Changes in the levels of free IAA and cytokinins in potato tubers during dormancy and sprouting

Changes in the levels of free indol-3-ylacetic acid (IAA) and free cytokinins were determined in the course of dormancy and sprouting period in potato tubers(Solanum tuberosum L., cv. Nevskii) stored at 4 °C. The same analyses were performed in potato tubers after Ethrel application, which prolongs dormancy. No significant changes were found in free IAA level during dormancy followed by a rapid decrease during sprouting. After Ethrel application a significant lower IAA level was found 3 weeks after application, but further on no changes in free IAA level between treated and non-treated tubers were detected. Cytokinin level was relatively low and constant till sprouting and increases then by about 55 %, mainly due to an increase in the level of zeatin riboside and isopentenyladenosine. Ethrel application decreased cytokinin level during dormancy slightly, but postpones the increase coupled with sprouting by about 1 month. Thus, IAA does not seem to have a significant effect on tuber dormancy, while cytokinins are probably necessary for sprouting initiation.     

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.     

Morphogenesis of potato plants in vitro. I. Effect of light quality and hormones

Stem cuttings of potato plants (Solanum tuberosum L., cv. Miranda) were cultured in vitro on MS medium with sucrose either without or with addition of indole-3-acetic acid (IAA) or kinetin (K) under red light (R) or blue light (B). Plants on medium without hormones under R were thin, long, with very small leaves, and produced no or only a few microtubers (after longer-lasting cultivations). In B, plants remained short, thick, with large, wellde-veloped leaves and produced a significant amount of microtubers. Darkening of both roots and shoots strongly promoted tuber formation; the tubers were formed on the darkened part of the plant. IAA had no pronounced effect on plant development in B except for slight lengthening of the stem, and, in longer cultivations, slightly enhanced tuber formation as well. In R, IAA brought about several significant effects: stem reduction and induction of tuber formation being the most significant. Kinetin in R increased tuber formation slightly. In B, kinetin not only strongly stimulated tuber formation, but also increased the total fresh weight and root (+ stolons)/shoot ratio. Results are discussed with regard to the possible role of auxins and/or cytokinins in mediating the morphogenetic effects of light.     

Endogenous cytokinins in the ribosomal RNA of higher plants

Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2-methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A₂₆₀ unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests.     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N⁶-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.     

Dynamics of IAA and cytokinins in flower tissues of transgenic tobacco mutant plants with mutant phenotype

A comparative analysis of IAA and total cytokinin contents at various developmental stages of floral morphogenesis (I–XII) in transgenic tobacco (Nicotiana tabacum L.) plants with mutant phenotypes was performed. Disturbances in flower structure were correlated with changes in the content and dynamics of these phytohormones. In plants with the phenotype “wavy corolla”, IAA content increased by three times as compared with control at stage VIII; in plants with the phenotype “longostyly”, IAA content increased gradually from VIII to XII stage and sharply (by nine times) at stage XII. In plants with the phenotype “wavy corolla”, total content of cytokinins was lower than in control at all developmental stages (except for stage I), whereas their dynamics coincided with that in control flowers. In plants with the phenotype “longostyly”, cytokinin content was twice lower than in control at early developmental stages, whereas it oscillated at later stages, approaching to control value at stage XII. It is supposed that differences observed could result from T-DNA insertion into plant own genes, which led to changes in the content and dynamics of phytohormones in developing flowers and production of anomalies in the flower structure.     

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis?trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.     

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.     

