Subcellular and tissue Mn compartmentation in bean leaves under Mn toxicity stress

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 (r(2) = 0.96; P = 0.003) but was not correlated with Mn in unfractionated tissue (r(2) = 0.026; P = 0.793) nor with Mn in the epidermis-enriched fraction (r(2) = 0.33; P = 0.314). Both cultivars showed high accumulation of Mn in the vacuoles as determined by the co-localization of alpha-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 mu g g(-1) dry weight in crystal-type structures whereas the unfractionated leaf tissue contained about 500 mu g 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.