Changes in Endogenous Cytokinins of Lettuce Seed during Germination

Using the soybean callus bioassay it has been shown that dormant lettuce seeds (Lactuca sativa L. cv. Grand Rapids) contain large amounts of water soluble cytokinins and small amounts of butanol soluble ones. When the seeds are irradiated with red light, or imbibed with 5 mg/1 gibberellic acid in the dark, the total cytokinin content of the seeds decreases, the level of water soluble cytokinins decreases, and the level of the butanol soluble cytokinins increases. Far-red light does not reverse this effect completely although cytokinin activity in the butanol extracts decreases following such irradiation. It is proposed that the interconversion of cytokinins initiated by red light, or gibberellic acid in the dark, is one of the primary events leading to radicle elongation in light-sensitive lettuce seed.     

Interactions between Abscisic Acid, Gibberellins and Cytokinins in Grand Rapids Lettuce Seeds Germination

Experiments with Grand Rapids lettuce seeds (Lactuca sativa L.) maintained in darkness or irradiated with red light have shown that the inhibition of germination induced by low concentrations of ABA (2, 4, 6 μM) could be overcome by gibberellins (GA₃ or GA₄). The same results were obtained, although to a lesser extent, under the influence of two out of the four cytokinins tested (K and BAP) for seeds maintained in darkness. To suppress the block induced by higher concentrations of ABA (for example 8 μM), it was necessary to apply a cytokinin (K, BAP, Z or 2iP) and a gibberellin (GA₄ or GA₃) simultaneously, or a cytokinin following a red light treatment. Experiments conducted in darkness in which ABA (8 μM) was applied together with a cytokinin (BAP) and a gibberellin (GA₄) showed that the gibberellin and the cytokinin played similar roles towards each other and towards ABA.     

Cytokinins in macroalgae

Thirty-one seaweeds were collected from the warmer KwaZulu-Natal coast and the cooler Cape waters (South Africa). Plant material was extracted with 70% ethanol supplemented with deuterium labelled standards of all known isoprenoid cytokinins. The samples were then centrifuged and purified by combined DEAE-Sephadex×octadecylsilica column and immunoaffinity chromatography and finally analysed for cytokinins by HPLC-linked mass spectrometry and a photodiode array detector. The cytokinin profiles were similar in all the macroalgae regardless of their taxonomy and growing locality. The main type of isoprenoid cytokinins present were zeatins with cis forms being more common than trans forms and isopentenyladenine (iP) derivatives. Only a few dihydrozeatin-type cytokinins were detected at very low levels in only nine species. Aromatic cytokinins were also present but at lower levels and were represented by benzyladenine (BA) and ortho- and meta-topolin derivatives. The topolins were present in greater diversity and concentrations than BA. For all the cytokinin types, the free bases, O-glucosides and nucleotides were the most common with no N-glucosides being detected and ribosides present at very low levels. The results suggest that different pathways for regulating cytokinin concentrations operate in macroalgae than in higher plants.     

Cytokinins in photoperiodic induction of flowering in Chenopodium species

Changes in cytokinin (zeatin – Z, zeatin riboside – ZR, isopentenyladenine – iP, isopentenyladenosine – iPA) levels were determined under light regimes inductive and non-inductive for flowering in leaves, stems, roots and apical parts of short-day Chenopodium rubrum and long-day Chenopodium murale. In leaves. stems and roots of both plant species the level of cytokinins (in C. rubrum of Z and ZR, in C. murale of Z. ZR, iP and iPA) decreased by about 50% during the dark period and increased again during the subsequent light period, No significant changes in cytokinin levels were observed in continuous light. In apical parts of C. rubrum cytokinin level (Z, ZR, iP) was dramatically increased (by 400–500%) at the end of the dark period and decreased to about the original value during the following light period, while no changes were observed in continuous light. In apical parts of C. murale the level of cytokinins doubled during floral induction consisting of 10 days of continuous light. A red (R) break (15 min at the 6th h of darkness), which prevents flowering in C. rubrum , has no significant effect on cytokinin levels in leaves at the end of darkness. Cytokinin levels increased 1 h after R and decreased again rapidly. On the other hand, the increase of cytokinin level in the apical parts of C. rubrum was largely prevented by the R break. These effects of R on cytokinin levels were not reverted by far-red (FR), while the effect on flowering was reverted. It may be concluded that there is no correlation between changes in cytokinin levels in leaves. Stems and roots and photoperiodic flower induction, as both species, representing different photoperiodic types, showed similar changes under the same light regime. The increase of cytokinin levels in apical parts of both photoperiodic species during floral induction suggests a role (increased cell division and branching) for cytokinins in apex evocation.     

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.     

