Effects Of Nitrate And Ammonium Supply Ratios On Growth,physiology And Gene Expression Of Oilseed Rape(Brassica Napus)

Oilseed rape（Brassica napus）is one of the four main oil crops worldwide,which requires a large amount of nitrogen（N）fertilizer.Rational application of N fertilizer can significantly promote the growth and improve the grain yield of oilseed rape.Meanwhile,different oilseed rape cultivars exhibit genotypic differences in N efficiency.Nitrate（NO₃^-）and ammonium（NH₄⁺）are available inorganic N sources for oilseed rape.Even so,little is known about the effects of NO₃^-and NH₄⁺ on oilseed rape and whether oilseed rapes differing in N efficiency have similar responses to NO₃^-and NH₄⁺.Based on Xiangyou 15（high N efficiency,denoted “15”）and 814（low N efficiency,denoted “814”）as test materials,we adopted sand culture and field test to study the effects of different NO₃^-and NH₄⁺ supply ratios on the biomass,root architecture,photosynthesis,grain yield and quality,uptake and accumulation of large nutrient elements,key enzymes activities for N metabolism in leaves and roots,and differentially expressed genes（DEGs）in roots of the two cultivars,in order to elucidate the physiological and molecular mechanism on NO₃^-and NH₄⁺ utilized by oilseed rape,and provide theoretical basis for rational application of N fertilizer to improve oilseed rape cultivation and promote oilseed yield.1.The effects of NO₃^-/NH₄⁺ ratio on the growth and yield of oilseed rape were studied.Under the conditions of equal amount of nitrogen（N 15 mmol/L）,compared with single application of NO₃^-treatment,75%NO₃^-+25%NH₄⁺ treatment promoted oilseed rape growth,enhanced photosynthesis and increased yield,while when the proportion of NH₄⁺ exceeded 50%,its growth was significantly inhibited.The combined application of NH₄⁺ and NO₃^-as N fertilizer significantly increased the chlorophyll content and photosynthesis in the leaves of oilseed rape,while the single application of NO₃^-or NH₄⁺ was not conducive to the increase of chlorophyll content and photosynthesis at the seedling period.Compared with single application of NO₃^-treatment,75%NO₃^-+25%NH₄⁺ treatment promoted root growth and increased root length,root surface area and root volume,while excessive NH₄⁺ inhibited root growth.2.The effects of NO₃^-/NH₄⁺ ratio on the uptake and accumulation of nitrogen（N）,phosphorus（P）,potassium（K）and activities of key enzymes of N metabolism in oilseed rape were studied.Compared with the treatments with more than 50% of NH₄⁺ in nutrient solution,appling only NO₃^-or NO₃^-/NH₄⁺ ratio was 75/25,it was beneficial to increase the activity of nitrate reductase（NR）and glutamine synthase（GS）in leaves at the seedling period,promote the uptake and accumulation of N,and significantlly improve the the N uptake efficiency,however 100%NO₃^-treatment was not beneficial to improve the GS activity in root system.Compared with 100%NO₃^-treatment,the 100%NH₄⁺ significantly increased the P content in the shoot and root system of oilseed rape at the flowering and harvest period,and promoted the transcriptional expression of the BnPHT1.4 encoding P transporter in the oilseed rape root system.Compared with 100% NO₃^-treatment,100% NH₄⁺ inhibited the uptake and accumulation of K in oilseed rape.3.Comparisons of gene expression in the roots of 15 and 814 between treatments（nitrogen-free（control,CK）,NO₃^-（NT）and NH₄⁺（AT）treatments）were conducted after tag mapping to the sequenced B.napus genome.The effects of different N treatments on DEGs in the root of oilseed rape were studied.A total of 14355 DEGs were identified with the following criteria: false discovery rate（FDR）<0.05 and an log₂（fold change）≥1.Two comparisons between the CK and NT treatments（15 and 814）revealed 4582 and 4203 upregulated and 1701 and 1506 downregulated DEGs in 15 and 814,while 501 and 271 upregulated and 867 and 746 downregulated DEGs were found between the CK and AT treatments（15 and 814）.Furthermore,compared with the NT treatment,the AT treatment resulted in many more downregulated DEGs,with 5016 and 4944 in 15 and 814 in the AT treatment,respectively.Among the enriched gene ontology（GO）terms and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway categories of these DEGs,carbohydrate metabolism,lipid metabolism,protein metabolism and cell wall biogenesis were inhibited in the AT treatment.Interestingly,DEGs,such as N transporters,genes involved in N assimilation and CESA genes related to cellulose synthase,were mostly also downregulated in the AT treatment.Based on these data,the biomass,N accumulation and root architecture of oilseed rape were inhibited under excessive NH₄⁺ as the sole N source due to the downregulation of genes related to N metabolism,cell wall biogenesis,and carbohydrate,lipid and protein metabolism.4.The difference of growth,physiology and gene expression between 15 and 814 treated with the same N treatment were studied.Except for 100% NH₄⁺ treatment,the chlorophyll content,photosynthetic,root length,root surface area,root volume,NR activity and grain yield of the 15 leaves were significantly higher than those of 814,while GS activity in 814 roots was significantly higher than that in 15 under the same N treatment.There was no significant difference in N accumulation between 15 and 814 at the harvest period.Transcriptome analysis showed that a total of 1877 DEGs were identified in 15vs814（NT）,in which 734 DEGs were upregulated and 1143 DEGs were downregulated.DEGs related to cell wall were significantly enriched in GO terms.DEGs related to six amino acid metabolic pathways were also significantly enriched in KEGG pathways.Deep analysis of GO terms related to cell wall manifested that there were 7 significantly enriched GO terms（q-value<0.05）,and the significantly enriched DEGs related to the 7 significantly enriched GO terms all showed that the number of downregulated DEGs was significantly higher than upregulated.Therefore,most of the downregulated DEGs related to cell wall synthesis resulted in the phenomena that the root length,root surface area and root volume of 814 were lower than those of 15 under 100%NO₃^-treatment.5.The effects of topdressing with different N fertilizers on the physiology and yield of oilseed rape were studied under field experiment.The seed yield of direct-sowing oilseed rape was the highest under 100%NH₄⁺ treatment（C treatment）,with the yield per unit area reaching 4120.8 kg/hm².C treatment was beneficial to increase the primary branch number and effective pod number per plant.The abovground biomass under C treatment was significantly higher than that of 100% NO₃^-treatment（A treatment）at flowering and harvest periods.The chlorophyll content and photosynthesis indexes of leaves under A treatment were significantly lower than those under B（50%NO₃^-+50%NH₄⁺）and C treatments at flowering and harvest periods,and the N,P and K accumulation in the shoot of oilseed rape under C treatment was significantly higher than that under A treatment.The NR activity of leaves under A treatment was higher than that under B and C treatmnets at the seedling period,while the NR activity of leaves under C treatment was higher than that under B and A treatments at the flowering periods.In upland soil,NH₄⁺ is easily converted to NO₃^-under the action of nitrifying bacteria,and NO₃^-is easily leached from rainfall or irrigation,which leads to the phenomena that the actual uptake of NO₃^-and yield of oilseed rape under C treatment are higher than that under A treatment.