Chloroindolyl-3-acetic Acid and Its Methyl Ester Incorporation of 36Cl in Immature Seeds of Pea and Barley

Immature seeds of pea and barley were harvested on plants grown in solutions containing ³⁶Cl^?, but no other chlorides. Autoradiography of two-dimensional thin layer chromatograms (silicagel) of butanol extracts of freeze-dried seeds showed the presence in both species of several radioactive compounds besides Cl^?. One compound, present in pea and probably in barley, cochromatographed with a mixture of 4- and 6-chloroindolyl-3-acetic acid methyl esters. Another, detected in pea, but probably not in barley, cochromatographed with a mixture of 4-and 6-chloroindolyl-3-acetic acids.     

Studies of the slowly exchanging chloride in Photosystem II of higher plants

³⁶Cl^- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of ³⁶Cl^-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10^-4 s^-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on ³⁶Cl^-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pK_a of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br^- and NO₃ ^-. The slowly exchanging Cl^- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S₂ state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S₂ state EPR signals.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - MWCO molecular weight cut off - PPBQ phenyl-p-benzoquinone - PS II Photosystem II     

盐氮处理下盐地碱蓬种子成熟过程中的离子积累和种子萌发特性

研究了盐氮处理条件下盐地碱蓬种子成熟过程中的离子积累以及种子萌发特性,以理解盐地碱蓬在种子发育及萌发过程中对高盐低氮生境的适应性。结果表明,种子成熟过程中,不同浓度盐氮处理下(0.5和5 mmol/L NO₃^--N;1和500 mmol/L NaCl),与果皮和果枝相比, 胚中Na⁺、K⁺、Cl^- 和NO₃^-离子含量几乎没有变化。所有盐氮处理下Na⁺ 和Cl^-都是果皮和果枝中高于胚中,尤其是在高盐处理下。高盐处理下,K⁺ 和NO₃^-含量呈现相反的趋势。高氮时无论高盐还是低盐,果皮中NO₃^-离子含量高于胚中,而果枝中NO₃^-离子含量低于胚中。而低氮时果皮及果枝中NO₃^-离子含量均显著低于胚中。与高氮环境下收获的种子相比,低氮环境下收获的种子萌发率,萌发指数,活力指数都要明显高。上述结果说明,盐地碱蓬种子成熟过程中存在完善的离子调控机制,保护胚免受Na⁺ 和Cl^-等有害离子的伤害并且促进K⁺ 和NO₃^-等营养离子的积累。低NO₃^--N下收获的种子对外界的NO₃^-含量比较敏感,施以较高浓度的NO₃^-能够促进种子萌发,提高萌发指数和活力指数,可能与盐地碱蓬长期适应高盐低氮生境有关。     

The relationship of the charasome to chloride uptake in Chara corallina: physiological and histochemical investigations

A possible role of the charasome in terms of chloride transport into Chara corallina Klein ex. Willd., em. R.D.W. is examined. The branches of Chara contain the most charasome material and are shown to be very effective in acquiring Cl^- to support continued shoot growth. The early maturation of the branches, the rather large Cl^- fluxes into these cells, and their ability of translocate Cl^- to growing cells of the shoot indicate a special role of these branches in Cl^- accumulation. The structure of the charasome, with its extensive periplasmic space, appears especially suited as a site for H⁺–Cl^- cotransport (influx). We show, by histochemical assay, that the charasomes of mature cells contain ATPase activity; such activity is absent in growing charasomes of very young cells. ATPase activity is also associated with the plasmodesmata of C. corallina. Charasome ATPase activity and Cl^- uptake are both inhibited by p-chloromercuribenzenesulfonic acid (1 mM) or diethylstibestrol (40 M; 45 min). The anion transport inhibitor, 4,4-diisothiocyano-2,2-disulfonic acid stilbene (1 mM) had no effect on Cl^- transport and inhibited ATPase activity only when applied after chemical fixation of the cells. Results of an attempt to demonstrate the presence of Cl^- within the cytoplasmic tubules of the charasome, using a silver precipitation technique, proved difficult to interpret because of a reaction between the silver and a cellular substance produced in the light.Abbreviations CPW Chara pond water - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-disulfonic acid stilbene - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid     

Effect of Water Stress on the Carbohydrate Metabolism of Citrullus lanatus Seeds during Germination

Gluconeogenesis in Citrullus lanatus seeds is a post germinative event. Increases in isocitrate lyase activity and incorporation of radioactivity from [2-¹⁴C]acetate into sugars occur only after radicle emergence. During germination, the seeds appear to rely on carbohydrate as the respiratory substrate. At this time, glycolysis, the pentose phosphate pathway, and the tricarbocyclic acid cycle seem to be functional. Utilization of raffinose during germination appears to be important.

Water stress, which completely inhibits germination, has a marked effect on carbohydrate metabolism. The rate of ¹⁴CO₂ release from [2-¹⁴C]acetate, [1-¹⁴C]glucose, and [6-¹⁴C]glucose is lower in the stressed seeds than the control seeds during the respiratory lag phase. However, in the stressed seeds neither glycolysis, the pentose phosphate pathway, nor the tricarboxylic acid cycle is completely inhibited. In contrast to the control seeds in which raffinose content sharply declines after 12 h of incubation, raffinose content in the stressed seeds remains fairly constant.

