Metabolomics and transcriptomics
Briefly, each sample (50 mg) was grounded in liquid nitrogen and homogenized with 40 µl of methanol/water (4:1, v/v) containing L-2-chloro-phenylalanine (0.3 mg/mL) as internal standard. The extraction was evaporation-dried and redissolved in 200 μL methanol for analysis. LC-MS instrumentation, metabolite identification, and data analysis are described in Method S2.
Total RNA was extracted from ultralow-temperature frozen leaves (approximately 0.1 g) using a plant RNA purification kit (Invitrogen, Carlsbad, CA, USA). cDNA libraries were constructed using the TruseqTM RNA sample prep Kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA sequencing, de novo assembly, functional annotation of unigenes, and data analysis are described in Method S3.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the enrichment pathways in MBROLE (http://Csbg.cnb.csic.es/mbrole2/) to obtain the KEGG ID of the significant differential metabolites (DMs) and differentially expressed genes (DEGs). The statistical significance of pathway enrichments was identified using Fisher’s exact test, andP values from the KEGG enrichment analyses were corrected by Benjamini and Hochberg as well. The corrected P values (false discovery rate, FDR) ≤0.05 were considered significantly enriched. Based on the results of KEGG pathway enrichment analysis of integrated transcriptome and metabolome, we collected the key DEGs groups involved in the biosynthesis of plant signaling molecules and phenylpropanoids (Table S1).