Regulators of Cell Division in Plant Tissues VUI. The Cytokinins of the Apple Fruit

The cytokinin activities of extracts of organs developed from the apple fruit bud were compared using the carrot phloem bioassay, and the identity of the cytokinins in the apple fruitlet was investigated. The activity of apple fruitlet extracts was slightly greater than the activity of pedicel extracts, and considerably greater than the activities of extracts of other organs. Extracts of the developing seeds of fruitlets were much more active than extracts of fruitlet flesh. Apple fruitlet extracts contained three principal cytokinins. One was identified as a 6-(substituted amino)purine and was either zeatin or some very closely related compound. The two other cytokinins exhibited the chromatographic behaviour of zeatin riboside and zeatin ribotide. A cytokinin extracted from vegetative apple shoots was chromatographically indistinguishable from zeatin.     

Regulation of cytokinin biosynthesis, compartmentalization and translocation

Cytokinins, a group of mobile phytohormones, play an important role in plant growth and development, and their activity is finely controlled by environmental factors in the control of morphogenic and metabolic adaptations. Inorganic nitrogen sources, such as nitrate, are a major factor regulating gene expression of adenosine phosphate-isopentenyltransferase (IPT), a key enzyme of cytokinin biosynthesis. Modulation of IPT and macronutrient transporter gene expression in response to nitrate, sulphate and phosphate, and cytokinin-dependent repression of the transporter genes suggest that cytokinins play a critical role in balancing acquisition and distribution of macronutrients. Biased distribution of trans-zeatin (tZ)-type cytokinins in xylem and N(6)-(Delta(2)-isopentenyl)adenine (iP)-type cytokinins in phloem saps suggest that, in addition to acting as local signals, cytokinins communicate acropetal and systemic long-distance signals, and that structural side chain variations mediate different biological messages. The compartmentalization of tZ- and iP-type cytokinins implies the involvement of a selective transport system. Recent studies have raised the possibility of subsets of the purine permease family as a transporter of cytokinin nucleobases and equilibrative nucleoside transporters (ENT) for cytokinin nucleosides. These biochemical and transgenic data suggest that AtENT6, an Arabidopsis ENT, could also participate in cytokinin nucleoside transport with a preference for iP riboside in vascular tissue.     

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

A quantitative fluorescence enzyme immunoassay for plant cytokinins

An enzyme-linked immunosorbent assay (ELISA) which used 4-methylumbelliferyl phosphate as an enzyme substrate was used to quantify two plant cytokinins. This assay detected as little as 0.03 pmol (approximately 10 pg) of cytokinin in microplate wells coated with a cytokinin-ovalbumin conjugate. The method measured competition between free cytokinin and the bound conjugate for reaction with monoclonal anticytokinin antibodies and used a standard curve prepared by use of known amounts of free cytokinin to quantify hormone levels in unknown samples. Standard curves which consisted of logit/log plots of fluorescence units versus picomoles of competing cytokinin measured from 0.03 to 256 pmol (approximately 10-85,000 picograms) of zeatin riboside (ZR) or isopentenyl adenosine. The fluorescence ELISA was compared with radioimmunoassay for the quantification of ZR in wheat (Triticum aestivum L., cultivar Stephens) seed samples. This fluorescence ELISA method is recommended for use in combination with a fractionation method, such as HPLC, to quantify cytokinins present in plant extracts.     

