Tobacco Callus Bioassay Research Articles

New aromatic 6-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives as potential plant growth regulators

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2′-deoxy-9-(β)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N6-substituted adenine-2′-deoxy-9-(β)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2′-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.

N9-Substituted N6-[(3-methylbut-2-en-1-yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays

Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for example cytokinin derivatives. A series of N6-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifically substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2–C4 alkyl chains terminated by various functional groups were prepared. The reason for this rational design was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substituted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of N6-[(3-methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of compound N6-[(3-methylbut-2-en-1-yl)amino]-9-(2-aminoethyl-amino)purine (12) when 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass spectral measurement (CI+ MS), and by 1H NMR spectroscopy. Biological activity of prepared compounds was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4 from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with the aim to reveal the way of the perception of the above mentioned derivatives in two different plant species, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-methylbut-2-en-1-yl)amino]-9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high concentration ⩾50μM.

Synthesis and cytokinin activity of fluorescent 7-Phenylethynylimidazo[4,5-b]pyridine and its riboside.

7-Phenylethynylimidazo[4,5-b]pyridine and its riboside have been newly developed as fluorescent carbon-substituted cytokinin analogues. Palladium-catalyzed coupling of 7-iodo-3-(tri-O-acetyl-beta-D-ribofuranosyl)imidazo[4,5-b]pyridine with phenylacetylene followed by ammonolysis afforded the 7-phenylethynyl riboside via its tri-O-acetate. Acid hydrolysis of the riboside provided its free base, which showed a marked enhancement in fluorescence intensity in an aqueous alkaline solution. The free base and its riboside were more active than the corresponding 6-phenylethynylpurine and its riboside, respectively, in Amaranthus betacyanin and tobacco callus bioassays. Surprisingly, the imidazo[4,5-b]pyridine base exhibited strong cytokinin activity comparable to that of N(6)-benzyladenine in the tobacco callus bioassay. This compound would be useful for studying localization and transport of cytokinins in cells or tissues of plants.

Purines. LXVIII. Trifluoroperoxyacetic Acid Oxidation of N6-Benzyladenine: Formation of the N(3)-Oxide and N(7)-Oxide, and Their Cytokinin Activities.

Treatment of N6-benzyladenine (2) with 15% aqueous H2O2 in trifluoroacetic acid at 65-70°C for 1 h was found to give the N(3)-oxide (3) and the N(7)-oxide (4) in 4% and 4% yields, respectively. The structure of 3 was established by its identity with a sample prepared from 6-chloropurine 3-oxide (6) and benzylamine, and the structure of 4 by its identity with a sample obtained from 1-benzyladenine 7-oxide (8) by Dimroth rearrangement. The N-oxides 3 and 4, together with previously reported N6-benzyladenine 1-oxide (1), were tested for cytokinin activity in the tobacco callus bioassay. Each of the three N-oxides was active at 4 μM concentration, being less active than the parent base 2 by a factor of 40.

Purines. LXII. Both enantiomers of N6-(1,3-dimethyl-2-butenyl)adenine and their 9-beta-D-ribofuranosides: synthesis and cytokinin activity.

Both enantiomers [(1'R)-6 and (1'S)-6] of N6-(1,3-dimethyl-2-butenyl)adenine and their 9-beta-D-ribofuranosides [(1"R)-16 and (1"S)-16] have been synthesized for the first time from both enantiomers of alanine (15) in nine steps. These aglycones and nucleosides, together with N6-(3-methyl-2-butenyl)adenine (5) and its 9-beta-D-ribofuranoside (18) as well as 9-beta-D-ribofuranosyl-cis-zeatin (20) and 9-(2-deoxy-beta-D-ribofuranosyl)-cis-zeatin (19), were tested for cytokinin activity in the tobacco callus bioassay. The order of their activity was 5 > (1'R)-6 > (1"R)-16 approximately 18 > (1'S)-6 > (1"S)-16 > 20 > 19. The bioassay results are compared with those obtained previously for the derivatives modified analogously in the N6-substituent in the cis- and trans-zeatin series.

Inhibition of dopamine-β-hydroxylase by cytokinins

The activity of cell-free preparations of dopamine-β-hydroxylase from a mammalian source was inhibited by a number of N6-substituted adenine derivatives that are hormonally active as cytokinins in plant systems. The synthetic cytokinin N6-cyclohexylmethyladenine exhibited inhibitory activity equivalent to that of 1-phenyl-3-(2-thiazolyl)-2-thiourea (PTTU), a compound known to be a potent inhibitor of dopamine-β-hydroxylase activity. PTTU itself was found to exhibit cytokinin activity in the tobacco callus bioassay and to inhibit the activity of the plant enzyme, cytokinin oxidase. The possible significance of these observations is discussed in relation to known effects of cytokinins on phenethylamine metabolism.

Cytokinin Production by Bradyrhizobium japonicum

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.

Purines. XXXV. Synthesis and cytokinin activity of racemic 1'-methylzeatin.

