up to 40%. About 70–85% of the procyanidins isolated from these cell suspension cultures are soluble in 70% methanol, but insoluble in ethyl acetate. They can be separated into a minimum of 4 fractions, all of which have apparent molecular weights greater than that of a tetramer. Dimers, trimers or tetramers are absent or present in only trace amounts, but large amounts of catechin, and lesser amounts of epicatechin, are found in the ethyl acetate-soluble fraction.     

DCPTA对盐胁迫下玉米苗期根系生长、渗透调节及膜透性的影响

黑龙江省西部地区盐碱土面积约有66.7×104hm2,制约着黑龙江省粮食生产水平的进一步提高。叔胺类活性物质2-(3,4-二氯苯氧基)-乙基-二乙胺(DCPTA)具有提高作物抗逆性作用,为探讨DCPTA提高玉米耐盐性机制,采用营养液水培试验,在前期试验基础之上,选取"先玉335"(耐盐性强)和"丰禾1号"(耐盐性弱)2个品种,研究DCPTA(15mg·L-1)对Na Cl胁迫下(150 mmol·L-1)玉米幼苗根系生长、渗透调节及质膜透性的影响。结果表明:DCPTA能增加盐胁迫下幼苗的根长、根表面积、根体积、根鲜重和根干重,缓解盐胁迫对根系生长的抑制;DCPTA维持了根系水分平衡,提高了盐胁迫下幼苗根系相对含水量,"先玉335"和"丰禾1号"分别提高3.6%和6.4%;DCPTA通过提高根系可溶性糖含量和可溶性蛋白含量,降低幼苗根系脯氨酸含量,增强了盐胁迫下玉米幼苗根系的渗透调节能力,并通过降低根系丙二醛(MDA)含量和相对电导率,保护了根系质膜结构和功能;与盐胁迫相比,"先玉335"丙二醛含量和相对电导率分别降低21.6%和24.2%,"丰禾1号"分别降低28.1%和30.4%;DCPTA缓解盐胁迫对根系伤害的效果表现为"丰禾1号""先玉335"。