Improvement of Antioxidant System and Decrease of Lignin by Nickel Treatment in Tea Plant

The effect of nickel (Ni) on the activities of antioxidant enzymes was evaluated in suspension-cultured cells of tea (Camellia sinensis L. cv. ‘Yabukita’) and two-year-old rooted cuttings of tea (Camellia sinensis L. assamica × sinensis). Suspension-cultured cells were grown in B5 medium and treated with 40 μM of Ni for 24 h. Tea plants were grown in a modified Hoagland solution and treated for one week with 40 μM of Ni. The activities of superoxide dismutase, catalase, ascorbate peroxidase, and ionically wall-bound peroxidase of both Ni-treated tea cells and Ni-treated tea plants increased compared with those of control groups. The activities of soluble peroxidase and covalently wall-bound peroxidase, as well as the lignin content of both tea cells and tea plants, decreased under Ni treatment. Thus it seems that Ni may have beneficial effects on tea plants via elevation of antioxidant activity and decrease of lignification of the walls.     

Alleviation of cadmium phytotoxicity by magnesium in Japanese mustard spinach

Abstract

To clarify the mechanism of Magnesium (Mg) in alleviating cadmium (Cd) phytotoxicity, Japanese mustard spinach (Brassica rapa L. var. pervirdis) was grown for 10 days after treatment in hydroponics in a growth chamber under natural light. The treatments were: (1) nutrient solution alone (Control), (2) 10 mmol L^?1 Mg (High-Mg), (3) 2.5 µmol L^?1 Cd (Cd-toxic), (4) 2.5 µmol L^?1 Cd plus 10 mmol L^?1 Mg (Mg-alleviated). The Cd-toxic treatment showed substantial growth retardation and chlorosis of young leaves, such symptoms were not observed in Mg-alleviated plants. Magnesium-alleviated plants showed higher shoot growth, more than twofold, and decreased shoot Cd concentration, approximately 40%, compared with Cd-toxic plants. This increase in shoot growth and simultaneous decrease in shoot Cd concentration may explain the alleviation of Cd toxicity with Mg in Japanese mustard spinach. In Cd-toxic plants, concentrations of K in shoots and Zn in both shoots and roots increased compared with the other three treatments. Concentrations and accumulations of Fe and Mn in shoots decreased significantly in the Cd-treated (Cd-toxic and Mg-alleviated) plants compared with the control and High-Mg plants. Thus, the application of high amounts of Mg in the nutrient solution can alleviate Cd toxicity in plants.     

Continual pH lowering and manganese dioxide solubilization in the rhizosphere of the Mn-hyperaccumulator plant Chengiopanax sciadophylloides

Abstract

The Japanese woody plant Chengiopanax sciadophylloides is well known for its extraordinary accumulation of manganese (Mn), and is used as a model for studying Mn uptake and utilization by plants. To clarify the role of manganese dioxide (MnO₂) solubilization for Mn acquisition and further Mn hyperaccumulation in this plant, we examined the lowering of pH in the rhizosphere and Mn accumulation of this plant using regenerated plants. Plants regenerated from C. sciadophylloides calli lowered the pH of the culture broth continuously and simultaneously solubilized MnO₂ added to the medium. The Mn content of the plant increased up to 1,300 mg kg^?1 within 4 weeks of culture. Release of protein or specific organic acid from the roots was not observed. The medium used for plant culture maintained MnO₂ solubilization ability after removal of the plant; however, this ability was lost by adjustment to the same medium pH of pre-culture conditions. In addition, pH lowering and MnO₂ solubilization were suppressed by adding 1 mmol L^?1 of the plasma H⁺-ATPase inhibitor Na₃VO₄ to the medium, and completely inhibited when 5 mmol L^?1 of Na₃VO₄ was added. These results suggested that H⁺ leaking from plasma H⁺-ATPase plays an important role in MnO₂ solubilization in the rhizosphere of C. sciadophylloides and in Mn accumulation in this plant.     

