Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus

Abstract

Background: Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus.

Results: The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log₂ FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and β-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination.

Conclusions: This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo.

Keywords: Coprinellus disseminatus; Cremastra appendiculata; Lignin degradation; Seed germination; Transcriptome analysis.

Fig. 1

Phenotypes of the seeds of C. appendiculata symbiotically germinated with C. disseminatus DJF-10 at different stage after sowing. Zero day, six days, twelve days, twenty-five days indicated symbiotic seeds at 0 day, 6 days, 12 days and 25 days after sowing, respectively

Fig. 2

Lignocellulose content and percentage of water in C. appendiculata seeds during symbiosis. CA, SY1, SY2 and SY3 indicated symbiotic germination at 0 day, 6 days, 12 days, and 25 days after sowing, respectively

Fig. 3

FTIR spectra of C. appendiculata seeds at different stages of symbiotic germination with C. disseminatus. CA, SY1, SY2 and SY3 indicated symbiotic germination at 0 day, 6 days, 12 days and 25 days after sowing, respectively

Fig. 4

Statistical analysis of the annotated and differetially expressed unigenes (DEGs) during C. appendiculata symbiotic seed germination. A. Numbers of unigenes annotated into Swissprot, Pfam, GO, KOG, KEGG, NT, NR and total number of annotated unigenes. B. Functional classification of unigenes annotated into KOG. Capital letters A-Y represent the functional categories. C. Venn diagram of all DEGs. D. Up/down-regulated unigenes in all development stages. The total number of DEGs peaked between SY1 and CA. CA, SY1, SY2 and SY3 indicated the symbiotic seeds at 0 day, 6 days, 12 days, and 25 days after sowing,respectively

Fig. 5

Cluster heatmap of response to water and ABA biosynthesis term-related gene expression in C. appendiculata during symbiotic germination. CA, SY1, SY2 and SY3 represented symbiotic seeds at 0 day, 6 days, 12 days and 25 days after sowing, respectively