Molecular Mechanisms Of Citrus Grandis Root Response To Excessive Copper Revealed By Transcriptome And Metabolome

Copper(Cu),as an essential micronutrient element in higher plants,is of great significance for maintaining plant metabolism and growth and development.Due to the long-term and excessive use of Cu containing fungicides,excessive Cu in the soil of Citrus orchards has become a common physiological obstacle limiting the yield and quality of Citrus in China.So far,there are few reports on the molecular and physiological mechanisms of plant roots in response to long-term copper(Cu)excess.Sand potted young seedlings of ‘Shatian’ pomelo [Citrus grandis(L.)Osbeck] were supplied six times weekly with nutrient solution at a Cu concentration of 400(excessive Cu)or 0.5(control)μM for six months.Thereafter,transcriptome,widely targeted metabolome and physiological methods were used to investigate the effects of Cu excess on seedling growth;Cu concentrations in roots and leaves;the expression levels of genes and the abundances of metabolites in roots;as well as the concentrations of cell wall(CW)materials(CWM)and components,total phenols(TPs),total free amino acids(TFAAs),total coumarins,total flavonoids and malondialdehyde(MDA)in roots.The objectives are to reveal the molecular and physiological mechanism of root response to Cu excess from the transcriptome,metabolome and physiological levels,identify the key genes,metabolites and/or metabolic pathways that play a role in Cu-tolerance,and clarify the similarities and differences between Cu-excess-responsive genes and metabolites between leaves and roots.The main research results are as follows:1.Excessive Cu reduced seedling growth,and increased the concentrations of Cu in roots and leaves.The young leaves turned yellow,the root system was sparse,and the fibrous roots became rotten and black brown.2.Excessive Cu increased root CWM,pectin and lignin contents,reduced hemicellulose 2(HC2)contents,had no significant effect on HC1,HCI+HC2 and cellulose contents on a dry weight(DW)basis.Excessive Cu reduced root HC2 and HC1+HC2 contents,but it had no significant effect on the contents of other CW components on a CWM basis.Excessive Cu increased the contents of total flavonoids,total coumarins,TPs,and MDA in roots,but had no significant effect on the contents of TFAAs in roots.3.Using widely targeted metabolome,142 [52 primary metabolites(PMs)and 90 secondary metabolites(SMs)] upregulated and 48(17 PMs and 31 SMs)downregulated metabolites were detected in 400 μM Cutreated roots(RCu400).A total of 65 differentially abundant metabolites(DAMs)were enriched to 59 KEGG pathways.Using RNA-Seq,350 upregulated and 156 downregulated genes were identified in RCu400.A total of 220 differentially expressed genes(DEGs)were enriched to 84 KEGG pathways.Conjoint analysis of transcriptome and metabolome showed that there were 40 common KEGG enrichment pathways between DEGs and DAMs.4.Further analysis demonstrated that the adaptive responses of roots to excessive Cu included:(a)elevated biosynthesis and accumulation of SMs(lignin,alkaloids,flavonoids,lignans and coumarins,phenolic acids and total phenolics);(b)increased Cu retention in root CW and reduced Cu translocation to shoots;(c)repressed and induced expression of Cu uptakeand Cu homeostasis-related genes,respectively;and(d)improved capacity to maintain Fe homeostasis in cytoplasm and energy homeostasis.Although the abundances of many metabolites and the expression of some genes related to ROS and reactive nitrogen species(RNS)detoxification were enhanced in RCu400,they could not protection these roots from oxidative damage.Some genes such as wall-associated kinase(WAK)1(WAK1),α-dioxygenase 1(DOX1),vacuolar iron transporter,ferric reduction oxidase 2(FRO2),Fe(II)transport protein 1(IRT1),basic helixloop-helix protein 29(b HLH29),Protein FERRIC REDUCTASE DEFECTIVE 3(FRD3),Fe-regulated transporter,member(IREG1)and ferritins genes,metabolites such as pectin,lignin,coumarins,alkaloids,phenolic acids(salicylic acid),organic acids(citric acid)and carbohydrates(glucose),and/or metabolic pathways(processes)such as phenylpropanoid biosynthesis,Fe ion homeostasis,cellular response to RNS and cellular response to ROS,might contribute to the adaptations of C.grandis roots to long-terms Cu excess.Also,some similarities and differences in excessive Cu-responsive genes and metabolites between leaves and roots.In conclusion,the results revealed some novel adaptive strategies of plant roots to Cu-toxicity.