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Isolation of Cytokininas from Corynbacterium fascians
... En el caso de Rhodococcus fascians se ha reportado que produce sustancias con capacidad fitoreguladora. En los sobrenadantes de los medios de cultivo donde se ha multiplicado este microorganismo, se han encontrado sustancias del tipo de las citocininas al utilizar manitol como fuente de carbono (5,6) . Esta bacteria es Gram positiva, aerobia, y se desarrolla en una escala de pH de 6 a 8 y crece a temperaturas desde 10 hasta 40 °C, donde su temperatura óptima de crecimiento es 26 °C (7) . ...
... In the case of Rhodococcus fascians, it has been reported that it can produce substances that act as phytoregulators. In overfloats of culture media where this microorganism was grown substances akin to cytokinins have been found when mannitol was used as carbon source (5,6) . This bacterium is gram positive, aerobic and grows in media with pH between 6 and 8 and at temperatures between 10 and 40 °C, being 26 °C the best. ...
... This treatment obtained a marginal rate of return slightly higher than 60 %, which can be considered quite acceptable (23) . However, the cost of the inoculum could be lowered if other less expensive C sources are used (5,6) for its multiplication, which is one of the main qualities necessary in an inoculum (32) . ...
The objectives were evaluating the combined effects of different rates of both inoculum (Rhodococcus fascians) and nitrogen on forage production of grasses and to perform a cost - benefit economic analysis. The species analyzed were Avena sativa L, Lolium multiflorum Lam, and Triticum durum L through the following treatments: inoculation, nitrogen fertilization (0, 30, 60 kg N ha -1) and a combination of inoculation and fertilization. The species were established under irrigated conditions in a clay loam soil with 6.7 pH, and 0.34 % organic matter. The microorganism was sprayed on the seed and later, on the plants after each forage harvest. Forage production was evaluated in terms of metric tons (t) of dry matter (DM) ha -1. The economic study was performed using marginal analysis, where the indicators were cost variables, forage production and net benefits. The experiment was carried out using a completely randomized design with a 2x3x3 factorial arrangement with four replications, where the first factor was inoculation; the second factor, species; and the third factor, N rate. The results show that the combined use of an inoculum and 30 kg N ha -1 produces 50 % more DM (approximately 11 t) than when only N is used, especially in Avena sativa L. and Lolium multiflorum Lam. The economic analysis showed that the best treatment was inoculation plus 30 kg N ha -1 for Avena sativa L, presenting a marginal return rate of over 60 %.
... Cytokinins are thought to attribute to many aspects of the infection phenotype because of the appearance of serrated or wrinkled leaves, the massive amplification of shoots, the inhibition of root development and the delayed process of senescence. Some of these symptoms can be mimicked by the exogenous application of cytokinins (Klämbt et al., 1966). R. fascians and cytokinins exert similar effects on shoot meristems and the expression of cell cycle markers albeit the consequences of cytokinin application are less pronounced (Vereecke et al., 2000). ...
... It seems therefore likely that the bacterium is responsible for an infection phenotype by the secretion of this hormone. Indeed, different cytokinins could be recuperated from the culture supernatant (Klämbt et al., 1966;Scarbrough et al., 1973;Armstrong et al., 1976;Eason et al., 1996) and a positive correlation between cytokinin production and the pathogenicity of a given strain has been made by Murai et al. (1980). However, the latter has been contested by other authors since the cytokinin production by virulent and non-pathogenic strains hardly showed any difference in cytokinin production Eason et al., 1996). ...
... Two of the compounds. N6-methylaminopurine and adenine, were shown to have a low cytokinin activity (9,19). The other compounds, guanine, N6' 6-dimethylaminopurine, and 2aminopurine, do not fulfill the structural requirement for cytokinin activity (6,19). ...
... These findings are in confirmation with those of Skoog and Miller (20) who showed that adenine overcame the kinetin inhibition of bud formation of tobacco stem segments. Furthermore, Klambt et al. (9) demonstrated that Nt-methylaminopurine, which had some cytokinin activity by itself, markedly depressed the response of tobacco callus tissue to the more active cytokinin Nt-(Qy,'ydimethylallylamino)-purine. In our system Nt-methylaminopurine, which was slightly inhibitory by itself, completely abolished kinetin inhibition. ...
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, N6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.
... Even prior to the discovery of the fas genes, cytokinins were implicated in the virulence of Rhodococcus (Klämbt et al., 1966;Thimann and Sachs, 1966). One of the extant models predicts that a mixture of three cytokinin types, isopentenyladenine (iP), trans-zeatin (tZ), and cis-zeatin (cZ), as well as modified variants, are synthesized in a fas-dependent manner and secreted into plant cells to cause disease (Pertry et al., 2010;Stes et al., 2011). ...
Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kb-sized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.
