Investigations on Cytokinins. III. Cytokinin Activity in Lemna minor tRNA Hydrolysates

tRNA was extracted from Lemna minor, grown on a cytokinin free medium. Alkaline hydrolysates of the tRNA were active in three cytokinin bioassays: mobilization test, tissue culture and growth of Lemna cultures. Some observations on the growth of Lemna as a bioassay for cytokinins, are given.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.     

Half-life of tRNA Containing N6(Δ2-isopentenyl)adenosine in Lactobacillus acidophilus ATCC 4963

tRNA containing N⁶-(Δ²-isopentenyl)adenosine may be precursors for the plant hormone cytokinin. To discriminate between tRNA containing and not containing cytokinin nucleotides, double labelling experiments were made by the use of [2¹⁴C]-mevalonic acid and [³H-methyl]-methionine. At a generation cycle of 2 h for Lactobacillus acidophilus ATCC 4963, the half-lives of tRNA labelled with [³H-methyl]-methionine and [2-¹⁴C]-mevalonic acid are similar, namely 3 h. Isopentenylation of tRNA could be measured to be maximally 1:10.     

Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Investigations on Cytokinins. II. Interaction of Light and Cytokinins as Studied in Lemna minor

In Lemna minor under non optimal growth conditions, due to light shortage, a cytokinin is able to supplement this shortage partly. The cytokinin seems to substitute the effect of non-photosynthetic light.     

Untersuchungen zur Biosynthese eines Cytokinins in Calluszellen von Laubmoossporophyten

Summary When callus cells derived from the sporogon of the hybrid Funaria hygrometrica x Physcomitrium piriforme are supplied with adenine-8-¹⁴C, they produce a labelled cytokinin which has the same chromatographic behavior as N⁶--(dimethylallyl)aminopurine. The cytokinin is the first radioactive product that can be detected in the culture medium. It is formed as long as labelled adenine is available. When callus cells are grown in an optimum culture medium containing amino acids, about 10% of the radioactivity supplied as adenine is found in the cytokinin. When the cells are grown in a medium without amino acids, the RNA-content of the cells and the total yield of cytokinin decrease, but about 18% of the radioactivity is taken up into RNA as adenine and guanine, which are both degraded to allantoin and urea but not converted to cytokinin. Free guanine is converted neither to adenine nor to cytokinin.Weak cytokinin activity can be detected in hydrolysates of sRNA, but no radioactive cytokinin can be isolated from sRNA of adenine-labelled callus cells. it is assumed that free cytokinin is not a degradation product of tRNA.     

Distribution of Cytokinin-active Ribonucleosides in Wheat Germ tRNA Species

The distribution of cytokinin activity in wheat (Triticum aestivum) germ tRNA fractionated by BD-cellulose and RPC-5 chromatography has been examined. As in other organisms, the cytokinin moieties in wheat germ tRNA appear to be restricted to tRNA species that would be expected to respond to codons beginning with U. Only a few of the wheat germ tRNA species in this coding group actually contain cytokinin modifications. Cytokinin activity was associated with isoaccepting tRNA^Ser species and with a minor tRNA^Leu species from wheat germ. All other wheat germ tRNA species corresponding to codons beginning with U were devoid of cytokinin activity in the tobacco callus bioassay.     

Growth retardation induced by nutritional deficiency or abscisic acid in Lemna gibba: The relationship between growth rate and endogenous cytokinin content

The relationship between endogenous cytokinin content and relative growth rate (RGR) was studied in cultures of Lemna gibba L. G3 supplied with daily doses of mineral nutrients that were increased exponentially over time. At the optimal level of nutrient supply the RGR was 30–35% day^-1. The RGR was regulated by adjusting the rate of nitrogen supply, or it was restricted by addition of 0.5 M abscisic acid (ABA). Another approach used to investigate the specific roles of nitrogen (N) and phosphorus (P), was to transfer optimally growing plants to media without N or P but otherwise complete. The plants were harvested at regular intervals for determination of the RGR and levels of cytokinins of the isopentenyladenosine (iPA) and zeatinriboside (ZR) types with an enzyme-linked immunosorbent assay (ELISA). Levels of both iPA- and ZR-type cytokinins decreased when nitrogen was applied to cultures in growth limiting amounts. The cytokinin levels decreased more rapidly than the RGR when either N or P was lacking in the medium, suggesting an early influence of nutrient availability on cytokinin levels which in turn may induce adaptive response by the plant. RGR retardation induced by ABA did not affect cytokinin levels during the first 4 days of the treatment, and the later effects were small. The experiments gave no indication that ABA is involved in the adaptation response of Lemna plants to nutritional stress.Abbreviations ABA - abscisic acid - BAP - benzylaminopurine - ELISA - enzyme-linked-immunosorbent-assay - iP - isopentenyladenine - iPA - isopentenyladenosine - PBS - phosphate-buffered saline - PVP - polyvinylpyrrolidone - RGR - relative growth rate - R_N - relative nitrogen addition rate - Z - trans-zeatin - ZR - trans-zeatin riboside     

