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

Nitrogen（N）is a key nutritional factor that affects plant production.In China,Citrus are mainly planted in hilly and mountainous areas,with poor nutrient conditions and insufficient nitrogen content in the soil.Therefore,it is important to explore the molecular and physiological mechanisms of Citrus sinensis leaves response to N deficiency.In this study,‘Xuegan’（Citrus sinensis）seedlings were used as the experimental materials,and two N levels were set:0 m M（N-deficiency）and 15 m M（control）.We used conjoint analysis of transcriptome,metabolome,and physiology to examine long-term N-deficiency-responsive genes and metabolites in leaves;as well as the effects of N-deficiency on seedling growth,N content,leaf gas exchange,superoxide anion production rate（SAPR）,malondialdehyde（MDA）,electrolyte leakage（EL）,total phenols,lignin and total coumarins contents.The main research results are as follows:1.N-deficient seedlings had reduced root,stem and leaf growth and N contents in roots and leaves,but increased ratio of root dry weight（DW）to shoot DW.Compared to the control,N-deficient leaves had decreased CO₂ assimilation,transpiration rate,stomatal conductance,EL and MDA content,but increased intercellular CO₂ concentration and SAPR,as well as the contents of total phenol,lignin and total coumarin.2.Extensive gene and metabolite reprogramming occurred in N-deficient leaves to cope with N-deficiency.A total of 1139 upregulated and931 downregulated genes were identified in N-deficient leaves.A total of224 upregulated[59 primary metabolites（PMs）and 165 secondary metabolites（SMs）]and 183 downregulated（133 PMs and 50 SMs）metabolites were identified in N-deficient leaves.3.Further analysis suggested that the molecular and physiological responses of leaves to long-term N-deficiency included:（a）decreased and increased accumulation of PMs（amino acids,lipids and organic acids）and SMs（lignin,phenols,phenolic acids,total coumarins,total phenolics and phenylpropanoids）;（b）enhanced capacity to maintain N homeostasis by causing shifts from N-rich amino acids and derivatives to carbon-rich carbohydrates and from N-rich alkaloids to carbon-rich phenylpropanoids and phenols,and increases in N compound degradation and N remobilization to N-demanding tissues;（c）enhanced capacity to maintain phosphate homeostasis by downregulating the expression levels of low-phosphate-responsive genes and increasing the abundances of compounds containing phosphorus but not N;and（d）activating MAPK signaling pathway.4.In addition to upregulating non-photochemical quenching and photorespiration,N-deficiency enhanced the expression levels of some genes（peroxidases,ascorbate peroxidase 2,temperature-induced lipocalin-1,galactinol synthase 2,glutathione S-transferases,alcohol dehydrogenases and aldo-keto reductases）related to reactive oxygen species and aldehyde removal,and many antioxidants（reduced glutathione,tryptophan,four vitamins,lignin and SMs）in leaves,thus upregulating the capacity to detoxifying reactive oxygen species and aldehydes and protecting N-deficient leaves from oxidative damage,and hence conferring leaf N-deficiency tolerance.Some genes such as nitrate transporter 2.5（NRT2.5）,NRT1.7,NRT1.4,NRT1.5,nitrate regulatory gene 2 protein（NRG2）,MAPKKK17 and MAPKKK5;metabolites such as reduced glutathione,tryptophan,plumerane,and phenylpropanoid;and/or metabolic pathways（processes）such as photorespiration,leaf senescence,senescence-associated vacuole,phenylalanine,tyrosine and tryptophan biosynthesis,glutathione metabolism,tryptophan metabolism,phenylpropanoid biosynthesis and MAPK signaling pathway-plant might be responsible for leaf N-deficiency tolerance.