Integration of physiology, metabolome and transcriptome for understanding of the adaptive strategies to long-term nitrogen deficiency in Citrus sinensis leaves

Abstract

The contribution of nitrogen (N)-deficiency-responsive metabolites and genes to long-term N-deficiency tolerance in Citrus is poorly understood. Using sweet orange (Citrus sinensis) seedlings as materials, we investigated long-term N-deficiency-responsive metabolites and genes, and long-term N-deficient impacts on some physiological parameters in leaves. Our findings demonstrated that extensive gene and metabolite reprogramming occurred in 0 mM N-treated leaves (LN0). The molecular and physiological responses of leaves to N-deficiency included: (a) leading to shifts from primary metabolites [amino acids and derivatives (AADs), lipids, and organic acids] to secondary metabolites (lignin, flavonoids, phenols, phenolic acids, total coumarins, total phenolics and phenylpropanoids), from N-rich AADs to carbon-rich carbohydrates, and from N-rich alkaloids to carbon-rich phenylpropanoids and phenols by upregulating phenylpropanoid pathway and downregulating alkaloid biosynthesis with the exceptions of indole alkaloids, as well as increases in N compound degradation and N remobilization to N-demanding tissues; (b) enhanced capacity to maintain phosphate homeostasis by downregulating the expression levels of low-phosphate-responsive genes and improving the abundances of compounds containing phosphorus but not N; and (c) activating MAPK signaling pathway. In addition to upregulating non-photochemical quenching and photorespiration, N-deficiency improved the expression levels of some genes related to reactive oxygen species (ROS) and aldehyde detoxification and the abundances of many antioxidants (reduced glutathione, tryptophan, four vitamins, lignin and secondary metabolites especially flavonoids) in leaves, thus upregulating the capacity to detoxifying ROS and aldehydes and protecting LN0 from oxidative damage, and hence conferring leaf N-deficiency tolerance. Also, we found that some metabolites such as reduced glutathione, tryptophan, plumerane and phenylpropanoid, and/or genes such as NRT2.5, NRT1.7, NRT1.4, NRT1.5, NRG2, MAPKKK17 and MAPKKK5 might be responsible for leaf N-deficiency tolerance. To conclude, our finding provided some new information on the adaptive strategies of leaves to long-term N-deficiency.