Efficiency of different methods of extraction and purification of cytokinins

The increasing use of advanced methods, such as mass spectrometry, for the determination of cytokinins has raised special requirements for the extraction and purification of this class of plant hormones. Extraction of Arabidopsis thaliana plants with three different solvents, [80% (v/v) MeOH, Bieleski's MCF-7, and modified Bieleski's] provided similar yields of most analyzed cytokinins determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). However, the extraction with a modified Bieleski's solvent (MeOH-HCO2H-H2O [15:1:4, v/v/v]) gave the highest responses of deuterated cytokinins (used as test compounds) in plant extracts as compared to the responses of pure deuterated standards (relative internal standard response, RISR). Purification of cytokinins using Oasis MCX sorbent with reversed-phase and cation-exchange characteristics, in comparison to the DEAE Sephadex RP-C18 method, provided higher levels of zeatin riboside monophosphate and similar levels of cytokinin bases, ribosides and glucosides. Using this method the content of UV-absorbing contaminates was decreased by about 90% and the RISR values of all tested cytokinin standards but riboside monophosphates were increased about two-fold. The former method provided preparations more suitable for HPLC/MS/MS analysis with respect to simplicity and sample purity.     

A procedure for quantification of cytokinins as free bases involving scintillation proximity immunoassay

Cytokinins occur in a diversity of forms and determination of their individual levels requires extensive purification. However, determination of the total level of each major base in free, riboside and nucleotide forms would often be adequate. Hence, a methanolysis procedure which releases cytokinin bases from 9-ribosyl derivatives was developed and applied to plant extracts. A simple procedure, involving low pressure column chromatography, for purification of the cytokinin bases in treated extracts, and a scintillation proximity immunoassay for their quantification, were developed. The total level of each cytokinin base [N⁶-(2-isopentenyl)adenine, zeatin and dihydrozeatin] in free and ribosylated forms determined by these methods is reported for several plant tissues and the results are compared with those obtained after additional purification by HPLC. Values for zeatin were not changed by HPLC but isopentenyl-adenine and dihydrozeatin levels were usually reduced indicating the presence of unknown compounds which cross-react in the immunoassay. Modifications to the above purification method to quantify O-glucosyl cytokinins are also described.
The methods described facilitate the quantification of the total amount of each cytokinin base in forms closely associated with cytokinin action, and the detection of cytokinin biosynthesis by labelled precursor incorporation.     

Occurrence of a Cytokinin Glucoside in the Leaves and in Honeydew of Salix babylonica

Honeydew and leaf extracts from Salix babylonica indicate that large quantities of cytokinin are present in the leaves and are transported through the phloem of this plant during late autumn. The active compound in the extracts could be hydrolysed with β-glucosidase, whereafter it showed the same chromatographic behaviour as zeatin. It is proposed that cytokinins in the leaves are converted to the glucoside and then redistributed to the rest of the plant organs where it is stored.     

Cytokinins in Rosa hybrida in relation to bud break

To assess the role of endogenous cytokinins in growth and development of Rosa hybrida , their concentrations in bleeding sap and in roots, stem, leaves, axillary shoots and bottom breaks in three stages of development were quantified. Cytokinins were purified by means of immunoaffinity chromatography and HPLC, and identified by retention time, UV spectrum and GC-MS. The major translocation form in the xylem was zeatin riboside (ZR). In all mature tissues, cytokinins of the zeatin-type were predominant, amounting to 80–90% of the total cytokinin concentration. The stems contained high concentrations of cytokinins, probably caused by lateral movement of ZR from the xylem to adjacent stem tissue and the ability of the stem to metabolize cytokinins. In young leaves the contribution of isopentenyl adenine (iP)-type cytokinins to the total cytokinin pool was about 50%, indicating that these leaves might be capable of de novo synthesis of cytokinins. In older leaves, the concentration of an unidentified cytokinin-like compound increased to more than 50% of total cytokinins. This compound, which was also found in the roots, might be a storage form of cytokinins. In young axillary shoots, about 50% of the cytokinins are iP-compounds, suggesting either import of iP-type cytokinins via the phloem or de novo synthesis of cytokinins. In buds forming bottom breaks, ZR and zeatin riboside monophosphate (ZRMP) are the main cytokinins, indicating that these buds receive their cytokinins from the roots.     