... Many of the symptoms triggered by R. fascians, including abundant shoot proliferation and inhibition of root development, are typical cytokinin effects and addition of cytokinins has been found to mimic some of the symptoms (Thimann and Sachs, 1966;Oduro and Munnecke, 1975;Depuydt et al., 2008). However, despite the detection of several cytokinins in the culture supernatant of R. fascians (Klämbt et al., 1966;Helgeson and Leonard, 1966;Rathbone and Hall, 1972;Scarbrough et al., 1973;Armstrong et al., 1976;Murai et al., 1980;Eason et al., 1996), to date, no clear positive correlation could be made between the amount of secreted cytokinins, the virulence of the producing strain and the kind of cytokinins detected in infected plants. Moreover, contradictory results in planta make it impossible to link cytokinins to symptom development Depuydt et al., 2008;Murai et al., 1980;Balázs and Sziráki, 1974;Eason et al., 1996;Crespi et al., 1992;de O Manes et al., 2001;Galis et al., 2005). ...
The fine-tuned balance of plant regulators plays a key role in growth and development of plants. Many plant-associated bacteria can influence their hosts either by modulating phytohormone production or by producing phytohormones themselves. The Actinomycete Rhodococcus fascians provokes the formation of differentiated leafy galls consisting of numerous shoot primordia that are inhibited in further outgrowth. Based on the shooty phenotype and the presence of an ipt gene on the linear virulence plasmid of R. fascians D188, the role for cytokinins in the pathology had been anticipated for a long time. Subsequent studies identified and characterized the fas operon as a key genetic determinant of virulence and likely cytokinin biosynthesis. Nevertheless, many aspects concerning regulation of fas gene expression, Fas protein function, and, importantly, the encoded cytokinin biosynthetic pathway and the identity of the produced morphogens remained to be uncovered. Therefore, the main objectives of this research were to identify the bacterial cytokinins responsible for the R. fascians pathology, to unravel how they exerted their function, and to elucidate the role of the fas locus and its expression in their production. In conclusion, our data have largely uncovered the role of cytokinins and the fas locus in the R. fascians pathology: the continuous challenge with defined ratios of synergistically acting cytokinins eventually defeats nearly all plants and transforms them into shooty niches. Many intriguing questions derived from the novel insights obtained during this work remain to be answered. Nevertheless, we feel that the results presented here have shed some light on the remaining secrets of this fascinating pathogen.
... The relevance of cytokinins as dominant pathogenicity factors of R. fascians has been recognized as early as 1965 [67] and has been corroborated repeatedly since then [27,29,31,[68][69][70]. Pathological cytokinin levels are synthesized de novo in strain D188 by the linear plasmid-encoded fas operon [27,29,30]. ...
... Biochemical and physiological tests were performed according to standard methods (Schaad et al., 2001; Suslow et al., 1982;Fahy and Hayward, 1983). Pathogenicity Test Pea seeds were surface sterilized, put in indirect sunlight on a sterile, wet filter paper. 2 to 3 days later, the germinated seeds were placed for one week in bacterial suspension with a concentration of 10 7 CFU (OD 600 =1) (Klambt et al., 1966). Sterile distilled water was used as control. ...
Rhodococcus fascians is a gram positive plant pathogenic bacterium that stimulates gall formation in
many monocot and dicot plants. In the present study, 27 isolates of Rhodococcus fascians were collected
from Shiraz, Jahrom and Darab (Fars, Iran). The phenotypic and genotypic characteristics of these
isolates, a standard strain (981011) and two isolates of petunia and geranium, from northern Iran were
assessed using RAPD-PCR. Analysis of phenotypic characteristics using NTsys-pc software, all isolates
showed 80% similarity. On the basis of our finding, petunia isolates were very similar and were placed in
a distinct cluster. Nasturtium isolates with northern petunia isolates, were placed in a separate cluster and
were separable from the other hosts. Standard isolate had the most and least similarity to the Shiraz’s
petunia isolate and Darab’s geranium isolate, respectively. Results showed that the nasturtium and petunia
isolates were homogeneous based on phenotypic characteristics and are separable from the other hosts;
geranium strains, on the other hand, showed more diversity and were placed in different groups. The
investigation of genotypic characteristics using six RAPD-PCR primers showed that all strains were
highly homogeneous. Genotypic dendrogram showed all isolates could be separated from each other at
the 90% similarity level. This held true, especially in the case of geranium and tobacco isolates and the
Iran’s northern strains. Analysis of RAPD-PCR fingerprints, showed that R. fascians isolates could be
distinguished from each other, based on the isolated host and geographical area. Comparison of the results
of phenotypic and genotypic analysis showed that there was no relationship between these two methods.