The respiratory lag phase of the control seeds coincides with a lower reducing substance content, glucose content, and fructose content than in the stressed seeds during the corresponding incubation period.

     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

CL−-Fluxe,Akkumulation und Transport in abgeschnittenen Mais-Wurzeln

Summary Fluxes, accumulation and transport rates of Cl^- in excised corn roots were investigated.Flux equilibrium in 5×10^-4M KCl was not reached within an experimental period of 28 hr.Transport of Cl^- (5×10^-4M KCl) through the xylem of 4–6 cm long excised corn roots had a lag of 1–2 hr. From 6 to 28 hr rates of accumulation and transport were nearly constant (5×10^-4M KCl). The velocity of volume-flow within the xylem was 1.5–2 cm hr^-1 (5×10^-4M KCl). ³⁶Cl^--efflux through the cut end of roots preloaded in K³⁶Cl of low concentration exhibits a discontinuity which is explained by addition of two fluxes: efflux of ions concentrated in the xylem (and symplasm) plus efflux from the vacuoles through the xylem.Accumulation of Cl^- by excised roots approaches a maximum level (Jackson and Edwards, 1966). Influx rates remain constant while efflux rates increase with time. It is shown in this paper that the flux of Cl^- through the xylem becomes a large proportion of the influx across the plasmalemma. Flux rates suggest that more than 50% of the Cl^- ions transported to the xylem passed through a vacuole (5×10^-4M KCl; 20–28 hr).     

Studies on the role of abscisic acid in the initiation of bud dormancy in Alnus glutinosa and Betula pubescens

Summary The effects of leaf-applied (+-)-abscisic acid on the growth and dormancy of Betula pubescens Ehrh. and Alnus glutinosa Gaertn. growing under long days provide no evidence that leaf-applied abscisic acid induces or promotes the formation of resting buds in these species. Radiotracer studies show that a small percentage of the radioactivity applied as [2-¹⁴C]abscisic acid to the leaves accumulates in the apical region of the shoot. Of the radioactivity that was recovered from this region after 8 days, less than 10% was chromatographically similar to [2-¹⁴C]abscisic acid. The significance of these results with respect to the role of abscisic acid in regulating the induction of bud dormancy is discussed.Abbreviation ABA abscisic acid     

Indoleacetic-acid-enhanced chloride uptake into coleoptile cells

Summary The enhancement by indoleacetic acid (IAA) of ³⁶Cl^- uptake into Avena coleoptile sections was used to study the effects of a hormone on a membrane-controlled phenomenon. Compared to sections in phosphate buffer only, Cl^- content of the cells increases 15 min after addition of IAA; the promotion is seen only with growth-active auxins and is saturated at 3 M IAA. The percent enhancement by IAA is the same over a wide range of Cl^- concentrations. The hormone effect is not observed at ice-bath temperature and is not correlated with growth or water movement into the cells. IAA does not influence the movement of Cl^- in the section. While auxin must be present within the tissue in order to maintain the enhancement, there is no relationship between the total amount of auxin and the accelerated Cl^- uptake that results. A polarity in the auxin effect is implied since only apical applications of IAA promote Cl^- uptake.     

The effect of glucose on chloride uptake by Chlorella

Active Cl^- uptake by Chlorella fusca was examined by using ³⁶Cl as a label. Under light/air conditions chloride influx from a 2.4·10^-5 M solution was 4.0±0.04 nmol m^-2s^-1. After 70±10 min a stationary 380±40 fold accumulation was reached. In dark/air and dark/argon influx and accumulation were reduced to 25±6%, respectively, 5±1.5% of the light/air control. Cl^- uptake had a broad optimum around pH 7 and showed saturation kinetics with a K _M of 1.25·10^-5 M and a v _max of 7.0 nmol m^-2s^-1 in light/air. Br^- inhibited Cl^- uptake strongly, J^-, ClO ₄ ^- , SO ₄ ^2- , and NO ₃ ^- had no inhibitory effect. Inhibitor studies with carbonyl cyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide resulted in a good correlation between Cl^- uptake and ATP level. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and darkness reduced transport activity without affecting the ATP level.The magnitudes of the pH gradient and the membrane potential across the cell membrane were determined and/or estimated under different conditions. It could be shown that in Chlorella Cl^- transport cannot proceed via secondary active H⁺/Cl^- cotransport. In addition, 2H⁺/Cl^- cotransport seems unlikely for energetic reasons. On the basis of the results of this and the following study, a primary active ATP-driven Cl^-/OH^- exchange pump is proposed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhyd razone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3.4-dichlorophenyl)-1.1-dimethylurea - DMO 5,5-dimethyloxazolidine-2,4-dione - Hepes N-2-hydroxyethylpiperazine-N ethane-sulfonic acid - POPOP 1.4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2.5-diphenyloxazole To whom correspondence should be addressed     

Evidence for differential action of indoleacetic acid upon ion fluxes in single cells of Petroselinum sativum

Summary The apparent influx of ³⁶Cl^- and ⁸⁶Rb⁺/K⁺ into cells from the higher plant Petroselinum sativum has been measured during the presence and absence in the culture medium of indolacetic acid (IAA) which is an essential auxin of these cells. While 10^-5 M IAA did not significantly affect the influx of ⁸⁶Rb⁺/K⁺, it substantially reduced that of ³⁶Cl^-, i.e. by a factor 0.25 within 30 min. This differential action of IAA, which holds for a reasonable range of external pH, is assumed to bear on current hypotheses that the primary events of auxin action involve plasmalemma functions.     

