Comparative Transcriptomics Analysis of the Symbiotic Germination of D. officinale (Orchidaceae) With Emphasis on Plant Cell Wall Modification and Cell Wall-Degrading Enzymes

Abstract

Orchid seed germination in nature is an extremely complex physiological and ecological process involving seed development and mutualistic interactions with a restricted range of compatible mycorrhizal fungi. The impact of the fungal species' partner on the orchids' transcriptomic and metabolic response is still unknown. In this study, we performed a comparative transcriptomic analysis between symbiotic and asymbiotic germination at three developmental stages based on two distinct fungi (Tulasnella sp. and Serendipita sp.) inoculated to the same host plant, Dendrobium officinale. Differentially expressed genes (DEGs) encoding important structural proteins of the host plant cell wall were identified, such as epidermis-specific secreted glycoprotein, proline-rich receptor-like protein, and leucine-rich repeat (LRR) extensin-like protein. These DEGs were significantly upregulated in the symbiotic germination stages and especially in the protocorm stage (stage 3) and seedling stage (stage 4). Differentially expressed carbohydrate-active enzymes (CAZymes) in symbiotic fungal mycelium were observed, they represented 66 out of the 266 and 99 out of the 270 CAZymes annotated in Tulasnella sp. and Serendipita sp., respectively. These genes were speculated to be involved in the reduction of plant immune response, successful colonization by fungi, or recognition of mycorrhizal fungi during symbiotic germination of orchid seed. Our study provides important data to further explore the molecular mechanism of symbiotic germination and orchid mycorrhiza and contribute to a better understanding of orchid seed biology.

Keywords: CAZymes; Serendipita sp.; Tulasnella sp.; comparative transcriptome; symbiotic germination.

Figure 1

Experiment designs for our study. Aymbiotic and symbiotic germination of medicinal orchid Dendrobium officinale inoculated with two mycorrhizal fungi Tulasnella sp. (S6) and Serendipita sp. (12825) at three development stages. (A) showing asymbiotic germination in 1/2 MS medium as control; (B,C) The free-living mycelium of two fungi on PDA medium, respectively. (D–H) showing the symbiotic germination assay with two fungal species, respectively. (I) Trypan blue staining showing the pelotons in embryo cell in symbiotic stage 4 of D. officinale seed inoculated with strain 12825. Scale bar = 20 μm in (F–H), and 50 μm in (I).

Figure 2

Venn diagram showing the numbers of plant differentially expressed genes (DEGs) in the adjacent development stage of two symbiotic groups of D. officinale seeds with Tulasnella sp. (S6) and Serendipita sp. (12825), respectively. (A,B) DEGs at adjacent development stage during D.officinale seed with Tulasnella sp. (A) and Serendipita sp. (B) and compared to the same stage in asymbiotic germination; (C) 1,003 common differentially expressed plant genes across the three development stages (stages 2, 3, and 4) in symbiotic process with two different mycorrhizal fungi compared asymbiotic germination. D2, D3, and D4 mean asymbiotic germination stages 2, 3, and 4, respectively; S2, S3, and S4 mean stages 2, 3, and 4 in symbiotic germination with Serendipita sp., respectively, T2, T3, and T4 mean stages 2, 3, and 4 in symbiotic germination with Tulasnella sp., respectively.

Figure 3

The expression patterns and functional classification of 452 differentially upregulated expressed plant genes (DEGs) at least one symbiotic stage during D. officinale inoculated two mycorrhizal fungi. (A) Heatmap showing the expression level of up-regulated genes in symbiotic germination of D. officinale. Expression value was calculated based on Log₂ Fold change. Red color means a gene with a high expression value (Fold change ≥ 8) and yellow color means a gene with a relatively low expression value (Fold change ≥ 2). The function annotation of the representative genes with high expression (Cluster I-Cluster VI) was marked in the heatmap right. (B) GO functional category of the 452 up-regulated genes in nine clusters. D2, D3, and D4 mean asymbiotic germination stage2, stage3, and stage4, respectively; S2, S3, and S4 mean stages 2, 3, and 4 in symbiotic germination with Serendipita sp., respectively, T2, T3, and T4 means stages 2, 3, and 4 in symbiotic germination with Tulasnella sp., respectively. The detailed information of these genes is in Supplementary Table S1.