... Surface sterilised seeds of Pisum sativum variety Bohatyr were imbibed for 4 h with stirring in Klambt medium [66] and placed in sterilised 500 mL containers with 0.6% (w/v) agar and 10% (w/v) Hoagland's mineral salts solution [67]. The containers were placed in a growth room at 22 • C with a 16-h photoperiod. ...
Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT), amino acid permease (AAP), Sugar Will Eventually be Exported Transporter (SWEET), cell wall invertase (CWINV), cytokinin biosynthesis (IPT), activation (LOG) and degradation (CKX) gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L.We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration.
... Bacterial cultures (three flasks for each strain) were grown over night in "523" broth (Kado and Heskett, 1970) at 26 • C with shaking at 150 rpm. Mid-log phase cultures were then inoculated into Klämbt broth (Klämbt et al., 1966), incubated for 4 h at 26 • C with shaking at 150 rpm. The cultures were then harvested by centrifugation at 15,000 rpm for 15 min at 4 • C. The supernatant was discarded and the pellets were re-suspended in TRIzol R reagent (Invitrogen, Carlsbad, CA, USA). ...
Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4 (dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms.
... Furthermore, the levels of cytokinins are altered in plant tissues infected with Rhodococcus (39,54). Likewise, cytokinins are detectable in Rhodococcus cultures (9,32,34,62,82,103,104). Most importantly, three of the six plasmid-borne fas virulence genes in Rhodococcus encode enzymes involved in synthesizing and modifying cytokinins (34,103,104). ...
Gram-positive bacteria are prominent members of plant-associated microbial communities. Although many are hypothesized to be beneficial, some are causative agents of economically important diseases of crop plants. Because the features of Gram-positive bacteria are fundamentally different relative to those of Gram-negative bacteria, the evolution and ecology as well as the mechanisms used to colonize and infect plants also differ. Here, we discuss recent advances in our understanding of Gram-positive, plant-associated bacteria and provide a framework for future research directions on these important plant symbionts.
... Bacterial cultures (three flasks for each strain) were grown over night in "523" broth (Kado and Heskett, 1970) at 26 • C with shaking at 150 rpm. Mid-log phase cultures were then inoculated into Klämbt broth (Klämbt et al., 1966), incubated for 4 h at 26 • C with shaking at 150 rpm. The cultures were then harvested by centrifugation at 15,000 rpm for 15 min at 4 • C. The supernatant was discarded and the pellets were re-suspended in TRIzol R reagent (Invitrogen, Carlsbad, CA, USA). ...
Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of
which can be mimicked by application of cytokinin. Both virulent and avirulent strains
produce a complex of cytokinins, most of which can be derived from tRNA degradation.
To test the three current hypotheses regarding the involvement of cytokinins as virulence
determinants, we used PCR to detect specific genes, previously associated with a
linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4
(dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum
(pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over
time and compared these to mock-inoculated controls. We used RT-qPCR to monitor
the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain
a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots
and shoots over time. The presence and expression of mt1 and mt2 was associated
with those strains of R. fascians classed as virulent, and not those classed as avirulent.
Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons
and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain
602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained
cytokinins likely to have been derived from tRNA turnover including the 2-methylthio
cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins,
the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-
methylated isopentenyladenine cytokinins were uniquely associated with infection by the
virulent strains and are, therefore, the likely causative factors of the disease symptoms.
For comparison with the corresponding N6-(ω-phenylalkyl)adenines, several N6-(ω-1,4-cyclohexadien-1-ylalkyl)adenines were synthesized by reducing the appropriate ω-phenylalkylamine with lithium in liquid ammonia followed by interaction of the resulting 1,4-cyclohexadienealkylamine with adenine at elevated temperatures. The methyl, ethyl, propyl, and pentyl members of this series of cyclohexadienylalkyladenines stimulate germination of lettuce seed and augment the inhibition of growth of Lactobacillus plantarum 8014 in the presence of folic acid analogs to a degree closely analogous to that observed with the corresponding phenylalkyl-adenines.
Eighteen amino purines have been synthesized and their phytohormonal properties have been studied by means of the tobacco-pith, pea-bud and Amaranthus tests. Only three compounds have been found active, all of them having their purine nucleus substituted at the 6-position. Activity has been observed, for the first time, in nitrogen-isosteric derivatives of N-(γ,γ-dimethyl-allyl)adenine. However, the cytokinin activities of the 3 derivatives are found to be lower than those of kinetin, N-benzyladenine and N-(γ,γ-dimethyl-allyl)adenine.
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 N(6)-(Δ(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.
