Limitations associated with conductivity measurement for monitoring growth in plant tissue culture

The suitability of conductivity measurement for monitoring growth in plant cell culture has been tested using suspended cells and genetically-transformed hairy roots of Atropa belladonna, and aggregated cells of Solanum aviculare. Other researchers have proposed that a constant ratio exists between increase in cell concentration (x) and decrease in medium conductivity (C). In all cases studied in this work, x/C was not constant over a wide range of cell densities tested in batch culture. With cell suspensions, x/C decreased continuously during the growth phase from 3.4 to 2.5 g cm l^–1 mS^–1. For the hairy roots, the ratio between x and C varied by as much as 4-fold during growth. The relationship between conductivity and growth for S. aviculare aggregates was found to vary depending on inoculum density. No simple correlation between conductivity change and cell growth was apparent for the plant-cell systems studied.     

A nontoxicPseudomonas exotoxin a induces active immunity and passive protective antibody againstPseudomonas exotoxin a intoxication

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced byPseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(576–613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(576–613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(576–613) at a dose of 250 µg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 µg, respectively. PE(576–613) was then used to immunize ICR mice. The minimum dose of PE(576–613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(576–613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(576–613) that could protect mice against a 2 µg PE challenge was found to be 15 µg. In addition, sera obtained from PE(576–613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(576–613) may be used as an alternative route to new PE vaccine development.     

Carbon isotope composition of N2-fixing and N-fertilized legumes along an elevational gradient

Dinitrogen-fixing legumes are frequently assumed to be less water-use efficient than plants utilizing soil mineral N, because of the high respiratory requirements for driving N₂ fixation. However, since respiration is assumed not to discriminate against ¹³C, any differences in water-use efficiency exclusively due to respiration should not be apparent in carbon isotope discrimination () values. Our objective was to determine if the source of N (N₂ fixation versus soil N) had any effect on of field-grown grain legumes grown at different elevations. Four legume species, Glycine max, Phaseolus lunatus, P. vulgaris, and Vigna unguiculata, were grown on five field sites spanning a 633 m elevational gradient on the island of Maui, Hawaii. The legumes were either inoculated with a mixture of three effective strains of rhizobia or fertilized weekly with urea at 100 kg N ha^-1 in an attempt to completely suppress symbiotic N₂-fixing activity. In 14 of 20 analyses of stover and 12 of 15 analyses of seed values were significantly higher (p=0.10) in the inoculated plants than the N-fertilized plants. Nitrogen concentrations were generally higher in the fertilized treatments than the inoculated treatments. The different values obtained depending on N-source may have implications in using as an indicator of water-use efficiency or yield potential of legumes.     

Response of effective quantum yield of photosystem 2 to in situ temperature in three alpine plants

The response of effective quantum yield of photosystem 2 (F/F_m) to temperature was investigated under field conditions (1 950 m a.s.l.) in three alpine plant species with contrasting leaf temperature climates. The in situ temperature response did not follow an optimum curve but under saturating irradiances [PPFD >800 µìmol(photon) m^–2s^–1] highest F/F_m occurred at leaf temperatures below 10°C. This was comparable to the temperature response of antarctic vascular plants. Leaf temperatures between 0 and 15°C were the most frequently (41 to 56%) experienced by the investigated species. At these temperatures, F/F_m was highest in all species (data from all irradiation classes included) but the species differed in the temperature at which F/F_m dropped below 50% (Soldanella pusilla >20°C, Loiseleuria procumbens >25°C, and Saxifraga paniculata >40°C). The in situ response of F/F_m showed significantly higher F/F_m values at saturating PPFD for the species growing in full sunlight (S. paniculata and L. procumbens) than for S. pusilla growing under more moderate PPFD. The effect of increasing PPFD on F/F_m, for a given leaf temperature, was most pronounced in S. pusilla. Despite the broad diurnal leaf temperature amplitude of alpine environments, only in S. paniculata did saturating PPFD occur over a broad range of leaf temperatures (43 K). In the other two species it was half of that (around 20 K). This indicates that the setting of environmental scenarios (leaf temperature×PPFD) in laboratory experiments often likely exceeds the actual environmental demand in the field.This revised version was published online in March 2005 with corrections to the page numbers.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Polymerase-chain-reaction-based detection of individual polyhydroxyalkanoate synthase phaC1 and phaC2 genes*,**

