A comparison of Zn, Mn, Cd, and Ca transport mechanisms in oat root tonoplast vesicles

Oat root tonoplast vesicles were used to determine if tonoplast transport of the divalent cations Zn and Mn occurs via an antiport mechanism, like that described for Ca and Cd. Also, inhibitors reported to affect Ca transport were tested for their effects on Cd versus Ca transport and tonoplast ATPase activity. The ability of Ca, Cd, Zn, and Mn to alter the proton gradient was monitored using both the fluorescent probe acridine orange and C-methylamine accumulation. After the proton gradient was established in MgATP-energized vesicles, addition of Ca, Cd, and Zn to the reaction restored the fluorescence of acridine orange, indicating dissipation of the proton gradient. Fluorescence recovery was linearly correlated with metal concentration and followed the order Ca>Cd≧Zn. Addition of Mn did not restore the fluorescence of acridine orange. All four ions released C-methylamine from MgATP-energized vesicles in an ion-concentration-dependent manner, and with relative initial rates in the order of Ca>Cd>Zn>Mn. The observed ion-concentration-dependent release of protons from sealed vesicles suggests that Zn and Mn, like Ca and Cd, can be antiported into the plant vacuole. In an effort to assess whether Ca and Cd use the same carrier, we tested the effects of verapamil, Do-Tea-Br, nifedipine, ruthenium red, and LaCl₂ on Ca versus Cd transport, and also on MgATPase activity. These compounds are shown to alter Ca transport in plants. Although some of the inhibitors had a negative effect on MgMAPase activity, the decrease in this activity did not account for the decrease in Ca or Cd transport observed in any case. Particularly verapamil had a much greater effect on Ca transport than Cd transport activity while not inhibiting ATPase substantially. Data presented provide evidence for Zn and Mn antiport activity in oat root tonoplast and show differences in responses of Ca and Cd antiport activities to several transport inhibitors.     

The properties of the Mn, Ni and Pb transport operating at plasma membranes of cucumber roots

To avoid metal toxicity, plants have developed mechanisms including efflux of metal ions from cells and their sequestration into cellular compartments. In this report, we present evidence for the role of plasma membrane efflux systems in metal tolerance of cucumber roots. We have identified the plasma membrane-transport system participating in Cd, Pb, Mn and Ni efflux from the cytosol. Kinetic characterization of this proton-coupled transport system revealed that it is saturable and has a different affinity for each of the metal ions used (with Km 5, 7.5 and 0.1 m M for Mn, Ni and Pb, respectively). Treatment of cucumber roots with 100 μ M Cd prior to the transport measurements caused a great increase (over 250%) in Cd antiport activity in plasmalemma vesicles. After decreasing the metal concentration to 50 μ M we still observed a large increase (over 150%) of this activity in comparison with the control. Moreover, the addition of 50 μ M Cd to the external solution stimulated not only Cd antiport in the plasmalemma vesicles but also the antiport of other metals used in the experiments. Treatment of cucumber roots with 50 μ M Ni revealed a similar effect: the antiport activity of Cd, Mn, Ni and Pb was stimulated, although to a lesser extent in comparison with stimulation by Cd. The data indicate that the root plasma membrane antiporter system is stimulated by the exogenous presence of heavy metals.     

The kinetics of calcium and magnesium entry into mycorrhizal spruce roots

 The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Piceaabies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers ²⁵Mg and ⁴⁴Ca. After labelling, samples of roots were excised from the plants, shock-frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t_1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t_1/2 of 100–120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While ²⁵Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at +6 °C were similar to those obtained at a root temperature of +22 °C. Received: 23 December 1998 / Accepted: 17 September 1999     

Tissue magnesium and calcium affect arbuscular mycorrhiza development and fungal reproduction

 Applications of high levels of MgSO₄ resulted in reduced root colonization and sporulation by Glomus sp. (INVAM isolate FL329) with sweet potato and onion in aeroponic and sand culture, respectively. Onion shoot-Mg concentrations were elevated when a nutrient solution containing 2.6 or 11.7 mm MgSO₄ was applied. Magnesium application depressed tissue-Ca levels. With lower Ca in the tissue, colonization was reduced from > 30% of root length to < 10%, and sporulation from > 1200 to ca. 200 spores per plant, 10 weeks after transplantation and the start of nutrient application. These effects on colonization and sporulation were independent of changes in tissue-P concentration. High Mg/low Ca tissue concentrations induced premature root senescence, which may have disrupted the mycorrhizal association. Our results confirm the importance of Ca for the maintenance of a functioning mycorrhiza. Accepted: 3 October 1997     

Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter β-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula × P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation. Received: 24 April 1997 / Accepted: 17 July 1997     

Enhanced Cd<Superscript>2+</Superscript>-selective root-tonoplast-transport in tobaccos expressing Arabidopsis cation exchangers

Several Arabidopsis CAtion eXchangers (CAXs) encode tonoplast-localized transporters that appear to be major contributors to vacuolar accumulation/sequestration of cadmium (Cd(2+)), an undesirable pollutant ion that occurs in man largely as a result of dietary consumption of aerial tissues of food plants. But, ion-selectivity of individual CAX transporter types remains largely unknown. Here, we transformed Nicotiana tabacum with several CAX genes driven by the Cauliflower Mosaic Virus (CaMV) 35S promoter and monitored divalent cation transport in root-tonoplast vesicles from these plants in order to select particular CAX genes directing high Cd(2+) antiporter activity in root tonoplast. Comparison of seven different CAX genes indicated that all transported Cd(2+), Ca(2+), Zn(2+), and Mn(2+) to varying degrees, but that CAX4 and CAX2 had high Cd(2+) transport and selectivity in tonoplast vesicles. CAX4 driven by the CaMV 35S and FS3 [figwort mosaic virus (FMV)] promoters increased the magnitude and initial rate of Cd(2+)/H(+) exchange in root-tonoplast vesicles. Ion selectivity of transport in root-tonoplast vesicles isolated from FS3::CAX4-expressing plant lines having a range of gene expression was Cd(2+)>Zn(2+)>Ca(2+)>Mn(2+) and the ratios of maximal Cd(2+) (and Zn(2+)) versus maximal Ca(2+) and Mn(2+) transport were correlated with the levels of CAX4 expression. Root Cd accumulation in high CAX4 and CAX2 expressing lines was increased in seedlings grown with 0.02 muM Cd. These observations are consistent with a model in which expression of an Arabidopsis-gene-encoded, Cd(2+)-efficient antiporter in host plant roots results in greater root vacuole Cd(2+) transport activity, increased root Cd accumulation, and a shift in overall root tonoplast ion transport selectivity towards higher Cd(2+) selectivity. Results support a model in which certain CAX antiporters are somewhat more selective for particular divalent cations.     

Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.     

Differential pH restoration after ammonia-elicited vacuolar alkalisation in rice and maize root hairs as measured by fluorescence ratio

Changes of vacuolar pH in hair cells of young rice (Oryza sativa L.) and maize (Zea mays L.) roots were measured after ammonia application at various levels of external pH. After loading the pH-sensitive, fluorescent dye Oregon green 488 carboxylic acid 6-isomer into the vacuoles of root hairs, ratiometric pH data of high statistical significance were obtained from root hair populations comprising hundreds of cells. The pH of the vacuole at external pH 5.0 was 5.32 ± 0.08 (±SD, n= 15) and 5.41 ± 0.13 (±SD, n= 15) in rice and maize, respectively. A moderate external ammonia concentration of 2 mM led to vacuolar alkalisation at both, low (pH 5.0) and high (pH 7.0–9.0) external pH, presumably due to NH₃ permeation into the vacuole. With increasing external pH, ammonia application did not cumulatively increase vacuolar pH. In rice, the increase in vacuolar pH ranged from 0.1–0.8 pH units; in maize a more constant increase of 0.5 pH units was observed. The vacuolar pH increase was efficiently depressed in rice (especially at high external pH), but not in maize. Inhibition of the tonoplast H⁺-ATPase by concanamycin A raised vacuolar pH and increased the ammonia-elicited vacuolar alkalisation in both species, proving that vacuolar H⁺-ATPase activity counters the ammonia-elicited alkalisation effect. However, even under conditions of vacuolar H⁺-ATPase inhibition, rice was still able to restore an ammonia-elicited pH increase. High vacuolar pH levels as found in maize under conditions of high NH₃ influx may derive from inefficient cytosolic ammonia assimilation and tonoplast proton pumping. Thus, in maize, prolonged reduction of the proton gradient between the cytosol and the vacuole may play an important role in NH₃ toxicity. Received: 12 September 1997 / Accepted: 19 January 1998     

Effects of Wastewater Irrigation on Chemical and Physical Properties of <Emphasis Type="Italic">Petroselinum crispum</Emphasis>