Comparative Effects of 6-Furfurylaminopurine and 6-(γ, γ-Dimethylallylamino) purine on Growth, Peroxidase, Chlorophyll and Carotenoid Content. — 6-Furfurylaminopurine (FAP) and 6-(γ, γ-dimethylallylamino) purine (IPA) both inhibit root growth and cause an increase in peroxidase activity. When used at 10−8 and 10−8M, IPA stimulates elongation of the coleoptile test, at 10−4M both FAP and IPA provoke an inhibition. FAP and IPA maintain a high level in chlorophylls and carotenoids in floating leaves discs. In all these experiments, IPA has thus a higher effect than FAP.
The production of N6-(◿2-isopentenyl)adenine in Agrobacterium tumefaciens strain B6 grown in logarithmic phase was estimated to be 0.3 μg/l in the medium and 2.3 μg in the bacteria from 1 l medium. The cytokinin activity of tRNA isolated from the bacteria was estimated to be 10-9 mol cytokinin/mg tRNA after alkaline hydrolysis and 4.6 × 10-10 mol cytokinin/mg tRNA after enzymic hydrolysis. It was calculated that the amount of cytokinin released from tRNA of 2–3 g bacteria present in the medium at early logarithmic phase is sufficient to account for all the cytokinin found. This calculation is based on the estimated half-life of 27.5 hours for tRNA in A. tumefaciens and on the assumption that every tenth macromolecule does contain a cytokinin. The data show that tRNA can be considered as an intermediate in cytokinin biogenesis although the possibility can not be excluded that a second cytokinin biosynthetic pathway independent of tRNA may exist.
To test the hypothesis that the growth inhibition occurring in seedlings of Ardisia species in the absence of their natural bacterial symbionts is due to a lack of cytokinins, we have studied two bacterial strains isolated from two Ardisia species as to a possible production of cytokinins. Extracts of an isolate from Ardisia crenata, of the culture filtrate of this organism, and of the culture filtrate of a Chromobacterium lividum strain that was isolated from A. crispa promoted growth in the soybean callus and the radish cotyledon bioassays for cytokinins. Both organisms caused malformations in pea seedlings exactly like those induced by Corynebacterium fascians. We conclude that the bacterial symbiosis in Ardisia species is based on the fact that the bacteria provide the host plant with cytokinins that it is unable to synthesize itself.
Protonema von Funaria hygrometrica wurde in Gegenwart der Pilze Aspergillus niger, Alternaria solani, Alternaria spec. und Penicillium spec. auf Agar mit Knop-Nährlösung kultiviert. In jedem Falle wurde das Wachstum durch die Pilze beschleunigt und bei frühzeitiger Zuimpfung der Pilze die Knospenbildung vorverlegt. Später zugegebene Mycelien haben in den meisten Fällen einen gleichartigen Effekt. Nur bei Penicillium spec. wird das Wachstum gehemmt, wenn der Pilz erst acht Tage nach der Keimung zugeimpft wird.
Eine Beschleunigung des Wachstums erhält man auch, wenn vor dem Beimpfen mit Moossporen auf dem Agar 13 Tage Pilze gewachsen waren, die dann vollständig entfernt wurden. Wird im Substrat Faktor H (Knospenbildung- und Wachstum-beschleunigender Faktor aus Moosprotonemen [Klein 1967]) durch Vorkultur von Moosen angereichert oder zugegeben, so wird dessen Wirkung durch die Pilzkultur nicht verändert. Die Wirkung der Pilze beruht daher auf einer von diesen abgegebenen Substanz und nicht auf einem Abbau mooseigener stofflicher Regulatoren.
Sixty-nine compounds, mostly purine derivatives and closely related substances, were tested for promotion of growth and regulation of organ formation in the tobacco bioassay to determine relationships between chemical structure and cytokinin activity. Forty-three substances were synthesized in this study, and 13 of these were reported for the first time. N6-Alkyladenines (I) varied in activity over a wide concentration range depending on the length of the alkyl chain. Starting with adenine, detectable at ⩾200 μM, activity increased with the chain length to an optimum for 6-pentylaminopurine detectable at ca. 0–001 μM, and then decreased to reach a barely detectable level for 6-decylaminopurine. The result of the incorporation of polar groups in the side chain was not necessarily reduction in activity. One hydroxyl group, as in zeatin (Id), improved the activity of 6-(γ,γ-dimethylallylamino)purine (Ib) if it affected it at all; two hydroxyl groups, as in 6-(2,3-dihydroxy-3-methylbutylamino)purine strongly reduced activity. Comparisons of 6-isoamylaminopurine with 6-(γ,γ-dimethylallylamino)purine and of other closely related pairs of compounds showed that a double bond in the side chain greatly increased cytokinin activity. Adenine derivatives with cyclic substituents in the N6-position (benzyl-Ic), cyclohexyl-, etc.) showed the same general range of activity, potentiation by unsaturation, and variation in activity with substituent size, etc. as did the alkyl derivatives. Heteroatoms in or on the substituent groups decreased activity (in the case of N or Cl) or had little effect (S for O in furfuryl). Of the mono-substituted adenines only the N6-derivatives definitely possessed cytokinin activity. The 1-(III), 3-(II), or 9-substituted adenines probably are inactive but could be activated by conversion to the N6-isomers. Except for slight activity in tests of high concentrations, which could be ascribed to contaminants, 7-substituted adenines were completely inactive. Modification in the adenine moiety lowered the cytokinin activity, often by 95 per cent or more. Substitution of N for the 8-C atom in kinetin and in 6-benzylaminopurine or S for the 6-amino N atom in 6-(γ,γ-dimethylallylamino)purine did not eliminate but drastically reduced activity in the tobacco bioassay. Elimination of the 6-amino group without substituting another group completely removed activity; thus, the purine derivatives, 1-benzylpurine and 1-(γ,γ-dimethylallyl)purine, were inactive in tests where the 1-adenine derivatives could be activated to give a positive response. Addition of a second substituent on the 1-or 3-position of N6-substituted adenines drastically reduced or eliminated cytokinin activity. It is suggested that the 1-position and possibly also the 3-position must be free. A second substituent in the N6-, 7-, or 9-position of N6-substituted adenine derivatives lowered but did not eliminate activity. Also, the disubstituted 1-adenine derivatives, 1,9-dibenzyladenine and 1,7-dibenzyladenine were active, presumably after rearrangement to the corresponding N6-substituted isomers.
