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.     

Physiological Comparisons of Pith Callus With Crown-Gall and Genetic Tumors of Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii Grown in Vitro. II. Nutritional Physiology

Explants of genetic tumors, tumors initiated by Agrobacterium tumefaciens strains B-6 and T-37, and excised pith plugs from Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii were cultured on Murashige and Skoog medium. All cultures, pith callus and tumors with the exception of N. langsdorffii pith grew on this medium. Addition of glutamine to the medium resulted in highly organoid growth in N. langsdorffii pith. In order to have material comparable to other pith cultures, N. langsdorffii was initiated on 2,4-dichlorophenoxyacetic acid medium, after which it grows on complete medium as amorphous pith callus. Except for the initiation of N. langsdorffii (and N. glauca) pith, the 2,4-dichlorophenoxyacetic acid medium, caused bleaching in cultures of T-37 induced tumors and death of B-6 induced tumors. Tumor cultures, except for the seedling tumor, grew well on a minimal medium lacking kinetin, indoleacetic acid, vitamins, glycine, and inositol. Glycine was necessary only in the growth of N. langsdorffii pith callus. A tissue culture model is presented which permits comparison of the various tissue types.     

Dominant lethal assay in female mice after oral dosing with dichlorvos or exposure to atmospheres containing dichlorovos.

The effect of a synthetic auxin-like substance (2,4-D) and a synthetic cell division factor (kinetin) on the induction of chromosome aberrations was studied on tissue cultures of Nicotiana glauca and the tumorous amphidiploid hybrid Nicotiana glauca × Nicotiana langsdorffii.The aberration frequencies in normal Nicotiana glauca tissue were proportional to the length of time of culture in the presence of 2,4-D. Moreover, both 2,4-D and kinetin increased chromosome breakage in the habiatouated Nicotiana glauca tissue but not in the amphidiploid hybrid tissue.The data are discussed in terms of genotype-hormone equilibria in long-term development of plant tissue culture.     

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.     

The influence of genomes on autonomous growth of pith cultures of Nicotiana glauca-Langsdorffii hybrids

Summary A differential influence of the two parental genomes on cell proliferation and morphogenesis in pith tissue explants can be observed among the various tumorous hybrid combinations between Nicotiana glauca Grah. and N. langsdorffii Weinm.: the F₁ hybrid (GL), its amphiploid (GGLL), and two different triploids (GGL and GLL). This influence was evident when the explants were cultured in the presence of exogenous auxin (indole-3-acetic acid, 2.5 M), supplied either continuously or for a brief period of time. Compared with the F₁ and the amphiploid, the higher proportion of N. glauca genomes in GGL cells resulted in greater growth, the higher proportion of N. langsdorffii genomes in GLL cells in lesser growth. In addition, shoots are produced on the GGL callus, while only roots are formed on calli of the other types in the same medium. When, in addition to auxin, a cytokinin [6-(3-methyl-2-butenyl-amino)purine] was added to the culture medium, the differential growth of the different tissue types was less pronounced; at 1.0 M of the cytokinin, all tissues grew at about the same rate and remained undifferentiated, regardless of their genomic composition.     

Evidence for an abundant 33,000-dalton polypeptide regulated by cytokinins in cultured tobacco tissues

When cloned pith and leaf tissues of Nicotiana tabacum L. cv. Havana 425 are subcultured for 3 d on auxin-containing medium and labelled for 18 h with [³⁵S]methionine, up to 10% of the labelled, soluble-protein fraction is found in a single band with an apparent molecular weight of approx. 32,000–34,000 dalton on sodium-dodecylsulfate polyacrylamide-gel electrophoretograms. The labelling of this band, designated P33, is dramatically inhibited by the cytokinin, kinetin, in some cell lines at concentrations as low as 1.4·10^-8 M. P33 is a major component of the protein fraction obtained from non-habituated clones, cytokinin-habituated clones, and revertant subclones of crown-gall-transformed clones, but cannot be detected in clones habituated for both auxin and cytokinin, or crown-gall-transformed clones. The evidence supports the hypothesis that cytokinin in the presence of auxin regulates the production of a specific, major polypeptide in the soluble-protein fraction of the tissue and that this protein is not produced in tissues autotrophic for both auxin and cytokinin.     

