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.     

Tissue-Specific Variation in the Cytokinin Habituation of Cultured Tobacco Cells

Cells of tobacco pith parenchyma sometimes lose their requirement for an exogenous supply of a cell division factor usually supplied as the synthetic cytokinin, kinetin. This change in phenotype, known as cytokinin habituation, is inherited by individual cells and appears to result from epigenetic changes rather than from rare, random, genetic mutations. We have found that tissues from different regions of the tobacco plant exhibit different states of habituation in culture. Pith tissues, as reported earlier, are usually cytokinin requiring and rapidly shift to the habituated state in culture. Leaf tissues are very slightly habituated and require kinetin for optimal rates of growth. Tissues from the stem-cortex are initially habituated. Both the leaf and cortex phenotypes are inherited by individual cells and persist for many cell generations in culture. These results show that certain tissue-specific phenotypes persist in culture and provide evidence that a process akin to habituation leading to different stable states of cytokinin requirement occurs in normal development.     

Cytokinin Habituation in Juvenile and Flowering Tobacco

The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38.Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.     

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.     

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     

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.     

Meiotic transmission of epigenetic changes in the cell-division factor requirement of plant cells

During the development of tobacco plants, cells undergo epigenetic changes that alter their requirement in culture for the cell-division factor cytokinin. Cultured leaf cells alternate between cytokinin-requiring (C-) and cytokinin-independent (C+) states at extremely high rates of approximately 10-2 per cell generation by a process called pseudodirected variation. Here we show that plants regenerated from most C+ clones express the Habituated leaf (Hl) trait, i.e., leaf tissues exhibit the C+ phenotype rather than the wild-type C- phenotype in culture. This new trait then segregates as a monogenic dominant trait indicating that conversion of C- cells to C+ cells is associated with a meiotically transmissible, genetic modification. Two independent mutants, Hl-2 and Hl-3, derived from C+ variants arising in culture were unstable in planta and reverted gametically at rates roughly comparable to pseudodirected variation in culture. Cells of the Hl-2 mutant, but not of a stable Hl-1 mutant, reverted phenotypically at high rates in culture. This revertant C- phenotype persisted in some plants regenerated from cloned revertant lines, and then showed irregular segregation in two successive sexual generations. These results show for the first time that meiotically transmissible epimutations can occur reversibly and at high rates in culture.     

Rapid Reversion of Cell-Division-Factor Habituated Cells in Culture

Abstract. The heritable conversion of cell-division factor (CDF)-requiring tobacco pith cells to the CDF-autotrophic state, known as CDF habituation, is a directed process that occurs at very high rates in culture. Here we show that tiny, leafy buds arising from habituated tissues placed on an inductive medium exhibit the non-habituated phenotype. The appearance of these buds was used to estimate the rate at which habituated cells revert. The rate obtained, approx. 4 × 10^-3 per cell generation, is at least 100 to 1,000 fold higher than expected for somatic mutation. Moreover, reversion is a directed process, i.e., it occurs at high rates only under conditions that promote bud formation. These findings provide compelling evidence that habituation of tobacco pith cells involves epigenetic changes rather than rare, random genetic mutations.     

Cytokinin Production by Rhizobium japonicum

Possible hormonal interactions between soybean roots and the Rhizobium initiating nodule proliferation in this genus were studied. A cytokinin has been isolated by column and paper chromatography from an effective strain of Rhizobium japonicum grown in pure culture. The substance promotes cell proliferation in a cytokinin-requiring soybean callus tissue. The bacteria are capable of conditioning a cylokinin-free soybean culture medium so that it is able to support the cytokinin-requiring tissue. It is concluded that the substance is a product of the bacteria rather than an artifact of purification. This unidentified cytokinin or a substance moving to a similar Rf value on the paper chromalogram produces polyploid divisions when tested on cultured pea root segments. Some of the division figures exhibit the diploclirornosomes typical of the root nodule primordium in pea.     

Activity and accumulation of cell division-promoting phenolics in tobacco tissue cultures

Dehydrodiconiferyl alcohol glucosides (DCGs) are derivatives of the phenylpropanoid pathway that have been isolated from Catharansus roseus L. (Vinca rosea) crown gall tumors. Fractions containing purified DCGs have been shown previously to promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins. In this study, we utilized synthetic DCG isomers to confirm the cell division-promoting activity of DCG isomers A and B and show that they neither promote shoot meristem initiation on Nicotiana tabacum L., cv Havana 425, leaf explants nor induce betacyanin synthesis in amaranth seedlings. Analysis of cultured tobacco pith tissue demonstrated that DCG accumulation was stimulated by cytokinin treatment and correlated with cytokinin-induced cell division. Thus, the accumulation of metabolites that could replace cytokinin in cell division bioassays is stimulated by cytokinins. These data support the model that DCGs are a component of a cytokinin-mediated regulatory circuit controlling cell division.     

Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth

The dehydrodiconiferyl alcohol glucosides A and B are factors isolated from transformed Vinca rosea tumor cells that can replace the cytokinin requirement for growth of tobacco (Nicotiana tabacum) pith and callus cells in culture. These factors, present in tobacco pith cells, have their concentrations elevated approximately 2 orders of magnitude after cytokinin exposure. Biosynthesis experiments showed that these compounds are not cell wall fragments, as previously suggested, but are produced directly from coniferyl alcohol. Their synthesis is probably associated with the existing pathway for cell wall biosynthesis in both Vinca tumors and tobacco pith explants. The pathway requires only two steps, the dimerization of coniferyl alcohol by a soluble intracellular peroxidase and subsequent glycosylation. Biosynthetic experiments suggested that dehydrodiconiferyl alcohol glucoside breakdown was very slow and control of its concentration was exerted through restricted availability of coniferyl alcohol.     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

Peptides of normal and variant cells of tobacco

Polypeptides solubilized from established normal and variant cell lines of Nicotiana tabacum L cv “Wisconsin 38” have been analyzed by one-dimensional and two-dimensional gel electrophoresis. There was little variability observed in the polypeptide profile in an established cell line; polypeptides present in different clonal lines of cells, all derived from an initial established cell culture, were very similar, if not identical. However, a large fraction of the observed polypeptides present in cytokinin-habituated cell lines (up to 3.8% of the total polypeptides analyzed by two-dimensional gel electrophoresis) were different from those found in the cytokinin-requiring cells from which they were selected. The habituated nature of the selected cell lines was demonstrated to be epigenetic; tissue cultures that were reisolated from plantlets regenerated from habituated cell lines did require cytokinin. Further observations demonstrate: (1) that epigenetic events that alter a cellular phenotype change the expression of a relatively large number of polypeptides, (2) that a single epigenetic phenotype may be the result of any one of a number of possible patterns of gene expression, and (3) that epigenetic events are not random events.     

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.     

Polyribosome Formation in Relation to Cytokinin-induced Cell Division in Suspension Cultures of Glycine max [L.] Merr

We have investigated the relationship between cell proliferation and protein synthetic capacity in a cytokinin-requiring strain of cultured soybean cells (Glycine max [L.] Merr. cv. Sodifuri, of cotyledonary origin) in suspension culture. When transferred to a defined medium lacking cytokinin, very little cell division or cell enlargement took place over the course of a 6-day culture period. Cells transferred to medium of the same composition, but containing 0.5 mum zeatin, exhibited rapid initial growth, with maximum mitotic activity occurring after 24 hours in culture, and a doubling of the cell population within the first 36 hours of the culture period. The polyribosomal RNA content of the cells decreased over the course of the first 24 hours of the growth cycle while the polyribosome to monoribosome (P/M) ratio increased. The increase in the P/M ratio was greater in the cytokinin-treated cells. This apparent relationship between cytokinin-induced cell proliferation and polyribosome formation was examined further. Polyribosome formation was stimulated when zeatin was added directly to cell populations which had been cultured for 24 hours in medium lacking a cytokinin. Transfer to fresh medium alone also stimulated polyribosome formation, whether this medium contained a cytokinin or not. The magnitude of transfer-induced polyribosome formation depended upon the initial cell density (number of cells/ml of medium). Regardless of the initial cell density and independent of the P/M ratios attained, the cytokinin-treated cell populations divided while the cytokinin-deprived cell populations did not. In vivo labeling with [(35)S]methionine and slab gel electrophoretic separation of sodium dodecyl sulfate derivatives of the labeled polypeptides demonstrated qualitative changes in the spectrum of proteins synthesized by the cytokinin-treated cells. These qualitative changes were independent of the cell density (and hence, independent of the P/M ratio) but they preceded cytokinin-induced cell division.     

5-Bromodeoxyuridine: A specific inhibitor of cytokinin-habituation in tobacco cell culture

Summary Cytokinin-habituated cells of Nicotiana tabacum L. cv. Havana 425 are able to grow in culture without added cytokinin. The thymidine analogue, 5-bromodeoxyuridine (BUdR), which selectively inhibits differentiation of animal cells, blocks expression of the cytokinin-habituated phenotype in culture. This effect is prevented by thymidine and is reversible. These findings suggest that habituation and animal cell differentiation have a common mechanism. BUdR provides a useful tool for investigating the metabolism of cell division factors and its regulation in higher plant cells.Abbreviation BUdR 5-bromodeoxyuridine - BU 5-bromouracil     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

Host and T-DNA determinants of cytokinin autonomy in tobacco cells transformed byAgrobacterium tumefaciens

The hormone autonomy of tobacco (Nicotiana tabacum L.) cells transformed byAgrobacterium tumefaciens containing mutations attmr (the “rooty” locus) of the pTiT37 plasmid has been examined. These cells require cytokinin, but not auxin for continuous growth in culture, indicating that the function of thetmr locus is to specify or induce cytokinin autonomy. Examination of tissues from plants regenerated from cells transformed by the mutant bacteria showed that the auxin independent phenotype is suppressed, but can be reinitiated in culture by exposure to an exogenous supply of auxin. In addition the developmental state of the cells from such regenerated plants can exert a profound influence on their cytokinin autonomy phenotype.