The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Inhibition of auxin-stimulated growth of pea stem segments by a specific nonasaccharide of xyloglucan

Hemicellulose extracted from cell walls of suspension-cultured rose (Rosa Paul's Scarlet) cells was digested with cellulase from Trichoderma viride. The quantitatively major oligosaccharide products, a nonasaccharide and a heptasaccharide derived from xyloglucan, were purified by gel permeation chromatography. The nonasaccharide was found to inhibit the 2,4-dichlorophenoxy-acetic-acid-induced elongation of etiolated pea (Pisum sativum) stem segments. This confirms an earlier report (York et al., 1984, Plant Physiol. 75, 295–297). The inhibition of elongation by the nonasaccharide showed a maximum at around 10^-9M with higher and lower concentrations being less effective. The heptasaccharide did not significantly inhibit elongation at 10^-7–10^-10M and also did not affect the inhibition caused by the nonasaccharide when co-incubated with the latter.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - XG xyloglucan - XG7 xyloglucan heptasaccharide (Glc₄·Xyl₃) - XG9 xyloglucan nonasaccharide (Glc₄·Xyl₃·Gal·Fuc)     

Measurement of internal pH in the coccolithophoreEmiliania huxleyi using 2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein acetoxymethylester and digital imaging microscopy

Internal pH (pH_i) was determined inEmiliania huxleyi (Lohmann) using the probe 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pH_i and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pH_i was 7.29 (±0.11) for cells in the presence of 2 mM HCO ₃ ^– . In the absence of HCO ₃ ^– the pH_i was decreased by 0.8 pH unit. The importance of these changes in pH_i is considered in relation to inorganic-carbon uptake.Abbreviations AM acetoxymethylester - BCECF 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - pH_i intracellular pH     

Synthesis of β(1–2)glucan in Rhizobium loti

Fast growing strains of Rhizobium loti isolated from nodules of Lotus tenuis of the flooding Pampas of Argentina produced cellular (1–2)glucans having a higher degree of polymerization and more anionic substituents than (1–2)glucans accumulated by Agrobacterium tumefaciens cells. Inner membranes of R. loti contained a 235 kDa (1–2)glucan intermediate protein indistinguishable by polyacrylamide gel electrophoresis from the intermediate protein present in A. tumefaciens inner membranes. Incubation of inner membrannes of R. loti with UDP-Gle led to the formation of neutral (1–2)glucans with a higher degree of polymerization than glucans formed by A. tumefaciens inner membranes.Introduction in R. loti strains of plasmid pCD523 containing A. tumefaciens chvA and chvB virulence regions yielded strains that accumulated 4 times more cellular (1–2)glucans than wild type cells. This glucan was, regarding anionic substitution and degree of polymerization, indistinguishable from A. tumefaciens (1–2)glucans. Furthermore inner membranes of these R. loti exoconjugant cells contained higher levels of the 235 kDa (1–2)glucan intermediate protein and formed in vitro 8 times more neutral (1–2)glucan with a degree of polymerization corresponding to A. tumefaciens (1–2)glucan than inner membranes isolated from wild type cells.It was concluded that A. tumefaciens chvB gene is expressed in R. loti and determined the degree of polymerization of (1–2)glucan.Abbreviations Nod⁺ effective nodulation - Vir⁺ virulent - Vir^- avirulent - Trp^r trimethoprim resistence - Tc^r tetracycline resistence - TCA trichloroacetic acid - PMSF phenyl methyl sulfonyl fluoride     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

ε-Caprolactam-degradation by Alcaligenes faecalis for bioremediation of wastewater of a nylon-6 production plant

Alcaligenes faecalis G utilized 95–97% of 5–15 g -caprolactam l^–1 in 24–48 h over a pH range of 6–8.5 and at 23–40 °C, without complex nutrient requirement. In the absence of KH₂PO₄ and K₂HPO₄/MgSO₄ in the medium, only 7.6% and 0.2% of 10 g caprolactam l^–1 was utilized, respectively. The chemical oxygen demand (COD) of the wastewater of nylon-6 plant was mainly due to its caprolactam content. A. faecalis G decreased the caprolactam content and COD of the wastewater by 80–90% of the original in spite of the wastewater having higher caprolactam content (3600 mg l^–1) and COD (7700 mg l^–1) than those of any of the previous reports.     