The present study was carried out to assess the impact of wastewater on parsley (Petroselinum crispum). The parameters determined for soil were pH, electrical conductivity (EC), soil organic matter (SOM), nutrient elements (Ca, Mg, Na, K, Mn, Cu, Zn, and Fe), and heavy metals (Cd, Cr, Ni, and Pb), while the parameters determined for the plant included pigment content, dry matter, nutrient element, and heavy metals. SOM, EC, and clay contents were higher, and pH was slightly acidic in soil treated with wastewater compared to control soil. The enrichment factors (EF) of the nutrient elements in contaminated soil are in the sequence of Na (2) > Ca (1.32) > Mn = Mg (1.17) > Cu (1.11) > Zn (1.08) > Fe (1.07) > K (0.93), while EF in parsley are Na (6.63) > Ca (1.60) > Mg (1.34) > Zn (1.15) > Fe (0.95) > Cu = K (0.90) > Mn (0.85). Application of wastewater significantly decreased dry matter, while photosynthetic pigment content increased in parsley. The enrichment of the heavy metals is in the sequence: Cd (1.142) > Pb (1.131) > Ni (1.112) > Cr (1.095). P. crispum shows a high transfer factor (TF > 1) for Cd signifying a high mobility of Cd from soil to plant. Thus, although the wastewater irrigation in parsley production aims to produce socioeconomic benefits, study results indicated that municipal wastewater is not suitable for irrigation of parsley because it has negative effects on plant and causes heavy metal accumulation.     

Remobilisation of vacuolar stored nitrate in barley root cells

Double-barrelled nitrate-selective microelectrodes have been used to measure the time course of the remobilisation of vacuolar stored nitrate in barley (Hordeum vulgare L. cv. Klaxon) root cells during 24 h of nitrate deprivation. These measurements showed that there are different time courses for this process in epidermal and cortical cells of the same root. The remobilisation was much slower from cortical cell vacuoles and had a time course which was similar to that obtained for tissue digests of the roots. The microelectrodes were also used to measure the nitrate concentration in sap exuding from detopped seedlings. These measurements showed that there was a gradual decrease in the delivery of nitrate to the shoot during this time. Root nitrate reductase activity of neither shoots nor roots changed significantly during the first 24 h. Direct measurement of the cytosolic nitrate in a root epidermal cell showed that during short-term changes, such as a 20-min exposure to zero external nitrate supply, cytosolic nitrate was maintained relatively unchanged. Net nitrate efflux from the roots was measurable during the initial 5 h of the zero-nitrate incubation period; after this time no further nitrate efflux was detectable. These measurements are discussed in relation to the nitrate budget of a root cell and we conclude that during the first 24 h of nitrate withdrawal vacuolar nitrate can be readily mobilised to supply the nitrogen demands of the seedling and to maintain the cytosolic nitrate concentration. Received: 31 July 1997 / Accepted 11 December 1997     

Variation in Cadmium Tolerance and Accumulation and Their Relationship in Wheat Recombinant Inbred Lines at Seedling Stage

In order to identify the variation of cadmium (Cd) tolerance and accumulation in wheat (Triticum aestivum L.), a study was conducted in hydroponic culture with or without Cd using recombinant inbred lines (RILs) consisting of 103 RILs derived from a cross of Chuan 35050 × Shannong 483 at seedling stage. The parameters of shoot height, secondary roots numbers, tiller numbers, shoot dry weights, root dry weights, and maximum efficiency of photosystem II photochemistry under dark-adopted conditions were measured. Cd-tolerant indexes were then calculated as relative the above traits under Cd stress to those under the control. Cd concentration in shoot or root was determined and Cd accumulation and translocation were calculated. Based on the Cd-tolerant indexes, membership function analysis was used to determine the variation of the above parameters. The results showed a continuous distribution among the RILs and then the RILs were divided into five groups according to their tolerance. Lines 76 and 17 were considered as the most Cd-tolerant lines while lines 103 and 51 were as the most Cd-sensitive lines. Meanwhile, lines 38 and 79 were with minimum Cd translocation ratio while lines 88 and 53 were with maximum Cd translocation ratio, respectively. The relationship between Cd tolerance and accumulation was not significant, indicating Cd tolerance and accumulation may be independent traits in the RILs. Thus, lines with high Cd tolerance and less Cd accumulation could be selected for wheat breeding.     

