Effects of metals on enzyme activity in plants

Abstract. Uptake of phytotoxic amounts of metal by higher plants or algae can result in inhibition of several enzymes, and in increase in activity (= induction) of others. Two mechanisms of enzyme inhibition predominate: (1) binding of the metal to sulphydryl groups, involved in the catalytic actionor structural integrity of enzymes, and (2) deficiency of an essential metal in metalloproteins or metal-protein complexes, eventually combined with substitution of the toxic metal for the deficient element. Metal accumulation in the cellular compartment of the enzyme is a prerequisite for enzyme inhibition in vivo. The induction of some enzymes is considered to play a significant role in the stress metabolism, induced by metal phytotoxicity. Peroxidase induction is likely to be related to oxidative reactions at the biomembrane; several enzymes of the intermediary metabolism might be stimulated to compensate for metal-sensitive photosynthetic reactions. The induction of enzymes and metal-specific changes in isoperoxidase pattern can be used as diagnostic criteria to evaluate the phytotoxicity of soils, contaminated by several metals. Lines for future research on metal phytotoxicity are proposed, involving the study of inhibition and induction of enzymes at the different cell membranes (especially the plasmamembrane) in vivo.     

双胸蚓纤溶酶的纯化及性质

 用硫酸铵分段盐析、超滤膜分级分离及DEAE-纤维素、Sephadex A-25和Sephadex G-50三种柱层析方法从双胸蚓组织的粗提取液中分离纯化出一种纤溶酶,分子量为29kD,由一条肽链组成。此晦具有强烈的溶解纤维蛋白的作用,对家兎实验性血凝块也具有明显的溶解作用。此酶的最适pH为8.0,在pH7.6～8.4之间活力相差不到2％;酶在PH4.7—11.0范围内稳定;酶作用的最适温度为57℃;此酶热稳定性较好,于25～50℃保温3小时,酶活力基本不变,60℃时,活力保留65％。金属离子Na~(+)、K~(+)、Mg~(2+)等可提高此酶的活力,而Hg~(2+)、Ca~(2+)等金属离子对此酶有不同程度的抑制作用。     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.     

Organogenesis andin vitro flowering ofEchinochloa colona. Effect of growth regulators and explant types

Echinochloa colona regeneration via organogenesis in callus cultures derived from leaf base and mesocotyl expiants andin vitro flowering were achived. Shoot bud regeneration was achieved on Murashige and Skoog’s (MS) basal medium supplemented with 6.66 μM 6-benzylaminopurine (BAP), 2.68 μM 1-naphthalene acetic acid (NAA) and 3 % (m/v) saccharose. Regenerated shoots were rooted on half strength basal MS medium with 2 % (m/v) saccharose devoid of growth regulators. About 90 -95 % of rooted plantlets survived in the greenhouse.In vitro flowering was induced in the regenerated shoots derived from callus on half strength MS medium supplemented with 4.4 μM BAP, 74.07 μM adeninesulphate, 0.72 μM gibberellic acid, and 3 % (m/v) saccharose. The frequency ofin vitro flowering was 80 – 90 % in three repeated experiments. Fertile seeds were recovered fromin vitro grown plantlets which were subsequently germinated into plants. Acknowledgement: The authors wish to thank to the Department of Environment and Forests, Government of India for financial assistance to undertake this investigation.     

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Growth analysis of UV-B-irradiated cucumber seedlings as influenced by photosynthetic photon flux source and cultivar

A growth analysis was made of ultraviolet-B (UV-B)-sensitive (Poinsett) and insensitive (Ashley) cultivars of Cucuumis satives L. grown in growth chambers at 600 μmol m⁻² s⁻¹ of photosynthetic photon flux (PPF) provided by red- and far-red-deficient metal halide (MH) or blue- and UV-A-deficient high pressure sodium/deluxe f HPS/DX) lamps. Plants were irradiated 6 h daiiy with 0.2 f-UV-B) or 18.2 C+UV-B) kJ m⁻² day⁻¹ of biologically effective UV-B for 8 or 15 days from time of seeding. In general, plants given supplemental UV-B for 15 days showed lower leaf area ratio (LARs, and higher specific leaf mass (SLM) mean relative growth rate (MRGR) and net assimilation rate (NAR) than that of control plants, but they showed no difference in leaf mass ratio (LMR), Plants grown under HPS/DX lamps vs MH lamps showed higher SLM and NAR. lower LAR and LMR. hut no difference in MRGR. LMR was the only growth parameter affected by cultivar: at 15 days, it was slightly greater in Poinsett than in Ashley. There were no interactive effects of UV-B. PPF source or cultivar on any of the growth parameters determined, indicating that the choice of either HPS/DX or MH lamps should not affect growth response to UV-B radiation. This was true even though leaves of UV-B-irradiated plants grown under HPS/DX lamps have been shown to have greater chlorosis than those grown under MH lamps.     

The binding of zinc,but not cadmium,by phytic acid in roots of crop plants

Plant species adapted to soils enriched with heavy metals often accumulate these metals in their above or below ground organs. In this study, electron probe microanalysis of fractured, quench-frozen root specimens of common crop species shows that an appreciable quantity of Zn can be bound as Zn phytate (myo-inositolkis-hexaphosphate) within small vacuoles of cells in the root elongation zone of lucerne, soybean, lupins, tomato, rapeseed, cabbage, radish, maize and wheat exposed to high levels of Zn (80–300 M). Globular deposits of Zn phytate are most frequently observed in the endodermis of dicotyledonous species and in the pericycle of monocotyledonous species, but may also occur in the stele and inner cortex after prolonged exposure to toxic levels of Zn. The deposits could not be found in Zn-treated sunflower, field peas and Italian ryegrass. In three crop species, lucerne, soybean and maize, Zn-induced phytate globules were frequent, but exposure of roots to 30 M Cd did not induce the formation of Cd-containing globular deposits as observed inLemna minor (Van Steveninck et al., 1990a, 1992). Simultaneous Zn and Cd treatment induced the formation of Zn phytate globules as effectively as Zn alone, and Cd was not detected in the deposits.     

Simultaneous extraction of soil phytoavailable cadmium,copper, and lead by chelating resin membrane

Cadmium, copper, and lead were extracted from suspensions of contaminated soils using metal chelating exchange resin membranes. Nine soils with widely varying properties and Cd, Cu and Pb levels were tested. Soil suspensions made up with 4 g in 40 mL deionized water were equilibrated with 5 cm² Ca-saturated Chelex exchange resin membrane which was retained inside a polypropylene bag and shaken at 150 rpm for 24 hrs. Resin membrane extractable Cd, Cu and Pb of the soils were correlated with Cd, Cu, and Pb uptake by young wheat seedlings grown in these soils and compared with soil Cd, Cu, and Pb extracted by 0.1 M HCl, 0.01 M CaCl₂, and 0.005 M Diethylenetriamine pentaacetic acid (DTPA). The amounts of Cd, Cu and Pb extracted by the Ca-saturated Chelex membrane from all tested soils correlated well with those absorbed by young wheat seedlings. The Ca-saturated Chelex membrane extractable Cd, Cu and Pb of the soil had the strongest correlation with plant uptake Cd, Cu and Pb among the extraction methods we tested. It was demonstrated that the Ca-saturated Chelex membrane extraction is an appropriate method in simultaneously estimating Cd, Cu and Pb phytoavailability of soil and is applicable to a wide range of soils.