Plant transcriptome analysis.

The total RNA extraction and purification of A. thaliana, cDNA library construction and quality control, and RNA-sequencing method were performed according to Cordovez’s research methods (27). For each treatment, three replicates were used. Each replicate consisted of three plates with five seedlings each to obtain enough biomass. To be consistent with the plants treated by MVOCs, all A. thaliana were collected by I-plate after 18 days of exposure under the same conditions. The whole plant was then used as a transcriptome sequencing sample. The AMPure XP system (Beckman Coulter, Beverly, USA), a NanoPhotometer spectrophotometer (Implen, CA, USA), and an Agilent Bioanalyzer 2100 system were used to purify and evaluate the quality of the cDNA libraries. Sequencing libraries were generated using an NEBNext Ultra RNA library prep kit for Illumina (NEB, USA) and were sequenced on the Illumina NovaSeq 6000 platform.

The software trim-galore was used to remove the adapters from the sequence, and low-quality and poly(N) sequences and sequences shorter than the length threshold (the default is 20 bp) were removed (if one sequence was shorter than the threshold, the corresponding two sequences were removed). A quality score of 20 (Q20) ≥ 85% and a Q30 ≥ 80% were qualified. Sequence alignment of the reference genome was performed using HISAT2 v2.0.5 to construct the index of the reference gene and the alignment of clean reads with the reference genome. Read count normalization used fragments per kilobase per million mapped reads (FPKM) to represent gene expression,

The Bioconductor package DESeq2 was used for normalization and differential gene expression analyses (107). The average FPKM ratio of the experimental group and the control group was fold change (FC); genes with a log⁡2|FC|of≥1 and an adjusted P value of <0.05 were defined as differentially expressed genes (DEGs). GO differential enrichment analysis of DEGs and statistical enrichment of DEGs in the KEGG pathway were performed with ClusterProfiler (3.4.4) (108).