Racemic 1'-methylzeatin [(±)-2] has been synthesized from (±)-alanine [(±)-3] through a 9-step route proceeding via the intermediates (±)-4-(±)-11. In the tobacco callus bioassay, (±)-2 exhibited a strong cytokinin activity at 0.04- 1μM concentration, a range between the optimum concentrations of both enantiomers (1'R)-2 and (1'S)-2.

Cytokinins in Azotobacter vinelandii Culture Medium

Azotobacter vinelandii OP was grown to stationary phase in defined medium. The cell-free culture medium was analyzed for cytokinin content by XAD-2 and Sephadex LH-20 chromatography, thin-layer chromatography, tobacco callus bioassay, and enzyme immunoassay. Three cytokinin-active fractions were detected and tentatively identified as trans-zeatin, isopentenyladenosine, and isopentenyladenine. The total cytokinin activity was equivalent to 0.75 mug of kinetin per liter.

Identity of cytokinins in Begonia leaves and their variation in relation to photoperiod and temperature

The identity of cytokinins extracted from leaves of Begonia×cheimantha Everett cv. Nova has been studied by purification and fractionation on Dowex 50 H’ and Sephade× L.H 20 columns followed by reverse‐phase HPLC separation and determination by enzyme immunoassay. The following four cytokinins were identified: trans‐zeatin, trans‐zeatin riboside, isopentenyladenin and isopentenyladenosin. At least three other compounds, possibly glucosides, were detected by activity in the tobacco callus bioassay but not yet identified. The quantity of the identified cytokinins was several times higher at 15 than at 24°C, the former temperature being optimal for regeneration of adventitious buds. At 15°C there was no clear effect of photoperiod whereas short days at 24°C increased the activity several‐fold compared with long day conditions.

Cytokinin activities ofN 6-benzyladenosine derivatives hydroxylated on the side-chain phenyl ring

Cytokinin activities ofN 6-benzyladenosine (bzl6Ado) and its derivatives hydroxylated on the side chain phenyl ring inortho, meta, andpara positions were compared in four bioassays based on stimulation of growth of tobacco callus, retention of chlorophyll in excised wheat leaves, dark induction of betacyanin synthesis inAmaranthus cotyledons, and release of lateral buds of pea from apical dominance. In all these bioassays hydroxylation of the phenyl ring of bzl6Ado inortho andpara positions significantly decreased cytokinin activity. Compared with bzl6Ado, the activity was decreased about 10× in the tobacco callus bioassay and wheat leaf chlorophyll retention test, 100× in theAmaranthus betacyanin bioassay, and 20× and 200×, respectively, in the pea bud test. Hydroxylation of the phenyl ring inmeta position increased activity in the tobacco callus, and wheat leaf chlorophyll retention bioassays, 9× and 1.7×, respectively, decreased activity about 2.4× in the pea bud test and was without effect in theAmaranthus bioassay. Cytokinin activity of themeta hydroxy derivative,N 6-(m-hydroxybenzyl) adenosine, was as high as that oftrans-zeatin in all four bioassays. Possible regulation of biological activities of cytokinins by positionally specific hydroxylation of the side chain phenyl ring is discussed with respect to the reported occurrence of natural purinyl cytokinins with aromatic side chains.

Quantitative structure-activity relationships in cytokinin agonistic and antagonistic pyrido[2,3-d]pyrimidine derivatives: insights into receptor topology.

2-(Methylthio)pyrido[2,3-d]pyrimidines having various alkylamino and anilino substituents at the 4-position were prepared and tested for their cytokinin agonistic and antagonistic activities by the tobacco callus bioassay. The alkyl series of compounds showed anticytokinin activity, whereas the anilino derivatives exhibited both cytokinin and anticytokinin activities depending on the structure and position of the benzene substituents. Quantitative structure-activity analyses were carried out for each class and for the combined set of compounds with use of physicochemical parameters and regression analysis, indicating that the quality of activity, agonistic or antagonistic, as well as the extent of activity, is significantly affected by the steric features of the molecule. On the basis of the present results and previous quantitative analyses on cytokinins and other classes of anticytokinins, a dimensional map for the cytokinin receptor site can be drawn, which can serve as the basis for the design of novel agonists and antagonists.

Effects of temperature and gibberellin on the activity of endogenous cytokinin‐like substances in leaves of Begonia x cheimantha

Changes in activity of endogenous cytokinin‐like substances were examined in intact plants and excised leaves of Begonia x chemantha Everett cv. Prinsesse Astrid (Christimas Begonia) by means of the tobacco callus bioassay. Cytokinin activity in the leaves of intact plants was higher in plants grown at 18°C than in those grown at 21° or 24°C. In excised leaves, an increase in cytokinin activity was observed during the first 4 days following leaf detachment. However, after the seventh day cylokinin activity decreased again. This decrease was more profound in leaves exposed to 24°C than in those exposed to 18°C.Treatment of detached leaves with gibberellic acid (2.8 mM) caused an increase in measurable cytokinin activity. This increase was more profound in the zones of activity which correspond with zeatin glucosides on paper and Sephadex LH‐20 chromatography. Additional zones of activity appeared after Sephadex chromatography. These were of a more slow moving nature with elution volumes corresponding to Nb‐(Δ2‐isopentenyl)adenine and its derivaties. Water‐treated control leaves had higher activity in the regions corresponding to zeatin and zeatin riboside.

