Comparative transcriptomics and proteomics analysis of the symbiotic germination of Paphiopedilum barbigerum with Epulorhiza sp. FQXY019

Abstract

Introduction: Paphiopedilum barbigerum is currently the rarest and most endangered species of orchids in China and has significant ornamental value. The mature seeds of P. barbigerum are difficult to germinate owing to the absence of an endosperm and are highly dependent on mycorrhizal fungi for germination and subsequent development. However, little is known about the regulation mechanisms of symbiosis and symbiotic germination of P. barbigerum seeds.

Methods: Herein, transcriptomics and proteomics were used to explore the changes in the P. barbigerum seeds after inoculation with (FQXY019 treatment group) or without (control group) Epulorhiza sp. FQXY019 at 90 days after germination.

Results: Transcriptome sequencing revealed that a total of 10,961 differentially expressed genes (DEGs; 2,599 upregulated and 8,402 downregulated) were identified in the control and FQXY019 treatment groups. These DEGs were mainly involved in carbohydrate, fatty acid, and amino acid metabolism. Furthermore, the expression levels of candidate DEGs related to nodulin, Ca²⁺ signaling, and plant lectins were significantly affected in P. barbigerum in the FQXY019 treatment groups. Subsequently, tandem mass tag-based quantitative proteomics was performed to recognize the differentially expressed proteins (DEPs), and a total of 537 DEPs (220 upregulated and 317 downregulated) were identified that were enriched in processes including photosynthesis, photosynthesis-antenna proteins, and fatty acid biosynthesis and metabolism.

Discussion: This study provides novel insight on the mechanisms underlying the in vitro seed germination and protocorm development of P. barbigerum by using a compatible fungal symbiont and will benefit the reintroduction and mycorrhizal symbiotic germination of endangered orchids.

Keywords: Paphiopedilum barbigerum; mycorrhizal fungi; proteome; symbiotic germination; transcriptome.

Figure 1

(A) Correlation analysis of the gene expression patterns in the FQXY019 treatment and CK groups. (B) Principal component analysis (PCA) of FPKM profiles in the FQXY019 treatment groups (group 1) and CK (group 2). The deeper the blue represents the stronger the positive correlation, and the deeper the purple the weaker the positive correlation (A). The blue and orange triangles represent the average coordinates of the three groups (B).

Figure 2

Differentially expressed genes (DEGs) for FQXY019 treatment groups vs. control (CK). (A) Volcano plot and (B) histogram; the number of up- and downregulated genes are represented by red and green, respectively.

Figure 3

Graphical representation of significantly enriched pathways. (A) GO enrichment analysis of DEGs for FQXY019 treatment groups vs. CK. The bubble diagram shows the top 20 significant enriched pathways in the upregulated (B) and downregulated (C) DEGs for FQXY019 treatment groups vs. CK obtained via KEGG analysis. (D) qRT-PCR validation of DEG results. Values of qRT-PCR are mean ± SD (n = 3). Bars indicate SD. The detailed primer information is shown in Supplementary Table S1. The X-axis was the rich factor, which was the ratio of the DEG number to the total gene number in a certain pathway; the Y-axis represents the name of the pathway. The bubble size represents the number of DEGs involved. The bubble color indicates the enrichment degree of the pathway.

Figure 4

Differentially expressed proteins (DEPs) for FQXY019 treatment groups vs. control (CK). (A) Volcano plot and (B) histogram; the number of up- and downregulated genes are represented by red and green, respectively. (C) Hierarchical clustering analysis of the DEPs in different treatments. CK was the control treatment and FQXY019 was the inoculation treatment. Blue represents the CK, and red represents the FQXY019 treatment groups. (D) Bubble diagram showing the top 20 pathways with significant enrichment of DEPs for FQXY019 treatment groups and CK on KEGG pathway analysis. The X-axis was the rich factor, which was the ratio of the DEP number to the total protein number in a certain pathway; the Y-axis represents the name of the pathway. The bubble size represents the number of DEPs involved. The color of the circle represents p-value, which indicates the enrichment degree of the pathway.

Figure 5

The expression levels of common DEPs in P. barbigerum involved in photosynthesis.

Figure 6

The expression levels of common DEPs in P. barbigerum related to photosynthesis-antenna proteins.

Figure 7

Schematic diagram of the potential mechanisms involved in the effect of Epulorhiza sp. FQXY019 symbiosis on the growth of P. barbigerum. Red and upward arrows indicate upregulated. Green and downward arrows indicate downregulated.