Molecular And Physiological Mechanisms Of Citrus Sinensis Root Response To Nitrogen Deficiency Revealed By Transcriptome And Metabolome

Nitrogen(N)is a macroelement that is necessary for plants to synthesize amino acids(AAs),proteins,nucleic acids,and other Ncontaining compounds to maintain their life cycle.It plays an important role in various metabolic processes in plants.There are different degrees of N-deficiency in Citrus production areas in China.So far,most studies on Citrus N deficiency have focused on physiological and biochemical processes,with little research on the molecular response of Citrus N deficiency.In this study,‘Xuegan’ [Citrus sinensis(L.)Osbeck] seedlings were irrigated with nutrient solution containing 15 m M(control)and 0 m M(N-deficiency)N for 10 weeks.Then,we used RNA-Seq-based transcriptome and ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry(UPLC-ESI-MS/MS)-based metabolome to investigate the impacts of N deficiency on the expression levels of genes and the abundances of metabolites in roots.At the same time,we investigated the effects of N deficiency on seedling growth and some related physiological parameters [N,lignin,total phenolics,malondialdehyde(MDA),superoxide anion production rate(SAPR)and electrolyte leakage(EL)] in roots.The objectives were to clarifying the molecular and physiological responses of Citrus sinensis roots to Ndeficiency at the physiological,transcriptome,and metabolome levels,and to identify the key genes and/or metabolites(pathways)that play a role in N-deficiency tolerance,ultimately improve Citrus sinensis N-deficiency tolerance,so as to provide theoretical support for high yield and quality cultivation of Citrus and sustainable development of the Citrus industry.The main research results are as follows:1.N-deficiency reduced the dry weight(DW)of roots,stems,and leaves,and increased the ratio of root DW to shoot DW(R/S).Compared with the control roots(RN15),the N-deficient root(RN0)had higher total phenol content and SAPR,but lower EL and contents of N,lignin and MDA.2.Using widely targeted metabolome,170 downregulated [81primary metabolites(56 N-containing and 25 non-N-containing metabolites)and 89 secondary metabolites(23 N-containing and 66 nonN-containing metabolites)] and 157 upregulated [84 primary metabolites(50 N-containing and 34 non-N-containing metabolites)and 73 secondary metabolites(12 N-containing and 61 non-N-containing metabolites)]metabolites were identified in RN0.Using RNA-Seq,2066 downregulated and 1879 upregulated genes were identified in RN0.Conjoint analysis of transcriptome and metabolome showed that there were 75 common enriched KEGG pathways between differentially abundant metabolites(DAMs)and differentially expressed genes(DEGs).3.Further analysis demonstrated that RN0 had extensive metabolic and gene reprogramming to combat with N-deficiency,including:(a)elevated ability to maintain phosphate(Pi)homeostasis by increasing the abundances of phosphorus-containing metabolites,Pi compartmentation in plastids,and/or lowering the expression levels of low-Pi-inducible genes,N homeostasis through enhancing N use efficiency and/or N remobilization efficiency,and energy homeostasis by increasing energy formation and decreasing energy consume;(b)enhancing the transmembrane transport of metabolites,thereby enhancing the remobilization and recycle of useful compounds,and(c)activating protein processing in endoplasmic reticulum,thus increasing protein folding and the degradation of misfolded or unfolded proteins accumulated in endoplasmic reticulum.RN0 could keep higher capacity to detoxify reactive oxygen species and aldehydes,thus protecting RN0 against oxidative damage.4.Some genes such as NRT2.4,NRT2.5,NRT1.1,NLP7,lipoxygenase and ABC transporters,and metabolic pathways such as protein processing in endoplasmic reticulum(ko04141),arginine and proline metabolism(ko00330),and ABC transporters(ko02010)might involve in root Ndeficiency tolerance.