Growth,mineral composition,leaf chlorophyll and water relationships of two cherry varieties under NaCl-induced salinity stress

Abstract

Growth, mineral nutrition, leaf chlorophyll and water relationships were studied in cherry plants (cv. ‘Bigarreau Burlat’[BB] and ‘Tragana Edessis’[TE]) grafted on ‘Mazzard’ rootstock and grown in modified Hoagland solutions containing 0, 25 or 50 mmol L^?1 NaCl, over a period of 55 days. Elongation of the main shoot of the plants treated with 25 or 50 mmol L^?1 NaCl was significantly reduced by approximately 29–36%, irrespective of the cultivar. However, both NaCl treatments caused a greater reduction in the dry weight of leaves and scion's stems in BB than in TE plants. Therefore, BB was more sensitive to salinity stress than TE. The reduction of leaf chlorophyll concentration was significant only when BB and TE plants were grown under 50 mmol L^?1 NaCl. Osmotic adjustment permitted the maintenance of leaf turgor in TE plants and induced an increase in leaf turgor of BB plants treated with 25 or 50 mmol L^?1 NaCl compared with 0 mmol L^?1 NaCl. Concerning the nutrient composition of various plant parts, Na concentrations in all plant parts of both cultivars were generally much lower than those of Cl. For both cultivars, leaf Cl concentrations were much higher than the concentrations in stems and roots, especially in the treatments containing NaCl. Finally, the distribution of Na within BB and TE plants treated with NaCl was relatively uniform.     

Effects of NaCl and silicon on ion distribution in the roots,shoots and leaves of two alfalfa cultivars with different salt tolerance

Abstract

The effects of exogenous NaCl and silicon on ion distribution were investigated in two alfalfa (Medicago sativa. L.) cultivars: the high salt tolerant Zhongmu No. 1 and the low salt tolerant Defor. The cultivars were grown in a hydroponic system with a control (that had neither NaCl nor Si added), a Si treatment (1 mmol L^?1 Si), a NaCl treatment (120 mmol L^?1 NaCl), and a Si and NaCl treatment (120 mmol L^?1 NaCl + 1 mmol L^?1 Si). After 15 days of the NaCl and Si treatments, four plants of the cultivars were removed and divided into root, shoot and leaf parts for Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Mn²⁺, Cu²⁺ and Zn²⁺ content measurements. Compared with the NaCl treatment, the added Si significantly decreased Na⁺ content in the roots, but notably increased K⁺ contents in the shoots and leaves of the high salt tolerant Zhongmu No.1 cultivar. Applying Si to both cultivars under NaCl stress did not significantly affect the Fe³⁺, Mg²⁺ and Zn²⁺ contents in the roots, shoots and leaves of Defor and the roots and shoots of Zhongmu No.1, but increased the Ca²⁺ content in the roots of Zhongmu No.1 and the Mn²⁺ contents in the shoots and leaves of both cultivars, while it decreased the Ca²⁺ and Cu²⁺ contents of the shoots and leaves of both cultivars under salt stress. Salt stress decreased the K⁺, Ca²⁺, Mg²⁺ and Cu²⁺ contents in plants, but significantly increased Zn²⁺ content in the roots, shoots and leaves and Mn²⁺ content in the shoots of both cultivars when Si was not applied. Thus, salt affects not only the macronutrient distribution but also the micronutrient distribution in alfalfa plants, while silicon could alter the distributions of Na⁺ and some trophic ions in the roots, shoots and leaves of plants to improve the salt tolerance.     

