Wendell A. Norvell

Copper solubility and speciation of in situ contaminated soils : Effects of copper level, pH and organic matter

This study attempts to identify the soil properties controlling the fractionation of copper into various soil pools and determine the influence of pH and metal loading on soil-solution free copper activity (pCu2+). The pCu2+ was determined in 0.01 M CaCl2 soil extracts using a copper ion selective electrode. We analyzed a wide variety of soils: urban, agricultural and forest soils from the Province of Québec, New York State and Denmark. The pCu2+ ranged from 12.21 to 6.18. The relationships among pCu2+, total soil copper, total dissolved copper and soil pH are studied for their variability within and between sites as well as for the whole data set. Regression equations are presented for predicting soluble copper as a function of total soil copper and also for predicting pCu2+ as a function of total soil copper and soil pH.

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe2+ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na2bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO4, Na3citrate, and Na2-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.

Growth and Nutrient Uptake by Barley (Hordeum vulgare L. cv Herta): Studies Using an N-(2-Hydroxyethyl)ethylenedinitrilotriacetic Acid-Buffered Nutrient Solution Technique (I. Zinc Ion Requirements)

The critical range of Zn2+ activity in nutrient solution required for optimum growth of barley (Hordeum vulgare L. cv Herta) was studied using the synthetic chelating agent N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid to buffer micronutrient metal ions. The activity of Zn2+ was varied over a wide range from approximately 0.1 x 10–)11 to 22 x 10–)11 M Zn2+. The dry weight of barley shoots reached a maximum at Zn2+ activities above approximately 3 x 10–)11 M and was clearly depressed when Zn2+ activities were below about 1 x 10–)11 M. The relationship in shoots between dry weight and Zn concentrations supports the view that there is a critical Zn concentration of about 25 [mu]g g-1 dry weight in whole shoots of barley seedlings. When Zn2+ activities in solution were near or below approximately 3 x 10–)11 M, barley shoots accumulated higher concentrations of P, Mn, Ca, Mg, and Na, whereas Cu concentrations were reduced. P and Mn began to accumulate in the shoots before differences in dry weights were apparent and provided the earliest index of Zn deficiency. In Zn-deficient roots, concentrations of Ca and Mg increased by 25 to 30%, and those of Fe and Mn more than doubled. Zn appears to play a special role in regulating uptake of several mineral nutrients in barley.

Growth and Nutrient Uptake by Barley (Hordeum vulgare L. cv Herta): Studies Using an N-(2-Hydroxyethyl)ethylenedinitrilotriacetic Acid-Buffered Nutrient Solution Technique (II. Role of Zinc in the Uptake and Root Leakage of Mineral Nutrients)

Barley seedlings (Hordeum vulgare L. cv Herta) were grown in N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid-buffered nutrient solutions with or without adequate Zn supplies. Fifteen-d-old Zn-deficient seedlings contained higher concentrations of Mn, Ca, Mg, and P in their shoots and more Fe, Mn, Cu, K, Ca, and P in their roots than did similar Zn-adequate seedlings, confirming results reported in our companion study (W.A. Norvell and R.M. Welch [1993] Plant Physiol 101: 619–)625). Zn-deficient roots leaked greater quantities of K, Mn, Cu, and Cl than did roots supplied adequately with Zn; they also leaked significant amounts of Zn even though the seedlings were not supplied Zn during growth. Calculated uptake rates of P, Mn, and Na were sharply reduced, but uptake rates of K and Mg were stimulated by increasing the Zn2+ activity in nutrient solutions. Intact roots of Zn-deficient seedlings contained lower concentrations of 5,5[prime] -dithio-bis(2-nitrobenzoic acid) reactive sulfhydryl groups in comparison to Zn-adequate roots. Apparently, Zn is required for the uptake and retention of several mineral nutrients by roots, possibly by playing a protective role in preventing the oxidation of sulfhydryl groups to disulfides in root-cell plasma membrane proteins involved in ion channel-gating phenomena.