Isolation and characterization of α-amylase derived from starchgrownClostridium acetobutylicum ATCC 824

Summary An extracellular -amylase was purified to homogeneity from the culture supernatant ofClostridium acetobutylicum ATCC 824 grown in synthetic medium containing starch by using a combination of ammonium sulfate fractionation, anion exchange chromatography and HPLC-gel filtration. The molecular weight of the 160-fold purified -amylase was determined by SDS-PAGE to be 61 kDa. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the -1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5–5.0 and temperature of 55°C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2–5 mM) increased -amylase activity by ca. 20%, while the addition of 19 g ml^–1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. TheV _max andK _m of -amylase were 2.17 mg min^–1 and 3.28 mg ml^–1 on amylose, and 1.67 mg min^–1 and 1.73 mg ml^–1 on soluble starch, respectively.     

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-β-(1→ 3),(1→ 4)-glucanase useful for the preparation of oligosaccharides from barley β-glucan

An endo--(13),(14)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo--(13),(14)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis, transformed with the recombinant plasmid pBLC771, produced an extracellular endo--(13),(14)-glucanase that was 130 times (7176 mU ml^–1) more active than that of the gene donor cells (55 mU ml^–1), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml^–1) more active than that of the gene donor cells. M _r of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble -glucan hydrolyzed by over-produced endo--(13),(14)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.     

The effect of exogenous N6-(Δ2-isopentenyl)adenine on aerobic energy generation in Zymomonas mobilis

A direct linear relationship between the rate of oxygen consumption and ATP content in starved Zymomonas mobilis cells was observed in the presence of ethanol (0.056–1.12 mM) as the substrate. Both the rate of oxygen consumption and the ATP content were significantly reduced by the exogenously added plant growth substance N⁶-(²-isopentenyl)adenine (i⁶Ade), directly proportional to the concentration (0.125–0.5 mM) of i⁶Ade in the incubation medium. The results obtained are consistent with the current view of ATP synthesis by oxidative phosphorylation in non-growing Z. mobilis cells and gives evidence that i⁶Ade can be used as a tool to affect in vivo the alcohol dehydrogenase reaction, which provides reducing equivalents for ethanol-dependent aerobic energy generation.     

Distinct Responses of Osphradial Neurons to Chemical Stimuli and Neurotransmitters in Lymnaea stagnalis L.

1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80–100 m), the small putative sensory neurons (SC; 20 m) and very small sensory cells (3–5 m).2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between –30 and –70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between –25 and –55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10^–2 M) and L-aspartate (10^–5 M) applied to the osphradium.3. 5-Hydroxytryptamine (5HT, 10^–6 M), -aminobutyric acid (GABA; 10^–6 M) and the GABA_B agonist baclofen (10^–6 M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10^–6 M) and FMRFamide (10^–6 M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10^–6 M) but picrotoxin (10^–5 M) failed to block the GABA-induced effect on the GC1-3 cells.4. The small putative sensory neurons (SCs) were excited by Ach (10^–6 M) and 5HT (10^–6 M) but were inhibited by GABA (10^–6 M). FMRFamide (10^–6 M) had a biphasic response. The Ach effect was blocked by hexamethonium (10^–6 M) and tetraethylammonium (10^–6 M), indicating the involvement of nicotinic cholinergic receptors.5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph.     

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble -(1–3),-(1–6) glucans. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg²⁺. Gel chromatography of soluble glucans resolved a cyclic -(1–3) glucan with a degree of polymerization of eleven from a family of -(1–3),-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce -(1–3),-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize -(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of -(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.Abbreviations EPS= exopolysaccharides - CPS= capsular polysaccharides - LPS= lipopolysaccharides - AMA= Yeast extract-mannitol medium - TY= tryptone-yeast extract - PMSF= phenyl methyl sulfonil fluoride

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Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies?

