Total nitrogen uptake by maize with Azotobacter inoculation

Summary The economic feasibility of using cultures of nitrogen fixing microorganisms in programmes to increase crop production, as a selfgenerating source of nitrogen, has been proved beyond doubtviz. Legume-Rhizobium symbiosis and blue green algal ‘fertilizer’ for rice. The extent to which the free living, N-fixing, aerobic, heterotropicAzotobacter chroococcum could replace the application of nitrogenous fertilizer to maize was investigatedin vivo. Total nitrogen uptake (kg ha⁻¹) by maize after inoculation with Azotobacter combined with moderate applications of nitrogen fertilizer and farmyard manure was influenced significantly and resulted in a higher nitrogen concentration in grain and stover along with a higher yield. Part of Ph.D. Thesis     

Assignment of the three disulfide bonds in ShK toxin: A potent potassium channel inhibitor from the sea anemone Stichodactyla helianthus

Summary ShK toxin, a 35-residue peptide isolated from the Caribbean sea anemone Stichodactyla helianthus, is a potent inhibitor of the Kv 1.3 potassium channel in lymphocytes. The natural toxin contains three disulfide bonds. The disulfide pairings of the synthetic ShK toxin were elucidated as a prerequisite for studies on its structure-function relationships. The toxin was fragmented at pH 6.5 using either thermolysin or a mixture of trypsin and chymotrypsin followed by thermolysin. The fragments were isolated by RP-HPLC and were identified by sequence analysis and MALDI-TOF mass spectrometry. The three disulfides were unambiguously identified in either proteolytic digest: Cys³ to Cys³⁵, Cys¹² to Cys²⁸ and Cys¹⁷ to Cys³². The Cys³-Cys³⁵ disulfide, linking the amino- and carboxyl-termini, defines the characteristic cyclic structure of the molecule. A similar disulfide pairing motif is found in the snake venom-derived potassium channel blocker dendrotoxin and the mammalian antibiotic peptide defensins.     

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

Energization of potassium uptake in Arabidopsis thaliana

Plant roots accumulate K⁺ from micromolar external concentrations. However, the absence of a firm determination of the trans-plasma-membrane electrochemical gradient for K⁺ in these conditions has precluded an assessment of whether K⁺-accumulation requires energization in addition to the driving force provided by the inside-negative membrane electrical potential (E_m). To address this question unequivocally, we measured E_m, and the cytosolic and external K⁺-activities in root cells of Arabidopsis thaliana (L.) Heynh. cv. Columbia in conditions in which net K⁺-accumulation occurs at low external K⁺ (10 M). In these conditions, net K⁺-uptake was about 0.1 mol · (g FW)^-1 · h^-1, E_m varied between-153 and -129 mV and the cytosolic K⁺-activity, determined with K⁺-selective electrodes, was 83 ± 4 mM. These values yield an outwardly-directed driving force on K⁺ of at least 6.5 kJ · mol^-1. Only if external potassium is raised to the region of 1 mM does E_m become sufficient to drive net K⁺-accumulation. It is therefore concluded that at micromolar external K⁺-activities which prevail in most soils, K⁺-uptake cannot be solely energized by E_m — as exemplified by a channel-mediated mechanism. The nature of the energization mechanism is discussed in relation to processes operating in fungal and algal cells.Abbreviations and Symbols AAS atomic absorption spectrometry - E_m membrane potential - electrochemical potassium gradient - F Faraday constant (96500 C · mol^-1) We thank Peter Barraclough, Roger Leigh, David Walker and Tony Miller (Rothamsted Experimental Station, Harpenden, UK) for helpful discussions. Financial support was provided by the Agricultural and Food Research Council (Grant PG87/529).     

The Antifungal Imidazole Clotrimazole and its Major In Vivo Metabolite are Potent Blockers of the Calcium-Activated Potassium Channel in Murine Erythroleukemia Cells

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca²⁺)-activated ⁸⁶Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of ¹²⁵I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell K_Ca2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca²⁺ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca²⁺. The extent of inhibition of whole cell MEL K_Ca2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single K_Ca2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca²⁺ currents in PC12 cells ≫ Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single K_Ca2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells. Received: 10 September 1996/Revised: 12 December 1996     

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K⁺ channels was investigated, in connection with the known functional diversity of K⁺ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K⁺ currents could be approximated by the algebraic sum of the 2 K⁺ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K⁺ channels.     

几种抑制剂对钾和IAA诱导的离体黄瓜子叶不定根形成的影响

应用离体黄瓜(Cucumis sativus L.)子叶生根试法测定了几种抑制剂对钾和IAA诱导生根的影响。实验结果表明:7×10~(-4)～7×10~(-1)mmol/L的5-氟尿嘧啶和3.5×10~(-4)～1.05×10~(-2)mmoL/L的环已亚胺明显抑制钾和IAA诱导的离体黄瓜子叶的生根;0.1～1.0mmol/L的钒酸钠显著抑制钾和IAA诱导的离体黄瓜子叶的生根;2×10~(-4)～2×10~(-1)mmol/L的2,3,5-三碘苯甲酸也明显抑制钾和IAA诱导的离体黄瓜子叶的生根。钾诱导的生根与IAA诱导的生根密切相关,有可能钾对生根的促进作用是通过影响内源IAA的水平而实现的。     

Annual nutrient budget for an alpine grassland in the Garhwal Himalaya

Abstract. N, P and K dynamics were investigated in grazed and ungrazed alpine forb and grassy meadows in the Garhwal Himalaya. The growth forms examined were dwarf shrubs, forbs and graminoides. N, P and K contents were determined for various plant components and soil. The contribution of plant parts to the total vegetation capital of N, P and K was 20–33% (live shoot), 6–8% (dead shoot), 2–3% (litter) and 56–71% (root) in ungrazed plots, and 16–27, 6–7, 1–2, and 64–76% respectively in grazed plots. Grazing removed between 41–69% of total uptake of nutrients from the grassland. In protected areas, however, 65 to 81% of all nutrients were retained by the vegetation. This retention of nutrients is due to translocation to roots and rhizomes and is considered beneficial during grazing as it aids resprouting of the vegetation.