Stability of the cytokinin requirement in Paul's scarlet rose cells in culture

Summary Cells ofRosa sp. cv Paul's scarlet have been reported to require cytokinin for growth in suspension culture. This report was verified in the present study. However, a rose cell line that was maintained for 2 yr in suspension culture by routine subculturing developed the capacity to grow without exogenous cytokinin. The stability of the cytokinin requirement and the basis for this altered response to cytokinin was investigated. The parental cell line, which has been maintained independently on agar-solidified medium, was subcloned and the cytokinin dependence of the subclones was determined. The subclones were found to exhibit a continuous spectrum of responses, ranging from a high degree of cytokinin dependence for growth to rapid growth upon the initial transfer to cytokinin-deficient medium. The average growth constant (K=1n W/Wo; Wo=initial fresh weight,W=fresh weight after growth for the stated time interval) of 30 subclones grown on medium containing 0.5 μM zeatin was 3.1, with a range of 1.1 to 4.0. The average growth constant of the same subclones when grown on medium lacking a cytokinin was 1.5, with a range of 0 to 3.9. By comparison, the parental cell line exhibited growth constants of 3.5 in the presence of 0.5 μM zeatin and 1.6 in the absence of exogenous cytokinin. Although the growth of some of the subclones after transfer to cytokinin-deficient medium suggested that they were cytokinin autotrophs, this was not the case because none of them grew after a second transfer to medium lacking cytokinin. Culture in medium containing cytokinin conferred upon the cells the capacity for a limited amount of growth after subsequent transfer to medium lacking cytokinin. The extent of this cytokinin-induced growth potential varied from subclone to subclone. Efforts to determine the frequency with which cytokinin autotrophs appeared in a subclone that required cytokinin suggested that it is a rare event and that the cytokinin requirement is a fairly stable phenotypic characteristic of these cells. This research was supported by Grant PCM 7722398 from the National Science Foundation.     

Hormone-mediated translational control of protein synthesis in cultured cells of Glycine max

Proliferative growth in cultured soybean cells (Glycine max cv. Sodifuri, of cotyledenary origin) is regulated to a substantial degree by the plant hormone, cytokinin. Previously we reported that stationary phase soybean cells exhibit a rapid increase in polyribosome formation upon transfer to fresh cytokinin-containing medium. Cytokinin plays a significant role in this expansion of the cell's protein synthetic capacity through a heretofore unidentified effect on protein synthesis at the translational level. The present study was undertaken to identify the deficiency in the translational process of stationary phase cells which is overcome by cytokinin treatment. We have demonstrated that cytokinin-induced polyribosome formation was brought about by the recruitment of existing monoribosomes into polyribosomal aggregates. Three hours of cytokinin stimulation nearly doubled the total amount of [³H]leucine incorporated into nascent polypeptides. Average polyribosome size was nearly identical in stationary phase cells and in cells which were induced to grow by cytokinin. Cytokinin-enhanced protein synthetic capacity was not accompanied by changes in polypeptide chain elongation or termination rates. Partial cycloheximide inhibition of elongation was used to demonstrate that initiation of polypeptide synthesis was limiting to a slight degree both before and after cytokinin stimulation. These results suggest that cytokinin stimulates protein synthesis by making more message available for translation. We obtained direct evidence to support this hypothesis by showing that labeled poly(A)-containing, nonpolyribosomal RNA, which was synthesized during stationary phase but not translated, moves into polyribosomes upon cytokinin treatment.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 M), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin.Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed.Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP.A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Cytokinins from a variant strain of cultured soybean cells

A strain of soybean cells capable of growing on a tissue culture medium lacking a cytokinin produced at least 3 compounds active in the soybean cytokinin assay. The characteristics of these compounds were consistent with their being zeatin in the free form, zeatin ribonucleoside and zeatin ribonucleotide. Although the conversion from a cytokinin dependent to independent condition in this strain parallels the change of normal cells to crown gall tumor state in terms of the capacity to synthesize cell division substances, the soybean factors are distinct from the nicotinamide derivatives reported for tumor cells of Vinca.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 μM), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin. Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed. Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP. A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Regulators of Cell Division in Plant Tissues

The cytokinins, 6-benzylaminopurine and kinetin, markedly enhanced the yield of both free and membrane-bound 80S ribosomes per unit weight of radish (Raphanus sativus) cotyledon tissue. The response was observed only after the induction of growth by cytokinin; during the lag period preceding cytokinin-induced growth, ribosome yields from both control and cytokinin-treated cotyledons were below detectable levels. Mannitol depressed both growth and ribosome yield to the same degree. The enhanced ribosome yield appeared to be an indirect effect of cytokinin and was probably a consequence of cytokinin-induced growth. The effect of 6-benzylaminopurine on ribosome yield was not reflected in enhanced levels of cytoplasmic ribosomal RNA, while recently synthesized ribosomes were found to be more readily recovered from cytokinin-treated tissue than from control tissue. It was concluded that cytokinin-enhanced ribosome yield resulted from enhanced ribosome recovery or extractability and that ribosome yield is an unreliable indication of ribosome level in plant tissue.     

