The role of cellulase in hormonal regulation of shoot morphogenesis in tobacco callus

A polyclonal antibody raised against cellulase (EC 3.2.1.4.) from callus ofNicotiana tabacum L. cv. Petit Havana SR1 reduced cellulase activity and induced shoot formation in tobacco callus in the presence of callus maintaining concentrations of auxin and cytokinin. Shoot induction as well as reduction of the cellulase activity was also obtained by withdrawing auxin from the callus medium. The effect of the two hormones on cellulase activity in the tobacco tissue was examined by varying the concentration of one of the hormones -naphthylacetic acid (NAA) or benzylaminopurine (BAP) at a time while the other was kept at a level sufficient for either callus growth or shoot induction. While NAA stimulated the enzyme activity increasingly with concentration in the range 5 × 10^–7 M to 5 × 10^–5 M at both levels of BAP, BAP only stimulated the cellulase activity at an optimum concentration of 5 × 10^–6 M when NAA was present at a level sufficient to induce callus growth. The results point to a pivotal role of the downward regulation of cellulase in the initiation of shoot induction. A series of events leading to oriented cell divisions as a result of the lowered cellulase level during the initial phase of the morphogenetic process is discussed.Abbreviations Ab Purified cellulase antibody - BAP benzylaminopurine - MS Murashige and Skoog medium - NAA -naphthylacetic acid - PS Purified preimmune serum We thank Mr. Poul Fabech for constructing the automatic viscosimetric equipment and Mr. Hans Hjorth for making the computer programme. This work was supported by The Danish Veterinary and Agricultural Research Council.     

Impermeant auxin analogues have auxin activity

Protein conjugates of 5-aminonaphthalene-1-acetic acid and of 5-azido-naphthalene-1-acetic acid have been prepared and evaluated for auxin activity in two types of assay. In standard elongation tests with pea (Pisum sativum L.) epicotyl sections the conjugates are inactive. However, if the epicotyls are abraded to perforate the cuticle, auxin activity is observed provided that the conjugates are not too large to traverse the cell wall. In a system lacking a cell wall — tobacco (Nicotiana tabacum L.) protoplasts — conjugates of widely differing size are able to induce membrane hyperpolarization. These results support other recent evidence that auxin receptors are exposed at the exterior face of the plasma membrane and indicate that auxins can produce both rapid and longer-term responses without entering the cell.Abbreviations ABP auxin-binding protein - BSA bovine serum albumin - E_m transmembrane potential difference - KLH keyhole limpet hemocyanin - NAA naphthalene-1-acetic acid This work was partly supported under the Biotechnology Action Programme of the European Economic Communities. We thank Mr. P. Cozens for technical assistance.To whom correspondence should be addressed.     

A specific radioimmunoassay for nanogram quantities of the auxin,indole-3-acetic acid

We have developed a specific radioimmunoassay [RIA] for indole-3-acetic acid (IAA) in the 0.2 ng to 12 ng range which, in principle, can be extended to other indole auxins as well. Methods are presented for obtaining suitable antibody, for the RIA procedure, and for measuring IAA in methanolic extracts of plant tissues. Antibody specific for IAA was obtained from rabbits immunized with IAA bound to bovine serum albumin by formaldehyde treatment. In assays with this antibody, 2,4-dichlorophenoxyacetic acid and indoles structurally related to IAA reacted from 300- to 3000-fold less than did IAA itself. However, -and -naphthaleneacetic acid reacted significantly and hence interfered with the assay. Extracts of tobacco (Nicotiana tabacum L.) tissue were immunoassayed after partial purification by buffer-ether partition. Crown-gall tumor tissue, which is auxin-autotrophic, and pith tissue depleted of auxin by the diffusion method contained, respectively, 26.7 ng and <0.5 ng extractable IAA per gram fresh weight.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - -NAA -naphthalenacetic acid - PBS phosphate-buffered saline - RIA radioimmunoassay     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Correlation between the nicotine content of tobacco plants and callus cultures

Callus cultures of two low-alkaloid lines of Nicotiana tabacum L. had considerably lower nicotine contents than cultures from the respective highalkaloid cultivars which were isogenic except for the two loci for alkaloid accumulation. Thus, there was a strong correlation between the nicotine content of callus cultures and the plants from which they were derived.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

Initiation of auxin autonomy in Nicotiana glutinosa cells by the cytokinin-biosynthesis gene from Agrobacterium tumefaciens