A semi-nested PCR method for the specific and individual amplification of type II phaC1 and phaC2 subgenomic fragments containing the catalytically important lipase box-like sequence and the phosphopantetheine binding site was developed. Direct sequencing of these phaC1 and phaC2 fragments is possible, thus greatly facilitating the characterization of these genes. The method was used to show that three strains of Pseudomonas oleovorans harbor different pha loci, while five strains of P. corrugata contain identical phaC1 and phaC2 subgenomic fragments.     

Role of guanosine tetraphosphate in gene expression and the survival of glucose or seryl-tRNA starved cells of Escherichia coli K12

The concentration of guanosine 3,5-bispyrophosphate (ppGpp) increases in bacteria in response to amino acid or carbon/energy source starvation. An Escherichia coli K12 relAspoT mutant lacking the ability to synthesize ppGpp lost viability at an increased rate during both glucose and seryl-tRNA starvation. Also, the deleterious effect of chloramphenicol on starved wild-type cells could be overcome by inducing expression of RelA from a plasmid carrying the relA gene transcribed from a tac promoter, prior to starvation and chloramphenicol treatment. As demonstrated by two dimensional gel electrophoresis, this induction of the RelA protein resulted in global alterations in gene expression including increased synthesis of some rpoS-dependent proteins. The relAspoT mutant maintained high expression of several ribosomal proteins during starvation and appeared to exhibit significantly decreased translational fidelity, as demonstrated by an unusual heterogeneity in the isoelectric point of several proteins and the failure to express higher molecular weight proteins during starvation. Moreover, both rpoS-dependent and independent genes failed to exhibit increased expression in the mutant. It is suggested that the deleterious effects on the cells of the relA, spoT deletions are not due solely to the inability of these cells to induce the sigma factor ^s, but also to deficiencies in translational fidelity and failure to exert classical stringent regulation.     

Constructed deletions in lumen-exposed regions of the D1 protein in the cyanobacterium Synechocystis 6803: Effects on D1 insertion and accumulation in the thylakoid membrane, and on Photosystem II assembly

Modified forms of the D1 protein with deletions in lumen-exposed regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in (N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in (V58-D61) or (D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For (P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the subunit of cytochrome b559 accumulate in the thylakoid membrane independently of the D1 protein, and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.Abbreviations PSI II Photosystem II - PCR Polymerase Chain reaction Present address: Université Joseph Fourier, Sciences Technologie Médecine, BP 53, 38041 Grenoble Cedex 9, France     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase     

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium,Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C161) of aerobically grown O. limnetica was shown to contain both the 7 (79%) and 9 (21%) isomers, while the octadecenoic (C181) acid was entirely the 9 acid. Incorporation of [2-¹⁴C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the 7 and 9 C161 and the 9 C181. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both 7 and 9 C161 and 9 and 11 C181. The synthesis of these isomers is characteristic of a bacterialtype, anaerobic pathway.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - MFA monounsaturated fatty acid     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Maintenance of plasmids in HU and 1HF mutants of Escherichia coli

Summary Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the hupA-hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the hupA-hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.     

D-Gluconate is an alternative growth substrate for cultivation of Schizosaccharomyces pombe mutants

Schizosaccharomyces pombe cells grow on d-gluconate as the sole carbon and energy source. d-Gluconate is taken up in symport with protons by a specific symporter, pH being the sole driving force. d-Gluconate uptake is independent of the sugar transporting system (e.g. for d-glucose) and of . The carrier is expressed constitutively, and its activity is not subject to glucose repression. Hence, d-gluconate is a suitable carbon and energy source for growth, when d-glucose or other hexoses have to be eliminated e.g. for selection of mutants deficient in hexose transport.Abbreviations 2-DG 2-deoxy-d-glucose - CCCP carbonylcyanide m-chlorophenylhydrazone - pH pH-gradient - electrical potential difference across the plasma membrane - SD standard deviation - SEM standard error of the mean - TPP⁺ tetraphenylphosphonium     

Hexose transport and membrane depolarization in Riccia fluitans

In the aquatic liverwort Riccia fluitans, the uptake of ¹⁴C-labeled 3-O-methyl glucose (3-OMG) and membrane depolarization ( _m ) caused by different hexoses has been studied as a function of time and concentration of hexose, K⁺ and H⁺, respectively. The rate of uptake of the non-metabolized 3-OMG shows two components: (A)A pH-dependent saturable uptake with a k_m value around 0.1 mM which saturates at 2.1 and 7.2 mol G _DW ^-1 h^-1 at pH 6.8 and 5.0, respectively; and (B) a pH-insensitive uptake component which increases linearly with the external 3-OMG concentration and does not saturate 4 mM. Hexoses rapidly depolarize the plasmalemma of the thallus cell and increase its electrical conductance. The maximal _m was 60±2 mV, the concentrations (mM) for half-maximal _m were 0.24 glucose, 0.32 galactose, 0.37 2-deoxy glucose, 0.38 3-OMG, 0.57 mannose, and 34 fructose. In terms of a hexose carrier model and an equivalent circuit for the hexose-induced depolarized state of the membrane, it is proposed that a hexose carrier operates either electrogenically in its protonated, pH-and voltage-sensitive state, or by transmembrane diffusion of its uncharged state.Symbols and Abbreviations _m membrane potential (mV) - g _m membrane (slope) conductance (Sm^-2) - 3-OMG 3-O-methyl glucose     

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTS^d) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTS^d phosphofructokinase defective (PFK^–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTS^d strain.Abbreviations BM basal medium - BM-G basal medium containing glucose - CM complex medium - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - P proton motive force - pH transmembrane pH gradient - transmembrane electrical potential difference - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PIPES piperazine-N,N-bis(-ethanesulfonic acid) - MES 4-morpholineethanesulfonic acid - G-6-P glucose 6-phosphate - F-6-P fructose 6-phosphate - FDP fructose 1,6-bisphosphate - EMP Embden-Meyerhof-Parnas pathway - PFK phosphofructokinase - GK glucokinase - HK hexokinase - IAA iodoacetic acid - II^man enzyme II component of man:PTS     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Effect of stereospecific hydroxylation of N6-(Δ 2-Isopentenyl)adenosine on cytokinin activity

The cytokinin activities of cis and trans ribosylzeatin isomers and that of N⁶-(²-isopentenyl)adenosine were compared in four bioassays. The trans isomer was found to be more active than the cis isomer in stimulation of cucumber cotyledon expansion (100x), retention of chlorophyll in detached leaf pieces (7x), induction and stimulation of chlorophyll synthesis in cucumber cotyledons (20x) and of betacyanin synthesis in Amaranthus caudatus seedlings grown in the dark (60x). The N⁶-(²-isopentenyl)adenosine adenosine was less active than the trans ribosylzeatin in all four bioassays and more active than the cis ribosylzeatin in induction and stimulation of betacyanin and chlorophyll synthesis. These results show that the hydroxylation of the trans methyl group in the N⁶ side chain of N⁶-(²-isopentenyl)adenosine increases the biological activity and that this activity is either decreased or not significantly changed when the cis methyl group is hydroxylated.Abbreviations i⁶Ado N⁶-(²-isopentenyl)adenosine or 6-(3-methyl-2-butenylamino)-9--D-ribofuranosylpurine - t-to⁶Ado trans-ribosylzeatin or 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9--D-ribofuranosylpurine - c-io⁶Ado cis-ribosylzeatin or 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine - HPLC high pressure liquid chromatography