The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl--2-butenylamino)-9-β--ribofuranosylpurine, i.e. ribosyl--zeatin, has been isolated from an hydrolysate of tRNA from . The identification of ribosyl--zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.
Cytokinin activities in the tobacco bioassay have been determined for four adenosine derivatives known to be components of wheat germ tRNA: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino)- 2-methylthio-9-β-d-ribofuranosylpurine, and 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-d-ribofuranosylpurine. Also determined and compared with the four natural components of tRNA were the activities of the four 3-methylbutylamino analogs of the naturally occurring species and the eight substituted purines corresponding to both sets of ribonucleosides. The systematic structural modifications within this group of sixteen compounds were reflected in the variations in cytokinin activity with the level of modification.
This chapter discusses and evaluates the experiments that appear to be promising steps toward explaining the mode of action of cytokinins. Skoog and Armstrong's thorough coverage of structure-activity relationships and the chemistry of cytokinins, both in free form and in tRNA make a detailed presentation of these subjects unnecessary. It summarizes some of the questions that need to be answered before a testable hypothesis of cytokinin action can be proposed. Three general areas require intensive investigation, which are (1) the physiological significance of cytokinins, (2) the site of action of cytokinins, and (3) the primary biochemical reactions that underlie the cytokinin responses. The chapter summarizes the achievements and points out the questions that need to be answered before attempts can be made to propose models for the mode of action of cytokinins.
Corynebacterium fascians causes a fasciation disease in a number of dicotyledons and this disease appears to be caused by compounds with cytokinin activity elaborated by the infecting bacteria. Extractions of C. fascians in late logarithmic phase under conditions where the pH never falls below 7.0 yield about 2 μg/l of N
6-(Δ2, a potent cytokinin. If a mild acidification step is included in the extraction procedure the yield increases to about 12 μg/l. This is due to release of N
6-(Δ2-isopentenyl)adenine from C. fascians tRNA during the extraction procedure. In terms of total cytokinin activity present in C. fascians cultures, N
6-(Δ2-isopentenyl)adenine appears to be a minor component.
Gas-liquid chromatographic retention times for 20 purines or purine nucleosides, 14 of which are highly active cytokinins, are reported. With one exception, all of the naturally occurring cytokinins are separated.
Ethyl acetate extraction of yeast transfer RNA hydrolysates and of culture filtrates of Agrobacterium tumefaciens gave sufficient concentration of the naturally occurring cytokinins for immediate gas-liquid chromatography. This procedure permitted the detection of 6-(3-methyl-2-butenylamino)-9-β, d-ribofuranosylpurine in yeast transfer RNA extracts. An active cytokinin was isolated from A. tumefaciens culture filtrates and was tentatively identified as 6-(3-methyl-2-butenylamino)purine.
With two extraction procedures differing in the very first extraction steps two cytokinins from a total amount of 180 kg sunflower leaves could be isolated. One of these cytokinins is identified as zeatin by Rf values, UV absorption, melting point of its picrate and its biological activity. The same data are given for the other cytokinin, which is very probably ribosylzeatin. The total free cytokinin content of sunflower leaves is calculated to be 50–90 μg of kinetin equivalent, or 5–9 μg of zeatin, per kg of fresh leaves.