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn₇–MT 1 and corresponding Zn₄-MT 1 and Zn₃-MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn₇–MT 1, Zn₄–MT 1 and Zn₃-MT 1 with AgClO₄ at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn₇–MT 1 at pH 7.5 has revealed the subsequent formation of Ag₄Zn₅–MT, Ag₇Zn₃–MT, Ag₈Zn₃–MT, Ag₁₀Zn₂–MT, Ag₁₂Zn₁–MT, Ag_x–MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag_x–MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the _xZn_y–MT (0y<7) species found, only M^I₄Zn₅–MT [(Zn₄)(₄Zn₁)] and M^I₇Zn₃–MT [(₃Zn₂)(₄Zn₁)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Oxygen transfer on β- D-galactosidase production by Kluyveromyces marxianus using sugar cane molasses as carbon source

The optimal initial volumetric oxygen transfer coefficient (K_La) was 60 h^–1 for the production of -d-galactosidase from Kluyveromyces marxianus CDB 002, using sugar cane molasses as carbon source. At this K_La applying an agitation/aeration relationship of 700 rpm/0.66 vvm resulted in 812 U l^–1 h^–1 for -d-galactosidase production. This was about 50% better than a relationship of 500 rpm/2 vvm at the same K_La.     

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.     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status

The effect of nitrogen on the cytokinin relations of Urtica dioica, the stinging nettle, has been investigated. The plants were grown in quartz sand and nutrient solutions providing levels of nitrate ranging from 1 to 22 mM. Nitrogen supply did not affect biomass production within the range of 3–15 mM NO ₃ ^- . However, the shoot: root ratio of biomass was significantly higher at 15 mM (standard plants) than at 3 mM (low-nitrogen plants) nitrate supply. The cytokinin patterns of the roots, stems and adult, as well as meristematic leaves of plants grown at these two levels of nitrate supply, were determined by means of high-performance liquid chromatography (HPLC) and immunoassays. Enzyme-linked immunosorbent assays (ELISAs) for zeatin riboside, dihydrozeatin riboside, isopentenyladenosine, benzyladenosine and o-hydroxybenzyladenosine enabled the quantification of 17 cytokinins, 13 of which were found in the various tissues of Urtica. trans-Zeatin and its conjugates were the predominant cytokinins in all examined samples. While the free base trans-zeatin and its O-glucoside were the major cytokinins in adult leaves, trans-zeatin riboside was prominent in the other tissues of at least the standard plants. Glucosides of the trans-zeatin type cytokinins were present only in lower amounts. However, considerable amounts of a compound, tentatively identified as cis-zeatin riboside-O-glucoside, were found, particularly in roots and meristematic leaves. Comparatively high amounts of trans-zeatin nucleotide as well as isopentenyladenosine phosphate were also demonstrated in these tissues. Analysis of the root-pressure exudates similarly showed trans-zeatin riboside and, at a lower concentration, trans-zeatin to be the only substantial components. In the low-nitrogen plants, shortage of nitrogen was manifest only in the roots; the nitrogen contents of the shoots did not respond to the nitrogen supply. Likewise, the total content of cytokinins in the shoots of the low-nitrogen plants equaled that of the standard-plant shoots, while it was lower by about 25% in the roots of the low-nitrogen plants. In the latter, the amounts of cytokinins exuded via the root-pressure fluid were also approximately 25% lower. Since the levels of only the trans-zeatin cytokinins in the roots showed a linear correlation with the shoot-to-root ratios, these cytokinins may play an important role in biomass partitioning in Urtica dioica.Abbreviations DHZ dihydrozeatin - ELISA enzyme-linked immunosorbent assay - -G glucoside - HPLC high-performance liquid chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - -N nucleotide (ribotide) - -OG O-glucoside - -R riboside - S/R shoot-to-root (ratio) - Z zeatin This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the SFB 137. The authors wish to thank Mrs. A. Fischbach for skilful technical assistence and Dr. Paul Ziegler (Lehrstuhl für Pflanzenphysiologie, University of Bayreuth, FRG) for linguistic suggestions.