Investigations on Cytokinins. IV. The Metabolism of 6-Benzylaminopurine in Lemna minor

The metabolism of ¹⁴C-labeled 6-benzylaminopurine in aseptic cultures of Lemna minor was investigated. This cytokinin is slowly taken up by the plants; part of it can be released and part of it is rapidly metabolized to several compounds, among which the corresponding nucleotides can be identified. In this connection the feasibility of locating the site of hormone receptors (sites of primary action) in plants is discussed. Incorporation of the labeled cytokinin into Lemna tRNA was not observed, although tRNA hydrolysates, isolated from plants grown on a cytokinin-free medium, contain a fair amount of cytokinin activity and therefore presumably cy okinin molecules.     

Cytokinins of sycamore spring sap

Summary The cytokinins present in the spring sap of Acer pseudoplatanus L. were investigated. Ribosyl-trans-zeatin, trans-zeatin and dihydrozeatin were isolated and identified by combined gas chromatography-mass spectrometry (GC-MS). A number of other cytokinin active fractions were observed. One of these was less polar than zeatin and did not behave as any known cytokinin. Two other fractions were more polar than ribosylzeatin and were unstable. A decomposition product of one of these was identified as ribosyl-trans-zeatin by GC-MS. The possible nature of the unstable compounds is discussed. Data on the changes in cytokinin activity of the various fractions during spring 1973 are presented and discussed.Abbreviations GLC gas-liquid chromatography - GG-MS combined gas chromatography-mass spectrometry - KE kinetin equivalents - TLC thin-layer chromatography - TMS trimethylsilyl - tRNA transfer RNA - i⁶ Ade 6-(3-methylbut-2-enylamino)-purine - i⁶ Ado 6-(3-methylbut-2-enylamino)-9--D-ribofuranosyl-purine     

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.     

Cytokinins in Populus x robusta (schneid): Light effects on endogenous levels

Summary Cytokinin levels in both attached and detached mature leaves of poplar (Populus x robusta) increase transiently after short periods of exposure to red light. The degree and rapidity of response seems dependent on the physiological condition of the leaves. The cytokinin, 6-(2-hydroxybenzyl)amino purine riboside, specifically increases after red light treatment. Diurnal changes of leaf cytokinins occur, with a pronounced peak of activity being present at daybreak.     

Cytokinins in Populus x robusta Schneid: A complex in leaves

Summary At least seven cytokinins have been detected in mature leaves of Populus x robusta Schneid after chromatography on Sephadex LH-20. Two of these have similar elution volumes to zeatin and zeatin riboside. A third appears to be a cytokinin glucoside. A fourth is a new, unidentified cytokinin, susceptible to mild oxidation, and yielding two cytokinin active products after acid hydrolysis. This cytokinin complex has been found in fully expanded leaves, a tissue in which cell division is completed.     

Cytokinins and Senescence in Lemon Leaves

The effect of leaf age on cytokinin level was studied in Citrus limon (L.) Burm. f. cv. Eureca. It was found that mature 1-year-old leaves and senescing 2-year-old leaves contained considerably more cytokinins than young 3-month-old leaves. No consistent distinct decrease in cytokinin activity was revealed when the leaf passed from the mature to the senescing state. It was concluded that the results cannot be regarded as supporting the hypothesis that leaf senescence is brought about, at least partially, by a decrease in the level of its cytokinins.     

The Location of Cytokinins and Gibberellins in Wheat Seeds

Wheat seeds (Triticum aestivum L. cvs. Yorkstar and Sirius) were cut transversely into embryoless and embryo-containing (embryonated) halves and the content of endogenous cytokinins and gibberellins in both halves determined before and after the seeds imbibed water for 12–15 h at 22°C in the light. Dry seeds contained little ethyl acetate-extractable gibberellin activity as measured by bioassay but n-butanol-soluble cytokinins were detected mainly in the embryoless halves. Dry, embryonated half-seeds contained water-soluble gibberellins which could be extracted into acidic ethyl acetate after treatment of the aqueous extract with either alkaline phosphatase, β-glucosidase or a crude pectolytic enzyme preparation. When half-seeds were allowed to imbibe water for 12 h and then extracted, cytokinin activity was largely lost from the embryoless halves and completely from the embryonated halves and water-soluble gibberellins were also lost from the embryonated halves. However, ethyl acetate-soluble gibberellins were present in the latter suggesting that “bound’ gibberellins were released during imbibition. The hormones present in normal and naturally embryoless dry grains of cv. Yorkstar were also determined. Both gibberellin and cytokinin activity was higher in normal grains suggesting that the presence of an embryo is essential for synthesis or accumulation of these hormones in the grain during development.     

Cytokinins: Biosynthesis metabolism and perception

Summary Cytokinins are essential hormones for plant growth and development. They are also of vital importance for in vitro manipulations of plant cells and tissues. The biological activities and chemistry of cytokinins are well defined but very little is known about their mode of action and it is only recently that cytokinin genes have been identified in plants. This review summarizes the current status of knowledge on cytokinin biosynthesis, metabolism and signal transduction, with an emphasis on genes encoding metabolic enzymes and putative receptors, and genes rapidly induced by cytokinins.     

