A Study On Mechanisms Of Uptake,Accumulation And Tolerance Of Cadmium In Apple Rootstocks

With increasing of the living standard of people,the proportion of human dietary of fruit is growing.China is the main producer of apple in the world,and the consumption of apple in our country is occupying an important position of fruit.However,due to the rapid development of industry and agriculture,sewage irrigation,and chemical fertilizers and pesticides in a large number of application makers the problems of heavy metal pollution increasingly prominent in orchard,especially Cd.Grafting is an important way to fruit tree propagation,and rootstocks is the basement of fruit production.The concentration of Cd in scion is directly affected by the uptake and transport capacity of Cd in rootstocks.Study on the variation in Cd accumulation,translocation and tolerance of apple rootstocks,which could provide a theoretical basis for the screening of low Cd accumulation and tolerance of apple rootstocks.First,M.baccata Borkh.,M.hupehensis Rehd.,M.micromalus 'qingzhoulinqin',and M.robusta Rehd.were used to detect spatial characteristics of Cd2+ uptake along root tips.Second,study on the physiological and molecular mechanism of Cd translocation and tolerance in four apple rootstocks.Third,the mitigation effects of exogenous melatonin of Cd stress in apple rootstocks were studied.Last,the variation in Cd uptake,transport and tolerance of different in four grafted apples were discussed.The main results were as follow:1.The results of the regular spatial characteristics of Cd2+ uptake along root tips and the transport of Cd in four apple rootstocks exposed 50?M Cd showed that the maximum net Cd2+ influxes at the root surface in four apple rootstocks were found at a distance of 600?m from the tips and decreased dramatically with increasing distance from the tips.Compared with other three apple rootstocks,the lowest Cd2+ influxes into roots and leaf petiole vascular bundles were observed in M.baccata Borkh.Metabolic inhibitors and channel blockers,such as vanadate,verapamil and N-ethylmaleimide(NEM),were significantly decreased the net Cd2+ influxes into roots of four apple rootstocks.Moreover,exogenous Fe3+ concentrations were also affected the net Cd2+ influxes into roots.Except in M.baccata,the photosynthesis and biomass increment were inhibited by Cd in four apple rootstocks.Among four apple rootstocks,the Cd concentration,total Cd,BCF and Tf in M.baccata was lowest.The lower transcript levels of genes involved in Cd uptake and transport,sucn as HA7,FR02-like,NRAMP1,NRAMP3 and HMA4,whereas stronger mRNA levels of NAS1 and MT2 associated with Cd detoxification were found in M.baccata.These data indicate that M.baccata could absorb and transport less Cd of 50?M Cd than the other three apple rootstocks.2.The results.of four apple rootstocks exposed to high concentration of Cd(300?M Cd)showed that Cd exposure caused decline in biomass,photosynthesis and chlorophyll in four apple rootstocks,which was less pronounced in M.baccata,and the highest Cd tolerance indexes(TI)of the whole plant were found.In comparison with the other three apple rootstocks,M.baccata displayed the lowest Cd concentrations in roots,xylem and leaves,the smallest total Cd amount and the lowest BCF.In apple rootstocks,it was found that to immobilize Cd in cell wall and vacuole and to convert it into pectates-or protein-integrated forms and undissolved Cd phosphate forms may be the primary strategies to reduce Cd mobility and toxicity.The physiological changes including ROS,carbohydrates and antioxidants were in line with the variations of Cd tolerance among four apple rootstocks.In comparison with the other three apple rootstocks,M.baccata had lower concentrations of ROS in roots and phloem,H2O2 in roots and leaves and MDA in roots,xylem and phloem,but higher concentrations of soluble sugars in phloem and starch in roots and leaves,and enhanced antioxidants.These results indicate that M.baccata are more tolerant to Cd stress than the other three apple rootstocks under the current experiment conditions,which is probably related to Cd accumulation,subcellular partitioning and chemical forms of Cd and well-coordinated antioxidant defense mechanisms.3.The result of add exogenous melatonin affected on the Cd uptake and transport,the antioxidant system and the gene expression in M.baccata and M.micromalus 'qingzhoulinqin'exposed to either 0 or 30?M Cd showed that the net Cd2+ influxes at the root surface,the Cd concentrations in leaves,and the Tf decreased dramatically,these data indicated that melatonin could inhibit the Cd uptake and transport,especially for M.baccata.Compare with 30?M Cd,the concentrations of O2·-,H2O2 and MDA in root,stem and leaf tissues were decreased,the Cd distribution ratios in organelle-rich fraction was declined,and the transcript levels of genes involved in Cd uptake and transport,sucn as NRAMP1 was reduced,whereas the antioxidant enzyme activities,such as CAT,GPX,APX and GR,and the concentrations of free proline in roots and leaves,the soluble phenolics and T-SH in roots,the ASC in stems,the GSH in roots and stems as well as the melatonin content in roots were increased,and enhanced mRNA levels of NASI associated with Cd detoxification were found of two apple rootstocks in 30?M Cd combined with 0.1?M melatonin.These data indicated that melatonin could inhibit the uptake and transport of Cd,enhance antioxidants,decrease the concentrations of ROS,increase the tolerance of Cd in apple rootstocks.But the activities of CAT and GR in roots and leaves,the GPX and APX in roots,the concentrations of free proline in roots and leaves,soluble phenolic and T-SH in leaves,GSH in stems and leaves of M.micromalus 'qingzhoulinqin' were lower than M.baccata.Moreover,the transcript levels of genes involved in Cd uptake,such as HA7 and NRAMP1,in M.baccata were lower than in M.micromalus 'qingzhoulinqin',while mRNA levels of NASI associated with Cd detoxification was higher in M.baccata.These data indicated that melatonin can significantly enhance the tolerance to Cd in M.baccata.4.The results of Cd uptake and transport,the antioxidants and the gene expression in four grafted apples showed that Cd diminished in biomass,inhibited in photosynthesis,and decreasd in chlorophyll contents,which was less pronounced in Malus domestica/M.baccata(HF/Mb).In comparison with HF/Mm and FJ/Mm,the Cd concentration in roots and scions,and the total Cd amount and BCF in root and aerial parts were lower than HF/Mb and FJ/Mb respectively,which indicated that the M.baccata as rootstock could reduce the Cd contents in grafted apple tree.Among the four grafted apples,the Cd concentration in xylem,phloem and leaves were the lowest,Cd concentrations in water soluble fraction were the smallest,the Cd distribution ratios of cell wall fraction were highest in roots and leaves,but the Cd concentrations and distribution ratios of organelle-rich fraction were lowest in roots and leaves of HF/Mb.These data indicate that the transport ability of Cd was lower in HF/Mb of four grafted apples.Furthermore,HF/Mb had the lowest concentrations of ROS and MDA in phloem and leaves,while had the highest concentrations of soluble phenolics,ASC and GSH,the highest transcript levels of genes involved in Cd detoxification,such as MT2.These data indicate the tolerance to Cd in HF/Mb is higher than the other three grafted apples.