Endogenous cytokinins in vegetative shoots of peas

In G2 peas senescence only takes place in long days. In order to determine the role of cytokinins in this process the endogenous cytokinins from vegetative shoots of G2 peas were characterized using gas chromatography-mass spectroscopy following purification by HPLC. Cytokinins were extracted and purified with and without the addition of ¹⁵N labelled internal standards of several cytokinins to estimate cytokin content by isotope dilution in the mass spectra. Samples without internal standards were bioassayed after HPLC. Bioassays showed the presence of zeatin, zeatin riboside and zeatin-0-glucoside. The presence of zeatin was confirmed by its mass spectrum of its permethylated derivative. Tentative identification of zeatin riboside, zeatin-0-glucoside, dihydrozeatin, and dihydrozeatin-0-glucoside was obtained by the coincidence of the major ion for the permethylated natural and ¹⁵N labelled internal standards on GC-MS, and the similar coincidence of ions for permethylated zeatin riboside-0-glucoside by direct probe MS. There was no indication of the presence of significant quantities of zeatin-7-glucoside or zeatin-9-glucoside. The amounts in the tissue ranged from 200–1000 ng/kg fresh weight for each cytokinin and about 2–4 g/kg fresh weight for total cytokinins. There was no apparent difference in the levels in mature but pre-senescent shoots grown in long days and short days indicating that apical senesecence in G2 peas does not appear to be induced by a decline in cytokinin level in the shoots.Cytokinin abbreviations CK Cytokinin - Z trans zeatin - [9R]Z t-zeatin riboside - [9R-5P] Z t-zeatin riboside-5-monophosphate - (OG)Z t-zeatin-0-glucoside - (OG)[9R]Z t-zeatin riboside-0-glucoside - [7Z]G t-zeatin-7-glucoside - [9G]Z t-zeatin-9-glucoside - (diH)Z dihydrozeatin - (diH)[9R]Z dihydrozeatin riboside - iP N6(²-isopentenyl) adenine - [9R]iP N6(²-isopentenyl) adenosine Work performed while PJD was on leave at the University College of Wales at Aberystwyth.     

Ethylene and IAA transport in potato

Although elongation growth is reduced by ethylene, swelling responses do not occur. Ethylene reduces neither transport nor metabolism of applied IAA in either mesocotyl or coleoptile. We propose that maintenance of high auxin levels within these tissues sustains polar transport and contributes to the relative insensitivity of maize to applied ethylene.     

Thidiazuron influences the endogenous levels of cytokinins and IAA during the flowering of isolated shoots of Dendrobium

This study reports the effects of thidiazuron (TDZ) on the endogenous levels of indoleacetic acid (IAA), zeatin, zeatin riboside ([9R]Z), isopentenyladenine and isopentenyladenosine ([9R]iP) as well as structural changes in the apical meristem of Dendrobium Second Love shoots during flower induction and initial development in vitro. The results revealed that the presence of 1.8microM TDZ had a profound effect on the endogenous cytokinins (CKs) and IAA levels of the explants, when compared to those grown on a TDZ-free medium. A significant increase in CKs (especially [9R]iP and [9R]Z) and IAA in the first samples (taken at day 5) grown on TDZ-enriched medium was associated with flower induction, while a second increase in the level of these hormones after 25d of culture was related to flower development. The histological changes detected in the shoot apical meristem of explants grown in the presence of 1.8microM TDZ during 30d of culture are also described. Based on these findings, it is suggested that both auxin and CKs seem to be involved with the floral transition of Dendrobium Second Love in vitro. However, a possible direct effect of TDZ on flower formation is not discarded.     

Endogenous IAA levels and development of coffee flower buds from dormancy to anthesis

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific water-soluble dye, azosulfamide, was used to quantify water uptake of buds after their release from dormancy by water stress. In non-stressed flower buds, the rate of water uptake was generally slower and variable compared to stressed flower buds, where the rate of uptake tripled from 1 to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester and amide IAA levels of developing flower buds were measured by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution technique with [¹³C₆]IAA as an internal standard. Throughout development, the majority of IAA was present as amide IAA. The proportions of amide and free IAA increased one day after plants were released from water stress, and preceded the doubling of fresh and dry weight. Free and conjugated IAA content per bud remained stable during the period of rapid flower growth until one day before anthesis.Abbreviations FW fresh weight - IAA indole 3-acetic acid - HPLC high performance liquid chromatography - GC-MS-SIM gas chromatography-mass spectrometry selected ion monitoring - NAA naphthalene acetic acid - IBA indole butyric acid     

In vitro lipid metabolism in the rat pancreas. II. Effects of secretagogues on fatty acid metabolism, net lipolysis and ATP levels.