Agrobacteria carrying mutations at the auxin-biosynthesizing loci (iaaH and iaaM of the Ti plasmid) induce shoot-forming tumors on many plant species. In some cases, e.g. Nicotiana glutinosa L., tumors induced by such mutant strains exhibit an unorganized and fully autonomous phenotype. These characteristics are stable in culture at both the tissue and cellular level. We demonstrate that the cytokinin-biosynthesis gene (ipt) of the Ti plasmid is responsble for the induction of both auxin and cytokinin autonomy in N. glutinosa. Cloned cell lines carrying an ipt gene but lacking iaaH and iaaM are capable of accumulating indole-3-acetic acid. Interestingly, non-transformed N. glutinosa tissues exhibit an auxin-requiring phenotype when they are grown on medium supplemented with an exogenous supply of cytokinin. These results strongly indicate that exogenously supplied cytokinin does not mimic all the effects of the expression of the ipt gene in causing the auxin-autonomous growth of N. glutinosa cells.Abbreviations FW fresh weight - IAA indole-3-acetic acid - I⁶ Ado isopentenyladenosine - kb kilobase - MS Murashige and Skoog (medium) - NAA -naphthaleneacetic acid - NAM -naphthaleneacetamide - T-DNA transferred DNA     

Stimulation by auxin of phospholipase A in membrane vesicles from an auxin-sensitive tissue is mediated by an auxin receptor

Microsomal vesicles were prepared from zucchini (Cucurbita pepo L.) hypocotyls containing radioactive phosphatidylethanolamine or phosphatidylcholine, and these lipids were used as substrates by phospholipase A which is activated by auxins. Phospholipase D and phospholipase C hydrolysed the same substrates but were not influenced by auxin. Phospholipase A was activated by the auxins indolyl-3-acetic acid, 2,4-dichlorophenoxyacetic acid and, to a lesser extent, by -naphthaleneacetic acid whereas the weak auxins 2,3-dichlorophenoxyacetic acid and -naphthaleneacetic acid were almost inactive. This hormone specificity was also found in growth tests with etiolated zucchini hypocotyls. Phospholipase A activation by auxin was blocked by a polyclonal antibody against the maize auxin-binding protein. We propose that phospholipase A activation is a primary reaction in the signal transduction leading from hormone-binding to the growth response.Abbreviations IAA indolyl-3-acetic acid - 2,3-D, 2,4-D 2,3- and 2,4-dichlorophenoxyacetic acid - -NAA; -NAA - and -naphthaleneacetic acid This work was supported by the Deutsche Forchungsgemeinschaft. We thank D. Klämbt (Botanical Institute, University of Bonn, FRG) for a generous gift of polyclonal antibody (IgG fraction) against auxin-binding protein and U. Kutschera (Botanical Institute, University of Bonn, FRG) for advice with the growth tests.     

DPX-3778 promotes proliferation of tobacco callus in vitro

DPX-3778, the triethanolamine salt of 3-(p-chlorophenyl)-6-methoxy-s-triazine-2,4(1H,3H) dione, at concentrations of 0.124–2.48 M enhanced ca. 4-5-fold the proliferation of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus cultured in the presence of indole-3-acetic acid and kinetin, and retarded its senescence.     

Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Relation between auxin autotrophy and tryptophan accumulation in cultured plant cells

Auxin autotrophy was studied in cultured carrot (Daucus carota L.), tobacco (Nicotiana tabacum L.), and potato (Solanum tuberosum L.) cell lines. Of 10 carrot lines resistant to 5-methyltryptophan (5MT), which accumulate free tryptophan (trp) because of an altered control enzyme, 5 were auxinautotrophic while the normal parent line was not. Carrot lines selected from the same parent line as resistant to other amino-acid analogs were not auxinautotrophic, like the parent. The only 5MT-resistant potato line studied was also auxin-autotrophic while the normal line was only partially auxin-autotrophic. The tobacco lines which accumulated free trp were not auxin-autotrophic, and no auxin-autotrophic tobacco lines were selected by screening for growth in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D). Several auxin-autotrophic carrot and potato lines were selected from the normal lines and none of these lines were resistant to 5MT. Length of time in culture and difficulty in selecting auxin-autotrophic lines were correlated on the 3 normal carrot lines studied. The addition of trp or indole to the culture medium would partially alleviate the auxin requirement of the normal lines studied. 2,4-D (0.4 mg/l) stimulated the growth of all auxin-autotrophic carrot lines.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PEP DL-p-fluorophenylalanine - IAA indole-3-acetic acid - 5MT DL-5-methyltryptophan - trp L-tryptophan     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Auxin binding to subcellular fractions from Cucurbita hypocotyls: In vitro evidence for an auxin transport carrier