Studies on the mechanisms of ozone tolerance: Cytokinin‐like activity of N‐[2‐(2‐oxo‐1‐imidazolidinyl)ethyl]‐N'‐phenylurea, a compound protecting against ozone injury

The cytokinin‐like activity of the growth regulating chemical EDU, N‐[2‐(2‐oxo‐1‐imidazolidinyl)ethyl]‐N′‐phenylurea, was determined and compared with the actitivity of kinetin using the tobacco callus bioassay. EDU has a pronounced stimulatory effect on callus growth at concentrations of 5 × 10−4 and 1 × 10−3M but was 5 000 times less potent than the synthetic cytokinin, kinetin. Senescence regulation and oxidant resistance induced by EDU and kinetin were also studied. EDU retarded the breakdown of chlorophyll, protein and RNA in 03‐sensitive tobacco leaf discs during senescence. EDU was much more effective in arresting senescence and in protecting against 03 injury than kinetin. Results indicate the EDU‐induced plant tolerance to 03 phytotoxicity may be indirect through enzyme induction regulation.

Changes in cytokinin in the fruits of parthenocarpic and normal tomatoes

Cytokinin in tomato fruits ( Lycopersicon esculentum Mill.) was detected by tobacco callus bioassay in both parthenocarpic and normal genotypes. Parthenocarpic fruit was found to contain lower levels of activity. A cytokinin peak occurred during the first week after anthesis. This activity was eluted in the position of isopentenyl adenine and its riboside when chromatographed on polyvinyl-pyrrolidone (PVP) column. High levels of activity were observed in normal fruit from the 15th to 35th day which declined during the change from the light green to the pink stage. The parthenocarpic fruit in the same period, 15–45 days after anthesis, not only contained lower levels of cytokinin but these did not decrease at the breaker and ripening stages.

Cytokinin Activity of p-Phenylthioureidosalicylic Acid

Summary In the tobacco callus bioassay p-phenylthioureidosalicylic acid possessed 7 times higher cytokinin activity than N’,N-diphenylurea and 18 times lower activity than kinetin.

Effects of 4-alkylaminopteridines on tobacco callus growth

In the tobacco callus bioassay, 4-benzylaminopteridine showed cytokinin activity, 4-cyclohexylaminoteridine no activity within the concentration range tested (∼ 40 μM) and 4-cyclopentylaminopteridine anticytokinin activity] against 0.05 μM kinetin, indicating that the pteridine analogs structurally related to the N 6-substituted adenines are biologically related to cytokinins.

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ( Nicotiana tabacum cv. Wisconsin #38) crown gall strains incited by Agrobacterium tumefaciens C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl- trans -zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.

Cytokinines fibres et liées dans les axes embryonnaires et les cotylédons des embryons dormants et non dormants de pommier

Summary The endogenous levels of free and bound cytokinins were studied in dormant and afterripened embryos of Pyrus malus L. cv. Golden delicious. The postmaturation treatment was a three months stratification at 4 °C in the seeds. Qualitative and quantitative studies were made using paper chromatography, sephadex gel chromatography and tobacco callus bioassay. In the embryonic axes from dormant or after-ripened embryos the cytokinins levels were found very similar: relatively important for the free forms, negligeable for the bound ones. In the cotyledons of dormant embryos the bound forms were found in very high amounts, whereas the free forms existed in negligeable quantities. The postmaturation treatment induced a spectacular decrease of the bound forms with an important increase of the free forms, the latter being found in lesser quantities than could be deduced from the losses of the bound forms. Our results showed that complex qualitative changes were also induced under the influence of the postmaturation treatment.

Active Cytokinins

FOUR SERIES OF AZIDOPURINES HAVE BEEN SYNTHESIZED AND TESTED FOR CYTOKININ ACTIVITY IN THE TOBACCO CALLUS BIOASSAY: 2- and 8-azido-N(6)-benzyladenines, -N(6)-(Delta(2)-isopentenyl)adenines, and -zeatins, and N(6)-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 x 10(-5) micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 x 10(-6) micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 x 10(-5) micromolar, representing a sensitivity rarely attained.All of the azido compounds have been submitted to photolysis in aqueous ethanol, and the photoproducts have been detected and identified by low and high resolution mass spectrometry. They are rationalized as products of abstraction and insertion reactions of the intermediate nitrenes. The potential of the major released products as cytokinins was also assessed by bioassay. 2-Azido-N(6)-(Delta(2)-isopentenyl)adenine competed with [(14)C]kinetin for the cytokinin-binding protein isolated from wheat germ. When the azido compound was photolysed in the presence of this protein, its attachment effectively blocked the binding of [(14)C]kinetin.