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.     

Turnover of Shikimate Pathway Metabolites during Shoot Initiation in Tobacco Callus Cultures

The turnover of shikimate pathway intermediates and end productswas examined in tobacco (Nicotiana tabacum L. Wisconsin 38)callus cultured under shoot-forming and non-shoot-forming conditions.In shoot-forming tissue there was a higher rate of net synthesisof quinic and shikimic acids than in proliferating callus. Post-incubation,there was a decrease in labeled quinate and an increase in shikimate.The changes in activity of quinate:NAD^$ oxidoreductase werein agreement with the above. The aromatic amino acids, tyrosine,phenylalanine and tryptophan, showed little turnover in theproliferating tissues. On the other hand, higher rates of netsynthesis and degradation, mainly of tyrosine, were observedin shoot-forming tissues. These findings are discussed in relationto the shoot-initiation process. (Received October 14, 1983; Accepted June 4, 1984)     

Non-autotrophic CO2 Fixation during Shoot Formation in Tobacco Callus

Non-autotrophic carbon fixation has been studied during growthof tobacco callus cultured in dark under shoot-forming (SF)and non-shoot-forming (NSF) conditions. The enzymes involvedin malate metabolism—phosphoenolpyruvate carboxylase,malic dehydrogenase, glutamic-oxalacetic transaminase, and malicenzyme—increased sharply during the first 4 d of cultureparticularly in SF tissue. The activities of the enzymes studiedwere considerably greater in SF than in NSF tissue. There wasa dramatic increase in malate content in SF tissue during thefirst 4 d of culture. Subsequently malate was rapidly depletedduring the time of organogenesis. In NSF tissue there was acontinuous build-up of malate content throughout the cultureperiod. We suggest that malate derived from dark fixation ofCO₂ plays differing roles in NSF (callus) and SF tissues. Inthe former, malate acts primarily as an osmotic solute regulating,at least in part, cell expansion between successive cell divisions.In shoot-forming tissue, on the other hand, malate preferentiallyprovides NADPH for reductive biosynthesis.     

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.     

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.     

Modulation of shoot formation in tobacco callus by tissue transfer between permissive and inhibitory media

Summary In an attempt to understand events involved in the cellular regulation of in vitro plant organogenesis, experiments were performed in which tobacco (Nicotiana tabacum L.) callus was transferred at different days in culture from a shoot-forming medium to a non-shoot-forming medium and vice versa. The transfers were made at key histologic stages of the shoot-forming process and known biochemical and biophysical correlates were examined. The changes in starch accumulation and disappearance supported the previously assigned functions, and could be correlated with the histologic changes that occurred in the callus after transfer at the different culture times. In contrast, the changes in respiration could not be correlated with these events. The changes in osmotic and turgor potentials after transfer showed that osmotic adjustment preceded both shoot initiation and development. This suggests that osmotic adjustment might play an important role in in vitro organogenesis. This research was supported by the Natural Sciences and Engineering Research Council of Canada grant A-6467 to T. A. T.     

Starch Metabolism in Shoot-forming Tobacco Callus

Starch, free sugars and protein contents, and the specific activitiesof enzymes of starch metabolism were determined in tobacco calluscultured under shoot-forming and non-shoot-forming conditions.Shoot-forming cultures contained higher levels of starch, freesugars and protein. Shoot-forming cultures had higher specificactivities for starch-synthesising enzymes throughout culture.On the other hand, higher levels of activity for starch-degradingenzymes in shoot-forming tissues were only observed during organizeddevelopment. The role of phosphorylase in the cultured tissuewas not clear.     

Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.     

Soluble and cell-wall peroxidases and auxin destruction in normal and habituated tobacco callus

Summary A comparison of the soluble and cell-wall-bound isoperoxidases of normal auxin-requiring and auxin-independent (habituated) tobacco callus revealed that normal tissues contained a higher level of isoperoxidases. There were also qualitative differences in these isoperoxidases. Partially purified soluble and ionically bound isoperoxidases of normal callus likewise exhibited higher auxin-oxidase activities. Normal tissues also were found to contain higher levels of auxin-oxidase inhibitors (auxin protectors). Overall, however, the data indicate that there is a higher rate of auxin destruction in normal tobacco callus than in habituated tissue. This presumably leads to insufficient endogenous auxin for growth. This study was supported in part by grants to T.G. from the Center IRSIA d'Etude de la Reproduction végétale and the FRFC Contract No. 2.9009. it Was carried out while T.A.T. was the holder of a senior Fellowship under the NATO senior Scientists Program.     

Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.     

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.     

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.     