Regulators of Cell Division in Plant Tissues

Procedures were devised for purification of the cytokinins in the milk of mature coconuts (Cocos nucifera fruits). One cytokinin isolated in crystalline form was unequivocally identified as 9-β-D-ribofuranosylzeatin. This compound appeared to account for a large proportion of the cytokinin activity in n-butanol extracts of coconut milk. The activity which was not extracted by n-butanol was largely due to unidentified compounds which could be partially purified by adsorption onto and elution from charcoal.     

Cytokinins in pathogenesis and disease resistance of Pyrenophora teres-barley and Dreschslera maydis-maize interactions during early stages of infection

Infection of Hordeum vulgare L. by Pyrenophora teres and of Zea mays by Dreschslera maydis were characterized by 'green island' formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine > zeatinriboside > zeatin > dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies, where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.     

The aromatic cytokinins

After the discovery of kinetin (Miller et al. 1956, J. Am. Chem. Soc. 78: 1345–1350) there was a flurry of syntheses that led to the finding of 6-benzylaminopurine (BA), an active and easily obtainable cytokinin. Much research into cytokinin physiology was subsequently done with this substance. Further, the isolation and unequivocal identification of natural BA and the high biological activity of its meta -hydroxylated analogues stimulated the search for other natural aromatic cytokinins. Screening was accomplished by ELISA of HPLC fractions using antisera against ortho - and meta -hydroxybenzyladenosine. Subsequent isolation and decisive identification by mass spectrometry led to discovery of a broad spectrum of endogenous plant growth substances structurally similar to a highly active compound, meta -topolin (6-[3-hydroxybenzyl-amino]purine), and to its less active analogue, ortho -topolin (6-[2-hydroxybenzyl-amino]purine). The structures of such aromatic cytokinins suggest considerably different biosynthetic pathways from that of zeatin and related isoprenoid cytokinins. From a physiological viewpoint, aromatic cytokinin metabolism can be classified under four main headings analogous to isoprenoid cytokinins: interconversion, hydroxylation, conjugation, and oxidative degradation. This review attempts to put into context what is known about 9-alkyl-BAs and compares their metabolism in regard to the practical use of cytokinins in agriculture and biotechnology. The recently discovered unusual specificity of additionally C2,N9-disubstituted aromatic cytokinins toward cell cycle kinases, suggests that these cytokinin-derived growth regulators may selectively inhibit certain steps of the cell cycle. The functional overlap of the aromatic cytokinins with those of their isoprenoid counterparts and cytokinin inhibitors, in relation to growth and developmental processes in plants, has yet to be determined.     

High Performance Liquid Chromatographic Analysis of Cytokinins in Sorghum bicolor Leaves

High performance liquid chromatography with octadecylsilica (Bondapak C₁₈/Poracil B) column packing was used to purify and separate cytokinins in Sorghum leaf extracts. The column size was 56 × 0.21 cm i.d. By gradient elution, using acidified water with increasing amounts of methanol, the major peaks of cytokinin activity, as determined by the callus tissue bioassay. were effectively separated from large amounts of extraneous impurities. These cytokinins were further separated on a microoctadecylsilica column (μBondapak C₁₈, 30 × 0.4 cm i.d.) with a gradient of acidified water-acetonitrile. Zeatin and zeatin riboside gave distinct ultra violet absorption peaks which could be used for quantitative estimation. Biological activity corresponded to the elution of these peaks. These two cytokinins are the major cytokinins in Sorghum leaves.     

Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix⁺), ineffective (nonnitrogen-fixing, Fix^–) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [³H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.     

Arabidopsis SOI33／AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtlPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that S0133 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryodc cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of^3Hlabeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Endogenous cytokinins in rose petals and the effect of exogenously applied cytokinins on flower senescence

In extracts from rose petals cytokinin activity was detected by Amaranthus bioassay in HPLC eluates corresponding to the standards: Z, ZR, 2iP and 2iPA; subsequently, the presence of two groups of endogenous cytokinins was confirmed by ELISA.Measurements of senesence indicators (cell sap osmolarity and conductivity) and observations of flower vase-life indicated that when the above cytokinins were applied as holding solutions they delayed flower senescence by 34–56% and prolonged rose longevity.Abbreviations B.H.T. 2.6-di-t-buytl-4-methyl phenol - ELISA Enzyme linked Immunosorbent Assay - HPLC High Performance Liquid Chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - Z trans-zeatin - ZR trans-zeatin riboside     

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.     