On the biogenesis of cytokinins in roots of Phaseolus vulgaris

Roots of intact bean plants were supplied with [¹⁴C]adenine by pulse-chase experiments. The rate of incorporation of radioactivity into tRNA and oligonucleotides of roots as well as the content of radioactive labeled cytokinin nucleotides in these RNA fractions were determined. On the average, 1/70 of the radioactivity incorporated into tRNA was localized in N⁶(²isopentenyl)adenosine. The half life of tRNA was estimated to be 65–70 h. Shortly after the pulse period, oligonucleotides contained zeatin riboside at a ratio of 1:800, on the basis of radioactivity. The half life of these oligonucleotides was determined to be about 8 h. The main free radioactive cytokinin of roots and leaves was zeatin. Comparing the rate of degradation of ¹⁴C-labeled tRNA and the oligonucleotides of roots and the rate of appearance of radioactive cytokinins in roots and leaves, we found strong indications for their dependency. The results contradict the hypothesis of de novo synthesis of cytokinins in roots of intact bean plants.Abbreviations AMP adenosine monophosphate - IPA N⁶(²isopentenyl)adenosine - IPAde N⁶(²isopentenyl)adenosine - Z zeatin - ZR zeatinriboside - TLC thin-layer chromatography - HPLC high performance liquid chromatography Part of the doctoral thesis, Bonn 1980     

Cytokinin-induced growth in the duckweeds <Emphasis Type="Italic">Lemna gibba</Emphasis> and <Emphasis Type="Italic">Spirodela polyrhiza</Emphasis>

Duckweeds, quick-growing aquatic plants, have been recently recognized as promising hosts for the large-scale production of recombinant proteins and as an ideal biomass feedstock for biofuel production. These possible wide-spread industrial uses of duckweeds intensified research aimed at understanding the mechanisms that control duckweed growth. Here, we describe how the hormone cytokinin affects growth. We performed a number of standard cytokinin growth- and physiological-response assays using sterile-grown colonies of Lemna gibba and Spirodela polyrhiza. Similar to land plants, cytokinin inhibited root elongation in duckweeds. Surprisingly, and in contrast to land plants, cytokinin promoted growth of aerial organs in both duckweed species, suggesting that the cytokinin growth response fundamentally differs between aquatic and land plants.     

Possible Involvement of Cytokinin in Nitrate-mediated Root Growth in Maize

Response of root system architecture to nutrient availability in soils is an essential way for plants to adapt to soil environments. Nitrate can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Nevertheless, less is known about the physiological mechanisms. In the present study, two maize (Zea mays L.) inbred lines (478 and Wu312) were used to study a possible role of cytokinin in nitrate-mediated root growth in nutrient solutions. Root elongation of 478 was more sensitive to high nitrate supply than that of Wu312. Medium high nitrate (5 mM) inhibited root elongation in 478, while, root elongation in Wu312 was only inhibited at high NO ₃ ⁻ supply (20 mM). Under high nitrate supply, the root elongation zone in 478 became swollen and the site of lateral root elongation was close towards the root tip. Both of the phenomena are typical of root growth induced by exogenous cytokinin treatments. Correspondingly, zeatin and zeatin nucleotide (Z + ZR) concentrations were increased at higher nitrate supply in 478, whereas they were constant in Wu312. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. Therefore, it is supposed that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots. High nitrate supply may have negative influences on root apex activity by affecting cytokinin metabolism so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex. Nitrate suppressed lateral root elongation in Wu312 at concentration higher than 5 mM. In 478, however, this phenomenon was not significant even at 20 mM nitrate. Although exogenous 6-BA (20 nM) could suppress lateral root elongation as well, the inhibitory effect of high NO ₃ ⁻ concentration of nitrate on lateral root growth cannot be explained by changes in endogenous cytokinin alone.     

Gamma-irradiated plant callus tissue: Cytokinins from transfer ribonucleic acid

Summary Callus tissue ofHaworthia mirabilis Haw. was irradiated with⁶⁰Co gamma rays. tRNA was isolated, hydrolyzed enzymatically, and cytokinin-active ribonucleosides were separated by Sephadex LH-20 column chromatography and assayed with the tobaccocallus cytokinin bioassay. Three cytokinins were detected in tRNA from irradiated tissue, two of which chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. The third cytokinin-active ribonucleoside was retained longer than the above compounds on the Sephadex column and may be 2-methylthio-N⁶-(Δ²-isopentenyl)adenosine. Two cytokinins were detected in tRNA from nonirradiated tissue—those chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. Relationships between cytokinins from tRNA and free cytokinins found in tissue earlier are discussed. This is paper 78-10-124 of the Kentucky Agricultural Experiment Staton and is published with approval of the Director.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Cytokinin metabolism in Physcomitrella patens – differences and similarities to higher plants