Ascorbic acid enhancement of organogenesis in tobacco callus

The effect of ascorbic acid on growth and shoot formation in callus cultures of tobacco (Nicotiana tabacum L.) was investigated, using young (4–12 subcultures) and old (more than 30 subcultures) tissue. It was found that ascorbate, at levels of 4–8×10^-4M, enhanced shoot formation in both young and old callus. Treatment with ascorbate also speeded up the shoot-forming process. In addition, ascorbate completely reversed the inhibition of shoot formation by gibberellic acid in young callus, but was less effective in old callus.     

Shoot histogenesis in tobacco callus cultures

Summary The earliest histological event observed in light-grown shoot-forming tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus was the deposition of safranin-stainable substances (probably suberin) on cut, exposed cell surfaces. This was followed by the initiation of cell files and the appearance of starch granules. Nodules with lignified tracheary elements also were observed in the upper part of the callus. Pronounced starch accumulation occurred in the lower part of the callus in which protrusions of tissue into the medium occurred. Meristemoids were found in these protrusions as well as elsewhere. In between meristemoids, parenchyma cells with starch granules of varying sizes were observed. Cell strands that connected with the meristemoids also were observed. These strands often terminated at the surface of the protrusion at which point shoot apices originated. The earliest shoots were formed in these protrusions. With time, additiional shoots were formed in other parts of the bottom of the callus and finally in the top part of the callus on prolonged culture. The determination of the loci at which shoot primordia were formed sequentially, was interpreted in relation to the physiological gradient concept. This work was carried out while E. M. was a Visiting Scientist at the University of Calgary under the Scientific Exchange Program between the National Research Council of Canada and the Japan Society for the Promotion of Science. Support for this study was provided by N.R.C. (Canada) Grant A-6467 to T. A. T.     

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.     

Changes in isoperoxidases during shoot formation in tobacco callus

Shoot formation in tobacco (Nicotiana tabacum L.) callus is accompanied by an increase in peroxidase activity which takes a form similar to a sigmoid curve. The "stationary" phase coincides with the period of organ formation. Characteristic changes in isoperoxidase pattern are found in the shoot-forming part of the callus. These changes are different from those in the nonshoot-forming part or in gibberellin-treated tissue, which does not form shoots.     

Changes in isoperoxidases during shoot formation in tobacco callus

Summary Shoot formation in tobacco (Nicotiana tabacum L.) callus is accompanied by an increase in peroxidase activity which takes a form similar to a sigmoid curve. The “stationary” phase coincide with the period of organ formation. Characteristic changes in isoperoxidase pattern are found in the shoot-forming part of the callus. These changes are different from those in the nonshoot-forming part or in gibberellin-treated tissue, which does not form shoots.     

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)     

Glutamate synthase in greening callus of Bouvardia ternifolia Schlecht

The distribution of the two glutamate-synthase (GOGAT) activities known to exist in higher plants (NADH dependent, EC 2.6.1.53; and ferredoxin dependent, EC 1.4.7.1) was studied in non-chlorophyllous and chlorophyllous cultured tissue as well as in young leaves of Bouvardia ternifolia. The NADH-GOGAT was present in all three tissues. Using a sucrose gradient we found it in both the soluble and the plastid fraction of non-chlorophyllous and chlorophyllous tissue, but exclusively in the chloroplast fraction of the leaves. Ferredoxin-GOGAT was found only in green tissues and was confined to the chloroplasts. Ferredoxin-GOGAT activity increased in parallel with the chlorophyll content of the callus during the greening process in Murashige-Skoog medium (nitrate and ammonium as the nitrogen sources), while NADH-GOGAT was not affected by the greening process in this medium. Furthermore, both activities were differentially affected by either nitrate or ammonium as the sole nitrogen source in the medium during this process. It is suggested that each GOGAT activity is a different entity or is differently regulated.Abbreviations GOGAT glutamate synthase - MS Murashige-Skoog (1962) medium - PMSF phenylmethylsulfonyl fluoride     

Effect of sulfur and nitrogen nutrition on derepression of ferredoxin-sulfite reductase in leek seedlings

The ferredoxin-sulfite reductase (Fd-SiR; hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1) activities of shoot and root of leek (Allium tuberosum) were increased by sulfate limitation in the early stage of growth. Western blot analysis demonstrated an increased amount of SiRs in root under sulfate limitation, suggesting that SiRs were derepressed. The derepression was observed in shoot when 1.5 mM nitrate was supplied to the plants under sulfate limitation, and clearly in root when 15 mM nitrate was supplied under sulfate limitation. When nitrate was absent from the nutrient solution, the SiR activity in both tissues was very low. Combined with the results of the sulfate- or nitrate-limitation experiments, it is suggested that the degree of the derepression of SiR in both tissue under sulfate limitation is affected by the concentration of nitrate, and further that the mechanism of regulation of the SiR activity is different in each tissue. The decreases in the ratios of the total SiR activities (shoot/root) in the latter stage of seedling growth indicate that root play a very important role in sulfate assimilation.