Comparative estimation of the neurotropicity of trace elements in the organism of children living in industrial towns of Ukraine

In 10- to 16-year-old children, inhabitants of industrial towns of Ukraine, an X-ray fluorescence analysis of the content of some chemical elements in hair samples allowed us to find a deficiency of Zn and Cu against the background of excesses of Ca, Cr, Ni, Mo, and Cd. Comparison of the parameters of EEG frequency components and levels of Ca, Zn, Cu, Fe, Mn, Co, Cr, Mo, Ni, Sr, Pb, and Cd showed that there are significant correlations between the normalized values of spectral powers of many frequency components of the ongoing EEG recorded in different functional states (eyes closed/open, solving an arithmetic task, and phono-/photostimulation) and concentrations of ten of the above-mentioned trace elements. Comparative estimation of the neurotropicity of the elements showed the following sequence of numbers of significant correlations (shown in parentheses) from the total set of possible comparisons: Cd (35) > Ni (31) > Cr (19) > Sr (17) > Pb (16) > > Ca (10) > Cu (7) > Mo (3) > Zn (2) > Fe (1). The intensity of correlations (values of the correlation coefficients) varied from 0.26 to 0.42 at 0.05 < P < 0.001; more frequently, such correlations were observed under conditions of EEG recording with the eyes closed (39) and upon solving an arithmetic task (33).     

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca²⁺/H⁺ and Cd²⁺/H⁺ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd²⁺/H⁺ antiport specific activity (at 10 µM Cd²⁺), and almost complete loss of Ca²⁺/H⁺ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd²⁺ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca²⁺ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd²⁺ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd²⁺/H⁺ activity and verapamil binding to vesicles, while Ca²⁺ pre‐treatment was much less protective of Ca^2+/H⁺ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd²⁺/H⁺ and Ca²⁺/H⁺. Our observation that solubilization and reconstitution have similar effects on both Cd²⁺/H⁺ and Ca²⁺/H⁺ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.     

The tropic response of plant roots to oxygen: oxytropism in Pisum sativum L.

Plant roots are known to orient growth through the soil by gravitropism, hydrotropism, and thigmotropism. Recent observations of plant roots that developed in a microgravity environment in space suggested that plant roots may also orient their growth toward oxygen (oxytropism). Using garden pea (Pisum sativum L. cv. Weibul's Apollo) and an agravitropic mutant (cv. Ageotropum), root oxytropism was studied in the controlled environment of a microrhizotron. A series of channels in the microrhizotron allowed establishment of an oxygen gradient of 0.8 mmol · mol⁻¹ · mm⁻¹. Curvature of seedling roots was determined prior to freezing the roots for subsequent spectrophotometric determinations of alcohol dehydrogenase activity. Oxytropic curvature was observed all along the gradient in both cultivars of pea. The normal gravitropic cultivar showed a maximal curvature of 45° after 48 h, while the agravitropic mutant curved to 90°. In each cultivar, the amount of curvature declined as the oxygen concentration decreased, and was linearly related to the root elongation rate. Since oxytropic curvature occurred in roots exposed to oxygen concentrations that were not low enough to induce the hypoxically responsive protein alcohol dehydrogenase, we suspect that the oxygen sensor associated with oxytropism does not control the induction of hypoxic metabolism. Our results indicate that oxygen can play a critical role in determining root orientation as well as impacting root metabolic status. Oxytropism allows roots to avoid oxygen-deprived soil strata and may also be the basis of an auto-avoidance mechanism, decreasing the competition between roots for water and nutrients as well as oxygen. Received: 14 January 1998 / Accepted: 10 February 1998     

Effect of zinc and cadmium on physiological and production characteristics in <Emphasis Type="Italic">Matricaria recutita</Emphasis>

Effects of zinc (12–180 μM) alone and in mixtures with 12 μM Cd on metal accumulation, dry masses of roots and shoots, root respiration rate, variable to maximum fluorescence ratio (F_V/F_M), and content of photosynthetic pigments were studied in hydroponically cultivated chamomile (Matricaria recutita) plants. The content of Zn in roots and shoots increased with the increasing external Zn concentration and its accumulation in the roots was higher than that in the shoots. While at lower Zn concentrations (12 and 60 μM) the presence of 12 μM Cd decreased Zn accumulation in the roots, treatment with 120 and 180 μM Zn together with 12 μM Cd caused enhancement of Zn content in the root. Presence of Zn (12–120 μM) decreased Cd accumulation in roots. On the other hand, Cd content in the shoots of plants treated with Zn + Cd exceeded that in the plants treated only with 12 μM Cd. Only higher Zn concentrations (120 and 180 μM) and Zn + Cd mixtures negatively influenced dry mass, chlorophyll (Chl) and carotenoid content, F_V/F_M and root respiration rate. Chl b was reduced to a higher extent than Chl a.     