Potential for phytoextraction of137 Cs from a contaminated soil

Potential for phytoremediation of a soil contaminated with radiocesium was investigated in three phases: (1) hydroponic screening for plant species capable of accumulating elevated levels of cesium in shoots, (2) investigation of several amendments for their potential to increase the bioavailability of 137Cs in the contaminated soil, and (3) bioaccumulation of radiocesium in shoots of plants grown in137 Cs-contaminated soil.The bioaccumulation ratio for Cs in shoots of hydroponically grown plants ranged between 38 and 165. From solution, dicot species accumulated 2- to 4-fold more cesium in shoots than grasses. In studies investigating the bioavailability of 137Cs in aged contaminated soil, ammonium salts were found to be the most effective desorbing agents, releasing approximately 25% of the137 Cs. The extent of 137Cs desorption from the soil increased with ammonium concentration up to 0.2 M. In a pot study conducted in a greenhouse, there was significant species-dependent variability in the ability to accumulate 137Cs in the shoot from contaminated soil. The ability to accumulate 137Cs from the soil increased in the order: reed canarygrass (Phalaris arundinacea) < Indian mustard (Brassica juncea) < tepary bean (Phaseolus acutifolius)< cabbage (B. oleracea var. capitata). It was also found that addition of NH4NO3 solution to the soil elicited a two- to twelve-fold increase in 137Cs accumulation in the shoot. The greatest amount of 137Cs (40 Bq g-1 dw) was removed in shoots of cabbage grown in contaminated soil amended with 80 mmols NH4NO3 kg-1 soil. Bioaccumulation ratios of 2–3 were obtained with the best performing plant species. These values are significantly greater than those previously reported in the literature (usually <0.1) for plants grown on aged contaminated soil. These results indicate that careful species selection along with amendments that increase the bioavailability of137 Cs in the soil could greatly enhance the prospects for the use of plants to remediate 137Cs-contaminated soils.

Advances in Solution Culture Methods for Plant Mineral Nutrition Research

Publisher Summary This chapter reviews some of the advances in solution culture methodology for plant mineral nutrition research. It focuses primarily on methodological advancements for the specific study of mineral nutrition, but recognizes that the methods may be useful for other types of physiological research as well. The methods are described for overcoming one of the most fundamental limitations of traditional solution culture, the absence of chemical “buffering” of nutrient concentrations. In soils, the soil solution is depleted of nutrients by plant uptake, but continuously replenished via chemical and biological reactions of the solid phase. The uses of flowing solution cultures are discussed in detail. The chapter provides an overview of methods wherein nutrients are continuously or intermittently added in proportion to plant growth. For convenience, abbreviations and chemical names for the chelating agents and pH buffers discussed throughout this paper are summarized.

Effects of nutrient solution zinc activity on net uptake, translocation, and root export of cadmium and zinc by separated sections of intact durum wheat (Triticum turgidum L. var durum) seedling roots

Cd accumulation in durum wheat presents a potential health risk to consumers. In an effort to understand the physiological mechanisms involved with Cd accumulation, this study examined the effects of Zn on Cd root uptake and phloem translocation in a split– root system. Durum wheat seedlings were grown in chelate-buffered nutrient solution with intact root systems divided into two sections. Each root section grew in a separate 1 l pot, one of which contained 0.2 μM CdSO4. In addition, each two-pot system contained ZnSO4 in the following combinations (in μm) (for -cd root system: +cd root system): 1:1, 1:10, 10:1,10:10, 1:19, and 19:1. Harvested plant material was analyzed for Cd and Zn. In addition, rates of Cd and Zn net uptake, translocation to the shoot, and root export (translocation from one root segment to the other) between days 8 and 22 were calculated. Results show that Zn was not translocated from one root section to its connected root section. Uptake rates of Cd increased as solution Zn concentrations increased. Cd translocation from one root section to the other decreased significantly when the Zn concentration in either pot was greater than 1 μM. These results show the potential of Zn to inhibit movement of Cd via the phloem, and suggests that providing adequate Zn levels may limit phloem loading of Cd into wheat grain. Increasing the rhizosphere activity of Zn2+ in Cd-containing soils may therefore result in reduced Cd accumulation in grain even while net Cd uptake is slightly enhanced.

