Mn Accumulation And Resistance In Hyperaccumulator Phytolacca Americana

Pokeweed (Phytolacca americana Linn.) has drawn much attention for its hyperaccumulation of manganese (Mn) in recent years. However, the mechanisms of its Mn resistance are still not well clarified. The present study carried out series of experiments, including hydroponic culture, photosynthesis detection, scanning electronic microscopy (SEM) and transmission electronic microscopy (TEM), energy dispersive analysis of X-rays (EDX), atom absorption spectrometer (AAS), subcellular fractioning, high performance liquid chromatography (HPLC), extraction of different chemical forms, and phase analysis of X-ray diffraction (XRD), to investigate the responses of pokeweeds treated with different levels of Mn, detect Mn uptake, accumulation and subcellular distribution in pokeweed, analyze the correlation between the possible factors involved with Mn resistance and Mn accumulation and detoxification, and further explore the potential mechanisms of Mn resistance in the plant. This research could advance our understanding of Mn resistance in plant, and provide theoretical evidence and scientific directions for the correction of Mn toxicity to crops and phytoremediation of acid soils or relative heavy metal-contaminated soils. The major results obtained are as follows.Pokeweed showed great tolerance to Mn, and it grew well with no discernable Mn phytotoxicity under different Mn treatment. Detection of its photosynthesis found that pokeweed is not a Mn-philic plant, and that Mn can not enhance its metabolism and growth. Mn-containing crystals were found to appear time sequently on the roots of pokeweeds treated with different concentrations of Mn, which suggested that pokeweed could exclude Mn through rhizodeposition. Observation with electronic microscopy found that Mn had minor toxicity to pokeweed cells in which, meanwhile, two kinds of crystals were found, calcium oxalate crystals and Mn-containing crystals. EDX detection showed Mn could be complexed with hydroxyl, carboxyl or phosphate groups. Pokeweed was also found to have a high content of oxalic acid and thiols. The primary study indicated that Mn resistance in pokeweed could be closely related to factors, including oxalic acid, calcium, thiols and phosphate.The plant was found to accumulate excess Mn in the leaves, mainly in the water soluble fraction and over 80% existing in a water-soluble chemical form, while accumulating excess Mn in the fraction of cellular organelles and membrane was observed to cause phytotoxicity. In addition, pokeweed was found with intrinsically high oxalate content. In all cases, there were sufficient levels of oxalate to chelate Mn in leaf water extracts throughout different levels of Mn. Phase analysis of X-ray diffraction detected the oxalate-Mn chelate complexes in the freeze-dried soluble fraction of pokeweed leaves. The results suggest that pokeweed accumulates excess Mn in the soluble fraction of leaf cells, most likely in vacuoles, in which the detoxification of Mn could be achieved by chelation with oxalate.Ca has an important impact on divalent metal accumulation in plants, and hence, the accumulation of Ca and Mn and their interaction in pokeweed were investigated. Exogenous Ca was observed to have a distinctive impact on the Mn phytotoxicity and accumulation in pokeweed, but exogenous Mn had little influence on the accumulation of Ca. Both Ca and Mn accumulated in pokeweed were detected to be mainly in the form of oxalate. Investigation with SEM and TEM found there were two kinds of crystals in the leaves, Ca oxalate crystals and Mn-containing crystals. Further detection showed that there was no inclusion of Mn inside the Ca oxalate crystals, and that other elements, such as C, 0 and P, were present in the Mn-containing crystals. These results suggest that Ca oxalate crystals in pokeweed have no direct effect on the detoxification of Mn. In addition, the finding of element P and 0 in the Mn-containing crystals indicates that excess Mn could be deposited by phosphate, which could contribute to Mn accumulation and detoxification in pokeweed.Mn phosphate crystals were observed to appear on the root surface, and phosphate was investigated to have a distinctive effect on the Mn accumulation and toxicity in pokeweed. The finding of Mn-containing crystals in the leaf and root indicated that Mn could be precipitated by phosphate, and the crystals in the tissue of root cortex, epidermis, root hair and root surface showed that pokeweed could exclude excess Mn by rhizodiposition with phosphate. The results suggest that phosphate plays a very important role in Mn tolerance and exclusion in pokeweed.Investigation of Mn accumulation and total sulfhydryl (TSH) and their interaction in pokeweeds treated with different levels of Mn and sulphate found that treatment with different concentrations of sulphate and Mn had an impact on the content of TSH, Mn accumulation and tolerance. In particular, Mn accumulation showed a much positive correlation to the content of TSH in the root. The results suggest that sulfhydryl compounds in the root act as an important role in the Mn hyperaccumulation and resistance in pokeweed. Considering the great adaptability and multiple tolerances to heavy metals, thiols in the pokeweed root could play a vital role which protects the plant from the damages from heavy metal ions.