Besides the leaf extraction, 100 l of root exudate of sunflowers were collected and the cytokinin was isolated. The root exudate contains 5–6 μg of kinetin equivalent, or 0,5–0,6 μg zeatin per 1 bleeding sap. The Rf values and UV absorption are similar to the corresponding data recorded for zeatin.
In addition to the four cytokinins, 6-(3-methyl-2-butenylamino)purine, 6-methylaminopurine and the cis and trans isomers of 6-(4-hydroxy-3-methyl-2-butenylamino)purine, reported earlier from our laboratories, three cytokinin-active fractions have been obtained from the aqueous medium of 6-day-old Corynebacterium fascians cultures. One of these has been identified as 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine (2-methylthio-cis-zeatin, c-ms²io⁶ Ade).
The elution volumes of the other two fractions correspond to those of authentic 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosyl-purine and 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, indicating the presence of trace amounts of these two ribonucleosides.
Rhodococcus fascians, causative agent of the leafy gall syndrome, produces a mixture of cytokinins to modify the hormone landscape of its broad range of plant hosts leading to tissue deformations and developmental alterations. Recent developments indicate that the pathogenic nature of these bacteria is superimposed on its plant growth-promoting effect. In the last two decades, its unique position as the only species within the genus able to interact with plants has been overthrown. Indeed, Pistachio Bushy Top Syndrome is an emerging disease linked to the presence of two Rhodococcus species, R. fascians and R. corynebacterioides. Both bacteria would act synergistically to cause the symptoms, giving the prospect of virulence strategies that differ from those of the leafy gall inducers. Additionally, as a result of microbiome research, it is clear that many Rhodococcus species live in close association with plants, and several of them exhibit plant growth-promoting activities. Finally, genome analyses of a collection of R. fascians isolates imply that the taxonomic position of this group of bacteria within the genus will have to be reevaluated, and likely a new genus consisting of several species will be proposed soon.
Cet article est destiné à définir la nature des substances d'origine bactérienne qui, dans la rhizosphère, sont capables d'intervenir sur la croissance des plantes. Parmi ces substances, les acides aminés et vitamines hydrosolubles, souvent identifiables dans les milieux de cultures bactériennes, ont un rôle difficile à préciser au niveau des plantes et interviennent plutôt sur l'équilibre microbien de la flore rhizosphérique. Par contre, on a pu démontrer que les synthèses bactériennes de substances phytohormonales telles que acide indolyl acétique, gibbérellines et cytckinines agissent directement surla croissance des plantes.
The carrot-root tissue culture assay for cytokinin activity has been improved by changing the site of explant excision and eliminating certain vitamins from the basal medium. These modifications increased its sensitivity and enabled zeatin [6-(4-hydroxy-3-methylbut-trans-2-enyl)aminopurine] to be detected at concentrations less than 5×10(-5)μM. In the improved assay, zeatin was markedly more active than kinetin, 6-benzylaminopurine, 6-(o-methylbenzyl)aminopurine and 6-(3-methylbut-2-enyl)aminopurine.The activity of zeatin also exceeded that of kinetin in the etiolated bean-leaf disk expansion assay. Zeatin was markedly more effective than kinetin and 6-(3-methylbut-2-enyl)aminopurine in promoting frond expansion and increasing frond number of Spirodela oligorrhiza cultures grown under continuous illumination. Zeatin was also more active than kinetin and 6-(3-methylbut-2-enyl)aminopurine in increasing frond number of Spirodela cultures grown in darkness. In retarding the senescence of disks of leaves of several species, kinetin was considerably more effective than zeatin which was more active than 6-(3-methylbut-2-enyl)aminopurine. The allylic hydroxyl group in zeatin is therefore a structural feature associated with high cytokinin activity.The relative activities of cytokinins can be very different and even in reverse order in different bioassays. It is suggested that this is due to the mechanism of cytokinin action varying in the different biological systems used.
Our present understanding of the nature of the molecules that promote cytokinin activity is based on the finding by Miller et al. (1–3) of the growth factor kinetin (1; 6-furfurylaminopurine) in old and heated samples of DNA. The first active analog, 6-benzylaminopurine (2) (4–6), was prepared within days and presaged the synthesis of large numbers of compounds that have helped to define the structure-activity relationships for such species with considerable precision [see, e.g., (7–12)]. Although kinetin is presumably an artifact formed by rearrangement of 2′-deoxyadenosine, structurally related species such as 6-(3-methyl-2-butenylamino)purine (3) were subsequently identified as the growth factors in certain plant pathogens (13–15) and have now been shown to occur more generally in plants at the purine (16–25) and purine ribonucleos(t)ide levels (26 – 28).
I would like to report significant issues of concern regarding this paper (Savory et al., 2017).
Plant growth and differentiation are regulated by growth regulators. Pathogenic infection may cause a departure from normal levels of one or more growth regulators in the infected plant. Imbalance of growth regulation could alter the growth habit of the plant and result in symptoms such as stunting, overgrowth, epinasty, and premature leaf drop.
