Gene Expression Profiles And Co-Expression Network Of Brassica Napus In Response To Nitrogen Starvation

Nitrogen（N）is essential for plant growth and development.N deficiency causes special symptoms such as yellow leaves,thin stems,and dwarf plant,and leads to a reduction of the yield.In China,crop yield relies on a large amount of nitrogenous fertilizer input due to a broad range of N deficiency in the soil.However,the elevated input of N fertilizer doesn’t enhance N use efficiency（NUE）.There is a strong relationship between the N loss of farmland and the damage to the environment,for instance,the emission of greenhouse gas to the atmosphere and water contamination.One effective strategy of NUE is to investigate the uptake and utilization mechanism and to breed crops with high NUE.Oilseed rape（Brassica napus,AACC,2n=38）,a hybrid heterotetraploid derived from B.oleracea（CC,2n=18）and B.rapa（AA,2n=20）,is a major oil crop in China.The oilseed rape seeds have considerable oil content and protein proportion,which can be a remarkable source of edible oil and livestock feed.Nonetheless,oilseed rape requires extensive input of N fertilizer per year and has relatively lower NUE.Depending on the germplasm resource screening of new-type B.napus of high NUE,this research concentrated on the transcriptome responses of oilseed rape to N starvation and their differences between the N-efficient genotype D4-15 and the N-inefficient genotype D2-1 by a timepoint gene expression profiling analysis.The main results were as follows:1.Transcriptional profile of B.napus in response to N starvationThe gene expression profile of short-term（6 h,24 h）and long-term（3 d,7 d）N starvation of the N-efficient genotype D4-15 and the N-inefficient genotype D2-1 was obtained by RNA-Seq.A total of 24435 differentially expressed genes（DEGs）between N deficient and sufficient conditions were detected,among which 19097 genes were differentially expressed in the shoot and 11106 in the root（|log₂（fold change）|>1 and p.adj<0.05）.17786 DEGs were detected between the N-efficient genotype D4-15 and the N-inefficient genotype D2-1,including 13298 DEGs in the shoot and 9137 DEGs in the root.In addition,the number of genes response to N starvation and expressed differentially between genotypes was 8811 in which 6271 in the shoot and 2521 in the root.DEGs between N deficient and sufficient conditions were enriched in the biological process（BP）ontology of cellular N compound biosynthetic process,peptide biosynthetic process,and amide biosynthetic process.DEGs between the N-efficient genotype and the N-inefficient genotype were enriched in the biological process ontology of the oxidation-reduction process,alpha-amino acid metabolic process,organic acid metabolic process,and ion transport.KEGG enrichment analysis identified the significantly enriched metabolic pathways,such as photosynthesis,starch and sucrose metabolism,alanine,aspartate and glutamate metabolism,and fatty acid degradation among DEGs between N starvation and supply,and glucosinolate biosynthesis,glutathione metabolism,tryptophan metabolism,and phenylpropanoid among DEGs between genotypes.2.Weighted gene co-expression network analysis of N-starvation-responsive genesA weighted gene co-expression network was constructed based on the expression profile of genes differentially expressed both in treatments and genotypes in D4-15.12modules were identified in the root,in which 6 modules have a contrasting gene expression profile between D4-15 and D2-1.Functional annotation of module genes indicated a relationship with ribosome biosynthesis,starch and sucrose metabolism,tricarboxylic acid cycle,pyruvate metabolism,photosynthesis,tyrosine metabolism,fatty acid degradation,and organic N compound metabolic process.13 modules were detected in the shoot,among them 5 modules expressed differentially between D4-15 and D2-1.Genes of those modules were enriched in N metabolism,glutamate biosynthesis,pyruvate metabolism,fatty acid metabolism,and peroxidase activity.The intramodular connectivity of each gene was calculated and used for ranking members in each module,with the top 10 members selected as hub genes.Most hub genes were related to phytohormone signals,such as WAT1,PGP4,IAA26 isoform X1 involved in auxin transport,GASA1 that response to gibberellin,abscisic acid and brassinosteroid,DAG2 and LTI65 induced by gibberellin and abscisic acid respectively,indicated an important role of plant hormone in N starvation stress responses.3.The expression profiles of genes related to short-term and long-term N starvationN uptake and assimilation,photosynthesis and phytohormone signal transduction were found to respond to the short-term N starvation（6 h,24 h）.The expression of some nitrate transporters and ammonium transporters were repressed by short-term N starvation.In N assimilation,nitrate reductase（NR）,nitrite reductase（NiR）and NADH-glutamate synthase（GOGAT）were repressed.Short-term N starvation also has an impact on genes involved in light harvesting and energy transfer of photosystemⅠ,photosystemⅡ,and organic compound biosynthesis in the Calvin-Benson cycle.Besides,the expression of genes related to phytohormone biosynthesis and signal transduction were influenced by short-term N starvation.Carbohydrate metabolism,amino acid metabolism,and fatty acid biosynthesis were responded to long-term N starvation（3 d,7 d）.The expression of genes in starch and sucrose biosynthesis were induced by long-term N starvation.PEPCK was repressed in the shoot but PPDK was induced.Genes involved in the accumulation of glutamate were induced in long-term N starvation,while the expression of most genes related to fatty acid biosynthesis was inhibited by long-term N starvation.There was a significant difference in gene expression profiles between the N-efficient genotype D4-15 and the N-inefficient genotype D2-1.In short-term N starvation,some nitrate transporters,ammonium transporters,and nitrate reductases were induced only in D4-15,which might contribute to the high uptake and utilization efficiency of D4-15.Several genes in photosystemⅠwere also induced only in D4-15,which might lead to a high photosynthetic rate of D4-15.Genes involved in ABA,IAA,CTK and ethylene synthesis and signal transduction also have a distinct expression profile between D4-15 and D2-1.In long-term N starvation,several genes related to starch-synthesis,degradation,and sucrose synthesis were induced only in the D4-15 shoot.Those genes might contribute to the high dry weight of D4-15 during N starvation.Meanwhile,genes involved in glutamate accumulation and fatty acid biosynthesis were also induced in D4-15 but not in D2-1,indicating a better N starvation stress resistance of D4-15 than D2-1.