ORIGIN OF ADVENTITIOUS SHOOTS REGENERATED FROM CULTURED TOBACCO LEAF TISSUE

Leaf discs from Nicotiana tabacum L., N. glauca Grahm., and a series of interspecific periclinal chimeras were cultured on Murashige and Skoog (MS) medium containing either 2.5 mg/1 6-benzylaminopurine(BA) or 3.0 mg/16-furfurylaminopurine (kinetin). Most shoots regenerated from chimeral leaf discs were non-chimeral but 51 of 658 shoots were chimeral. The histogenic composition of plants regenerated from leaf discs of periclinal chimeras indicated that any cell layer in a leaf can be the ultimate source of shoots, and that shoots can be multicellular in origin. Certain periclinal arrangements were recovered more frequently and their chimeral nature was more stable during subsequent shoot growth. While N. tabacum leaf discs regenerated shoots on MS medium supplemented with either BA or with kinetin, N. glauca leaf discs did not form shoots on the medium containing kinetin. However, chimeras which possessed cells of both species arose on medium containing BA or kinetin, indicating that the morphogenetically competent (i.e., able to produce shoots in culture), N. tabacum cells either interacted with N. glauca cells leading to their competence or incorporated non-competent N. glauca cells into nascent shoot apical meristems.     

Cytokinin autotrophy and differentiation in tissue cultures of haploid Nicotiana tabacum L.

Explants from the apical region (10 cm from the tip) of haploid Nicotiana tabacum cv. Wisconsin-38 were cultured on media with and without kinetin. Cell lines were selected in the dark and in the light. Cytokinins were extracted from the apical region of haploid plants and from callus tissues after 84 days of growth (third transfer culture). Chlorophyll was extracted from callus grown under light after 21 days of growth at each of the four cell line selection steps. Kinetin (+) cell lines and cytokinin autotrophic tissues grown in the light showed a compact growth pattern. Microscopic examination of these callus showed the presence of large numbers of nodules consisting of tracheary elements, parenchymatic cells, sieve elements and meristematic cells. Cytokinin-autotrophic callus grown in the dark showed an irregular growth pattern presenting regions of compact tissue and friable tissue. The compact tissue contained large amounts of nodules similar to those of kinetin (+) tissues and of cytokinin autotrophic tissues grown in the light. Extraction of the compact and the friable callus components showed high cytokinin activity in the compact region and low activity in the friable portion. It is suggested that cytokinin synthesis is related to the differentiation of the nodular structures. The amount of chlorophyll increased during the process of cytokinin autotrophic cell line selection.     

L'Indole et le Tryptophane "régulateurs" de croissance, en culture "in-vitro", pour les tissus de parenchyme médullaire de Tabac, normaux et tumoraux

Indoleacetic acid, tryptophan and indole promote growth of pith tissues of tumorous Nicotiana glauca×N. langsdorffii amphidiploïd hybrid, but only indoleacetic acid gives the potentiality of continuous development to the tumorous tissues after some days of contact on a medium without growth substances. In combination with 6-benzylaminopurine, tryptophan and especially indole are promoting substances like auxin for the non-tumorous pith tissues of tobacco, and allow a considerable organogenesis.     

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.     

Introduction of xylem differentiation in lactuca by ethylene

Evidence was obtained to support the hypothesis that ethylene is involved in xylem differentiation in primary pith explants of Lactuca sativa L. cv Romaine cultured in vitro. Xylem elements differentiated when explants were supplied indole-3-acetic acid (IAA) in combination with either the ethylene biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene-releasing agent 2-chloroethylphosphonic acid (CEPA), or kinetin. In contrast, no xylem elements differentiated in the presence of IAA, kinetin, ACC, or CEPA alone, or when kinetin was supplied together with ACC or CEPA. These results show that ethylene will substitute qualitatively for cytokinin during auxin-induced xylogenesis, and suggest that both ethylene and auxin are required for xylem differentiation in Lactuca.     