An auxin binding sive, with characteristics different from the previously described auxin binding sites I and II in maize coleoptiles, is reported in homogenates of zucchini (Cucurbita pepo L. cv. Black Beauty) hypocotyls. Evidence from differential centrifugation and sucrose and metrizamide density gradients indicates that the site is localized on the plasma membrane. The site has a K_D of 1–2×10^–6 M for indole acetic acid and has a pH optimum of 5.0. Binding specificity measured with several auxins, weak auxins, and anti-auxins generally parallels the activities of the same compounds as inhibitors of auxin transport. 1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid (2,3,5-TIBA), both auxin transport inhibitors in vivo, increase specific auxin binding to this site. 3,4,5-TIBA, which can partially reverse 2,3,5-TIBA's transport inhibition when the two substances are added together in vivo, partially reverses 2,3,5-TIBA's increase in specific auxin binding to the plasma membrane site when added with 2,3,5-TIBA in vitro. Preliminary investigations indicate that a similar plasma membrane site exists in maize (Zea mays L.) coleoptiles. It is suggested that different conformations of this site may function during active auxin transport.Abbreviations IAA indole-3-acetic acid - NPA 1-N-naphthylphthalamie acid - 2,3,5-TIBA 2,3,5-triiodobenzoic acid - 3,4,5-TIBA 3,4,5-triiodobenzoic acid - 1-NAA 1-naphthaleneacetic acid - 2-NAA 2-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - DTE dithioerythritol - MOPS N-morpholino-3-propansulfonic acid - CCO cytochrome c oxidase - CCR NADH: cytochrome c reductase - glu I glucan synthetase I - ER endoplasmic reticulum     

Furofuran lignans from a callus culture of Cichorium intybus

Three new and one known furofuran lignans—syringaresinol derivatives—along with the known phenylpropanoids cichoriin and syringin were isolated from a callus tissue of Cichorium intybus. The compounds were characterised by spectral methods. This is the first report on the presence of furofuran lignans in Cichorium species.     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.     

Auxin-binding by particulate fractions from tobacco leaf protoplasts

In vitro binding of 1-naphthaleneacetic acid (NAA) to particulate fractions from tobacco leaf protoplasts was studied. In freshly isolated protoplasts no specific binding could be detected, whereas it was present in particulate fractions from tobacco leaves. It is concluded that the NAA-binding-sites are probably located at the external face of the plasma membrane; they are destroyed during protoplast isolation by proteolytic enzymes in the cellulase and macerozyme preparations. After culturing the protoplasts for 3–4 d, the first cell divisions were observed and at the same time specific NAA-binding became detectable. The affinity constant for NAA was approx. 2·10⁶ mol^-1 and the number of binding sites increased during further culture.Abbreviations MES 4-morpholinoethanesulfonic acid - NAA 1-naphthaleneacetic acid     

Improved plant regeneration from barley callus cultures by increased copper levels

Incorporation of cupric sulfate into callus induction, maintenance, and regeneration media significantly enhanced plant regeneration from callus cultures of barley (Hordeum vulgare L.) immature embryos. Embryos from the cultivars Hector and Excel were cultured on MS medium containing 0, 0.1 (MS level), 0.5, 1.0, 5.0, 10.0, 50.0, or 100.0 M cupric sulfate. Plants were regenerated beginning at 8 weeks and continuing through 36 weeks. For Hector, medium containing 50 M copper regenerated significantly more plants than any other medium, with an average of 17 plants per embryo. In comparison, medium with MS copper levels (0.1 M) regenerated only 5 plants per embryo. For Excel, medium containing 5.0 M copper was the best, regenerating 1.4 plants per embryo. No Excel regenerants were obtained on medium with MS copper levels. Increased copper levels also increased the percentage of embryos that regenerated at least one plant, in both cultivars. The results indicate that MS copper levels are not optimized for barley callus cultures, and that improved plant regeneration can be obtained at higher copper concentrations.Abbreviations MS Murashige & Skoog (1962) - 2,4-d 2,4-dichlorophenoxyacetic acid The US Government's right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged