Extracellular ATP stimulates volume decrease in Necturus red blood cells

This study examined whether extracellular ATP stimulatesregulatory volume decrease (RVD) in Necturusmaculosus (mudpuppy) red blood cells (RBCs). Thehemolytic index (a measure of osmotic fragility) decreased withextracellular ATP (50 µM). In contrast, the ATP scavenger hexokinase(2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, theATP-dependent K⁺ channelantagonist glibenclamide (100 µM) increased the hemolytic index, andthis inhibition was reversed with ATP (50 µM). We also measured cellvolume recovery in response to hypotonic shock electronically with aCoulter counter. Extracellular ATP (50 µM) enhanced cell volumedecrease in a hypotonic (0.5×) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml)inhibited cell volume recovery. The inhibitory effect of hexokinase wasreversed with the Ca²⁺ ionophoreA-23187 (1 µM); it also was reversed with the cationophore gramicidin(5 µM in a choline-Ringer solution), indicating that ATP was linkedto K⁺ efflux. In addition,glibenclamide (100 µM) and gadolinium (10 µM) inhibited cell volumedecrease, and the effect of these agents was reversed with ATP (50 µM) and A-23187 (1 µM). Using the whole cell patch-clamp technique,we found that ATP (50 µM) stimulated a whole cell current underisosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide(100 µM), and gadolinium (10 µM) inhibited whole cell currents thatwere activated during hypotonic swelling. The inhibitory effect ofapyrase was reversed with the nonhydrolyzable analog adenosine5'-O-(3-thiotriphosphate) (50 µM), and the effect of glibenclamide or gadolinium was reversed withATP (50 µM). Finally, anionic whole cell currents were activated withhypotonic swelling when ATP was the only significant charge carrier,suggesting that increases in cell volume led to ATP efflux through aconductive pathway. Taken together, these results indicate thatextracellular ATP stimulated cell volume decrease via aCa²⁺-dependent step that led toK⁺ efflux.

     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102

The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perfusing with 10 µM forskolin (FOR) and 1 mM IBMX, and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl^– conductance was inhibited 69% by 1 µM mercuric chloride and 78% by 5 µM mercuric chloride (IC₅₀ of 0.8 µM). Despite comparable stimulation of conductance, hCFTR was insensitive to 1 µM HgCl₂ and maximum inhibition was 15% at the highest concentration used (5 µM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50–200 µM) also reversibly inhibited sCFTR (40–75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site-specific mutations introducing the first but not the second shark unique cysteine in hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark vs. human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue. chloride transport; Xenopus laevis oocytes; dithiothreitol; glutathione; p-chloromercuriphenylsulfonic acid; cystic fibrosis transmembrane regulator     

Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

Subcellular Distribution of Inorganic Phosphate, and Levels of Nucleoside Triphosphate, in Mature Maize Roots at Low External Phosphate Concentrations: Measurements with 31P-NMR

Maize plants were grown in nutrient solution without phosphate,or in which inorganic phosphate (Pi) was maintained at nearlyconstant concentrations of 1 µM, 10µM or 0·5mM. In vivo ³¹P-NMR measurements showed that there was no discernibledifference in the cytoplasmic Pi content (µmol cm^–3root volume) of the mature roots of plants exposed to 1 µM,10µM or 0·5 mM external phosphate for up to 12d. However, the vacuolar Pi content of the mature roots variedabout 10-fold between these three groups. The cytoplasmic Pi content of roots receiving no external phosphatedecreased significantly after about 7 d total growth, and atabout this time the vacuolar pool of Pi became too small foraccurate measurement. The presence of 1 µM Pi in the nutrientsolution completely prevented this decline in cytoplasmic Pi,and there was some evidence that it also raised the Pi contentof the root vacuoles above the almost undetectable level foundin the totally P-starved roots. During the first 7–9 d of growth, the nucleoside triphosphatecontent of the mature roots was unaffected by the concentrationof phosphate in the nutrient solution. The results highlight the close control of cytoplasmic concentrationsof certain important phosphorus metabolites in roots growingin soil of normal agricultural fertility. Key words: Vacuole, cytoplasm, intracellular compartmentation, NTP, P-nutrition     

Effect of Paraquat on Growth of Nitrosomonas europaea and Nitrobacter agilis

Paraquat at 4µM strongly inhibited the growth of Nitrobacteragilis but had no effect at all on the growth of Nitrosomonaseuropaea. However, nitrite did not accumulate in the presenceof 4µM paraquat when ammonia was oxidized by a mixed cultureof the two nitrifiers. (Received May 21, 1983; Accepted August 15, 1983)     

Tentoxin Inhibits Both Photophosphorylation in Thylakoids and the ATPase Activity of Isolated Coupling Factor F1 from the Cyanobacterium Anacystis nidulans

Tentoxin strongly inhibited the ATPase activity of isolatedcoupling factor 1 (AF₁) from the cyanobacterium Anacystis nidulans,with 50% inhibition occurring at 0.3 µM. When thylakoidsfrom A. nidulans were preincubated with 0.3 µM tentoxinfor 30 min, photophosphorylation was inhibited by 50%. Measurementsof fluorescence from 9-aminoacridine indicated that tentoxininhibited the utilization of the proton gradient by ATP formationin thylakoids. These results indicate that tentoxin is a strongenergy-transfer inhibitor of photophosphorylation in A. nidulans.Tentoxin decreased the level of ATP in intact cells both inthe light and in darkness, its effects being much stronger inthe dark. Tentoxin at 50 µM strongly inhibited the growthof the cells. ³Present address: Corporate Research and Development Laboratory,Tonen Co. 1-3-1 Nishi-tsurugaoka, Ohi-machi, Saitama, 354 Japan ⁴Present address: Technology and Engineering Laboratories, AjinomotoCo., Inc. Suzuki-cho 1, Kawasaki, 210 Japan     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

Direct and Indirect Shoot Organogenic Pathways in Epicotyl Cuttings of Troyer Citrange Differ in Hormone Requirements and in their Response to Light

Bud differentiation by direct organogenesis at the apical endof Troyer citrange (Citrus sinensis[L]. OsbeckxPoncirus trifoliata[L].Raf.) epicotyl cuttings inserted vertically in a semi-solidculture medium did not require hormone additions. The numberof buds regenerated was slightly, but significantly, increasedwhen the incubation was performed in the light as compared tothe dark, and by the addition of benzyladenine (BA; 2.2 to 22µM) to the medium. Bud sprouting and subsequent shootformation required the addition of BA and was increased by lightto a higher extent than bud formation. The best response wasobtained with the highest BA concentration tested (22 µM).Regeneration through the indirect organogenic pathway at thetwo edges of the epicotyl cuttings when in contact with theculture medium did not occur in the absence of benzyladenine,which was an absolute requirement for callus development. Thebest regeneration response was obtained when the explants wereincubated in the light in the presence of 4.4 µM BA andan auxin. Indole-3-acetic acid (IAA; 5.8 µM) was moreeffective in increasing shoot formation than naphthaleneaceticacid (NAA; 0.54 µM). Higher NAA concentrations inhibitedshoot formation. Incubation in the dark or increasing the BAconcentration (22 µM) increased markedly callus growth,but inhibited both bud differentiation and sprouting, almostcompletely suppressing shoot formation. The conditions duringregeneration affected the rooting of the regenerated shoots.Rooting of 86% of the shoots was achieved in a medium with 2.7µM NAA and 2.6 µM indole-3-butyric acid. All therooted explants acclimated and survived transplanting. Underthe optimal conditions tested, the proliferation rate obtainedthrough the indirect regeneration pathway ranged from 60 to86 plants per seedling. Copyright 2000 Annals of Botany Company Troyer citrange, Citrus sinensisxPoncirus trifoliata, auxins, benzyladenine, direct organogenesis, hormone requirement, indirect organogenesis, light, morphogenesis, rooting.     

Flower-Promoting Effects of Iron and EDDHA in Lemna paucicostata 151

Lemna paucicostata 151 cultured in 1/10 strength M medium containing50 µM FeCl₃ easily flowered in response to short days,although it scarcely flowered under any photoperiod when themedium contained the standard amount of iron (2 µM FeCl₃).The flowering response was accomparied by an increase in theiron content of the plants, which was maximal at pH 5.0. Instandard M medium containing 50 µM FeCl³, this plant didnot flower even though it had a high iron content. Ethylenediamine-di (o-hydroxyphenylacetic acid) (EDDHA) inducedflowering of this strain under continuous light even in theabsence of iron and copper, and its effect was slightly loweredby the presence of iron in the medium. Thus the flower-inducingactivity of EDDHA could not be attributed to the action of ironor copper. EDTA inhibited both the iron uptake and floweringin Fe-rich medium under short-day conditions. (Received May 16, 1986; Accepted July 25, 1986)     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Nuclear and Cell Sizes in Different Regions of Root Meristems of Zea mays L.

Nuclear volumes and cell areas were determined for seven regionsof the meristem of roots of Zea mays. Roots were fixed in 10per cent neutral buffered formalin, in 3 per cent glutaraldehydeor in acetic acid/alcohol; they were prepared as sections oralls were teased apart. Mean volumes of interphase nuclei weresimilar in all regions of the root except the vascular tissueof the stele. Mean nuclear volumes and the overall range ofvolumes were similar in sub-populations of cells with differentproportions of G¹, S and G² cells, e.g. in row I of root capinitials, whose cells lack a G¹ phase, and in quiescent centrecells, which are mainly in G¹. Nuclear volume does not appearto be closely correlated with DNA content. Nuclear volumes covereda 6 to 12-fold range within a meristem and even within specificregions, in which cells are part of the same cell lineages,there was a 4- to 9-fold range. Nuclear volumes were comparedin sister cells in rows I and II of the root cap initials. In10 per cent of the pairs, sister nuclei had identical volumes;the other pain had different volumes and mean difference was68 µm³. Mechanisms by which this variability could begenerated are discussed, particularly asymmetry, at mitoses,of factors that regulate nuclear growth. Zea mays L., nuclear volume, cell size, root mcristem, DNA content, mitosis     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Modification of the root plasma membrane lipid composition of cadmium-treated Pisum sativum

The effects of in vivo Cd treatments on pea root plasma membrane(PM) lipid composition were studied. In the long-term experiment,plants were supplied with Cd: moderate stress (10 µM)or strong stress (50 µM) for 10 d. Growth of root andshoot was severely affected in 50 µM Cd-treated plants,as evidenced by the approximately 7-fold reduction in theirRelative Growth Increment (RGI). Treatment with Cd (10 µM)resulted in changes to the lipid composition of the pea rootPM, including increases in the degree of unsaturation of phospholipid-associatedfatty acids and in the relative amount of stigmasterol (30–42%).This change was accompanied by a reduction in sitosterol content(26.8 to 17.4 µg mg^–1 protein). However, the sterolcomposition was not altered in plants treated with 50 µMCd for 10 d. The content of phosphatidylethanolamine and phosphatidylcholine(major phospholipids present in pea root PM) decreased as Cdlevel increased, but the ratio between them remained unaffected.In the short-term experiment, plants exposed to Cd (50 µM)accumulated less sitosterol (from 27.7 to 14.0 µg g mg^–1protein) over 72 h, but no significant effect on other measuredlipids was observed. The physiological repercussions of changesin plasma membrane lipid composition, as a result of Cd exposureare discussed. Key words: Cadmium, lipids, pea, Pisum sativum, plasma membranes     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

Changes in Nuclear DNA Content, Cell and Nuclear Size, and Frequency of Cell Division in the Cotyledons of Brassica napus L. during Embryogenesis