1. The concentration of carbamylcholine, bombesin, pancreozymin, pentagastrin and secretin evoking a similar 4--5-fold maximal increase in amylase secretion from rat pancreatic fragments were 3.10(-6), 10(-7), 10(-8), 3.10(-6), and 3.10(-6) M, respectively. The maximal concentration of vasoactive intestinal peptide tested (3.10(-6) M) increased amylase secretion by 250%. The six secretagogues could be separated into two groups according to their effects on lipid metabolism and ATP levels. 2. When used at their optimal concentrations, carbamylcholine, bombesin, pancreozymin, and pentagastrin lowered pancreatic ATP levels by 18-26% and increased net release of free fatty acids by 68-105%. 3. The effects of 3.10(-6) M carbamylcholine and 10(-8) M pancreozymin on the metabolism of 3H2O, D-[U-14C]glucose and [1-14C]acetate were similar; the incorporation of radioactivity in the fatty acid moiety of glycerolipids decreased by 20--50% whereas the incorporation of 3H from 3H2O and of 14C from [U-14C]glucose increased by 20--35% in the glycerol moiety. In addition, the oxidation of [U-14C]glucose, [1-14C]acetate and [1-14C]palmitate to 14CO2 increased by 15--32% while the esterification of [1-14C]palmitate, [1-14C]-linoleate, and [1-14C]arachidonate was inhibited by 14--23%. The spectrum of fatty acids labeled with [1-14C]acetate indicated an inhibition of the malonic acid pathway whereas the elongation of polyenoic fatty acids was unaltered.     

Acclimation of potato plants to polyethylene glycol-induced water deficit II. Contents and subcellular distribution of organic solutes

Potato plants (Solanum tuberosum cv. Désirée) were grown hydroponically and subjected to water deficit induced by addition of 10% (w/v) PEG 6000. The potato plants were able to grow under water deficit by accumulating organic solutes (osmoregulation). Osmoregulation occurred in two phases. During the initial 2d hexoses were accumulated, and after 7 d of PEG treatment osmotic adjustment was mostly due to the accumulation of amino acids, especially proline, which accumulated up to 150 times the control content. Sucrose contents remained unchanged in leaves of PEG-treated plants compared with controls, whereas the starch content decreased during PEG treatment.In control leaves, the hexoses and malate were compartmented in the vacuole and sucrose was found in the cytosol and vacuole. Amino acids were distributed between the cytosol and stroma, but only minor amounts of amino acids could be detected in the vacuole. Under water deficit the subcellular distribution of hexoses, malate and sucrose remained unchanged. Most amino acids showed a slight to moderate higher concentration in the vacuole under water deficit. Proline, the metabolite contributing mainly to osmoregulation, was concentrated mostly in the chloroplast and the cytosol. This underlines the important role of proline as the osmolyte under water deficit.     

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     

Endogenous levels of free and conjugated forms of auxin, cytokinins and abscisic acid during seed development in Douglas fir

Endogenous levels of free and conjugated forms of three classes of planthormones were quantified at various stages of megagametophyte development inDouglas fir. Megagametophytes were excised weekly from 8–16 weeks pastpollination (WPP), a period encompassing the central cell to the earlymaturation stage of seed development. The hormones indole-3 acetic acid (IAA),indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine(iP), isopentenyladenosine (iPA), abscisic acid (ABA) and abscisic acid glucoseester (ABA-GE) were extracted, purified, fractionated by high- performanceliquid chromatography (HPLC), and then quantified using an enzyme-linkedimmunosorbent assay (ELISA) method. Z levels ranged from 0–25ng/g dry weight (DW) and were highest in megagametophytes at thecentral cell stage (8 WPP). During embryogenesis, Z levels peakedduring week 13. In contrast, the ZR conjugate was not detected over the periodstudied. The iP content of megagametophytes increased at 10 and 13WPP, while the iPA concentration increased at 13 WPP.Prior to fertilisation, IAA was highest in megagametophytes at 9WPP. During embryogenesis, the major IAA accumulations occurred at11, 13 and 15 WPP, the concentration ranging from 0–0.43g/g DW. IAAsp concentrations reached their highest level duringembryogenesis at 14 WPP. ABA content increased at 11 and 13WPP, with a concentration range of 0.1–13 g/gDW. In contrast, ABA-GE levels were relatively constant over the 9-weekperiod analyzed. The endogenous levels of plant hormones varied withmegagametophyte development and were associated with morphological changes.     

