Transcriptome Analysis of Gene Expression Patterns Potentially Associated with Premature Senescence in Nicotiana tabacum L

Abstract

Senescence affects the remobilization of nutrients and adaption of the plant to the environment. Combined stresses can result in premature senescence in plants which exist in the field. In this study, transcriptomic analysis was performed on mature leaves and leaves in three stages of premature senescence to understand the molecular mechanism. With progressive premature senescence, a declining chlorophyll (chl) content and an increasing malonaldehyde (MDA) content were observed, while plasmolysis and cell nucleus pyknosis occurred, mitochondria melted, thylakoid lamellae were dilated, starch grains in chloroplast decreased, and osmiophilic granules increased gradually. Moreover, in total 69 common differentially expressed genes (DEGs) in three stages of premature senescing leaves were found, which were significantly enriched in summarized Gene Ontology (GO) terms of membrane-bounded organelle, regulation of cellular component synthesis and metabolic and biosynthetic processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the plant hormone signal transduction pathway was significantly enriched. The common DEGs and four senescence-related pathways, including plant hormone signal transduction, porphyrin and chlorophyll metabolism, carotenoid biosynthesis, and regulation of autophagy were selected to be discussed further. This work aimed to provide potential genes signaling and modulating premature senescence as well as the possible dynamic network of gene expression patterns for further study.

Keywords: plant hormone; premature senescence; tobacco; transcriptome; ultrastructure.

Figure 1

Plant phenotypes, Chlorophyll (chl) and malonaldehyde (MDA) measurements at the four stages of premature senescence. M, mature, EA early senescence, MA middle senescence, LA late senescence. (A) Appearance of tobacco leaves at four stages of premature senescence. (B) chl content. (C) MDA content. Error bars indicate the means ± SD (n = 3).

Figure 2

Ultrastructure of mesophyll cell at four stages. (A,E,I,M) Leaves of mature stage (M). (B,F,J,N) Leaves of early premature senescence stage (EA). (C,G,K,O) Leaves of middle premature senescence stage (MA). (D,H,L,P) Leaves of late premature senescence stage (LA). A–D, scale bars in bottom right corner of every picture correspond to 10 μm; E–L, scale bars correspond to 500 nm; L–P, scale bars correspond to 2 μm. Sg, starch grain; Tl, thylakoid lamellae; Og, Osmiophilic granules; Ch, Chloroplast; Cn, Cell Nucleus; Mi, Mitochondria; Cy, cytoderm.

Figure 3

Venn diagram of differentially expressed genes (DEGs) and heat map of the 69 common DEGs. (A) Venn diagram of DEGs at three premature senescence stages compared with the mature stage. (B) Expression patterns of 69 common DEGs in the process of premature senescence. The x-axis represents each stage of premature senescence. The y-axis refers to gene expression levels. Log₁₀(FPKM + 1) was used for the heat map to normalize the gene expression level. The gradient from blue to red indicates that the relative levels of gene expression range from low to high. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced.

Figure 4

Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common DEGs in the process of premature senescence. (A) Barplot of significantly enriched GO terms. The x-axis represents the number of genes belonging to the left GO terms. The y-axis indicates GO terms. (B) Enriched KEGG pathways. Rich factor represents the ratio of the number of enriched DEGs in the KEGG pathway and the number of annotated background genes in this pathway. The x-axis shows rich factor of each pathway. The y-axis refers to KEGG pathways.

Figure 5

Expression patterns of DEGs in different KEGG pathways. (A) Plant hormone signal transduction. (B) Porphyrin and chlorophyll metabolism. (C) Carotenoid biosynthesis. (D) Regulation of autophagy. * represents a significant difference of gene expression levels at p ≤ 0.05 compared with M. The x-axis refers to each stage of premature senescence. The y-axis indicates gene expression levels. Log₁₀(FPKM + 1) was used for the heat map to normalize the gene expression level. Blue and red represent low and high gene relative expression levels respectively.

Figure 6

qRT-PCR validation of selected transcripts in tobacco leaves. The green columns represent the relative intensity of real-time quantitative RT-PCR (left y-axis), the red lines represent the FPKM value of the transcripts (right y-axis). The x-axis refers to the four stages of premature senescence. Error bars refer to standard error (n = 3). The values of r mean the Pearson correlation coefficient of data between RNA-seq and qRT-PCR.