| Molybdenum(Mo) is an essential trace element for plant growth and development.Oilseed rape(Brassica napus L.)is mostly cultivated in Mo deficient soils.The effects of molybdenum deficiencies have been documented in oilseed rape growth,seed yield,and quality,and further on human health.Therefore,the most economic and effective way to increase Mo in oilseed rape is by obtaining a highly Mo efficient genotype cultivar.Based on the genotype diversity of oilseed rape,high and low Mo accumulation cultivars were obtained by using a two-step-screen method,and analysing the differences in their mechanisms through the entire growth period.First we compared the physiological basis of Mo utilization efficiency between two cultivars first and in further studies examined the responses of molybdenum uptake,transport and subcellualar distribution under different NO3-:NH4+ ratios,or competing ion.In addition,the identification of molybdate transporters from oilseed rape,gene expression patterns,and heterologous expressions in Saccharomyces cerevisiae were also investigated.Furthermore,the changes in matabolic profiling of xylem sap were analyzed under Mo deficiency and supply using metabolomics method in hydroponic experiment.The main results are as follows:1.Screening for high or low Mo accumulation cultivars.The preliminary screening experiment was investigated using 30 varieties grown under field conditions in an acid yellow-brown earth with no Mo or added Mo(0.75 kg/hm)treatments.The high,middle,and low Mo efficient clusters contained 4,20,and 6 cultivars,respectively.The re-screening and expanded experiments were executed using 4 high cluster varieties,4 low cluster varieties,and 10 other varieties.The highest and lowest Mo accumulation cultivars were ZS11 and L0917,respectively.Overall,we defined ZS11 as a high Mo accumulation cultivar and L0917 as a low Mo accumulation cultivar.2.The Mo efficiency and physiological differences between ZS11 and L0917.We investigated the growth differences during all growth periods of ZS11 and L0917 by using a pot culture experiment.The results showed that the biomass and Mo accumulation per plant of both cultivars were higher under the +Mo treatment at the seedling stage.The transpiration rate,soluble sugars,free amino acids,soluble protein,and nitrate reductase were all higher under the +Mo treatment at the stem elongation period.The seed yield,content of unsaturated fatty acid(UFA)and saturated fatty acid(SFA),and UFA/SFA were higher under the +Mo treatment at maturity.Additionally,the biomass and Mo accumulation in plants of the cultivar ZS11 were higher than that of cultivar L0917 at the seedling stage.The pigment contents(Chlorophyll a,Chlorophyll b,Carotenoid),photosynthetic parameters(transpiration rate,intercellular CO2 concentration),soluble sugars,free amino acids,and soluble protein of ZS11 were higher than that of L0917 at the stem elongation stage.The seed yield,Mo efficiency ratio,and UFA/SFA of ZS11 were higher than that of L0917 at maturity.3.The expression differences of molybdate transporters in ZS11 and L0917.At MOT1 and At MOT2 homologous genes were cloned in oilseed rape using a homologous gene sequence method.Molybdate transport was determined from molybdate uptake in S.cerevisiae.Results showed that S.cerevisiae with empty vector,Bn MOT1,and Bn MOT2 take up molybdate after NO3--N induction.The uptake rates were 6.21% and 25.43% higher in Bn MOT1 and Bn MOT2 compared with empty vector,respectively.The expression profiles of Bn MOT1 and Bn MOT2 were investigated at the seedling,elongation,flowering,and maturity stage using RT-PCR.Expressions of Bn MOT1 and Bn MOT2 were significantly up-regulated under Mo deficiency.In ZS11,up-regulation expression of Bn MOT1 in root and Bn MOT2 in shoot at seedling stage were found under-Mo treatment.Moreover,gene expression levels of each of Bn MOT2 of ZS11 were much higher than that of Bn MOT1 at elongation,flowering,and mature stage.Therefore,we speculated that the higher experssion of Bn MOT2 was one of the reasons why the ZS11 accumulated more Mo than L0917.4.Responses of molybdenum uptake,transport and distribution to combined Mo and W treatments in cultivars ZS11 and L0917.We assessed the effects of tungsten(W)on growth,Mo accumulation,transport,and subcellular distribution in cultivars ZS11 and L0917 using a hydroponics experiment at high or/and low equal concentrations of Mo and W.The maximum biomass and root parameters in both cultivars occurred with the Mo1 treatment,and decreased with the W addition.The ultrastructure of roots and leaves changed with the low W concentration,however,resuslts were more serious with high W concentration.Plant Mo accumulation was inhibited by W addition.It also found that the Mo1+W1 treatment increased xylem and phloem Mo concentrations,while the xylem Mo concentrations were decreased by the high W concentration compared with the Mo200 treatment.The phloem Mo concentration of cultivar L0917 increased with high W concentration compared with the Mo200 treatment,but decreased for cultivar ZS11.For cultivar ZS11,the Mo percentage in cell walls increased with low W concentration,and the Mo percentage in the soluble fraction of root increased with high W concentration,indicating that the main forms of stored Mo are the soluble fraction in roots,and cell walls in leaves.In conclusion,W is not beneficial to oilseed rape growth and Mo uptake.There were different protection mechanisms between cultivars ZS11 and L0917 under combined Mo and W exposure.5.Molybdenum uptake,transport and distribution in response to different NO3-:NH4+ ratios in two oilseed rape cultivars ZS11 and L0917.We investigated the effects of different nitrate(NO3-)/ammonium(NH4+)ratios on the Mo uptake,transportation,and distribution between L0917 and ZS11 in hydroponic culture.The biomass and Mo accumulation of both cultivars treated with 7.5:7.5 of NO3-:NH4+ were the highest,but decreased with the 14:1 and 1:14 treatments.However,the leaf/stalk transport coefficient with 1:14 treated plants was high,suggesting that low NO3-:NH4+ ratios promote Mo transportation from stalks to leaves.The xylem sap Mo concentration and phloem sap Mo accumulation of cultivar L0917 treated with the 1:14 ratio of NO3-:NH4+ decreased,while the xylem sap Mo concentration and phloem sap Mo accumulation of cultivar ZS11 treated with the same 1:14 ratio were higher.Furthermore,the percentage of Mo in cell walls and soluble fraction decreased significantly for cultivar L0917 with the 1:14 treatment,but not for cultivar ZS11.This indicates that the processes of Mo translocation and re-translocation by xylem and phloem were the determining factors for Mo balance in leaf subcellular fractions of cultivar ZS11.6.The metabolites profiling and pathway in xylem sap were analyzed of oilseed rape before and after Mo supplied.There were 45 and 39 metabolites showing significantly difference in ZS11 and L0917 after Mo supplied,respectively.There were 29 metabolites detected in both cultivars.The detected metabolites were mainly amino acids,alkaloid,organic acid,hormone,fatty acid and aromatic.The significantly different metabolites indicated that Mo affects the pathways in plant,such as amino acid biosynthesis and metabolism,alkaloid biosynthesis,organic acid metabolism,hormone metabolism,fatty acid and aromatic metabolism,especially for amino acid,indoleacetic acid and salicylic acid.The increased levels of colnelenic acid,9(S)-HOTr E,matrine,oleamide,N-acetyltyramine,MG(18:0/0:0/0:0),12-ketodeoxycholic acid,methyl jasmonate,citric acid,dehydroascorbic acid,3-furoic acid and L-arginine were observed in xylem sap after Mo supplied.Therefore,we speculated that part of the compound involved in the transportation of Mo in xylem.We also found that the fatty acid metabolism was the key different metabolic pathway between high Mo accumulation cultivar and low Mo accumulation cultivar. |