The effect of drought on levels of abscisic acid,cytokinins, gibberellins and ethylene in aeroponically-grown sunflower plants

Abscisic acid (ABA), cytokinins and gibberellin-like substances (GAs) were extracted from the roots and shoots of 17-day-old sunflower seedlings which had been droughted or were unstressed. Plants were grown in an aeroponic chamber which allowed for good control over degree of water stress and easy access to roots. Following methanolic extraction of lyophilized material, cytokinins were separated from the acidic growth-regulators on a cellulose PO₄ cationic exchange column. The cytokinins were analysed by paper chromatography and HPLC and the soybean hypocotyl section assay. Semipurified acidic regulators were chromatographed on SiO₂ columns and HPLC and aliquots assayed with the dwarf rice cv. Tan-ginbozu bioassay for GAs. Fractions known to contain ABA were purified by sequential reverse-phase HPLC of the acid and then of the methyl ester forms followed by quantitation as Me-ABA on GLC-EC. ABA losses were measured by using an internal standard [³H]-ABA). Ethylene production was also monitored in stressed and unstressed seedlings.The effect of drought on GAs and ethylene was minimal. The ABA levels were markedly higher in droughted plants. Stressed roots had 32 times more ABA than controls. The levels of cytokinins in the shoots of droughted plants were about half those in unstressed shoots, and qualitative differences occurred in the roots. Under stress a large peak of activity was present similar to zeatin glucoside which was not present in the unstressed condition. The results are discussed in relation to drought-effects on metabolism.     

Dependence of root biomass accumulation on the content and metabolism of cytokinins in ethylene-insensitive plants

Diverse functions of ethylene in plants may depend on its ability to interact with other hormones. We studied the participation of ethylene in the regulation of accumulation and metabolism of cytokinins comparing ethylene-insensitive mutant plants of arabidopsis (Arabidopsis thaliana [L.] Heynh., etr1-1) with the plants of original ecotype Columbia (Col-0). Because cytokinins can regulate growth of both leaves and roots, we determined the weights of these organs and the ratio between them. The content of zeatin and its riboside in the roots of etr1-1 plants was two times greater than in Col-0 plants, which could be accounted for by inhibition of conversion of these forms of cytokinins into 9-N-glucosides. In the leaves of mutant plants, expression of IPT3 gene responsible for the synthesis of cytokinins was more intense than in Col-0 plants, which could also contribute to a rise in the content of cytokinins. In this case, the weight of roots in etr1-1 mutants was lower than in the plants of original ecotype. Because high concentrations of cytokinins can inhibit root growth, suppression of accumulation of their biomass in mutant plants may be related to a greater content of cytokinins therein. The obtained results suggest that ethylene can suppress accumulation of cytokinins and, thereby, maintain redistribution of biomass in favor of the roots, which is important for plant adaptation to a shortage of water and ions.     

Endogenous IAA and morphogenesis in tobacco petiole cultures

Short duration (48 h) dark or high light treatment of the donor plants has been shown to influence the pattern of auxin metabolism in tobacco petioles in culture. In explants from dark treated donor plants there was a peak of IAA detectable at day 3 in culture. This was associated with reduced morphogenetic potential of the explant. Inclusion of 2,3,5-triiodobenzoic acid in the medium prevented this IAA accumulation and eliminated the inhibitory effect of the dark pretreatment. Inclusion of IAA in the culture medium reduced the morphogenetic potential of explants from high light treated donor plants but had no effect on the morphogenetie potential of explants from dark treated donor plants. Explants cultured for one day on auxin-free medium and then transferred to IAA-containing medium were sensitive to auxin; those transferred after five days were insensitive. Studies on the interaction between media sucrose concentrations, endogenous IAA and peroxidase, and morphogenesis are reported. The results are discussed in relation to the influence of endogenous auxin on the receptivity of explants to exogenous (media) morphogenetic stimuli.     

Alterations in growth and metabolism of potato plants by calcium deficiency

Summary Experiment in water culture was conducted to evaluate the calcium deficiency symptoms and their cause inSolanum tuberosum L. var. Chandramukhi. Meristematic regions at stem and roots were severely affected and ultimately ceased to grow. Plants remained stunted with few and smaller tubers. Reducing sugar, non-reducing sugar and starch accumulated more in the leaves and stems and less in roots and tubers of calcium deficient plants. Deficiency caused decrease in protein nitrogen, RNA, DNA and increase in soluble nitrogen in all the plant parts. Potassium, phosphorus, calcium and sodium contents were lower and magnesium content higher in the deficient plant, than that of the healthy ones. Morphological symptoms of calcium deficiency can be established by ionic balance and accumulation of oxalic acid in potato plants.