Metabolomics combined with physiology and transcriptomics reveals how Citrus grandis leaves cope with copper-toxicity

Limited data are available on metabolic responses of plants to copper (Cu)-toxicity. Firstly, we investigated Cutoxic effects on metabolomics, the levels of free amino acids, NH4+-N, NO3--N, total nitrogen, total soluble proteins, total phenolics, lignin, reduced glutathione (GSH) and malondialdehyde, and the activities of nitrogenassimilatory enzymes in 'Shatian' pummelo (Citrus grandis) leaves. Then, a conjoint analysis of metabolomics, physiology and transcriptomics was performed. Herein, 59 upregulated [30 primary metabolites (PMs) and 29 secondary metabolites (SMs)] and 52 downregulated (31 PMs and 21 SMs) metabolites were identified in Cutoxic leaves. The toxicity of Cu to leaves was related to the Cu-induced accumulation of NH4+ and decrease of nitrogen assimilation. Metabolomics combined with physiology and transcriptomics revealed some adaptive responses of C. grandis leaves to Cu-toxicity, including (a) enhancing tryptophan metabolism and the levels of some amino acids and derivatives (tryptophan, phenylalanine, 5-hydroxy-L-tryptophan, 5-oxoproline and GSH); (b) increasing the accumulation of carbohydrates and alcohols and upregulating tricarboxylic acid cycle and the levels of some organic acids and derivatives (chlorogenic acid, quinic acid, D-tartaric acid and gallic acid Ohexoside); (c) reducing phospholipid (lysophosphatidylcholine and lysophosphatidylethanolamine) levels, increasing non-phosphate containing lipid [monoacylglycerol ester (acyl 18:2) isomer 1] levels, and inducing low-phosphate-responsive gene expression; and (d) triggering the biosynthesis of some chelators (total phenolics, lignin, L-trytamine, indole, eriodictyol C-hexoside, quercetin 5-O-malonylhexosyl-hexoside, N-caffeoyl agmatine, N '-p-coumaroyl agmatine, hydroxy-methoxycinnamate and protocatechuic acid O-glucoside) and vitamins and derivatives (nicotinic acid-hexoside, B1 and methyl nicotinate). Cu-induced upregulation of many antioxidants could not protect Cu-toxic leaves from oxidative damage. To conclude, our findings corroborated the hypothesis that extensive reprogramming of metabolites was carried out in Cu-toxic C. grandis leaves in order to cope with Cu-toxicity.

Automated summary

The study revealed that copper toxicity in 'Shatian' pummelo leaves led to increased levels of amino acids and derivatives, accumulation of carbohydrates and alcohols, decreased phospholipid levels, and induction of biosynthesis of chelators and vitamins. The upregulation of antioxidants in response to copper toxicity did not prevent oxidative damage in the leaves.