Autoradiographic and microspectrophotometric studies of DNA synthesis in excised tobacco pith tissue

Summary Pith tissue was cultured on modified White’s nutrient medium supplemented, except for controls, with 2 mg/l of IAA and/or 0,5 mg/l of kinetin. For autoradiographs sections were used from tissue grown on medium containing tritiated thymidine. Nuclear DNA contents (Feulgen) were measured by the microspectrophotometric two-wavelengths method. No fading of Feulgen dye in nuclei was found in 11 weeks, in contrast to considerable fading observed in earlier work when a different batch of basic fuchsin had been employed. Counts of radioactive nuclei in autoradiographs agreed well with microspectrophotometric results on the occurrance of DNA synthesis. In control cultures, with or without tritiated thymidine, DNA doubling took place in about 20% of the nuclei during the first two days but in few, if any, thereafter. It was confirmed that kinetin, as well as IAA, increases the frequency of nuclei undergoing DNA synthesis. However, IAA, in contrast with kinetin, still induced considerable DNA doubling after two days. Continued cell reproduction was maintained only in the presence of both substances. This work has been supported in part by research grants toK. Patau from the US Public Health Service (grant No. C-3313) and the American Cancer Society; and by grants toF. Skoog from the American Cancer Society and the Research Committee of the University of Wisconsin Graduate School with funds from the Wisconsin Alumni Research Foundation.     

Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue

Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation.     

Tracheid differentiation in tobacco pith cultures

Summary Sterile pith cultures of Nicotiana tabacum have been induced to form localized regions of differentiating tracheids. These localized regions have been examined by phase, fluorescence, and electron microscopy, and polarization optics. Fixation for electron microscopy was with glutaraldehyde-osmium. The differentiating tracheids develop characteristic thick cell walls which are eventually lignified. The lignifications appear to be uniform throughout the secondary wall and little or no lignin appears to be deposited in the primary walls or intercellular layer. At all stages of secondary wall deposition, the peripheral cytoplasm contains a system of microtubules which form a pattern similar to that of the developing thickenings. Within this system the microtubules are oriented, the direction of orientation mirroring that of the fibrils in the most recently deposited parts of the wall. The observations support the view that the microtubules are somehow involved in microfibril orientation. The microtubules appear to be attached to the plasma membrane which has a triple layered structure. The two electron dense layers of the plasma membrane have a particulate structure. In the differentiating tracheids at regions where secondary wall thickening has not yet been deposited numerous invaginations of the plasma membrane are observed which contain loosely organized fibrillar material. It is suggested that these are areas of localized activity of the plasma membrane and that the enzymes concerned with the final organization of the cellulose microfibrils are situated at the surface of the plasma membrane. Dictyosomes in the differentiation cells give rise to vesicles which contain fibrous material and the contents are incorporated into the cell wall. Numerous profiles characteristic of plasmodesmata are evident in sections of the secondary thickenings.Part of this work was carried out at the Osborne Memorial Laboratories, Yale University.     

Structural physiological, and biochemical gradients in tobacco pith tissue

Explants of tobacco pith taken at various distances from the apex of a mature stem show a sharp gradient in growth potential in vitro; growth is highest in the extreme apical and basal explants, and is minimal in explants removed ca. 75 cm from the apex. Calluses produced by the vigorously growing basal explants are harder and more compact than those produced from more apical explants. The gradient in growth potential is directly correlated with gradients in RNA, protein of cell sap and soluble N per unit fresh weight, but is inversely correlated with peroxidase activity. Cell size increases from apex to base of plants.

The peroxidase activity of pith explants is electrophoretically resolvable into 2 isoperoxidases, moving anodically at pH 9.0. During in vitro culture, this activity rises, due to the formation of several new isozymes moving toward the cathode. The appearance of these isozymes occurs most rapidly in apical and extreme basal explants.

     

Utilization of exogenous sugars by excised maize embryos in culture

Sucrose was markedly superior to fructose and glucose in promoting growth of plantlets from immature maize embryos. The elongation of roots is shown to be more sucrose dependent than that of shoots. On the other hand, the exogenous sucrose was less effective than fructose as substrate for carbohydrate catabolism and for the synthesis of alcohol-insoluble compounds at the beginning of embryo cultivation. The absorbed fructose was found to be rapidly converted to sucrose and the level of endogenous sucrose derived from sugar supplied to the medium was higher in fructosethan in sucrose-fed embryos. The preferential utilization of fructose over sucrose, however, declined with the progress of germination which may be related to the decrease in proportion of scutellum in total mass and physiological activity of the embryo.     

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. Havana 425 are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

Repression of secondary metabolite production by exogenous cAMP in Monascus

The cAMP signal pathway controls various biological functions, including secondary metabolism of filamentous fungi. We found that exogenous cAMP represses the production of lovastatin, red pigments, and citrinin in Monascus. Interestingly, a mutant MK-1 with increased lovastatin and red pigments production was not influenced by cAMP on these productions, indicating that cAMP signaling might be lacking in MK-1.     