Cadmium accumulation in Medicago sativa seedlings treated with salicylic acid

Growth parameters and cadmium accumulation were investigated in alfalfa seedlings treated with 10 μM salicylic acid (SA) at the beginning of seed imbibition. Shoot and root growths were accelerated by SA treatment and suppressed by Cd both in presence and absence of SA. Cd accumulation was stimulated by SA in alfalfa seedlings in dependence of the treatment duration. K, Mg, Ca and Fe contents in roots are decreased in the presence of Cd alone, while SA induces a decrease of Mg, Ca and Fe. Shoot K, Mg and Ca concentrations are increased by Cd only in the absence of SA, while SA induces also an increase of these concentrations, but only in the absence of Cd. High negative correlation of Cd concentration with K and Ca concentrations in root indicates a competition for the same carrier not regulated by SA. Positive correlation between Cd and Mg concentrations in shoots, which is decreased by SA pre-treatment, together with the increase of positive correlation between Cd and Fe concentrations in shoots under the influence of SA, indicates a possible mechanism of SA action through maintenance of ionic homeostasis.     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

Contributions of apoplasmic cadmium accumulation,antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium-accumulating cultivar of black oat (<Emphasis Type="Italic">Avena strigosa</Emphasis> Schreb.)

The contributions of cadmium (Cd) accumulation in cell walls, antioxidative enzymes and induction of phytochelatins (PCs) to Cd tolerance were investigated in two distinctive genotypes of black oat (Avena strigosa Schreb.). One cultivar of black oat ‘New oat’ accumulated Cd in the leaves at the highest concentration compared to another black oat cultivar ‘Soil saver’ and other major graminaceous crops. The shoot:root Cd ratio also demonstrated that ‘New oat’ was the high Cd-accumulating cultivar, whereas ‘Soil saver’ was the low Cd-accumulating cultivar. Varied levels of Cd exposure demonstrated the strong Cd tolerance of ‘New oat’. By contrast, low Cd-accumulating cultivar ‘Soil saver’ suffered Cd toxicity such as growth defects and increased lipid peroxidation, even though it accumulated less Cd in shoots than ‘New oat’. Higher activities of ascorbate peroxidase (EC 1.11.1.11) and superoxide dismutase (EC 1. 15. 1. 1) were observed in the leaves of ‘New oat’ than in ‘Soil saver’. No advantage of ‘New oat’ in PCs induction was observed in comparison to Cd-sensitive cultivar ‘Soil saver’, although Cd exposure increased the concentration of total PCs in both cultivars. Higher and increased Cd accumulation in cell wall fraction was observed in shoots of ‘New oat’. On the other hand, in ‘Soil saver’, apoplasmic Cd accumulation showed saturation under higher Cd exposure. Overall, the present results suggest that cell wall Cd accumulation and antioxidative activities function in the tolerance against Cd stress possibly in combination with vacuolar Cd compartmentation.     

Enhanced cadmium accumulation in maize roots—the impact of organic acids

Low molecular weight organic acids are important components of root exudates and therefore, knowledge regarding the mechanisms of cadmium (Cd) uptake and distribution within plants under the influence of organic acids, is necessary for a better understanding of Cd behavior in the plant–soil system. In this study, acetic and malic acids increased the uptake of Cd by maize (Zea mays L. cv. TY2) roots and enhanced Cd accumulation in shoots under hydroponic conditions. Concentration-dependent net Cd influx in the presence and absence of organic acids could be resolved into linear and saturable components. The saturable component followed Michaelis–Menten kinetics, which indicated that Cd uptake across the plasma membrane was transporter-mediated. While the K _m values were similar, the V _max values in the presence of acetic and malic acids were respectively 6.0 and 3.0 times that of the control. Zinc transporters were the most probable pathways for Cd accumulation. It was hypothesized that Cd(II)–organic acid complexes associated with the root zone, could decompose and liberate Cd²⁺ for subsequent absorption by maize roots; and that in the layer of the roots or within the root free space, depletion of Cd²⁺ was buffered by the presence of Cd(II)–organic acid complexes. Plant response to elevated Cd levels involved overproduction of organic acids in maize roots as a resistance mechanism to alleviate Cd toxicity.