| Molybdenum(Mo)is an essential microelement for plant growth and development.Soybean(Glycine max)is a main crop which demands high Mo,and its yield and quality would decrease seriously under Mo deficient condition.Currently,an effective approach to conquer Mo deficiency is to screen high Mo uptake and utilization efficiency cultivars,which could impove Mo nutrient,yield and quality of soybean.In this research,using Mo-efficient and Mo-infficient cultivars as materials,we explored the physiological and molecular mechanisms of high Mo uptake and utilization efficiency by transcriptomics,metabolomics and gene clone technologies basing on the characteristics of Mo-efficient and Mo-infficient cultivars uptake,transport and distribution,and the main results were summarized as following:1.Propounded the evaluation indexes of high Mo uptake efficiency,obtained Mo-efficient and Mo-inefficient soybean cultivars by screening.In this experiment,26 soybean cultivars were used as experimental materials for primary screen in the field containing low Mo.First,we set the Mo concentration of seed and shoot,shoot Mo accumulaition and shoot/root Mo translocation coefficient as screening standards by principal component analysis.Then,we obtained two Mo-efficient and three Mo-inefficient cultivars by using these screening standards.Soil culture and hydroponics experiments were conducted for re-screening and verification to obtain Mo-efficient and Mo-inefficient soybean cultivars.Finally,we obtained Mo-efficient cultivar(1301)and Mo-inefficient cultivar(1304),respectively.2.Identify of the differences of Mo uptak,translocation and accumulation between Mo-efficient and Mo-inefficient soybean cultivars.In this study,we explored the differences of Mo uptake,translocation,accumulation and distribution between 1301 and 1304 cultivars at different growth periods.The results indicated that the Mo concentration and accumulation of shoot in 1301 cultivar were higher than 1304 cultivar under Mo deficient and sufficient conditions,which exhibited significant differences at filling stage.The filling stage was the crucial period of high Mo uptake efficiency.The higher Mo transfer coefficient of shoot/root and Mo accumulation percentage of seed in 1301 cultivar could explain its high Mo uptake and utilization efficiency under Mo deficient condition.3.RNA-seq based comparative transcriptome profiling analysis between Mo-efficient and Mo-inefficient soybean cultivars in response to Mo deficiency.In this study,we performed the transcriptome profiling analysis to explore the differences between 1301 and 1304 cultivars under 6 h and 6 d Mo deficient condition.There were 559 and 172 differential genes in 1301 and 1304 cultivars under Mo deficient condition for 6 h individually,there were 675 and 745 differential genes responded to Mo deficiency for 6 d in 1301 and 1304 cultivars respectively.There were 6 and 276 common DEGs between two cultivars in response to 6 h and 6d Mo deficiency,respectively.The change times of up-regulated DEGs were higher in 1301 cultivar than in 1304 cultivar,such as molybdate transport related genes(molybdate transporter-2,inorganic phosphate transporter 1-7,PHO1,sulfate transporter 1.3),nitrate transporter genes(high affinity nitrate transporter 2.4),flavonoid and isoflavone biosynthesis related genes(DFR,7IMOT),GSH metabolism related genes(spe/SRM,ANPEP/CD13)and signaling transduction genes(GH3,PYR,GAR1,MYC2,TGA).The higher expression of these genes and related pathways might enhance the ability of molybdate transport,nitrogen assimilation and stress tolerance in Mo-efficient soybean cultivar 1301.4.Differences of molybdate transporter candidate genes expression between two soybean cultivars.First,we cloned the molybdate transporter homologous genes(Gm MOT1.1,Gm MOT1.2 and Gm MOT2)of soybean,and determined their molybdate transport function by yeast heterologous expression experiment.The results indicated that Gm MOT2 exhibited higher Mo transport ability than Gm MOT1.1 and Gm MOT1.2.In addition,the Mo transport regulated by Gm MOT2 might be an energy-dependent process,and nitrate might activate Gm MOT2 expression indirectly.Furthermore,we found that the expression of Gm MOT1.1,Gm MOT1.2 and Gm MOT2 were all inhibited by adding sulfate and tungstate,but inhibited more significantly by tungstate.Furthermore,transient expression of Gm MOT2 assays shown that Gm MOT2 localized in the plasma and vacuole membranes.The expression of Gm MOT2 was induced under Mo deficient condition.Under Mo deficient condition,Gm MOT2 expression of pod,seed and root in 1301 cultivar were 1.47,2.21 and 2.26 times than in 1304.Thus,we speculated that the higher expression of Gm MOT2 in 1301 might be one character of Mo-efficient soybean cultivar.5.Metabonomics analysis of Mo-efficient soybean cultivar in response to high Mo level.In this research,we comprehensively analyzed the metabolic profile changes of leaves and roots in soybean seedlings treated with 100 mg/L Mo for 48 h.There were 42 differential metabolites identified in roots,38 were up-regulated and 4 were down-regulated;nineteen differentially produced metabolites were identified in leaves,15 were up-regulated and 4 were down-regulated.In roots,the levels of 2-oxoarginine,L-nicotine,gluconic acid,D-glucarate and citric acid elevated significantly,these organic compounds might chelate with Mo to enhance the Mo accumulation ability in Mo-efficient soybean cultivar.In addition,signal molecules(salicylic acid,jasmonic acid and some lipids)and oxidation-resistant patwhays(As A-GSH metabolism and flavonoid and isoflavones biosynthesis)might strengthen the excess Mo tolerance ability of Mo-efficient soybean cultivars.Taken together,we suggested that the sequestration of Mo and organic compounds and other oxidation-resistant pathways might act synergistically to enhance the ability of high Mo uptake and utilization efficiency in Mo-efficient soybean cultivar 1301. |
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