Zeatin-induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes

* Here, cytokinin-induced nitric oxide (NO) biosynthesis and cytokinin responses were investigated in Arabidopsis thaliana wild type and mutants defective in NO biosynthesis or cytokinin signaling components. * NO release from seedlings was quantified by a fluorometric method and, by microscopy, observed NO biosynthesis as fluorescence increase of DAR-4M AM (diaminorhodamine 4M acetoxymethyl ester) in different tissues. * Atnoa1 seedlings were indistinguishable in NO tissue distribution pattern and morphological responses, induced by zeatin, from wild-type seedlings. Wild-type and nia1,2 seedlings, lacking nitrate reductase (NR), responded to zeatin with an increase within 3 min in NO biosynthesis so that NR does not seem relevant for rapid NO induction, which was mediated by an unknown 2-(2-aminoethyl)2-thiopseudourea (AET)-sensitive enzyme and was quenched by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO). Long-term morphological responses to zeatin were severely altered and NO biosynthesis was increased in nia1,2 seedlings. As cytokinin signaling mutants we used the single-receptor knockout cre1/ahk4, three double-receptor knockouts (ahk2,3, ahk2,4, ahk3,4) and triple-knockout ahp1,2,3 plants. All cytokinin-signaling mutants showed aberrant tissue patterns of NO accumulation in response to zeatin and altered morphological responses to zeatin. * Because aberrant NO biosynthesis correlated with aberrant morphological responses to zeatin the hypothesis was put forward that NO is an intermediate in cytokinin signaling.     

Benzyladenine-controlled protein synthesis and growth in apple cell suspensions

Prolific growth in cytokinin-requiring apple cell suspensions was achieved during 10 days of culture with appropriate concentrations of benzyladenine (BA). Unlike the controls, BA-treated cells showed a well developed sub-cellular organization and protein synthesis. Upon transfer to fresh cytokinin-free medium, however, these cells exhibited a rapid decrease in polyribosome formation, but with unchanged nuclear and nucleolar activities. Cells lacking added cytokinin showed a reduced metabolic activity and failed to divide. Deficiency in the translation process of cytokinin-deprived cells was overcome by exogenous BA-treatment. In cells maintained on a cytokinin-free medium for 5 days, BA-treatment enhanced protein synthesis from pre-existing mRNA in a specific way.     

Kinetics of Determination in the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar α-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells.     

Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

Changes in ribosome-polyribosome balances in chick muscle cells during tissue dissociation, development in culture, and exposure to simplified culture-medium

The populations of polyribosomes, monomeric ribosomes, and ribosomal subunits are described from the time of tissue explantation to the time of complete muscle differentiation in primary cultures of chick muscle cells. There is extensive degradation of polyribosomes, and a net loss of ribosomes recovered, as cells of embryonic muscle are dissociated with proteolytic enzymes. The cells rapidly restore a high polysome: monomeric ribosome ratio. This recovery of the polyribosome population occurs before there is any detectable net increase in ribosome number. Ribosome production begins after a lag of approximately 15 hours in culture. Number of ribosomes/cell triples by 60 hours, at which time cell fusion (myotube formation) is complete. Unlike developing muscle in vivo, cultured cells have a very reduced pool of monomeric ribosomes. Medium simplification experiments done with fully differentiated cultures show, however, that monomers accumulate during starvation. These monomers reassociate to form polyribosomes during medium replenishment. Subunit complements are maintained at a constant level regardless of nutritional conditions. These features of cultured muscle are discussed as possible tools for further study of muscle development.     

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea ) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).     

Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: Correlation of metabolic differentiation with morphological differentiation

A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot ( Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10⁻⁷ M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10⁻⁷ to 10⁻⁸ M zeatin, 2,4-D at higher concentrations than 10⁻⁷ M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (>14% of Ficoll) in a medium containing 10⁻⁷ M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (<12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.     

Factors affecting in vitro shoot formation from vegetative shoot apices of apple and relationship between organogenic response and cytokinin localisation

ABSTRACT

The effects of macro- and micro-elements, benzyladenine (BA) concentration, and the period of auxin application on adventitious shoot formation from callus originating from vegetative shoot apices were tested on apple (Malus domestica Borkh) rootstock Jork 9. The putative relationship between organogenic response and cytokinin localisation was also studied by an immunolocalisation technique for in situ determination of free cytokinins. The use of MS (Murashige & Skoog, 1962) salts in the medium instead of those of LP (Quoirin & Lepoivre, 1977) had a strong positive effect both on shoot formation rate and on the number of shoots produced. The highest organogenic response from callus was induced using 17.8 μM BA in the presence of 2.7 μM NAA and by maintaining the explants for 20 days in darkness, then transferring them to fresh auxin-free medium and to the light. The in situ localisation studies, performed using antibodies with a marked specificity against zeatin and isopentenyladenine, revealed changes in the localisation of free zeatin in the tissues during the shoot-forming process, in particular during the active cell division phase leading to callus formation, and in the initial phase of bud formation. Changes in zeatin distribution in the tissues of the vegetative shoot apex during shoot formation may indicate a role for this cytokinin free base in cell differentiation and organogenesis.     

