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. 2021 May 17;12(5):754.
doi: 10.3390/genes12050754.

Comprehensive Transcriptome Analysis of Rare Carpinus putoensis Plants under NO2 stress

Qianqian Sheng  1   2 , Congzhe Liu  1   2 , Min Song  1   2 , Jingyuan Xu  1   2 , Zunling Zhu  1   2   3
Affiliations

Comprehensive Transcriptome Analysis of Rare Carpinus putoensis Plants under NO2 stress

Qianqian Sheng et al. Genes (Basel). .

Abstract

We evaluated a transcriptome using high-throughput Illumina HiSeq sequencing and related it to the morphology, leaf anatomy, and physiological parameters of Carpinus putoensis putoensis under NO2 stress. The molecular mechanism of the C. putoensis NO2 stress response was evaluated using sequencing data. NO2 stress adversely affected the morphology, leaf anatomy, and total peroxidase (POD) activity. Through RNA-seq analysis, we used NCBI to compare the transcripts with nine databases and obtained their functional annotations. We annotated up to 2255 million clean Illumina paired-end RNA-seq reads, and 250,200 unigene sequences were assembled based on the resulting transcriptome data. More than 89% of the C. putoensis transcripts were functionally annotated. Under NO2 stress, 1119 genes were upregulated and 1240 were downregulated. According to the KEGG pathway and GO analyses, photosynthesis, chloroplasts, plastids, and the stimulus response are related to NO2 stress. Additionally, NO2 stress changed the expression of POD families, and the HPL2, HPL1, and POD genes exhibited high expression. The transcriptome analysis of C. putoensis leaves under NO2 stress supplies a reference for studying the molecular mechanism of C. putoensis resistance to NO2 stress. The given transcriptome data represent a valuable resource for studies on plant genes, which will contribute towards genome annotations during future genome projects.

Keywords: NO2 stress; gene expression; high-throughput sequencing; molecular mechanism; resistance; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphological changes in C. putoensis leaves receiving NO2 treatment. CK: control group. NO2 treated: 1 h, 6 h, 24 h, 72 h, and recovery for 30 days.
Figure 2
Figure 2
Images of cell structures from the primary leaf under a transmission electron microscope. (a), control; (b–e), NO2-treated plants; and (f), recovery plants. From 1 h to 72 h, the plastoglobuli in the cells gradually increase, and the chloroplasts gradually shrink, and they become slender and sticky. Slowly, the cell wall is separated. The red arrows in (e) indicate plasmolysis. V, vacuole; P, plastoglobuli; PM, plasma membrane; S, starch grain; CW, cell wall; and Chl, chloroplasts.
Figure 3
Figure 3
POD activity after different NO2 stress times or 30 days of recovery. Six replicates for each group.
Figure 4
Figure 4
GO (a), euKaryotic Ortholog Group (KOG) (b) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (c) classification of all the identified genes.
Figure 5
Figure 5
Venn diagram analysis of the expressed genes in two samples (A: Control, B: NO2 stressed).
Figure 6
Figure 6
Volcano plots (a) of RNA-seq data for an A vs B pairwise comparison; (b) the top 30 differentially-regulated TF families were identified among all the genes. A: NO2 control. B: NO2 stressed. q-value, <0.05; fold change, >2.
Figure 7
Figure 7
GO enrichment factor analysis of the DEGs. (a) Upregulated genes: the top three upregulated genes are involved in multicellular organism development, plastids, and chloroplasts; (b) downregulated genes: the downregulated genes predominantly reflected response to stimulus, response to stress, and oxidoreductase activity.
Figure 8
Figure 8
Upregulated genes of KEGG pathway categories (a) and enrichment factor analysis (b) of the DEGs. The upregulated genes are functionally assigned to 76 biological pathways, and the top upregulated genes are involved in photosynthesis.
Figure 9
Figure 9
Downregulated genes of KEGG pathway categories (a) and enrichment factor analysis (b) of the DEGs. The downregulated genes predominantly represent amino acid biosynthesis and carbon metabolism.
Figure 10
Figure 10
There are 4 genes (Psb, Psa, Pet, and F-type ATPase a) involved in photosynthesis in C. putoensis under NO2 stress. Green represents the downregulated expression of the gene, red represents the upregulated expression of the gene, and yellow indicates no significant difference in gene expression.
Figure 11
Figure 11
RT-qPCR validations of 8 candidate genes involved in NO2 stress in C. putoensis based on RNA-seq data. Hypothetical protein, chloroplastic, peroxidase, and allene oxide synthase represent different gene types.
Figure 12
Figure 12
The expression profiles according to RT-qPCR (relative expression) and RNA-seq (FPKM values).
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