Silicon Improves Water Use Efficiency in Maize Plants

Abstract

The influence of silicon (Si) on water use efficiency (WUE) in maize plants (Zea mays L. cv. Nongda108) was investigated and the results showed that plants treated with 2 mmol L^?1 silicic acid (Si) had 20% higher WUE than that of plants without Si application. The WUE was increased up to 35% when the plants were exposed to water stress and this was accounted for by reductions in leaf transpiration and water flow rate in xylem vessels. To examine the effect of silicon on transpiration, changes in stomata opening were compared between Si-treated and nontreated leaves by measuring transpiration rate and leaf resistance. The results showed that the reduction in transpiration following the application of silicon was largely due to a reduction in transpiration rate through stomata, indicating that silicon influences stomata movement. In xylem sap of plants treated with 2 mmol L^?1 silicic acid, the Si concentration was 200-fold higher, while the Ca concentration which is mainly determined by the transpiration rate, was 2.5-fold lower than that of plants grown without Si. Furthermore, the water flow rate in xylem vessels of plants with and without Si was compared. Flow rate in plants with 2 mmol L^?1 Si was 20% lower than that without Si, which was accounted for by the increased affinity for water in xylem vessels induced by silica deposits. These results demonstrated the role of Si in improving WUE in maize plants.     

Calcium Effects on Acetic Acid Toxicity in Rice

Recent soil‐management practices such as no‐tillage and minimal tillage, when applied to the irrigated rice crop, promote changes in soil composition as a result of anaerobic degradation of organic matter. Several short‐chain organic acids are formed, such as acetic acid. The objective of this work was to determine the effect of calcium (Ca) on plant development under stress by acetic acid toxicity. The experiment was conducted in hydroponics by testing different Ca (0.2, 1.0, and 5.0 mmol L^?1) and acetic acid (0 and 2.5 mmol L^?1) concentrations. The variables evaluated were the root system morphological parameters (total length, radius, area, dry‐matter weight, and main root growth), shoot parameters (shoot dry matter, plant height), and concentration and total accumulated nitrogen (N), phosphorus (P), potassium (K), Ca, and magnesium (Mg) in the plants. The growth of the root system and the shoots of rice plants were not affected by the addition of Ca to the treatments containing acetic acid.     

低浓度NaCl对水培韭菜生长、产量及品质的影响

NaCl一直以来被认为是一个胁迫因子,且是造成植物盐害的主要因素,而低浓度NaCl对植物生长不仅没有危害,反而具有促进植物生长,提高品质的作用。韭菜营养液水培作为一种新型的栽培方式,很好的解决了韭蛆等病虫的为害。为了探究低浓度NaCl添加到营养液中对韭菜生长、品质和产量的影响,试验设定了0 mmol·L-1(对照)、5 mmol·L-1、10 mmol·L-1、20 mmol·L-1、30 mmol·L-1 5个NaCl浓度,研究不同浓度NaCl处理下韭菜生长、生理和产量的变化。研究结果表明,韭菜的株高、假茎粗和叶长均以20 mmol·L-1 NaCl处理下最高,各处理的叶片数均多于对照,叶片宽度随着NaCl浓度的增加呈现下降趋势。低浓度NaCl增加了韭菜的光合色素含量,10 mmol·L-1处理下叶绿素a、叶绿素b、类胡萝卜素和叶绿素a+b含量最高。NaCl处理能有效降低韭菜中硝酸盐的积累,各NaCl处理硝酸盐含量分别比对照下降12.42%、13.17%、15.16%和26.76%。Vc含量随添加NaCl浓度的增加表现出先上升后下降的趋势,其中各NaCl处理含量分别比对照高12.2%、12.8%、5.1%、-15.4%,可溶性糖与粗纤维均在10 mmol·L-1时达到最大值。产量以10 mmol·L-1处理最高,3茬产量及总产量分别比对照处理增加6.7%、6.6%、6.9%、6.7%。综合分析认为,韭菜营养液中添加10 mmol·L-1的NaCl,在促进韭菜生长,降低硝酸盐,改善品质和提高产量上综合表现最佳。     

Behavior of Metals Under Different Seasonal Conditions: Effects on the Quality of a Mexico–USA Border River

