Objective  Molecular mechanisms of Citrus grandis (L.) Osbeck. cv. Guanximiyou in response to simulated acid rain stress were investigated.

  Method  The llumina HiSeq 4000 system, a high-throughput transcriptome sequencing technology, was applied to reveal the differential expressions of the grapefruit transcriptome after a 24 h simulated acid rain treatment. The unigenes obtained were compared to the Nr and KEGG database. Abundance of gene expression of the samples were screened according to transcriptome data by using PRKM method. Differentially expressed genes (DEGs) among the treated samples were estimated by referring to the standard of FDR ≤ 0.05 and |log₂FC| ≥ 1. Functions and pathways of those DEGs were analyzed using the Gene Ontology (GO) and KEGG pathway database.

  Result  Significant lumpy lesions began to appear on the young grapefruit leaves 24 h after the artificial acid rain spray with 21 497 fully described unigenes obtained. In comparison to control, 879 of the genes were significantly upregulated and 588 downregulated. The top 50 DEGs were all in the upregulated category and mainly associated with metabolic pathways, secondary metabolism, phenylpropyl propane biosynthesis, or terpenoid biosynthesis. The GO enrichment analysis showed the DEGs being largely located in extracellular region and, among various molecular functions, the catalytic activity being the most significantly enriched, followed by oxidoreductase activity, while the DNA metabolism being the most significant of DEGs in the GO term on biological process. The KEGG enrichment analysis indicated that DNA replication was the most significant enrichment pathway, followed by secondary metabolic biosynthesis, and lignin synthesis. The PCR fluorescence quantitative analysis on the 4 DEGs in the secondary metabolic biosynthesis pathway i.e., POD isozyme cg3g018770 and cg2g001440, cinnamyl coenzyme A reductase (CCR) isozyme cg1g021310, and 4-coumaric acid-coenzyme A ligase (4CL) isozyme cg3g029290 confirmed that the acid rain stress indeed significantly affected the expressions of these genes.

  Conclusion  C. grandis (L.) Osbeck. cv. Guanximiyou was strongly tolerant to acid rain. The stress response of the plants involved numerous genes regulated by various collaborative biological processes. Among them, the regulation of secondary metabolism appeared to play a major role in coping with acid rain stress by the plant.