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 controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

Excised Tobacco Pith Bioassays for Root-knot Nematode-produced Plant Growth Substances

Root-knot nematodes, Meloidogyne incognita, were grown in tobacco pith tissue cultured in vitro on modifired white's medium. The nematodes did not secrete detectable amounts of auxin or cytokinin, but did promote growth in the presence of both types of growth substance. When both IAA and kinetin were supplied, the nematode-infected tissue was larger, more irregularly shaped than control tissue; nematodes induced syncytia and developed past the second larval stage. When either IAA or kinetin was omitted from the medium, nematodeinfected and control tissue failed to grow and the nematodes failed to induce syncytia or develop.     

Kinetin inhibition of h-uracil and C-leucine incorporation by tobacco cells in suspension culture

The rate of ¹⁴C-leucine and ³H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 × 10⁻⁵m. Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N⁶-methyl-aminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N^6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.     

Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

Variation in the Competence of Tobacco Pith Cells for Cytokinin-Habituation in Culture

Pith parenchyma tissues of tobacco sometimes lose their exogenous requirement for a cell division factor such as the cytokinin, kinetin. This process, known as cytokinin habituation, appears to involve epigenetic changes since it is a heritable change in cell phenotype which is directed, regularly reversible, and leaves the cell totipotent. In this report, we show that pith cells in culture consist of at least two types of cytokinin-requiring cells. The first type habituates rapidly under inductive conditions. The second type continues to express the cytokinin-requiring phenotype for many cell generations in culture but retains the capacity for habituation. These findings suggest that pith cells differ in their competence to habituate and that different states of competence are inherited by individual cells.     

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.     

N9-substituted derivatives of kinetin: effective anti-senescence agents

The first isolated cytokinin, 6-furfurylaminopurine (kinetin or Kin), was identified almost 55 years ago. Its biological effects on plant cells and tissues include influences on such processes as gene expression, cell cycle, chloroplast development, chlorophyll biosynthesis, stimulation of vascular development, delay of senescence, and mobilization of nutrients. In the present study we prepared a series of eight N9-substituted Kin derivatives, and characterized them with available physicochemical methods such as CI+ mass spectrometry and ¹H NMR spectroscopy. All compounds were tested in three classical cytokinin bioassays: a tobacco callus assay, an Amaranthus assay, and a senescence assay with excised wheat leaves. The ability of the compounds to interact with Arabidopsis cytokinin receptors CRE1/AHK4 and AHK3 was tested in a bacterial receptor assay. Prepared derivatives with certain substitutions of the N9-atom of the purine moiety enhanced the cytokinin activity of the parent compound in the bioassays to a remarkable degree but negatively affected its perception by CRE1/AHK4 and AHK3. The ability of compounds to delay the senescence of excised wheat leaves in both dark and light conditions, was highly correlated with their ability to influence membrane lipid peroxidation, which is a typical symptom of senescence. Our results were corroborated by gene expression profiling of those genes involved in cytokinin metabolism and perception, plant senescence, and the stress response, and suggest that prepared kinetin derivatives might be used as potent anti-senescence agents.     

Correlation between Auxin Resistance and the Lack of a Membrane-Bound Auxin Binding Protein and a Root-Specific Peroxidase in Nicotiana tabacum

The levels of a membrane-bound auxin binding protein (MABP) and a root-specific peroxidase (RSP) were studied in several tobacco (Nicotiana tabacum L.) cell lines including an auxin-resistant variant. Groups of cell lines were distinguished which behaved differentially with respect to MABP and RSP depending on the hormonal composition of the medium. In cell lines in which there existed a correlation between the presence or absence of MABP and that of RSP both phenotypes were expressed if kinetin (1-2 micromolar) was supplied. In contrast, neither MABP nor RSP could be detected under any hormonal conditions tested in the auxin-resistant variant which retains the ability to differentiate shoots but lacks the ability to differentiate roots. About an eightfold increase in the concentration of MABP and a dramatic increase in the activity of RSP occurred in a transformant by a mutant strain of Agrobacterium tumefaciens lacking an intact cytokinin gene when it was grown on medium containing 1 to 2 micromolar kinetin. A correlation between auxin resistance and the lack of MABP and RSP suggests that MABP might be involved in auxin-mediated root differentiation in tobacco.