Spatial and seasonal mobilization trends of metals in surface water were evaluated in the US–Mexico San Pedro River (SPR). Water samples were collected at five sampling stations for the analysis of dissolved oxygen, pH, electric conductivity, sulfates, and metals (Cd, Cu, Fe, Mn, Pb, and Zn). Quality of the water was characterized through Ecological Criteria of Water Quality (ECWQ) established in Mexico and Water Quality Criteria (Environmental Protection Agency (EPA)). High total metal concentrations were detected as follows: Fe?>?Cu?>?Mn?>?Zn?>?Pb?>?Cd. Metal concentrations were slightly higher in dry season than in rainy season: Cd (below detection limit (BDL)–0.21 mg L^?1), Cu (BDL–13 mg L^?1), Fe (0.16–345 mg L^?1), Mn (0.12–52 mg L^?1), Pb (BDL–0.48 mg L^?1), and Zn (0.03–17.8 mg L^?1). Low pH and dissolved oxygen values as well as high sulfate content were detected in both seasons. High values of metals (Cd, Cu, Fe, Mn, Pb, Zn) were detected at station E1 representing pollution source, as well as at stations E2 (Cd, Cu, Fe, Mn), E3 (Fe, Mn, Pb), and E4 and E5 (Fe, Mn). Detected concentrations exceeded maximum permissible established in ECWQ and Water Quality Criteria (EPA). Efflorescence salts on sediments in the dry season could increase levels of metals in water column. This study provides valuable information on the potential mobility of metals in surface water of SPR located in an arid environment where transport processes are strongly linked to climate. The information derived from this study should help the regional and national authorities to address present environmental regulations.     

Excess Nickel Modulates Oxidative Stress Responsive Enzymes in Groundnut

Groundnut plants exposed to excess nickel (Ni) produced visual symptoms of toxicity that intensified with increasing level and duration of metal supply. Decreased concentrations of pigments along with a marked increase in the activities of anti-oxidative enzymes such as superoxide dismutase, ascorbate peroxidase, and peroxidase suggest strong induction of oxidative stress due to excess Ni. The decreased activity of catalase may suggest interference of excess nickel in iron metabolism of plants. The appearance of metal specific toxicity is a likely result of damage predominantly due to enhanced generation of reactive oxygen species (ROS) at higher (300–400 μM) nickel supply. The threshold of toxicity (10% growth reduction) and toxicity (33% growth reduction) values of Ni in groundnut were 26 and 72 μg g^?1 in leaves, 17 and 94 μg g^?1 in stem and 45 and 240 μg g^?1 in roots respectively.     

Beneficial effects of silicon nutrition in alleviating salinity stress in hydroponically grown canola,Brassica napus L., plants

Abstract

Silicon (Si) is the second most abundant element in soil and effectively counteracts the effects of various abiotic stresses, such as drought, heavy metal toxicity and salinity, on plants. In the present study the ameliorating effects of Si nutrition supplied as 2?mmol?L^?1 sodium silicate were investigated on hydroponically grown canola (Brassica napus L.) plants under salinity stress (i.e. 150?mmol?L^?1 sodium chloride). Salinity decreased plant growth parameters such as tissue fresh and dry weights. These decreases were accompanied by increased lignin contents, Na⁺ ion accumulation, increased lipid peroxidation and decreased chlorophyll contents in plants. Silicon nutrition, however, enhanced plant growth parameters and led to the prevention of lignin and the Na⁺ accumulation in shoots, reduced levels of lipid peroxidation in the roots and higher levels of chlorophyll. As a result of salinity, catalase activity in the whole plant and both soluble and cell wall peroxidase activities in the shoots decreased. Silicon nutrition, however, increased the reactive oxygen species scavenging capacity of salt-stressed plants through increased catalase and cell wall peroxidase activities. Thus, silicon nutrition ameliorated the deleterious effects of salinity on the growth of canola plants through lower tissue Na⁺ contents, maintaining the membrane integrity of root cells as evidenced by reduced lipid peroxidation, increased reactive oxygen species scavenging capacity and reduced lignification.     