Cellular behaviour was examined during embryogenesis in Brassicanapus to test whether or not polyploidy occurs in the cotyledonsduring the phase of oil deposition. Nuclear DNA content, nuclearand cell size, and the mitotic index were measured in the cotyledonson various days post anthesis (dpa). In squashed monolayersfrom 15 dpa cotyledons, a polyploid (>5C) population wasdetected together with a substantial number of cells in G2 (4C).Nuclear volume was measured on sectioned tissues and, at 15dpa, the range of values from the cotyledons (40–500 *m³)contrasted with that in the vestigial suspensor and endosperm(50–> 600 µm³). At 15 dpa the nuclear volumedata suggest that whilst cells in the cotyledons were in Gland G2 many endosperm and suspensor cells were polyploid. Thus,polyploidy observed in the squashed monolayers was probablydue to contaminating endosperm/suspensor cells. At 25 and 35dpa, polyploidy was not detected; all cells were in Gl (2C)and cell area increased. The mitotic index peaked at 20 dpabefore declining and given the narrower distribution of nuclearvolumes at 25 and 35 dpa (50–300 µm³), these dataare consistent with cell arrest in Gl. Thus, polyploidy wasnot detected in the cotyledons of B. napus which differs fromwhat is known about cellular development in legume cotyledons. Key words: Brassica napus L., DNA, nuclear volume     

Novel inhibitors of ethylene production in higher plants

Of a number of O-substituted hydroxylamine derivatives, N-benzyloxycarbonyl-L-a-aminooxy-propionicacid and -aminooxyacetic acid inhibited ethylene productionby etiolated mung bean hypocotyls by 50% at 3 and 6 µmconcentrations, respectively. Their potency is thus similarto that of aminoethoxyvinylglycine (50% inhibition at 2 µM),the most potent inhibitor of ethylene production hitherto known.Methionine partially alleviated inhibition of ethylene productionby a-aminooxy-acetic acid. The results are in agreement withthe postulated involvement of pyridoxal phosphate in ethylenebiosynthesis. (Received August 31, 1979; )     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

High-frequency Organogenesis from Direct Seed Culture in Lathyrus

Culture conditions were developed for inducing a high frequencyof direct shoot morphogenesis and whole plant regeneration incultures of intact seedlings of Lathyrus cicera L., L. ochrus(L.) DC., L. sativus L., and L. tingitanus L. The procedureof shoot regeneration involved culturing of whole, mature seedson MS medium containing cytokinins, or thidiazuron (TDZ), asubstituted phenylurea with cytokinin-like activity. Differentiationof shoots occurred without an intervening callus phase fromthe cotyledonary node and surrounding tissues of the intactseedlings developed from seeds germinated on media containingkinetin, BAP or TDZ. An average of 19·0, 15·8,28·8 and 43·0 shoots were regenerated at optimalconcentrations of 50·0, 50·0 and 80·0 µMBAP in L. cicera, L. tingitanus, L. ochrus and L. sativus, respectively.TDZ enhanced the shoot formation at the concentration of 10µM to an average of 33·1 and 33·7 shootsper seedling in L. cicera and L. tingitanus and at 50 µM,to 57·4 shoots per seedling in L. sativus. Regeneratedshoots developed roots on a modified MS medium containing 2·5µM NAA and the surviving plantlets were transferred tosoil. Histological studies revealed de novo formation of shoot budsfrom the epidermal or subepidermal cells of the basal and nodalregion of multiple epicotyls. Several meristematic centres consistingof actively-dividing cells developed in the subepidermal celllayer of the nodal tissue and adjacent areas within 7 d of seedculture on a cytokinin- or TDZ-supplemented medium. The patternof the development of these meristematic centres and shoot developmentwas similar in all four species.Copyright 1993, 1999 AcademicPress Seed culture, direct shoot morphogenesis, Lathyrus, thidiazuron, grass pea, vetch ochrus