1-aminocyclopropane-1-carboxylic Acid concentrations in shoot-forming and non-shoot-forming tobacco callus cultures

Shoot-forming tobacco (Nicotiana tabacum var. Wisconsin 38) callus tissues contain significantly lower concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid compared to non-shoot-forming callus tissues. This difference is evident 1 day after subculture to shoot-forming or non-shoot-forming medium, and is maintained through the first week of growth. The lack of auxin in shoot-forming medium is the probable cause for this difference in ACC concentrations.     

ANALYSIS OF POLYPEPTIDES ASSOCIATED WITH SHOOT FORMATION IN TOBACCO CALLUS CULTURES

Callus lines of Nicotiana tabacum were selected for competence and lack of competence in shoot formation. Changes in total and chromosomal polypeptides in these shoot-forming and nonshoot-forming tobacco cultures were examined by twodimensional polyacrylamide gel electrophoresis. Qualitative and quantitative differences in total, nonhistone chromosomal, and basic chromosomal polypeptides were evident throughout the 7-d test period. The analysis of total proteins identified polypeptides specific to shoot-forming and nonshoot-forming tissue during the 7-d sampling period. A small number of basic chromosomal proteins were found solely in shoot-forming or nonshoot-forming tissue. One basic chromosomal protein was detected in only nonshoot-forming tissue at all sampling times. Two proteins, although present in shoot-forming tissue, were present at elevated levels in the nonshoot-forming cultures. No temporal changes in basic proteins over the 7-d incubation period were observed. Qualitative differences in total nonhistone chromosomal polypeptides in the shoot-forming and nonshoot-forming tissue were also observed. Differences in chromosomal polypeptides were observed. In contrast to the basic chromosomal proteins, temporal variation in the nonhistone chromosomal polypeptides was demonstrated. Throughout the 7-d sampling period, 29 and 12 nonhistone chromosomal polypeptides varied qualitatively in shoot-forming and nonshoot-forming callus cultures, respectively. In vitro labeling with ³²P-orthophosphate indicated that approximately 1.0% and 0.3% of the nonhistone chromosomal proteins were phosphorylated in the shoot-forming and nonshoot-forming cultures. Of these phosphorylated polypeptides, one was present in nonshoot-forming tissue and three were detected only in the shoot-forming tissue. Phosphorylation occurred at serine or threonine residues.     

Nitrate assimulation in shoot-forming tobacco callus cultures

Summary The contents of nitrogen-containing compounds and the activities of nitrate assimilating enzymes were determined in tobacco callus cultured under shoot-forming and non-shoot-forming conditions. Whole tissue and tissues cut into top and bottom portions were examined. Highes levels of total-N, protein-N, nitrate and ammonium-N, as well as higher activities of nitrate and nitrite reductases were found in shoot-forming whole tissue and in the shoot-forming bottom portion of tobacco callus in comparison to the non-shoot-forming proliferating tissues throughout the culture period. These findings indicate that enhanced nitrogen assimilation occurs during de, de novo shoot organogenesis. This work was supported by NSERC of Canada Grant No. A-6467 to T.A.T.     

Enzymes of starch metabolism in Nicotiana tabacum callus

Activities of enzymes of starch metabolism were determined in tobacco callus grown in light or darkness and in the presence or absence of gibberellic acid. There was a higher rate of starch turnover in light-grown cultures, as judged by the activities of synthetic and degradative enzymes. Gibberellic acid-treated tissues contained a lower level of starch than the corresponding control tissue. This decrease could be correlated with the activity of phosphorylase. Cultured tissue and seedling material were found to have comparable levels of activity for the starch metabolizing enzymes.     

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; α-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter.     

Photoautotrophic Culture of Undifferentiated Cells and Shoot-Forming Cultures of Digitalis purpurea L.