Cytokinins in Populus×robusta: Qualitative Changes during Development

Qualitative changes of cytokinins in leaves of different ages from Populus x robusta (Schneid.) have been determined, together with seasonal changes in cytokinin activity in mature leaves and xylem sap. Chromatography on Sephadex LH-20 has shown that total cytokinin activity and diversity are at a maximum in expanding leaves. As leaves age, the amount and number of cytokinins decrease, with yellow senescent leaves having only one detectable cytokinin, thought to be a glucoside. Seasonal changes were followed by chromatography of the extracts on paper in butan-2-ol: 25 % NH₄OH (4:1). Maximum cytokinin levels, due to Fraction Z (Rf 0.5–0.8), in leaves and xylem sap were found in mid-summer. Prior and subsequent to cessation of shoot elongation growth, fraction Z decreased and fraction N (Rf 0–0.2) increased to predominate in senescent leaves. Removal of the apex resulted in an increase of fraction N in leaves from decapitated plants when compared to similar leaves from intact plants. It is suggested that, once apical sink activity has ceased, cytokinins in the xylem sap are diverted into leaves and converted to a cytokinin glucoside, possibly a storage form of the hormone.     

Cytokinins in plant senescence: From spray and pray to clone and play

Three approaches have been used to investigate the inhibitory role of the cytokinin class of phytohormones in plant senescence: external application of cytokinins, measurement of endogenous cytokinin levels before and during senescence, and manipulation of endogenous cytokinin production in transgenic plants. In transgenic plant studies, endogenous cytokinin levels are manipulated by expression of IPT, a gene encoding isopentenyl transferase. Transgenic plants expressing IPT from a variety of promoters exhibit developmental and morphological alterations and often display retarded leaf senescence. A recently developed autoregulatory senescence-inhibition system targets cytokinin production quantitatively, spatially and temporally, and results in transgenic plants that exhibit significantly delayed senescence without abnormalities. These transgenic studies not only confirm the regulatory role of cytokinins in plant senescence, but also provide a way to manipulate senescence for potential agricultural applications.     

The Effect of Photoperiod on Levels of Endogenous Cytokinins in Xanthium strumarium

The cytokinin content of Xanthium strumarium L. plants decreased markedly when they were exposed to short days (SD). There was a significant decrease in the content of the butanol-soluble cytokinins of the mature leaves after only 5 SD cycles, and after 10 SD there was no significant cytokinin activity in butanol extracts; the changes in the young leaves were less marked. Most of the cytokinin activity in mature leaves appears to be present in the aqueous fraction, whereas in young leaves most activity occurs in the butanol-soluble fraction. SD treated plants produced less root exudate than LD plants, but there were no significant differences in the amounts of cytokinin in the root exudates from LD and SD plants collected over an equivalent time period. The cytokinin levels of SD-induced leaves remained low even when transferred back to LD. The observed differences in cytokinin levels did not appear to be the result of photosynthetic differences. Exposure of detached leaves to LD or SD did not result in differences in cytokinin content. It is not clear whether the observed changes in cytokinin levels in the leaves under SD are involved in the flowering response, but they may be causally related to a reduced chlorophyll content observed in SD-induced leaves.     

Modern aspects of studying phytohormones. Cytokinins

The review presents current data on mechanisms of cytokinin action in plants. By analogy with the first part (Ivanova et al., 1999), in which general principles of phytohormone action and cardinal trends of phytohormone investigations were examined, here the relevant information on mechanisms of action of auxins and gibberellins has been given, and taking cytokines as example an attempt has been done to summarize the literature data on the number of questions offered for analysing hormones of high animals (Gudwin, Merser, 1986). The review demonstrates that mechanisms of cytokine action at the cellular level are not known in many cases. One of the most significant factors in the action of phytohormones of this class on plants is their concentration, determined by their synthesis, transportation and further chemical conversions. This paper points to a poor knowledge of the relative role of these processes in regulation of cytokinin contents and their distribution among plant organs. Two possible ways of studying cytokinin action at the present day stage of investigations have been designated: 1) revealing the cytokinin expressed genes and establishing mechanisms of their action; 2) estimation of endogenous cytokinin alteration and the influence of this alteration on definite processes in the cell with the help of ipt-gene from t-DNA of Agrobacterium tumefaciens.     

Cytokinins from the Moss Physcomitrella patens

Gametophore-over-producing mutants of the moss, Physcomitrella patens, when grown in liquid culture export high levels of cytokinin into their culture medium. The cytokinin produced by these mutants is postulated to account for their peculiar phenotype, that of mosses treated with exogenous cytokinin. N⁶-(Δ²-isopentenyl)adenine, the major cytokinin, has been identified previously in two of these mutants (Wang, Cove, Beutelmann, Hartmann 1980 Phytochemistry 19: 1103-1105) and now in additional representatives. A second cytokinin, zeatin, has been identified by its chromatographic behavior and mass spectrum including chemical ionization mass spectrometry of its permethyl derivative.