An improved selective extraction method for Mn oxides and occluded metals with emphasis on applicability to Andisols

Abstract

It has been showed that Chao’s method [extraction with 0.1 mol L^?1 hydroxylamine hydrochloride (NH₂OH-HCl) at pH 2.0 for 30 min], which is commonly used to extract manganese (Mn) oxides and occluded heavy metals from soil samples, is not suitable for Andisols because of low solubility, and thus low extractability, of Mn oxides in such soils. Therefore, a new method is evaluated here, for extracting Mn oxides and occluded heavy metals from Andisols, Entisols and Inceptisols. The method has three steps: (1) reduction of Mn oxides with 0.01 mol L^?1 NH₂OH-HCl (pH 5.0) for 16 h, (2) recovery of re-adsorbed metals by short-time extraction with 0.5 mol L^?1 ammonium chloride in 0.02 mol L^?1 hydrochloric acid, and (3) washing with ultrapure water. This method achieves a higher rate of extraction of Mn oxides than does Chao’s method, especially from Andisol samples. Standard addition experiments showed that both the new method and Chao’s method can successfully extract released cadmium (Cd), cobalt (Co), nickel (Ni) and zinc (Zn) from Mn oxides with little re-adsorption. The selectivity of Mn oxide extraction by the new method, indicated by the rate of extraction of iron (Fe) oxides and the aluminum (Al)/Mn and silicon (Si)/Mn extraction ratios, is comparable to that of Chao’s method. Thus, the new method should be useful for extracting Mn oxides and occluded Cd, Co, Ni, and Zn from soil samples. Moreover, because the new method achieved nearly complete extraction of NH₂OH-HCl reactive Mn oxides even from Andisol samples, the method is more applicable to Andisol samples than Chao’s method.     

Effects of nitrogen and phosphorus on the microstructure and ultrastructure of tomato leaves (Solanum lycopersicum)

This study aims to evaluate the effects of nitrogen (N) and phosphorus (P) on tomato leaves at the microscale in order to propose new methods to detect N and P conditions of plants. N and P solutions composed of three N levels (N0.25, 1.75; N1, 7; and N1.5, 10.5 mmol L^?1) and three P levels (P0.25, 0.17; P1, 0.67; and P1.5, 1.01 mmol L^?1) with ten replications. Results showed a significant decrease in the leaf thickness among plants treated with N0.25, N1.5N and P0.25, whereas showed an increase among plants treated with P1.5 (p < 0.05). Stomatal density was reduced in P0.25- and N0.25-treated plants, whereas increased in N1.5- and P1.5-treated plants (p < 0.05). Moreover, N0.25-treated plants showed reduced trichome density, whereas N1.5-treated plants exhibited the highestamount of trichome density (p < 0.05). The densest veins occurred in the leaves of P1.5P-treated plants, then density reduced in plants with N1.5, N1P1, N0.25, P0.25 treatments, in a decreasing order (p < 0.05).     

Influence of the Modification of Nutritional Parameters in Aglaonema commutatum: K+, Ca2+, Mg2+ and Na+

Abstract

This trial was carried out to establish an appropriate nutrient solution for Aglaonema commutatum and to investigate the nutritional effects generated by modifications in the solution. Six treatments were tested: control (T₀; pH 6.5, E.C. 1.5 dS m^?1, 6 mmol L^?1 NO₃ ^?‐N, and 6 mmol L^?1 K⁺); high nitrogen (N) level (T₁; 9 mmol L^?1 6:3 NO₃ ^?–NH₄ ⁺); N form (T₂; 6 mmol L^?1 N‐NH₄ ⁺); high K⁺ level (T₃; 12 mmol L^?1 K⁺); high electrical conductivity (T₄; E.C. 4 dS m^?1, 25 mmol L^?1 NaCl), and basic pH (T₅; pH 8). At the end of the cultivation, leaf, shoot, and root dry weights and elemental concentrations were determined. Nutrient contents and total plant uptake were calculated from the dry weights and nutrient concentrations. Plant K⁺ uptake increased with application of K⁺ or basic nutrient solution. The uptake and transport of calcium (Ca) were enhanced by the use of NO₃ ^?‐N and inhibited by the presence of other cations in the medium (NH₄ ⁺, K⁺, Na⁺) and by basic pH. Magnesium (Mg) uptake increased with NO₃ ^?‐N application and with pH. Sodium (Na) uptake was the highest in the saline treatment (T₄), followed by the basic pH treatment. Sodium accumulation was detected in the roots (natrophobic plant), where the plant generated a physiological barrier to avoid damage. Dry weight did not differ significantly (p<0.05) among treatments except in the NaCl treatment. These results may help in the formulation of nutrient solutions that take into account the ionic composition of irrigation water and the physiological requirements of plants.     

