| Phytolacca acinosa Roxb. (P. acinosa) was found as Mn hyperaccumulator plant in south China. The P. acinosa is easy to cultivate due to its perennial herbaceous characteristic, and which made it a useful material for studying. Nutrient solution cultivation and absorption inhibitor experiments were applied in this study for exploring the mechanism of Mn absorption and hyperaccumulation for P. acinosa. The distribution patterns and combination forms of Mn at organ, tissue and cellular levels were investigated by using chemical analysis, gel filtration chromatography, liquid chromatogram (HLPC), synchrotron radiation EXAFS, and ect, results showed that the compartmentation distribution of Mn and the combination of Mn with organic substances were the major mechanism for P. acinosa. to hyperaccumulate Mn and resistant to Mn toxicity. Physiological and biochemical analysis were also employed in this study for exploring physiological response of P. acinosa to Mn. The results throughout the paper were summarized as follows:P. acinosa is of high ability for accumulating Mn and the Mn is mainly accumulated in the leaves of P acinosa. With the hydraulic cultivation of 2000 μM Mn, the Mn content in leaves is as high as 10,036 mg/kg dry weight without toxicity symptom. The P. acinosa is still of high accumulating ability even under the treatment of relatively low Mn concentrations. Both uncoupling agents 2, 4 - dinitrophenol (DNP) and P-type ATP enzyme inhibitor is of inhibitions for absorptions of Mn in P. acinosa, which suggested that the absorption of Mn in P. acinosa may be an active absorption process. The absorption of Mn in P. acinosa is also related to Ca~2+ channeldue to the inhibitions of Mn absorption fromThe main organ for P. acinosa to hyperaccumulate Mn is the leaf. Leaves with different ages have different Mn content. The Mn content in mature leaves is higher than that in old leaves and young leaves. Within a leaf, the Mn content in marginal areas was the highest, followed by central areas and midrib, such distribution pattern was mainly controlled by transpiration rate. After stopping the supplies of external Mn, both the Mn content and Mn concentration in the newly developed leaves were extremely low, whereas the Mn content in old leaves increased continuously. The increased Mn in old leaves was translocated from the remaining Mn in the roots.As for tissue levels, Mn in vascular bundles of the cross section of root was the highest, followed by epidermis, and cortex. Regarding stems, Mn in vascular bundles was also the highest. Within the cross section of leaves of P. acinosa, the Mn content in epidermis is higher than that in mesophyll, besides Mn content in upper epidermis is also higher than in lower epidermis. Concerning cell levels, the Mn content in cell walls and soluble parts of cytoplasm of P. acinosa roots covered 19-59% and 33-66% of the total Mn, respectively. Soluble Mn in stems was 59-81% of total Mn. Within leaves, most Mn (74-82%) was stored in vacuoles, the Mn content in cell walls of leaves covered 15-20%, whereas the Mn distribution ratio fororganelle isthe least, only 2.7-3.4% of total Mn in leaves. Higher Mn and soluble Mn concentrations in vascular bundles of stems would make for translocating of Mn to above-ground part and accumulating in leaves. The accumulation of Mn in the non-active metabolism parts (vacuole and cell wall) of P. acinosa leaves is one of the main mechanisms for hyperaccumulating Mn.The results from extraction experiments by using different extractants showed that the Mn in the roots of P. acinosa was transforming from water extractable Mn to diluted acid extractable Mn with the increase of Mn concentrations in the treatment, which indicates that the Mn content combined with cell walls of roots increased as the treatment Mn concentrations increased. As for stems, the water extractable Mn content is 55-86%, and the proportions of water extractable Mn increased with the increasing of Mn concentrations in the treatment, which suggested that the Mn combined with cell walls of roots restrict the up translocation of Mn. Once the Mn arrived at above-ground part of stems, the Mn mobility will become strong due to the increasing of water extractable Mn, which would make the translocation of Mn to leaves easy.The water extractable Mn covered 83-91% of total Mn in leaves of P. acinosa. Further evidence from the gel filtration chromatography experiments suggested that the probability of Mn in P. acinosa leaves combined with proteins was very small. Amino acid analysis showed that amino acid is not the main combination form for Mn. The components obtained from total organic acid analysis of leaves indicated that Mn probably be combined with oxalic acid, and the separation results of Sephades G-10 provided the further evidences simultaneously. The ultraviolet-visible spectrum and synchrotron radiation EXAFS scanning proved that the dominant form of Mn in leaves of P. acinosa was Mn2+, and combined mainly with oxalic acids.The adsorption experiment of cell wall showed that pH is an important factor affecting the absorbability of cell wall. The optimal pH for Mn adsorption by cell wall is from 5 to 6. In the case of pH is 5, the adsorption patterns of Mn in cell walls of roots, stems and leaves were in accord with the Longmuir and Freundlich equations very well. Infrared spectral analysis suggested that -OH played a very important role during the combination process of Mn and cell walls. Results from XAFS analysis indicated that both bond length and coordination number of Mn-0 are of great differences, which suggested that the Mn form is of certain differences.There are not significant differences of plasma membrane permeability and MDA (the peroxidating substance of cell membrane) content under different Mn treatments indicating that the cell membrane is still not be injured. The content of soluble protein changed with different Mn treatment suggested that the adjust of metabolism and structure of the plant. The activity of SOD and POD increased as the increase of Mn concentration in the treatments, the improvement of protection enzyme activities would help to eliminate the active oxygen free radicals, which may be one of the response mechanisms for P. acinosa to resist high Mn concentrations.
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