Direct Measurement of 59Fe-Labeled Fe2+ Influx in Roots of Pea Using a Chelator Buffer System to Control Free Fe2+ in Solution

Fe2+ transport in plants has been difficult to quantify because of the inability to control Fe2+ activity in aerated solutions and non-specific binding of Fe to cell walls. In this study, a Fe(II)-3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4[prime]4″-disulfonic acid buffer system was used to control free Fe2+ in uptake solutions. Additionally, desorption methodologies were developed to adequately remove nonspecifically bound Fe from the root apoplasm. This enabled us to quantify unidirectional Fe2+ influx via radiotracer (59Fe) uptake in roots of pea (Pisum sativum cv Sparkle) and its single gene mutant brz, an Fe hyperaccumulator. Fe influx into roots was dramatically inhibited by low temperature, indicating that the measured Fe accumulation in these roots was due to true influx across the plasma membrane rather than nonspecific binding to the root apoplasm. Both Fe2+ influx and Fe translocation to the shoots were stimulated by Fe deficiency in Sparkle. Additionally, brz, a mutant that constitutively exhibits high ferric reductase activity, exhibited higher Fe2+ influx rates than +Fe-grown Sparkle. These results suggest that either Fe deficiency triggers the induction of the Fe2+ transporter or that the enhanced ferric reductase activity somehow stimulates the activity of the existing Fe2+ transport protein.

Induction of the Root Cell Plasma Membrane Ferric Reductase (An Exclusive Role for Fe and Cu).

Induction of ferric reductase activity in dicots and nongrass monocots is a well-recognized response to Fe deficiency. Recent evidence has shown that Cu deficiency also induces plasma membrane Fe reduction. In this study we investigated whether other nutrient deficiencies could also induce ferric reductase activity in roots of pea (Pisum sativum L. cv Sparkle) seedlings. Of the nutrient deficiencies tested (K, Mg, Ca, Mn, Zn, Fe, and Cu), only Cu and Fe deficiencies elicited a response. Cu deficiency induced an activity intermediate between Fe-deficient and control plant activities. To ascertain whether the same reductase is induced by Fe and Cu deficiency, concentration- and pH-dependent kinetics of root ferric reduction were compared in plants grown under control, -Fe, and -Cu conditions. Additionally, rhizosphere acidification, another process induced by Fe deficiency, was quantified in pea seedlings grown under the three regimes. Control, Fe-deficient, and Cu-deficient plants exhibited no major differences in pH optima or Km for the kinetics of ferric reduction. However, the Vmax for ferric reduction was dramatically influenced by plant nutrient status, increasing 16- to 38-fold under Fe deficiency and 1.5- to 4-fold under Cu deficiency, compared with that of control plants. These results are consistent with a model in which varying amounts of the same enzyme are deployed on the plasma membrane in response to plant Fe or Cu status. Rhizosphere acidification rates in the Cu-deficient plants were similarly intermediate between those of the control and Fe-deficient plants. These results suggest that Cu deficiency induces the same responses induced by Fe deficiency in peas.

Measurement of thiol-containing amino acids and phytochelatin (PC2) via capillary electrophoresis with laser-induced fluorescence detection.

An analytical method for determining thiols and phytochelatins using high‐performance capillary electrophoresis coupled with laser‐induced fluorescence detection is presented. The technique utilizes the labeling of thiols with the fluorescent reagent 5‐bromomethylfluorescein (5‐BMF), which is excited by a 488 nm argon ion laser and fluoresces at 515 nm. The paper describes the determination of the optimal conditions for reaction of 5‐BMF with thiols as well as the parameters for electrophoresis runs that produce optimal electropherogram peaks. The technique is shown to be very sensitive for cysteine, cysteinyl‐glycine, γ‐glutamyl‐cysteine, glutathione and (γ‐glutamyl‐cysteinyl)2‐glycine (PC2). Concentrations as low as 25 nmol/L and amounts as low as 1 fmol were detected for glutathione. Sensitivity for detection of PC2 was somewhat lower. The method was shown to be simple, rapid and accurate and should facilitate measurement of thiol‐containing amino acids, peptides and phytochelatin (PC2) in small volumes of extracts obtained from biological tissue.