Physiological and mineralogical properties of arsenic-induced chlorosis in rice seedlings grown hydroponically

Abstract

A hydroponic experiment was conducted to observe the effect of arsenic (As) on a number of physiological and mineralogical properties of rice (Oryza sativa L. cv. Akihikari) seedlings. Seedlings were treated with 0, 6.7, 13.4 and 26.8 µmol L^?1 As (0, 0.5, 1.0 and 2.0 mg As L^?1) for 14 days in a greenhouse. Shoot dry matter yield decreased by 23, 56 and 64%; however, the values for roots were 15, 35 and 42% for the 6.7, 13.4 and 26.8 µmol L^?1 As treatments, respectively. Shoot height decreased by 11, 35 and 43%, while that of the roots decreased by 6, 11 and 33%, respectively. These results indicated that the shoot was more sensitive to As than the root in rice. Leaf number and width of leaf blade also decreased with As toxicity. Arsenic toxicity induced chlorosis symptoms in the youngest leaves of rice seedlings by decreasing chlorophyll content. Concentrations and accumulations of K, Mg, Fe, Mn, Zn and Cu decreased significantly in shoots in the 26.8 µmol L^?1 As treatment. However, the concentration of P increased in shoots at 6.7 and 13.4 µmol L^?1 As levels, indicating a cooperative rather than antagonistic relationship. Arsenic and Fe concentration increased in roots at higher As treatments. Arsenic translocation (%) decreased in the 13.4 and 26.8 µmol L^?1 As treatments compared with the 6.7 µmol L^?1 As treatment. Arsenic and Fe were mostly concentrated in the roots of rice seedlings, assuming co-existence of these two elements. Roots contained an almost 8–16-fold higher As concentration than shoots in plants in the As treatments. Considering the concentration of Mn, Zn and Cu, it was suggested that chlorosis resulted from Fe deficiency induced by As and not heavy-metal-induced Fe deficiency.     

锌在再力花体内的富集性及亚细胞分布和化学形态研究

为研究Zn在再力花(Thalia dealbata Fraser)体内的富集性及亚细胞分布和化学形态,依次设定0(CK)、0.075 mmol.L 1、0.250 mmol.L 1、0.500 mmol.L 1、1.000 mmol.L 1、2.000 mmol.L 16个Zn处理浓度,对再力花进行水培培养的胁迫试验。结果表明:再力花不能有效地将Zn运输到地上部,Zn主要积累在根部。在系列Zn浓度处理下,转运系数均<1,得出再力花并非超富集植物。在此基础上,采用差速离心技术和化学试剂逐步提取法,研究Zn在该植物根系中的亚细胞分布和化学形态。结果表明:低浓度[0(CK),0.075mmol.L 1,0.250 mmol.L 1]Zn处理下,Zn主要分布于再力花根部的细胞壁、细胞核及叶绿体中;Zn在再力花根部的存在形态主要是乙醇提取态,占50%以上,其次是氯化钠提取态。随着Zn处理浓度的提高(0.500 mmol.L 1,1.000 mmol.L 1,2.000 mmol.L 1),细胞溶质成为Zn的最主要分布位点,分别占31.15%、45.12%和56.44%,其次为细胞壁;乙醇提取态的比例有所下降,氯化钠提取态、水提取态的比例则随着处理浓度的提高而提高至30%以上,成为Zn在再力花根部的三大存在形态。     

