Integrated transcriptome and endogenous hormone analysis provides new insights into callus proliferation in Osmanthus fragrans

Abstract

Osmanthus fragrans is an important evergreen species with both medicinal and ornamental value in China. Given the low efficiency of callus proliferation and the difficulty of adventitious bud differentiation, tissue culture and regeneration systems have not been successfully established for this species. To understand the mechanism of callus proliferation, transcriptome sequencing and endogenous hormone content determination were performed from the initial growth stages to the early stages of senescence on O. fragrans calli. In total, 47,340 genes were identified by transcriptome sequencing, including 1798 previously unidentified genes specifically involved in callus development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) was significantly enriched in plant hormone signal transduction pathways. Furthermore, our results from the orthogonal projections to latent structures discrimination analysis (OPLS-DA) of six typical hormones in five development stages of O. fragrans calli showed jasmonic acid (JA) could play important role in the initial stages of calli growth, whereas JA and auxin (IAA) were dominant in the early stages of calli senescence. Based on the weighted gene co-expression network analysis, OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b were selected as hub genes from the modules with the significant relevance to JA and IAA respectively. The gene regulation network and quantitative real-time PCR implied that during the initial stages of callus growth, the transcription factors (TFs) OfERF4 and OfMYC2a could down-regulate the expression of hub genes OfSRC2 and OfPP2CD5, resulting in decreased JA content and rapid callus growth; during the late stage of callus growth, the TFs OfERF4, OfMYC2a and OfTGA21c, OfHSFA1 could positively regulate the expression of hub genes OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b, respectively, leading to increased JA and IAA contents and inducing the senescence of O. fragrans calli. Hopefully, our results could provide new insights into the molecular mechanism of the proliferation of O. fragrans calli.

Figure 1

Analysis of endogenous hormone content in callus of O. fragrans at different developmental stages. (A) Content of six endogenous hormones in callus of O. fragrans at five developmental stages. ABA abscisic acid, BR brassinosteroids, GA₃ gibberellin, IAA auxin, JA jasmonic acid, ZR zeatin. Different letters denote significant differences according to the Tukey’s test (P < 0.05). (B) Score plot of endogenous hormone content of O. fragrans according to different developmental stages using OPLS-DA analysis.

Figure 2

Number of DEGs. Y-25d: callus at 25 days; Y-45d: callus at 45 days; Y-55d: callus at 55 days; Y-65d: callus at 65 days; Y-75d: callus at 75 days.

Figure 3

Thermal diagram of endogenous hormones and characteristic modules. Each column represents a physiological indicator, and each row represents a genetic module. The number in each grid represents the correlation between the module and the trait. The number in parentheses represents the P-value. The smaller the P-value, the stronger the significance of the representativeness and module correlation. ABA abscisic acid, BR brassinosteroids, GA₃ gibberellin, IAA auxin, JA jasmonic acid, ZR zeatin.

Figure 4

qRT-PCR validation of the transcriptome data results for 14 selected genes. (A) Expression levels of 14 genes and FPKM values. Different letters denote significant differences according to the Tukey’s test (P < 0.05). (B) Correlation analysis of the gene expression ratios between qRT-PCR and FPKM.

Figure 5

Regulatory networks of hub genes and edge genes in feature modules. (A) Construction of a regulatory network of JA-related upregulated genes and transcription factors in ‘royalblue’ module. (B) Construction of a regulatory network of IAA-related upregulated genes and transcription factors in the ‘brown’ module. Hub genes are red ellipses, transcription factors are blue ellipses, and TFs with the highest correlation are blue triangles.

Figure 6

qRT-PCR validation of hub genes and edge genes in feature modules. (A) The expression levels of 12 genes (four genes from the ‘royalblue’ module and eight from the ‘brown’ module) and FPKM values. Different letters denote significant differences according to the Tukey’s test (P < 0.05). (B) Correlation coefficient analysis of hub genes and edge genes in feature modules.

Figure 7

Proposed model of callus proliferation regulation in O. fragrans.

Figure 8

Different developmental stages of O. fragrans callus. Callus at (A) 25 days, (B) 45 days, (C) 55 days, (D) 65 days, and (E) 75 days of growth.