Uptake, Ultrastructure Localization And Chemical Form Distribution Of Lead In Radish (Raphanus Sativus L.)

Radish (Raphanus sativus L.) which belongs to the Brassicaceae family is an important biennial root vegetable crop with long cultivated history in our country. Furthermore, the cultivation area has increased year by year, China has become the biggest radish producer in the world. Lead (Pb) is considered to be highly toxic and wide-spreading heavy metal elements. It has no biological function but it is toxic to human beings and other creatures when they exposure to lead for a long time. Whist lead has been considered a possible carcinogen by the international agency for research on cancer (IARC). In recent years, lead pollution has attracted widespread attention, for lead can harm human health via food chain. Many studies had confirmed that various plant species can absorb Pb and accumulate in edible parts. However, species and organs differed significantly in Pb absorption, translocation and distribution. Although knowledge of Pb uptake by plants has developed rapidly, there is little information about how Pb is translocated and distributed within radish plants after its absorption, as well as Pb chemical forms of distribution and ultrastructure localization. In this study, we studied the genotypic differences of Pb absorption among 36 radish genotypes under lead stress, and the variations of Pb concentrations among different parts of radish under different Pb levels; subcellular distribution and localization of Pb among different parts of radish under different Pb regimes; chemical form distribution of Pb among different tissues of radish. The main results were summarized as follow:A hydroponic culture with Pb(NO3)2 supplied as Pb stress was carried out to study the genotypic differences of 36 radish cultivars. The results showed that significant (p <0.05) differences in root Pb concentration were observed among the 36 tested genotypes under Pb treatments 500 mg/L, ranging from 2108.67 to 15850 mg/kg, with the mean of 7364.27 mg/kg DW. The concentration for the highest Pb accumulation genotype was about 6.5 times higher than that for the lowest Pb accumulation genotype. Some high accumulation genotypes such as ’NAU-SG’, ’NAU-CH’ and low accumulation genotypes such as’NAU-JLCX’,’NAU-JYCX,’NAU-TDQT and ’NAU-YH’ were screened out.A hydroponic experiment was carried out to study the characteristic of Pb accumulation and transport in intact radish. The results exhibited that lateral roots enriched a large amount of Pb when exposured to Pb in the solution for the first time, but the intake by lateral roots was less. However the rate of the enrichment decreased, the Pb intake by lateral roots increased with the increasing of treatment duration. The skins of taproot accumulated a relative larger portion of Pb (1.92-10.80% and 0.15-1.74%) than root flesh (0.44-1.56% and 0.11-0.19%) in ’NAU-XHT’ and ’NAU-YH’. Moreover, the skins with lateral roots developing parts accumulated more amount lead (1.44-7.88%) than the smooth skins without lateral roots growing (0.48-2.92%). The root heads of radish also accumulated a lot of Pb in ’NAU-XHT’ and ’NAU-YH’. Most of Pb was retained by underground part (83.37-99.19%), and little could be transported to aboveground part, which was demonstrated by TFs (<0.17). Generally, the concentration in leaves was higher than that in petioles under Pb treatment 200 and 500mg/L, but the Pb concentration in petioles increased faster than that in leaves with the increasing of treatment duration and concentration, and finally the Pb concentration in petioles got higher than in leaves in ’NAU-XLM’ and ’NAU-RG’ under Pb treatment 1000mg/L. The phenomenon indicated that the lateral roots of radish firstly enriched a large amount of Pb, and then a portion of the Pb was absorbed and a little was transported upward (0.81-16.63%). Radish skins accumulated plenty of Pb, but the root flesh without skin just absorbed little Pb.The method of chemical reagent extraction step by step was carried out to determine the chemical forms of lead in radish. The result showed that in different radish tissues of ’NAU-XLM1, the majority of Pb was integrated with the undissolved Pb oxalate (extracted by 0.6M HC1), protein and pectates (extracted by 1M NaCl) and Pb-phosphate complexes (extracted by 2% HAC), representing 88.11-99.59%. In lateral roots and skin of ’NAU-XHT’, the majority of Pb was also associated with Pb oxalate, protein and pectates and Pb-phosphate complexes, representing 90.97-97.12%. In leaf and root flesh of ’NAU-XHT’, the majority of Pb was integrated with Pb oxalate, protein and pectates and water soluble Pb-organic acid complexes, representing85.84-94.29%. Moreover ’NAU-XHT’, a relatively low accumulation genotype, was found to have higher Pb concentration in water-soluble forms, and lower Pb concentration of undissolved Pb phosphate than ’NAU-XLM’ which was a relatively high accumulation genotype in various tissues under all the three Pb treatments.Differential centrifugation and transmission electron microscopy (TEM) were performed to study the subcellular distribution and localization of lead in radish. The results indicated that in lateral roots, most of Pb (87.62-93.76% and 93.72-95.29% in ’NAU-XLM’ and ’NAU-XHT’, respectively) was present in the cell wall fraction and nucleus and chloroplasts fraction, while a minor portion of Pb was found in other parts. In skins, a large proportion of Pb (54.44-77.94% and 46.22-77.73% in ’NAU-XLM’ and ’NAU-XHT’, respectively) was stored in the cell wall fraction, but a relative high proportion of Pb also accumulated in soluble components containing ribosomes under the Pb treatment 200mg/L. In roots, most of Pb was localized in cell wall fraction and soluble components containing ribosomes, representing 74.55-78.31% and 68.28-77.10%, respectively, and the proportion of cell wall fraction increased, but the proportion of soluble components containing ribosomes decreased with increasing Pb treatment strength. The similar phenomenon was also found in leaves, the proportions of cell wall fraction and soluble components containing ribosomes in ’NAU-XLM’ and ’NAU-XHT’ were 67.51-74.61% and 69.05-80.46%, respectively. Moreover ultrastructural observation also proved plentiful Pb deposit on the cell wall in the roots and lateral roots. Ultrastructural observation of roots and lateral root found Pb deposited mainly on intercellular space, and other sites also found Pb particles, such as cell wall, plasmalemma, vacuole, lumen and epidermis.