Aromatic cytokinins in micropropagated potato plants

Endogenous cytokinins were studied in three micropropagated Solanum tuberosum L. cultivars (Kennebec, Turia and Jaerla) differing in survival after transplanting. Leaf and stem cytokinins were determined both in vitro and 10 d after being transferred to ex vitro conditions by a combination of high-performance liquid chromatography and enzyme-linked immunosorbent assay. Nine aromatic and nine isoprenoid type cytokinins were identified. Higher levels of total cytokinins mainly aromatics (92%) were detected in Kennebec, the cultivar showing better in vitro growth and 99% survival. On the contrary, a predominance of isoprenoid cytokinins (up to 57%) was observed after transplanting in Jaerla, the cultivar showing lower viability. Significant survival improvement was obtained in the Jaerla cultivar after addition to the culture medium of the aromatic cytokinin meta-topolin riboside (mTR). We also report here isolation and identification of this cytokinin by several sophisticated techniques including mTR-specific immunoaffinity chromatography, diode-array high-performance liquid chromatography (HPLC), and gas chromatography–mass spectrometry of permethylated HPLC fractions. The occurrence of the aromatic cytokinins in potato plants is described for the first time.     

The role of adenine in the synthesis of cytokinins in tomato plants and in cell-free root extracts

The incorporation of labelled adenine into cytokinin-like compounds was investigated in intact tomato plants, decapitated tomato roots and cell-free root extracts. In all three cases evidence was found for the incorporation of adenine into endogenous cytokinins. In intact plants and decapitated root systems no evidence was found for the incorporation into cytokinin nucleotides. Cytokinin bases and nucleosides were however, labelled. In the cell-free root extract there was some evidence for the incorporation of labelled adenine into cytokinin nucleotides. This suggests that the biosynthetic process may be strictly compartmentalized. The present results provide no evidence for the relative importance of cytokinin nucleotides in the biosynthetic process. What is clear is that the rate of adenine incorporation into cytokinins is extremely low and that only a small proportion of the cytokinins which became labelled were exported to the shoot via the root exudate.The financial support of the CSIR/Israel Collaborative Programme is gratefully acknowledged.     

On the "activation" of cytokinins.

A number of cytokinin analogs containing modifications in the heterocyclic moiety were prepared. These compounds were tested for activity as cytokinins and anticytokinins in the tabacco bioassay and the results were used to determine whether any position(s) of the heterocyclic nucleus of cytokinins may require derivatization as part of an over-all "activation" process. 3-substituted 4-alkylaminopyrazolo [3,4-d]pyrimidines and 4-alkylaminopyrrolo[2,3-d]pyrimidines, for example, have (substituted) carbon rather than nitrogen atoms at positions 3 and 5, respectively (analogous to position 7 in purines) and would be predicted to be metabolically stable at these positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable position, and by extension at position 7 in cytokinin analogues which are purines, is not a prerequisite for the expression of cytokinin activity. Similar consideration of other heterocyclic analogs which have cytokinin activity suggests that the active form of a cytokinin can be the exogenous compound itself. Certain structural analogs of cytokinins were found to inhibit the growth of tobacco callus promoted by 6-(3-methyl-2-butenylamino)purine. These compounds were studied as potential cytokinin antagonists, i.e. having activity analogous to the 7-alkylamino-3-methylpyrazolo[4,3-d]pyrimidines (Hecht, S. M., 2068-2610; Skoog, F., Schmitz, R.Y., Hecht, S.M., and Bock, R. M. (1973) Phytochemistry 12, 25-37). The activity of these compounds is discussed and criteria are proposed to distinguish between those species which are specific anticytokinins and those which otherwise inhibit growth.