Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

Responses of stomata of senescing and nonsenescing leaves of Nicotians glauca to changes in intercellular concentrations of leaf carbon dioxide

Abstract. Gas exchange measurements were performed to test the hypothesis that failure of stomata to open in senescing leaves of Nicotiana glauca is caused by elevated concentrations of carbon dioxide in the intercellular spaces of leaf mesophyll tissue (c_i). Senescing leaves selected for experiments were completely chlorotic and lacked positive rates of photosynthesis. When stomata in detached epidermis from senescing leaves were illuminated in CO2-free air, they opened to similar apertures as those in detached epidermis from nonsenescing leaves. To compare the effects of changes in c_i on stomatal responses of the two leaf types, leaf 'flags' of either nonsenescing or senescing leaves were illuminated at a photosynthetic photon flux density of 500 μmol m⁻² s⁻¹ in a gas exchange cuvette. Leaf temperatures were maintained at 23.5 ± 0.5°C, and vapour pressure differences between leaves and the air were maintained between 0.70 and 0.75kPa. C_i was adjusted by changing external concentrations of carbon dioxide in air circulating through the cuvette. Conductances and photosynthetic rates of nonsenescing leaves changed in response to changes in c_i, but neither the conductances nor the photosynthetic rates of senescing leaves were affected significantly by changes in q. We conclude that guard cells of senescing leaves of Nicotiana glauca do not lose the capacity to respond to changes in carbon dioxide concentration and that increases in c_i resulting from declining rates of mesophyll photosynthesis are not the sole cause of maintenance of stomatal closure during leaf senescence. The data suggest that factors external to guard cells may prevent them from responding to changes in carbon dioxide concentrations in intact senescing leaves.     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Increased anthraquinone production in Galium vernum cell cultures induced by polymeric adsorbents

Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.Abbreviations DW dry weight - MS Murashige & Skoog[7]medium - NAA 1-naphthaleneacetic acid     

Increasing in vitro germination of Musa balbisiana seed

Seeds of Musa balbisiana were soaked in water for five days prior to excision of embryos. Embryos with their longitudinal axis laid flat and half-way embedded on agar-solidified medium produced the highest germination and the most desirable plantlet characteristics. Germination in vitro was 94% within 7 days compared to 50% after 54 days for greenhouse-sown seeds.     

Effect of potassium humate on apple cv. ‘Golden Delicious’ cultured in vitro

The effects of humic substances on in vitro culture of Golden Delicious apple are reported. Potassium humate (KH) when used in proliferation showed a negative interaction with BA while it enhanced rooting when IBA was not present in the culture medium. In the presence of IBA, KH increased root number and reduced root growth. The highest concentration tested, 500 mg l^-1, caused a drastic reduction in root system development. 50 mg l^-1 KH hastened rooting and plants grew more rapidly when transferred to soil.     

Culture of soybean fruit explants: growth conditions and efficiency of nitrogen sources for reserve protein synthesis

A system was devised for the in vitro culture of soybean fruits. The culture system consisted of a single fruit attached to a short piece of stem through which the nutrients were supplied. The fruit explants were taken when pods were fully expanded and the seeds at initial stages of growth. During a 7-day culture period, the seeds accumulated dry matter and protein in quantities comparable to those in situ. Omission of the C source (sucrose) from the medium resulted in no dry matter accumulation in the seeds, but omission of the N source (glutamine) still led to some protein accumulation, indicating mobilization of N from other parts of the fruit explant. Optimum protein accumulation occurred when glutamine was supplied at 1.2 mg N ml^-1. Protein accumulation in the seeds was highly dependent on the nature of the N source. Glutamine, asparagine and the ureide, allantoin, were equally the most efficient sources, whereas several other amino acids tested showed lower degrees of efficiency. The data indicate a high metabolic capacity of the fruit tissues for principal N transport compounds of soybean, namely allantoin, asparagine and glutamine. The culture system described should prove useful for developmental and metabolic studies where the complex influence of the rest of the plant is to be avoided.Abbreviations ALN allantoin - ALC allantoic acid Preliminary report presented at the IV World Soybean Research Conference, Buenos Aires, Arggentina, March 1989.     

Analysis of pollen-tube growth in cultured maize silks

Summary In vitro pollen-tube growth in maize was studied using an in vitro pollination system. In the cut-silk method, ovaries with silks were placed on medium in vitro, whereafter the silk was cut and the upper part of the silk was pollinated. Pollen tubes were not able to bridge the space between the two silk parts. Even when silk parts were tightly connected, pollen tubes still were not able to pass the cut ends and reach the lower silk part. Pollen-tube growth rates and the direction of tube growth were not influenced by the presence or absence of an ovary. Prepollination did not have any influence on pollen-tube growth rate. Measurements of pollen-tube growth rate also showed that there was no population effect, i.e. growth rate was not stimulated by pollination with an excess of pollen grains. We found that the direction in which maize pollen grew was determined only by the positioning of the silk hairs.