Chloride transport in Anacystis nidulans

Summary Anacystis nidulans will take up and accumulate chloride ions. When the external concentration was 0.2 mM Cl^- the level in the cells was 2.8 mM Cl^- and under these conditions the flux across the cell surface was in the region of 10^-13equiv Cl^-·sec^-1·cm^-2. It is suggested that this Cl^- influx is active and operates against an electrochemical potential gradient estimated to be 117 mV or 2.68 kcal/mole. The uptake of ³⁶Cl was inhibited by low temperatures and there was a net loss of Cl^- from the cells with the level decreasing towards the equilibrium value as estimated from K⁺ distribution. Although the active influx of Cl^- was often stimulated by light this was not always the case. Dark storage treatment and regulation of the chlorophyll a/phycocyanin ratios as well as total pigment content of the cells did not clarify the conditions which brought about light stimulation. Moreover, the metabolic inhibitors CCCP and CMU and also the use of anaerobic conditions did not clearly indicate the relationship between the influx mechanism and light-dark metabolism and no firm conclusions could be made about the nature of the energy source. The variation in the degree of light stimulation probably reflects the fact that in this procaryotic organism the photosynthetic and respiratory units are located on the same membrane systems and are in very close proximity to the probable site of the Cl^- pump, the plasmalemma.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - CMU 3-(4-chlorophenyl)-1,1-dimethylurea     

Two types of chloride channels in hen colon epithelial cells identified by patch-clamp experiments

Summary Freshly isolated epithelial cells from hen colon were investigated using the patch-clamp technique. The aim of this investigation was to characterise the cellular conducting site for Cl^- secretion. In cell-attached mode two types of Cl^--channels were found. Both showed distinct outward rectification. The channel types differed in single channel conductances and the marked voltage dependence of the open probabilities. A low conductance Cl^--channel was observed with a mean conductance at negative holding potentials of g_-=9 pS, and of g₊=34 pS at positive potentials. This channel was predominantly open at negative potentials, corresponding to cell hyperpolarization. The second channel type observed had conductances of g_-=35 pS and g₊=77 pS, and showed increasing open probabilities with increasing holding potentials (cell depolarisation). Both channel types were blockable by the Cl^--channel blocker NPPB. These data in combination with previously published transepithelial transport data on hen colon indicate that these channels are the Cl^- secretory sites in colon epithelium.Abbreviations DNSO dimethylsulfoxide - EGTA ethyleneglycol triacetic acid - g₊, g_- single channel conductance at positive and negative voltages - HEPES N-(2-hydroxy-ethyl)piperazine-N-(2-ethane-sulfonic acid) - i single channel current - NMDG N-methyl-d-glucosamine - NPPB 5-hitro-2-(3-phenylpropylamino)-benzoate - P_o open probability - V_p holding potential     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Fluxes and compartmentation of potassium and chloride in the green alga Mougeotia

Summary Some ionic relations of the filamentous green alga Mougeotia sp. have been analyzed under different light conditions. Data from influx and efflux measurements using ⁸⁶Rb⁺ and ³⁶Cl^- fit the model of three cellular compartments (cell wall, cytoplasm, vacuole) in series. This result is remarkable, since in a Mougeotia cell at least two thirds of the cytoplasmic compartment are occupied by the cell-filling, flat and nearly rectangular chloroplast which is axially oriented. The chloroplast is concluded to be part of the cytoplasmic flux compartment.Photosynthetically saturating irradiances of continuous white light enhance the active and passive fluxes of K⁺ and Cl^- at the plasmalemma by a factor of 3. Photosystem II is responsible for the light-dependent increase of the uptake of Cl^- (³⁶Cl^-) whereas the uptake of K⁺ (⁸⁶Rb⁺) depends additionally on energy from photosystem I.Ion flux measurements performed after irradiations with red and far-red, respectively, show that the fluxes of K⁺ and Cl^- across the plasmalemma are not affected by the state of phytochrome.     

Conversion of DOPA to tetrahydroisoquinolines and stizolobic acid in a callus culture of Stizolobium hassjoo

Isolation and identification of l-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and l-1-methyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline from seeds and callus of S. hassjoo are described. Administration of [β-¹⁴C]-labelled DOPA to a callus culture of this legume resulted in the incorporation of radioactivity into l-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, l-1-methyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and stizolobic acid, which was confirmed by constant specific radioactivity after co-crystallization with authentic samples of each compound.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop