Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.)

Abstract

Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish.

Figure 1. The quality assessment of sequencing data from 0_DAP, 7_DAP and 15_DAP libraries.

The large portion per bar showed total number and percentage of reads mapped to the reference sequences.

Figure 2. Differentially expressed transcripts during radish embryogenesis (0_DAP, 7_DAP and 15_DAP).

(A) Number of differentially expressed transcripts in three libraries (0_DAP, 7_DAP and 15_DAP) based on pairwise comparison. Numbers of up- and down-regulated genes were summarized; (B, C) Number of commonly differentially expressed transcripts among the three libraries (0_DAP, 7_DAP and 15_DAP). (B, C) showed the number distribution of up- and down-regulated transcripts based on pairwise comparison, respectively.

Figure 3. Cluster diagram of DEGs based on pairwise comparison among the three libraries (0_DAP, 7_DAP and 15_DAP) in radish.

Expression levels for DEGs for the three groups of pairwise comparisons were hierarchically clustered and shown in a heat-map. Level of expression was represented by color scale from green (down-regulated expression) to red (up-regulated expression), as indicated by a scale bar in the upper right corner. The dendrogram of distances were also shown for genes, names of which were presented in Supplementary Table S3. The two small heat-maps placed lower showed the enlarged view of the two sections in red braces.

Figure 4. Histogram of level 2 Gene ontology classification of DEGs during embryogenesis in radish.

Results are summarized for three main GO categories: biological process (P), molecular function (F), and cellular component (C). The x-axis and y-axis indicate the names and the number of each GO term, respectively.

Figure 5. RT-qPCR validation of some DGEs during radish embryogenesis.

The relative expression of DGEs between 0_DAP and 7_DAP libraries (A), 0_DAP and 15_DAP libraries (B) and 7_DAP and 15_DAP libraries (C) were analyzed by the 2^−ΔΔCT method.

Figure 6. Relative expression levels of DEGs in four developmental stages of radish embryogenesis.

The normalized expression levels at 0 DAP were arbitrarily set to 1. Different letters indicate significant differences at P < 0.05 according to Duncan’s multiple range tests. Each bar shows the mean ± SE of triplicate assays.

Figure 7. A proposed model of genetic and molecular interactions in the regulatory network during embryogenesis in radish.

Embryo development consists of two phases, morphogenesis and maturation. Arrows in blue and black indicate regulation and affiliation, respectively, whereas red lines between genes indicate transcriptional repression. The circle with solid and dotted line represented genes identified or not in radish and the corresponding miRNAs are shown in red. Genes in yellow boxes were connected with miRNAs by purple arrows using Cytoscape, which were visualized as miRNA-mRNA interactions.