Corynebacterium fascianscauses a disease in a wide range of dicotyledonous plants typified by the release from apical dominance and the outgrowth of lateral buds (Lacey 1936), a syndrome known as witches’ broom. Thimann and Sachs (1966) showed that the symptoms displayed after they had induced localized infection by puncture could be closely duplicated by treatment with pure kinetin, and from these observations deduced that “the disease is due to the production in the host tissue of a cytokinin.”
The olefinic gas ethylene (ethene) is the most unusual and perhaps powerful of the growth-regulating chemicals produced by microorganisms and by healthy and diseased plants. The earliest observations of the physiological effects of the gas by Fahnstock (1858) and Girardin (1864, Abeles 1973) were in relation to damage to coleus plants and lime (Tilia vulgaris) trees by fuel (coal) gas contaminated by ethylene. The effects of ethylene on lime trees closely simulated the disease syndrome associated with vascular wilt pathogens such as the Dutch elm disease fungus (Ceratocystis ulmi) on elm, but it was many years later that its biosynthesis by microorganisms (including fungal and bacterial vascular pathogens) was established (Pegg 1976b).
It may be expected that an attack on a plant by a pathogen may alter the normal hormonal balance in the plant and in this way change the growth habit of the host. Specific morphogenetic changes of leaves or stems and the production of tumors indicating interference with growth hormone regulation are well known responses (Sequeira, 1963, 1973). Within the context of this book, this Chapter deals primarily with the role of cytokinins in disease symptom expressions. Cytokinins are plant hormones which may be defined as compounds which induce cell division in plant cells in cooperation with an auxin (Skoog et al., 1965; Srivastava, 1967).
High concentrations of kinetin (400–2,000 μg/l) permit continuous growth of tobacco callus cultures (Nicotiana tabacum, var. Wisconsin No. 38) in the absence of exogenous thiamine. On the optimum concentration (1,000 μg/l) the tissue has been maintained through 21 bimonthly passages without change in vigor or other growth characteristics.
The effect of kinetin is general, not “mutagenic”, because tissue returned to low-kinetin, thiamine-free medium failed to grow.
Kinetin-thiamine interactions in “cytokinin mutant” strains which were grown without cytokinin in light and darkness suggest that the endogenous content of cytokinins may markedly affect the requirement for thiamine and possibly the tissue content of this vitamin and other growth factors.
The viability of tissue on low-kinetin media in enhanced by thiamine, but the addition of this vitamin does not eliminate the requirement for a cytokinin.
The great divergence in minimum kinetin concentrations required for growth of the tissue in the presence and absence of thiamine indicates that the growth promoting action of cytokinin must be different in the two cases.
The purpose of this chapter is to acquaint the reader with the chemical structures of various plant growth regulators and reports of their detection from plant sources. In general only plant growth substances from higher plants are considered. Growth substances from algae have been recently reviewed by Augier (see Augier, 1978). Kochert (1978) has reviewed sexual pheromones in algae and fungi. This topic will be dealt with in detail also in a forthcoming volume of this Encyclopedia on Interactions Between Plant Cells. Secondary metabolites from bacteria and fungi that affect the growth of higher plants have been reviewed by Strobel (1974, 1977), Patil (1974), Rudolph (1976), Harborne (1977) and Tamura (1979). Letham (1978 b) has reviewed growth substances (other than the principal hormones) from both higher and lower plants.
Publisher Summary Alkaloids bearing a purine nucleus form a small but important group of natural products. They are often not classified as alkaloids because of their almost universal distribution in living matter and their mode of biosynthesis which shows no relationship to amino acids from which most alkaloids arise. The chapter explores that some purine alkaloids are major constituents of plants such as tea and coffee which are used throughout the world as stimulating beverages. Many of the physiologically important purine alkaloids possess a xanthine nucleus. Xanthine itself has not been found naturally, but several simple N-alkyl derivatives of xanthine are of considerable significance. Prime among these are 1, 3, 7-trimethyIxanthine (caffeine), 1, 3-dimethylxanthine (theophylline), and 3, 7-dimethylxanthine (theobromine). Other purine alkaloids are derivatives of adenine and guanine, with some of the N 6 -alkylated adenines possessing considerable cytokinin-like activity. The cytokinins are plant growth substances which promote cell division.
visible morphological distortions of the plants they infect. Past studies on the effects of these particular pathogen-host interactions have been mainly descriptive and the precise mechanisms which cause these distortion were not well understood. Many of the early efforts have been to examine the diseased plant at the site of infection, and few workers have emphasized the detailed study of the pathogen itself. As stressed previously (Kado, 1976), the rewards will be great when we gain a comprehensive knowledge of the pathogen of interest. This is clearly evidenced by the studies of Agrobacterium tumefaciens, the results of which have yielded a wealth of new information not only in plant pathology, but also in molecular biology and molecular genetics. Furthermore, the discovery that genetic information is naturally transferred from a prokaryote to an eukaryote during the interaction sets a new precedence in biology.