Trace Element Concentrations in Saltmarsh Soils Strongly Affected by Wastes from Metal Sulphide Mining Areas

Soil and water samples were analysed for trace metals and As in two watercourses and 14 sampling plots in a salt marsh polluted by mine wastes in SE Spain. Groundwater levels, soil pH and Eh were measured ‘in situ’ for a 12-month period in each sampling plot, and total calcium carbonate was also determined. Low concentrations of soluble metals (maximum Mn 1.089 mg L^?1 and maximum Zn 0.553 mg L^?1) were found in the watercourses. However, total metal contents were extremely high in the soils of a zone of the salt marsh (maximum 1,933 mg kg^?1 of Mn, 62,280 mg kg^?1 of Zn, 16,845 mg kg^?1 of Pb, 77 mg kg^?1 of Cd, 418 mg kg^?1 of Cu and 725 mg kg^?1 of As), and soluble metals in the pore water reached 38.7 mg L^?1 for Zn, 3.15 mg L^?1 for Pb, 48.0 mg L^?1 for Mn, 0.61 mg L^?1 for Cd and 0.29 mg L^?1 for As. Variable concentrations with depth indicate a possible re-mobilisation of the metals, which could be related to spatial and temporal variations of water table level, pH and Eh and to the presence of calcium carbonate. A tendency for the Eh to decrease in the warmest months and to increase in the coldest ones was found, especially, in plots that received water with a high content of dissolved organic carbon. Hence, the existence of nutrient effluent-enriched water may modify the physical–chemical conditions of the soil–water system and influence metal mobility.     

Physiology of manganese toxicity and tolerance in Vigna unguiculata (L.) Walp.

In cowpea (Vigna unguiculata (L.) Walp.) tolerance of manganese (Mn) excess depends on genotype, silicon (Si) nutrition, form of nitrogen (N) supply, and leaf age. The physiological mechanisms for improved Mn leaf-tissue tolerance are still poorly understood. On the basis of the density of brown spots per unit of leaf area and the callose content which are sensitive indicators of Mn toxicity, it was confirmed that cultivar (cv.) TVu 1987 was more Mn-tolerant than cv. TVu 91, young leaves were more Mn-tolerant, Si improved Mn tolerance, and NO₃^—-grown plants were more Mn-tolerant than NH₄⁺-grown plants. A close positive relationship existed between the bulk-leaf Mn content and the vacuolar Mn concentration from the same leaves. Since no clear and consistent differences existed between leaf tissues differing in Mn tolerance, the results suggest that accumulation of Mn in the vacuoles and its complexation by organic anions do not play a role in Mn leaf-tissue tolerance in cowpea. A near linear relationship was found between leaf Mn contents and concentrations of free (H₂O-soluble) and exchangeable-bound (BaCl₂-extractable) Mn in the apoplastic washing fluid (AWF) extracted from whole leaves by an infiltration and centrifugation technique. There were no differences in apoplastic Mn concentrations owing to genotype and form of nitrogen nutrition. However, Si decreased the Mn concentration in the AWF. With increasing bulk-leaf Mn contents, concentrations of organic anions in the AWF also increased. The results suggest that complexation of Mn by organic anions in the leaf apoplast contribute to Mn tolerance due to genotype and more clearly due to NO₃-N nutrition. Cell wall-bound peroxidase activity increased with leaf age and was higher in the Mn-sensitive cv. TVu 91 than in cv. TVu 1987. This was in agreement with a higher H₂O₂ production rate in cv. TVu 91. Also, a lower ratio of reduced to oxidized ascorbic acid in the AWF revealed that in Mn-sensitive leaf tissue, the apoplastic reduction capacity was lower than in Mn-tolerant leaf tissue when genotypes and leaves of different age were compared. We interpret our results as strong circumstantial evidence that Mn tolerance depends on the control of the free Mn²⁺concentration and of Mn²⁺-mediated oxidation/reduction reactions in the leaf apoplast.