Undifferentiated cells and shoot-forming cultures of Digitalispurpurea L. were grown photoautotrophically under 1% CO₂. During3 weeks of culture, the undifferentiated cells multiplied 3-foldand the shoot-forming cultures 2-fold on a fresh weight basis.The chlorophyll content, ribulose 1,5-bisphosphate carboxylaseactivity, Hill reaction activity of the isolated chloroplastsand photosynthetic O₂ evolution of the photoautotrophicallygrown cultures were somewhat higher than the values of the correspondingphotomixotrophic cultures. The digitoxin contents, however,were not improved by photoautotrophic culture. (Received November 9, 1983; Accepted June 11, 1984)     

Mitochondrial activity during shoot formation and growth in tobacco callus

Mitochondria isolated from tobacco ( Nicotiana tabacum L. cv. Wisconsin 38) callus growing on either shoot-forming or non-shoot forming medium show an increase in state 3 and state 4 respiration and a drop in respiratory control and ADP/O ratios after subculture. the protein content of the mitochondria fraction and the activity of succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and catalase also increase after subculture but there is no apparent difference between shoot-forming and non-shoot-forming tissue. For mitochondria assayed at their native osmolarities, a trend of higher respiration rates and respiratory control as well as lower levels of cyanide-resistant respiration was observed for shoot-forming tissue. Generally, differences were greatest after day 9 in culture, the time during which primordia formation occurred in the shoot-forming callus. These patterns are in concert with the view that the shoot-forming process has a high energy requirement which must be realized during the time of primordia formation.     

Carbohydrate Availability and Shoot Formation in Tobacco Callus Cultures

Tobacco callus grown on a shoot-forming medium containing sorbitol or no carbon source survived, but did not produce shoots. Transfer of tissue from a sucrose medium to carbohydrate-deficient media and vice versa suggested that the growth of the tissue was a function of a total period in contact with available carbohydrate. Both starch and free sugars in the tissue were utilized during shoot initiation. Furthermore, it appeared that the continuous availability of carbohydrate was required for shoot primordium growth and/or their development into leafy vegetative shoots in dark-grown cultures.     

Starch turnover in shoot-forming tobacco callus

Tobacco callus grown under shoot-forming conditions or in the presence of gibberellic acid, which inhibits shoot formation, was incubated in [¹⁴C]-sucrose at three different periods in culture and then replanted. Evolution of ¹⁴CO₂ occurred during the 10 day post-incubation period. Most of the radioactivity was incorporated into the ethanol-soluble fraction, which lost most of its label after 24 h. Starch was the major ethanol-insoluble component and post-incubation synthesis occurred in this fraction for 24 h or longer. Greater net synthesis of starch occurred in shoot-forming tissue and the loss of label from starch began later than in tissue cultured in the presence of gibbe-rellic acid. Newly synthesized starch was not immediately utilised in the organogenic process, but its utilization could be correlated with the shoot-forming process.     

Digitoxin Biosynthesis in Isolated Mesophyll Cells and Cultured Cells of Digitalis

In the laminae of Digitalis, most of the digitoxin present isfound in the mesophyll. A new method for determining the amountof digitoxin biosynthesis using a digitoxin antibody was devisedto estimate this activity in isolated mesophyll cells and culturedcells. Isolated mesophyll cells showed significant activity,which suggests that the site of biosynthesis and the accumulationof cardenolides in a lamina of Digitalis is mainly in the mesophyllcells. Of five liquid cultures of D. purpurea; green shoot-formingcultures, white shoot-forming cultures, root-forming cultures,undifferentiated green cells and undifferentiated white cells,the green shoot-forming cultures had the highest activity. Thewhite shoot-forming cultures had about one-third the activityof the green shoot-forming cultures, and the other three cultureshad very low activity. No stimulatory effect of light was foundduring the 48-h incubation. (Received January 19, 1984; Accepted June 8, 1984)     

[14C]-Glucose Metabolism during Shoot Bud Development in Cultured Cotyledon Explants of Pinus radiata

Excised cotyledons of Pinus radiata D. Don cultured under shoot-forming(plus benzyladenine) and non shoot-forming (minus benzyladenine)conditions for 10 and 21 days were fed U-[¹⁴C]-glucose for 3h in the light followed by a 3 h chase period. The labellingof individual metabolites as well as ¹⁴C incorporation intoprotein was assessed. It was found that the general metabolicpatterns were qualitatively the same in shoot-forming and nonshoot-forming conditions, however, metabolism leading to respirationas well as to the synthesis of some amino acids and proteinsynthesis was enhanced in the shoot-forming cultures. (Received February 16, 1987; Accepted July 8, 1987)     