Corynebacterium fascians causes fasciation disease or witches’ broom in dicotyledonous plants. The disease is characterized by release of apical dominance and outgrowth of lateral buds, thus giving rise to the characteristic witches’ broom syndrome (Roussaux, 1965). The symptoms of the disease can be duplicated by treatment of seedlings with cytokinins, indicating that the disease may be caused by cytokinins produced by the microbe (Thimann and Sachs, 1966). This work deals with the kinds and quantities of cytokinins released into culture media and present in tRNA of five strains of C. fascians, ranging from highly virulent MW2, moderately virulent Cf2 and Cfl, weakly virulent Cf 15 to avirulent Cfl6. It examines the relationships of the presence of plasmids to cytokinin production and to pathogenicity. It also reports the isolation from highly virulent MW2 of activity of cytokinin synthase that catalyzes the formation of N6-(Δ2-isopentenyl) adenosine-5′-monophosphate (i6 AMP) from 5′-AMP and Δ2-IPP.
The definitive discovery of the cytokinins occurred in 1955 when C. O. Miller, Folke Skoog, M. H. von Saltza, and F. M. Strong, working in the laboratories of Skoog and Strong at the University of Wisconsin, isolated a substance called “kinetin” (6-furfurylaminopurine, C10H9N5O) (Fig. 4-1) from an autoclaved sample of herring sperm DNA and demonstrated it to be very active in promoting mitosis and cell division in tobacco callus tissue in vitro.
Chemical conversions of 6‐(3‐methyl‐2‐butenylamino)purine, 2iP, and 6‐(3‐methyl‐3‐butenylamino)purine, 3iP, to common products have been effected. A number of compounds related to these isomers and their ribosides have been synthesized, including 6‐(3‐hydroxy‐3‐methylbutylamino)‐9‐β‐D‐ribofuranosylpurine, 6‐(3‐chloro‐3‐methylbutylamino)purine, 6‐(3, 4‐dihydroxy‐3‐methylbutylamino) purine, and 6‐(3,4‐dihydroxy‐3‐methylbutylamino)‐9‐β‐D‐ribofuranosylpurine, and the cytokinin activities have been compared in the tobacco bioassay. The effect of 4‐hydroxyl substitution on the side chain has been noted. Diagnostic fragmentations of the side chains have been observed in the mass spectra.
The varied abnormal growths in plants are characterised by extensive alterations and overgrowths due to the plant organ losing control over the growth potential of the affected area. Amongst the different types of abnormal growth, galls, hypertrophies, malformations and witches-brooms are worth mention. The various agents or conditions reported to act as incitants of abnormal growth in plants are: physical and chemical agents, genetic constitution, bacteria, viruses, fungi, insects, mites and nematodes. The abnormal growths are unique examples of complex interactions and mutual adaptation between the host and the pathogen. As a result of an attack on a plant by a pathogen the normal growth hormone balance is disturbed which brings about the change in the growth habit of the host. Within the context of this volume the text will be confined to a consideration of some of the important abnormal growths in plants and their hormonal regulation.
4-Furfurylamino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]-pyrimidine, the 7-deaza analog of kinetin riboside, has been synthesized and found to be a potent anticytokinin in the tabacco callus bioassay.
A substance which markedly promotes cell division in various plant tissue cultures in concentrations as low as one microgram per liter has been isolated in pure form from heated deoxyribonucleic acid preparations, and has been shown by degradation and synthesis to be 6-furfurylaminopurine. The specific name kinetin has been applied to this substance, and the generic term kinin is suggested for any substance which similarly stimulates cytokinesis.
Evidence from chemical degradations and physical methods has indicated that the structure of triacanthine is 6-amino-3-(γ,γ-dimethylallyl)-purine (V). Use of the "exchange animation" reaction has been introduced in the structure proof, and the acid cleavage of substituted N-allylic adenines has been documented. A combination of ultraviolet spectral and dissociation constant data has now provided a general method for distinguishing between 3-, 7- and 9-substituted adenines. Triacanthine and several of its isomers have been synthesized by alkylation of adenine, the alkylation on N3 representing an unusual departure from the hitherto expected course of purine alkylations. Ozonolysis studies of substituted N-allylic adenines have revealed a new feature potentially important when this degradation of hemiterpenic side chains is used as a means of structure proof. Finally, the formation of pyrotriacanthine chloride (XXV) provides a starting point for the study of allylic ring closures and rearrangements on the purine nucleus.