Figure 4

KEGG and GO enrichment analysis of differentially expressed plant genes (DEGs) during germination of D. officinale seeds with two mycorrhizal fungi. (A) GO enrichment analysis; red color indicated that typical Go functional enrichment for these genes involved in symbiotic germination compared to asymbiotic germination. (B) KEGG pathway enrichment analysis showed that the significant enrichment pathway exists as overlaps between symbiotic and asymbiotic germination. It means these pathways could be most important during seed development. And the symbiotic germination with two fungal species have similar metabolic pathway but also display difference e.g., lysosome in Tulasnella sp.-D.officinale and peroxisome in Serendipita sp.- D.officinale; (C) heatmap showing the expression profile of genes in the significantly enriched four metabolic pathways during symbiotic germination of D.officinale (such as plant hormone signal transduction, phenylpropanoid biosynthesis, starch, and sucrose metabolism and plant-pathogen interaction); expression value was calculated based on Log2 Fold change. Significant expression changes were labeled in colors: blue represents down regulated and yellow represents up regulated. The functional annotation of representative genes in each enriched pathway was recorded in heatmap aside.

Figure 5

Heatmp showing the differentially expressed genes (DEGs) between symbiotic germination at the same stage during D. offcinale inoculated with Tulasnella sp. (S6) and Serendipita sp. (12825). (A) The number of DEGs when D. officinale seed inoculated with Tulasnella sp. compared to with Serendipita sp. at the same development stage; (B–D) KEGG enrichment analysis of up-regulated DEGs at various germination stages during D.officinale symbiotic germination with two fungi. (E) Heatmap showing the expression pattern of 350 common differentially expressed genes in D. officinale × Tulasnella sp. compared to D. officinale × Serendipita sp. Expression value was calculated based on Log2 Fold change, red color means up-regulated expression and green color means down-regulated expression. S2, S3, and S4 mean stages 2, 3, and 4 in symbiotic germination with Serendipita sp., respectively, T2, T3, and T4 mean stages 2, 3, and 4 in symbiotic germination with Tulasnella sp., respectively.

Figure 6

Heatmap showing the expression profile of fungal DEGs encoding CAZyme proteins during symbiotic germination of D.officinale with Tulasnella sp. and Serendipita sp. ∙ means genes differentially expression, blank means no significant differential expression; yellow color means genes up-regulated with fold change ≥ 5 and green color means genes down regulated with fold change ≥ 5. Ft2, Ft3, and Ft4 represent Tulasnella sp. in symbiotic statuses 2, 3, 4 stages while Fs2, Fs3, and Fs4 represent Serendipita sp. in symbiotic statuses 2, 3, and 4 stages (according to germination stage) and Ft and Fs means free-living mycelium of Tulasnella sp. and Serendipita sp., respectively.

Figure 7

Heatmap showing the expression profile of fungal orthologous genes encoding CAZyme, secreted proteins, and protease during symbiotic germination of D. officinale with Tulasnella sp. and Serendipita sp. The expression level was evaluated using fold change in different colors. Green color (dark green means expression level with fold change ≥ 10) means genes down-regulated expression and red and orange color means genes up-regulated expression (red color means expression level with fold change ≥ 10). Ft2, Ft3, and Ft4 represent Tulasnella sp. in symbiotic statuses 2, 3, 4 stages while Fs2, Fs3, and Fs4 represent Serendipita sp. in symbiotic statuses 2, 3, and 4 stages (according to germination stage) and Ft and Fs means free-living mycelium of Tulasnella sp. and Serendipita sp., respectively.