Gibberellic Acid Inhibits Dendrobium nobile- Piriformospora Symbiosis by Regulating the Expression of Cell Wall Metabolism Genes

Abstract

Orchid seeds lack endosperms and depend on mycorrhizal fungi for germination and nutrition acquisition under natural conditions. Piriformospora indica is a mycorrhizal fungus that promotes seed germination and seedling development in epiphytic orchids, such as Dendrobium nobile. To understand the impact of P. indica on D. nobile seed germination, we examined endogenous hormone levels by using liquid chromatography-mass spectrometry. We performed transcriptomic analysis of D. nobile protocorm at two developmental stages under asymbiotic germination (AG) and symbiotic germination (SG) conditions. The result showed that the level of endogenous IAA in the SG protocorm treatments was significantly higher than that in the AG protocorm treatments. Meanwhile, GA₃ was only detected in the SG protocorm stages. IAA and GA synthesis and signaling genes were upregulated in the SG protocorm stages. Exogenous GA₃ application inhibited fungal colonization inside the protocorm, and a GA biosynthesis inhibitor (PAC) promoted fungal colonization. Furthermore, we found that PAC prevented fungal hyphae collapse and degeneration in the protocorm, and differentially expressed genes related to cell wall metabolism were identified between the SG and AG protocorm stages. Exogenous GA₃ upregulated SRC2 and LRX4 expression, leading to decreased fungal colonization. Meanwhile, GA inhibitors upregulated EXP6, EXB16, and EXP10-2 expression, leading to increased fungal colonization. Our findings suggest that GA regulates the expression of cell wall metabolism genes in D. nobile, thereby inhibiting the establishment of mycorrhizal symbiosis.

Keywords: cell wall; hormones; protocorm; symbiosis.

Figure 1

Morphological characters and seed germination stages of D. nobile under symbiotic germination (SG) and asymbiotic germination (AG) conditions. (A–C): symbiotic germination on the OMA with mycorrhizal fungi P. indica, (a–c): asymbotic germination. (A,a): Ten days after sowing seeds: embryo swollen, turned light green, rupture of testa; (B,b): Twenty days after sowing seeds: the appearance of protomeristem (protocorm stage1); (C,c): Forty days after sowing seeds: leaf differentiation, shoot appearance in protocorm (protocorm stage 2); scale bar = 100 µm. (D): Effect on protocorm formation of symbiotic and asymbotic treatments t-test (two-tailed), **, p < 0.01.

Figure 2

Histology of germination D. nobile seeds under symbiotic germination (SG) condition. (A) embryo swollen, the uncolonized embryo covered by testa; (B) protocorm with the appearance of protomeristem; (C) protocorm with a developing shoot pole; scale bar = 50 µm. Black arrows: peloton hyphae; red arrows: collapsing and degenerated peloton hyphae.

Figure 3

Overview of DEGs analysis of samples at different stages. (A) The number of up/down-regulated DEGs in each compared group. (B) Venn diagram of the number of DEGs in each compared group. (C) The analysis of KEGG enrichment in the comparison group A-S1 vs. S-S1. (D) The analysis of KEGG enrichment analysis in the comparison group A-S2 vs. S-S2.

Figure 4

Heatmap of DEGs related to GAs and IAA metabolism and signaling. (A) GAs biosynthesis and signaling, (B) IAA biosynthesis, transduction and signaling. S-S1 and S-S2: 20 and 40 days after sowing of symbiotic germination; A-S1 and A-S2: 20 and 40 days after sowing of asymbiotic germination.

Figure 5

Effect of exogenous GA₃ and PAC on P. indica colonization in D. nobile protocorm. (A–C): Morphological characters of symbiotic germination inoculated with P. indica at different treatments with GA₃ and PAC. (D–F): Histology observation of protocorm inoculated with P. indica at different treatments with GA₃ and PAC; scale bars = 50 µm. (G): Effect on protocorm formation of GA₃ and PAC treatments, five biological replicates were analyzed; t-test (two-tailed). (H): The number of hyphae peloton in protocorm at different treatments with GA₃ and PAC; five biological replicates were analyzed; t-test (two-tailed), **, p < 0.01.

Figure 6

Cell wall metabolism of D. nobile protocorms under symbiotic germination (SG) and asymbiotic germination (AG) conditions (A): the color scale bar is shown as the log10 (FPKM + 1) value in the right; the horizontal coordinates indicate the protocorm stages under AG and SG condition. SRC2 encoding leucine-rich repeat extensin-like protein, LRX4 encoding LLR extensin-like protein, EXP6 encoding expansin-A6-like, EXB16 encoding expansin-B16-like, EXP10-2 encoding expansin-A10-like. (B): Relative expression of cell wall metabolism-related genes after GA₃ and PAC treatment. Three biological repeats were analyzed: t-test (two-tailed), *, p < 0.05; **, p < 0.01.

Figure 7

The expression levels in AG and SG protocorm development by qPCR. The reference gene was Dnactin. The bar chart and the left y-axis represent the FPKM values. The line graph and y-axis represent the relative expression levels by qPCR. Three biological repeats were performed: t-test (two-tailed), *, p < 0.05.

Figure 8

Proposed working model for the role of GA₃ and GA inhibitor in D. nobile–P. indica symbiosis by regulating cell wall loosen-related genes expression. GA inhibited the symbiosis establishment and protocorm growth by regulating the level of cell wall metabolism gene expression. At high GA level conditions, the extensin code genes LRX4 and SRC2 are upregulated, and the expansin code genes EXP6, EXB16, and EXP10-2 are downregulated, leading to cell wall stiffening, resulting in the inhibition of fungi colonization. At low GA level conditions, the extensin code genes LRX4 and SRC2 are downregulated, and the expansin code genes EXP6, EXB16, and EXP10-2 are upregulated, leading to cell wall loosening, resulting in promoting fungi territory expansion and inhibiting protocorm growth.