Glucose Oxidation during Shoot Initiation in Tobacco Callus Cultures

Involvement of the Embden-Meyerhof Parnas and the pentose phosphatepathways in glucose oxidation in glucose oxidation in tobaccocallus was examined. Marked changes in the activities of glucokinase,aldolase, glucose-6-phosphate dehydrogenase, and phosphogluconatedehydrogenase were observed during culture of tobacco callusunder shoot-forming and non-shoot-forming conditions. Activitiesof these enzymes were higher in shoot-forming tissue than innon-shoot-forming tissue. Furthermore, the activities of thepentose phosphate pathway enzymes showed greater differencesthan those of the Embden-Meyerhof-Parnas pathway. Confirmationof these findings was obtained by investigating the contributionsof ¹⁴C from [14C-1]- and [¹⁴C–6]-glucose to CO2 released.The significance of these findings on glucose oxidation in relationto the shoot-initiation process are discussed.     

Shoot initiation in light- and dark-grown tobacco callus: the role of ethylene

Shoot-forming tobacco ( Nicotiana tabacum L. cv. Wisconsin 38) callus produces less endogenous ethylene than non-shoot-forming tissue cultured in the light (16 h photoperiod) or the dark. In shoot-forming tissue more ethylene is produced early in culture (days 0–5) than later. Also dark-grown tissue produces much more ethylene than light-grown. On the basis of experiments in which (1) gaseous ethylene was added to or (2) CO₂ removed from the flasks, (3) Ethrel (an ethylene releasing agent) and (4) 1-aminocyclopropane 1-carboxylic acid (an ethylene precursor) were added to the medium, it was determined that this gaseous phytohormone had two contrary effects on shoot initiation (shoot primordium formation). Early in culture (days 0–5) endogenous or exogenous ethylene inhibited organogenesis, but later (days 5–10) exogenous ethylene or increased endogenous ethylene production speeded up primordium formation.     

ULTRASTRUCTURAL ASPECTS OF SHOOT INITIATION IN TOBACCO CALLUS CULTURES

An ultrastructural investigation of shoot initiation in tobacco (Nicotiana tabacum L. var. W. 38) callus cultures was made. Zones of preferential division were observed in the basal portion of the tissue by eight days in culture and these led, sequentially, to meristemoids, primordia, and shoots. During the initial stages of meristemoid formation, protein inclusions and large accumulations of plastid starch were present in the cells, while vacuoles were filled with membranous and cytoplasmic protrusions. At later stages of meristemoid development, these features were not observed in the cells, which were also smaller in size and possessed numerous small, peripheral vacuoles. It appears that the membranous and cytoplasmic protrusions are involved in vacuolar reduction during meristemoid formation. It would also appear that the storage materials supply the energy and other reserves needed for the organogenetic process. By contrast, tissue cultured under nonshoot-forming conditions and nonmeristemoid regions of shoot-forming tissue remained parenchymatous over the same time period.     

Isozymes of Glucose-6-phosphate Dehydrogenase and NAD+-malate Dehydrogenase in Shoot-forming Foliar Discs of Tobacco