Cytokinin-induced switch in development in excised cotyledons of radiata pine cultured in vitro

Cotyledons of Pinus radiata D. Don were cultured under shoot-forming (plus cytokinin) and elongating (minus cytokinin) conditions. Using. autoradiographic and precursor incorporation techniques, the sites and rate of macromolecular synthesis were examined during the first five days in culture. Active incorporation of ³H-thymidine, ³ H-uridine and ³H-leucine occurred. In shoot-forming cotyledons the incorporation became preferentially located in the epidermal and sub-epidermal cell layers in contact with the medium. In elongating cotyledons, in contrast, incorporation was randomly distributed, and the amount of incorporation declined with time. Biochemically, differences in DNA, RNA and total protein synthetic patterns were observed. In elongating cotyledons the rates of RNA and protein synthesis were higher during the first 48 h than in shoot-forming tissues, after which the synthetic rates were similar. Two peaks of newly formed DNA were observed in both tissues. These findings indicate that the cytokinin-induced changes in developmental pathways began within 24 h in culture.     

Plant regeneration from cell suspension cultures ofArabidopsis thaliana heynh

A technique is described for the establishment, maintenance, and regeneration of plants from cell suspension cultures ofArabidopsis thaliana (L) Heynh. Friable, rapidly growing cell suspension cultures were initiated from leaf or hypocotyl callus cultures and these have been maintained in liquid culture for 24 months. The cells grown in liquid culture were used to study the effects of growth regulators, medium salts composition, culture temperature, sucrose concentration and medium solidifying agents on morphogenesis. The most important parameters for plant regeneration were culture temperatures lower than 25°C, the medium solidification agent gelrite at 0.2% (w/v) and zeatin or thidiazuron as the choice of cytokinin. These cell suspensions continue to regenerate fertile plants with a total of over 200 plants having been rooted to date and they also serve as convenient sources of cells for protoplast isolation, biochemical, and molecular assays.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine     

Osmotic pretreatment promotes axillary shooting from cauliflower curd pieces by acting through internal cytokinin level modifications

In vitro propagation of cauliflower has generally been achieved through axillary shoot proliferation of curd explants on Murashige and Skoog (MS) medium supplemented with an auxin and a cytokinin. Recently, it has been shown (Vandemoortele 1999) that a soaking in sucrose (-2 MPa for 24 h) of cauliflower curd explants, before culture without any growth regulator, also induced axillary branching. The later procedure avoids the phenomenon of hyperhydricity in the shoots formed. Axillary shooting obtained by the two methods appears to be mediated by modifications of internal cytokinin levels. The osmotic pretreatment did not influence auxin levels, but induced a zeatin and a zeatin riboside levels increase. Curd explants cultured with the usual procedure (on MS medium supplemented with 5 μmol/L BA and 0.5 μmol/L NAA) showed a zeatin and zeatin riboside levels increase of the same magnitude and a higher one for isopentenyl adenine and isopentenyl adenosine. The modification of the cytokinin status in the curd explants subjected to a short osmotic pretreatment thus should be less favourable for hyperhydricity.     

Dependence of cytokinin distribution in plants on their physical and chemical properties and transpiration rate

The effect of transpiration on cytokinin accumulation and distribution in 7-day-old wheat (Triticum durum Desf.) seedlings grown on nutrient medium supplemented with zeatin or its riboside was studied. The content of cytokinins in plants and nutrient medium was measured by the immunoenzyme analysis; cytokinin distribution between root cells was assessed immunohistochemically using antibodies against zeatin derivatives. The rate of transpiration was reduced 20-fold by plant placing in humid chamber. At normal transpiration, after 6 h of plant incubation on the solution of zeatin, the level of cytokinins in plant tissues increased stronger than after incubation on the solution of zeatin riboside (by 7.3 and 3.5 times, respectively, as compared with control), although the rates of both cytokinin uptake were equal. Most portions of cytokinins were retained in the roots, which was stronger expressed in the case of free zeatin uptake. A decrease in the rate of transpiration did not affect substantially the zeatin absorption from nutrient medium and the total level of cytokinin accumulation in plants, but these indices were sharply decreased in the case of zeatin riboside. In the zone of absorption of both control roots and roots treated with cytokinins, more intense cytokinin immunostaining was observed in the cells of the central cylinder. The interrelation between cytokinin distribution between the cells and apoplast, their inactivation, and transport over the plant and their form (zeatin or zeatin riboside) used for treatment is discussed.