Cytokinins play an important role in plant development and occur informs with different hormonal activity. As the nucleotide forms of cytokininsare considered to have little or no biological activity, the conversion ofcytokinin bases and ribosides to their nucleotides can contribute to the tuningof cytokinin activity in plant cells. Cytokinin metabolism was monitoredin vivo by feeding either radiolabelledisopentenyladenosine (³H-[9R]iP) or isopentenyladenine(³H-iP) to liquid grown chloronema tissue ofPhyscomitrellapatens (Hedw.) B.S.G. wild type. The riboside ³H-[9R]iPwas rapidly converted to ³H-iP, which was released into the culturemedium. The intracellular concentration of the ³H-iP was twice ashigh as extracellular. From the overall amount of ³H-iP about 95%were present in the medium. Cytokinin nucleotides occurred as tritiated mono-,di- and triphosphates of ³H-[9R]iP. When feeding the base³H-iP however, its main metabolic fate was degradation and nosignificant amounts of radiolabelled cytokinin nucleotides were detected. Forthe cytokinin metabolism in P. patens it is concludedthat,in contrast to higher plants nucleotides are mainly formed from ribosidesvia the adenosine kinase pathway and not byribophosphorylation of the cytokinin base via adeninephosphoribosyltransferase.     

Untersuchung des Einflusses der Temperatur auf die Photosynthese-Induktion bei Chlorella vulgaris mit radioaktivem CO2

Summary The lateral bud of Solanum andigena has the potentiality to develop as a stolon or as a leafy, orthotropic shoot. Natural stolons are normally only produced from underground nodes, but aerial stolons can be induced to form by the application of a combination of indole-3-acetic acid and gibberellic acid (IAA/GA₃) paste to the cut surface; under some conditions both natural or induced stolons are converted to upright, leafy shoots. The presence of roots was found to be necessary for the conversion of a natural stolon to a leafy shoot, but this root effect could be replaced by the synthetic cytokinin, 6-benzylaminopurine (BAP). By using -¹⁴C-BAP it was demonstrated that cytokinin accumulates in the tip of an induced stolon, prior to its conversion to a leafy shoot caused by withdrawal of the IAA/GA₃ paste. The application of IAA/GA₃ to decapitated plants was shown to influence both the distribution and metabolism of the cytokinin. The possibility that the role of auxin in apical dominance, at least in part, is to control the distribution and metabolism of cytokinins is discussed.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Transactivation of a target gene using a suppressor tRNA in transgenic tobacco plants

The abundance of tRNAs, together with their central role in translation, has generated considerable interest in the use of tRNA genes for biotechnological applications. One such application is the use of suppressor tRNAs to transactivate target genes containing premature stop codons. Previous work has shown that such systems can work in transient expression experiments in plant protoplasts; here these experiments are extended to show that suppression of stop codons can occur in whole plants. Transgenic tobacco plants homozygous for a modified tRNA^Leu gene expressing a strong amber suppressor tRNA, and plants carrying a β-glucuronidase (gus) gene inactivated by a premature amber stop codon have been obtained. When the two types of plants are crossed, many of the F₁ hybrids show significant GUS activity. The GUS activity is dependent on the presence of both the suppressor tRNA gene and the gus gene. Tobacco plants carrying the suppressor tRNA gene are phenotypically normal, fertile and the gene shows normal Mendelian inheritance. The potential applications of such a system are discussed.     

Nitrogen Nutrition and Cytokinin Activity in Solanum tuberosum

In water culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), the influence of continuous nitrogen nutrition (constant supply of NO₃^?) and discontinuous nitrogen nutrition (interruption of NO₃^? supply, i. e., nitrogen withdrawal for 6 days) on the endogeneous cytokinin level in the roots, shoots and exudate of decapitated plants was studied. Harvests took place at intervals of 3 days. The chlorophyll formation test (cucumber cotyledons) and soya callus test were used to determine the cytokinin activity. With continuous nitrogen, the cytokinin activity decreased slightly with time in both roots and shoots but rose in the exudate. With discontinuous nitrogen, the nitrogen withdrawal led to a temporary, pronounced increase in cytokinin activity in the roots; at the same time, the cytokinin activity in the exudate decreased sharply. It is assumed that this temporary increase in cytokinin activity in the roots is a reflection of increased meristem activity in the roots. In the shoots, the cytokinin activity decreased during the nitrogen withdrawal period. These nitrogen-induced changes in cytokinin activity in the roots and shoots of potato should presumably have an important influence on the physiological age of the shoot, with all its consequences in the further development of the plant. Zeatin riboside was likely the main cytokinin component involved.