Discrete pale, meristematic, shoot-forming zones (SF) and green,relatively nondividing, non-shoot-forming zones (NSF) of cellswere obtained from leaf discs of tobacco cultured for 12 dayson a shoot-forming medium. Higher chlorophyll and starch content,increased rates of O₂ evolution and CO₂ fixation in light, andincreased activities of amylases and chloroplastic enzymes suchas ribulose 1,5-bisphosphate carboxylase and NADP⁺-linked glyceraldehyde-3-phosphatedehydrogenase (G3PDH) were characteristic of the cells constitutingNSF. On the other hand, active participation of sucrose hydrolysis,dark-mediated CO₂ incorporation, an oxidative pentose phosphatepathway, glycolysis and mitochondrial complements in shoot formationwere evident from the significantly high activities of phosphoenolpyruvatecarboxylase, invertase, glucose-6-phosphate dehydrogenase (G6PDH),NAD+-G3PDH and NAD+-linked malate dehydrogenase (NAD+-MDH) respectively,in SF cells. Detection of activity of the enzymes by stainingon polyacrylamide gels disclosed synthesis of additional isoenzyme(s)of G6PDH, NAD+-MDH and peroxidase in shoot initiation sites.The much pronounced activity and isozyme groups of G6PDH andNAD+-MDH in the photosynthetically incompetent shoot-formingcells, are considered to increase the carbon budget of the differentiatingcells through non-autotrohpic CO₂ fixation and to supplementreducing power (NADPH) for the organogenetic process which requiresmuch energy. The changes in isozymes of these enzymes, as inthe isoperoxidase system, probably can serve as useful markersof the differentiation process. (Received January 23, 1981; Accepted June 17, 1981)     

Starch Metabolism, Respiration, and Shoot Formation in Tobacco Callus Cultures

The starch content of shoot-forming and non-shoot-forming tobacco callus cultured in light and darkness was determined. A variety of carbohydrates and cytokinins incorporated into the culture medium were effective in bringing about starch accumulation and shoot formation in the tissue. In addition, the respiratory activity of the callus, grown in the presence or absence of gibberellic acid, was measured. A strong correlation between the starch content of the tissue, its rate of respiration, and shoot formation was observed.     

Molecular subtyping within a single Salmonella typhimurium phage type, DT204c, with a PCR-generated probe for IS200

Abstract Pullulan is an industrial biopolymer produced by the yeast-like fungus Aureobasidium , usually by direct fermentation of starch. Despite evidence that autogenous amylases produced during these fermentations are detrimental to the final molecular mass of the product, fundamental studies of these enzymes have not been reported. Total extracellular amylases were studied from the promising production strain NRRL Y-12,974. Growth rates and yields were equivalent in cultures grown on glucose, maltose, soluble starch, or cornstarch. Total amylase levels were low and varied only three-fold, from 0.01 IU ml⁻¹ in glucose-grown cultures to 0.03 IU ml⁻¹ in soluble-starch-grown cultures. All cultures showed both α-amylase activity and activity against pullulan. Synthetic oligosaccharide substrates were apparently attacked by an α-glucosidase, produced in highest levels by maltose-grown cultures.     

Physiological and metabolic enzymes activity changes in transgenic rice plants with increased phosphoenolpyruvate carboxylase activity during the flowering stage

To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO₂ into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C₃ plants possessing a C₄-like photosynthesis pathway worked by expression of PEPC.     

Variation of enzyme activities and metabolite levels in 24 Arabidopsis accessions growing in carbon-limited conditions

Our understanding of the interaction of carbon (C) metabolism with nitrogen (N) metabolism and growth is based mainly on studies of responses to environmental treatments, and studies of mutants and transformants. Here, we investigate which metabolic parameters vary and which parameters change in a coordinated manner in 24 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions, grown in C-limited conditions. The accessions were grown in short days, moderate light, and high nitrate, and analyzed for rosette biomass, levels of structural components (protein, chlorophyll), total phenols and major metabolic intermediates (sugars, starch, nitrate, amino acids), and the activities of seven representative enzymes from central C and N metabolism. The largest variation was found for plant weight, reducing sugars, starch at the end of the night, and several enzyme activities. High levels of one sugar correlated with high levels of other sugars and starch, and a trend to increased amino acids, slightly lower nitrate, and higher protein. The activities of enzymes at the interface of C and N metabolism correlated with each other, but were unrelated to carbohydrates, amino acid levels, and total protein. Rosette weight was unrelated or showed a weak negative trend to sugar and amino acid contents at the end of the day in most of the accessions, and was negatively correlated with starch at the end of the night. Rosette weight was positively correlated with several enzyme activities. We propose that growth is not related to the absolute levels of starch, sugars, and amino acids; instead, it is related to flux, which is indicated by the enzymatic capacity to use these central resources.