Induction of indoleacetic Acid synthetases in tobacco pith explants

Formation of indoleacetic acid synthetases in tobacco pith explants was determined by following the growth of tissue cultures under conditions of indole-3-acetic acid (IAA) deprivation and by measuring the enzymatic conversion of tryptophan to IAA in the cultures. The pith explants obtained from the parent plant (Nicotiana glauca) and from basal regions of the tumor-prone hybrid (N. glauca × N. langsdorffii) both show a requirement for exogenous IAA for growth initiation in culture. The parent pith requires the constant presence of added IAA for continued growth, but hybrid pith, after initial treatment with IAA, will grow without further additions. IAA synthetases are detected in the cell homogenates of hybrid pith explants cultured with either continuous or initial IAA addition. These observations indicate that IAA may induce its own production. In contrast, IAA synthetases are not found in the parent pith under comparable culture conditions. Besides IAA, nonhormonal compounds such as indole and tryptophan are also capable of stimulating growth of hybrid pith, possibly through the induction of IAA synthetases needed for IAA formation. Indole and tryptophan are, however, inactive in growth promotion of the parent pith. These results suggest that the genomic expression of IAA synthetase formation is more stringently controlled in N. glauca than in the tumorprone hybrid.     

Reversible, cell-heritable changes during the development of tobacco pith tissues

Cytokinin requiring cells of Nicotiana tabacum L. cv "Havana 425" can be induced in culture to become cytokinin autotrophic. This process is known as cytokinin habituation. Earlier we showed that pith parenchyma tissue consists of inducible cells, which habituate at high rates when treated with cytokinin, and noninducible cells, which remain cytokinin requiring under these conditions. The inducible and noninducible phenotypes are determined states that arise during the development of the tobacco plant and are inherited by individual cells. Here we show that pith tissue of plants regenerated from cloned lines of noninducible cells exhibits the inducible phenotype indicating that noninducible cells, or their descendants, can become inducible. This change in competence for habituation appears to have an epigenetic basis; it is reversible, occurs at high rates, and depends on the developmental state of the cells. The habituated state occurs in two forms that can be distinguished by their difference in developmental potential. Habituated cells derived from inducible pith cells give rise to normal plants whose leaf and pith tissues require cytokinin for growth in culture. In contrast, habituated cells obtained by transferring noninducible cells on media with progressively lower cytokinin concentrations give rise to plants whose leaf and pith tissues exhibit a cytokinin-habituated phenotype in culture.     

Responses of tobacco pith nucleic to growth substances

Summary A cytological study was made of excised segments of pith of tobacco (ev. Wisconsin 38) grown in vitro on media containing various growth substances. Explants were cultured on a basal medium and on media containing kinetin, indoleacetic acid, gibberellic acid, and combinations of these three growth substances. Controls consisted of pith segments chemically fixed immediately after excision. The nuclei of explants on the basal medium and those on media containing the growth substances showed a response as early as 1 day after the beginning of the experiment. Nucleoli were indicators of initial stimulation in that they became swollen and displayed internal vacuolation, a known characteristic of active cells. These fatures were more pronounced by the second day for cultures grown on media containing growth regulators. Nucleoli were centers of nucleohistone staining as shown by the ammoniacal silver method. Continued and sustained growth of explants was possible only on media containing combinations of IAA and kinetin, or a combination of kinetin, IAA and GA. The greatest initial nucleolar stimulation occurred with IAA alone, even though subsequent growth did not take place. There was no nucleolar response of explants grown on abscisic acid alone.Abbreviations ABA abscisic acid - GA gibberellic acid - IAA indole-3-acetic acid The senior author is most grateful for support by fellowships from the Fulbright Commission and the John Simon Guggenheim Memorial Foundation. The authors wish to thank Mrs. Colette Nitsch, Miss Simone Hamon and Miss Linda Robinson for their valuable technical assistance.     

Cytokinesis, cell expansion, and the potential for cytokinin-autonomous growth in tobacco pith

Pith tissue from Nicotiana tabacum L. cv `Maryland Mammoth' or `Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center.     

Cytokinin effects on growth of quiescent tobacco pith cells

Excised pith tissue from Nicotiana glauca or the tumor-prone hybrid N. glauca × N. langsdorffii has no growth requirement for exogenous cytokinins. Addition of kinetin to cultures of these lines results in growth inhibition at a kinetin concentration 1000-fold lower than the optimal level for kinetin-requiring lines. Cytological comparison of the kinetin-inhibited 2N hybrid and glauca tissues with pith from the kinetin-requiring N. tabacum var. Wisconsin 38 suggests that the nature of the cytokinin action is similar in both situations and that the primary function of cytokinin, when it stimulates growth, may be to curtail cell expansion, thereby facilitating a balance of cell expansion and division requisite for maximal growth.     

Flower formation in excised tobacco stem segments; I. Methodology and effects of plant hormones

The formation of flowers has been studied in stem tissue excised from flowering plants of Nicotiana tabacum variety Wisconsin No. 38, and cultured in vitro on Murashige and Skoog nutrient medium. A procedure for quantitative evaluation of factors influencing floral expression has been developed and effects of the growth substances, indole-3-acetic acid (IAA), kinetin and gibberellic acid (GA₃), on the process are reported.     

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. Havana 425 becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10^–3 per cell generation, was 100–1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10^–10 M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.     

Effect of N-phenyl-N'-(4-pyridyl)urea on shoot formation in tobacco pith disk and callus cultured in vitro

N-Phenyl-N'-(4-pyridyl)urea (4PU), which was originally synthesizedby Bruce et al. and also reported by Okamoto as a highly activecytokinin in the tobacco pith callus bioassay, showed a markedeffect on shoot formation in tobacco pith disk and callus witha single application. Details of experiments and results arereported and the interrelation between auxin and 4PU for organformation is discussed. (Received January 7, 1976; )