Non-aqueous fractionation was used to characterize subcellular and tissue Mn compartmentation of mature and immature leaves of two common bean (Phaseolus vulgaris L.) cultivars contrasting in their response to Mn toxicity. Excess Mn decreases leaf CO2 assimilation through a reduction of chlorophyll content in immature leaves with no effect detected on mature leaves. We hypothesized that differential accumulation of Mn in chloroplasts occurs at different leaf developmental stages. Chloroplasts of immature leaves accumulated at least three times as much Mn as those of mature leaves at equivalent total foliar Mn. Chlorosis was positively correlated with Mn concentration in chloroplasts from high-Mn plants (r2 = 0.96; P = 0.003) but was not correlated with Mn in unfractionated tissue (r2 = 0.026; P = 0.793) nor with Mn in the epidermis-enriched fraction (r2 = 0.33; P = 0.314). Both cultivars showed high accumulation of Mn in the vacuoles as determined by the co-localization of α-mannosidase and Mn content on a continuous density gradient. Cultivars differed significantly in Mn concentration in an epidermis-enriched fraction, with the tolerant cultivar Calima accumulating more Mn in this fraction than the sensitive cultivar ZPV-292. In both cultivars, Mn was accumulated up to 2400 µg g–1 dry weight in crystal-type structures whereas the unfractionated leaf tissue contained about 500 µg g–1 dry weight. The results demonstrate that Mn compartmentation occurs at both the tissue and the organelle level and that Mn accumulation in the epidermis-enriched fraction could contribute to Mn tolerance in common bean. The role of Mn accumulation in structures resembling oxalate crystals is discussed.

The effect of light intensity on antioxidants, antioxidant enzymes, and chlorophyll content was studied in common bean (Phaseolus vulgaris L.) exposed to excess Mn. Leaves of bean genotypes contrasting in Mn tolerance were exposed to two different light intensities and to excess Mn; light was controlled by shading a leaflet with filter paper. After 5 d of Mn treatment ascorbate was depleted by 45% in leaves of the Mn-sensitive genotype ZPV-292 and by 20% in the Mn-tolerant genotype CALIMA. Nonprotein sulfhydryl groups and glutathione reductase were not affected by Mn or light treatment. Ten days of Mn-toxicity stress increased leaf ascorbate peroxidase activity of cv ZPV-292 by 78% in low light and by 235% in high light, and superoxide dismutase activity followed a similar trend. Increases of ascorbate peroxidase and superoxide dismutase activity observed in cv CALIMA were lower than those observed in the susceptible cv ZPV-292. The cv CALIMA had less ascorbate oxidation under excess Mn-toxicity stress. Depletion of ascorbate occurred before the onset of chlorosis in Mn-stressed plants, especially in cv ZPV-292. Lipid peroxidation was not detected in floating leaf discs of mature leaves exposed to excess Mn. Our results suggest that Mn toxicity may be mediated by oxidative stress, and that the tolerant genotype may maintain higher ascorbate levels under stress than the sensitive genotype.

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.

Parameters related to leaf photosynthesis were evaluated in three genotypes of common bean (Phaseolus vulgaris L.) with contrasting tolerance to Mn toxicity. Two short‐term studies in solution culture were used to assess the effect of excess Mn on CO2 assimilation in mature and immature leaves. Mn toxicity decreased total chlorophyll content only in immature leaves, with a consequent reduction of leaf CO2 assimilation. Mature leaves that showed brown speckles characteristic of Mn toxicity, did not suffer any detriment in their capacity to assimilate CO2, at least in a 4‐day experiment. Stomatal conductance and transpiration were not affected by the presence of high levels of Mn in leaf tissue. Lower stomatal conductance and transpiration rates were observed only in leaves with advanced chlorosis. Differences among genotypes were detected as increased chlorosis in the more sensitive genotype ZPV‐292, followed by A‐283 and less chlorosis in the tolerant genotype CALIMA. Since CO2 assimilation expressed per unit of chlorophyll was not different between high‐Mn plants and control plants, we conclude that the negative effect of Mn toxicity on CO2 assimilation can be explained by a reduction in leaf chlorophyll content.

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