Lignocellulose degradation and water uptake of C. appendiculata seeds during symbiotic germination

To investigate the degradation of lignocellulose by C. disseminatus, the loss of lignocellulose dry weight and water absorption during symbiosis were measured. After 6 days of symbiotic germination of C. appendiculata with C. disseminatus, the lignin content significantly dropped from 119.48 mg g− 1 (CA) to 38.55 mg g− 1 (SY1) and the lignin degradation rate in the seeds was 73.15% at 12 days (SY2) (Fig. 2). Subsequently, with cell differentiation and protocorm development, lignin accumulation increased to 68.84 mg g− 1 at SY3, but remain much lower than that of CA. The hemicellulose content was also decreased from 55.04 mg g− 1 in CA to 38.65 mg g− 1 in SY1. The hemicellulose degradation rate was 29.78% in SY1, and significantly increased to 61.61 mg g− 1 in SY2. With protocorm formation, the hemicellulose content increased to 126.89 mg g− 1 in SY3. The cellulose content did not change significantly among different stages.

Under symbiosis, the water content increased sharply significantly from CA to SY1 (13.94 and 91.66%, respectively) and then unchanged at SY2 and SY3 (92.54 and 91.58%, respectively) (Fig. 2). These results revealed that the lignified seed coat was broken by C. disseminatus, resulting in enhanced water permeation to the embryo. The results also demonstrate that the seed coat limitation is one of the important components of seed dormancy.

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

Identification of lignocellulose in symbiotic seeds

To further validate the lignocellulose degradation in C. appendiculata seeds by C. disseminatus, the chemical compositions of different samples was characterized by FTIR and Py-GC/MS. The samples showed remarkably different spectra (Fig. 3). Bands of C-lignin in CA were evident at 1650 (strong) and 782 cm− 1. Bands of G/S lignin existed at valleys of 1592, 1515 (sharp) and 871 cm− 1 (sharp). Under symbiosis with C. disseminatus, significant reduction in the lignin signals were recorded at 1650, 1592 and 1515 cm− 1 in SY1, SY2 and SY3, respectively as compared to CA. The xylan band of 2923 cm− 1 was significantly decreased at SY1, SY2 and SY3. The absorption band at 1621 cm− 1 (arrow 1) may be associated with carbonyl and acetyl groups in xylan degradation intermediates. The band, which was associated with cellulose, disappeared at 1000 cm− 1 (arrow 2) during symbiosis. In addition, strong and sharp signals were recorded at 1766 and 1749 cm− 1, though it was not clear whether or not they were associated with lignocellulose.

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

Pyrolysis of the seed symbiotic samples released various families of compounds (Table 1). The fraction of phenolic compounds decreased significantly, while the concentrations furanics and other compounds varied in different samples. These results suggested that the lignocellulose fraction of the seeds was degraded by the symbiotic fungus. In particular, lignin degradation was more significant as demonstrated. Other non-phenolic compounds such as acetic acid and ketone groups resulted from lignin and from the efficient release of phenolic monomers.

Table 1 Part of the chemical compounds released after Py-GC/MS of C. appendiculata seeds and seedings of CA, SY1, SY2 and SY3

GC/MS analysis of low-molecular-weight products of degradation of lignocellulose of C. appendiculata seeds symbiotically germinated with C. disseminatus

To identify the products of lignocellulose biodegradation, GC-MS was used to detect the low-molecular-weight compounds in OMA symbiotic medium collected at different stages during the symbiotic germination of C. appendiculata seeds with C. disseminatus (Table 2; Fig. S1). The degradation products are listed in Table 2. More than 10 degradation products were detected. However, the CA group contained only dibutyl phthalate, suggesting that the compound might have originated from the OMA medium.

Table 2 The chemical composition of lignocellulose degradation products analyzed by GC-MS

It is well documented that benzene compounds result from degradation of lignin. Five relative compounds (benzeneethanamine, benzene, benzeneacetic acid, benzamide and benzenedicarboxylic acid) were identified in the OMA symbiotic medium of SY1 and SY2. This suggested that the lignin component of the seed coat was degraded, in agreement with FTIR and Py-GC/MS data. Furfural, acetic acid and butanoic acid are degradation products of xylan. Acetic acid was detected in SY1, SY2 and SY3, whereas butanoic acid was detected only in SY2. Furfural was not detected at any stage. Some other acids and eaters resulting from lignocellulose degradation were detected in SY1 or SY2. These results confirmed that the lignocellulose fraction of the seed coat was degraded by C. disseminatus during symbiotic germination.

RNAseq analysis, de novo assembly, and functional annotation

Analysis of RNA-seq of C. appendiculata at four developmental stages of symbiotic germination was performed to characterize the transcriptome changes during seed germination. A total of 41.06 Gb clean data were generated from each library after filtering out the low-quality data. Among raw reads in all samples, the Q30 values ranged from 91.47 to 92.28% indicating high-quality reads appropriate for further analysis (Table S1). Since no reference genome was available for C. appendiculata, all 359,510,000 reads were do novo-assembled into 48,750 CDS with an N50 length of 1398 bp and 97,800 unigenes with an average length of 1120 bp (Table S2). Samples of different biological replicates were clustered separately based on their distinct developmental stages.

For annotation, 97,800 unigenes were subjected to BLASTX search against the sequences in NR, Nt, Pfam, Swissprot, GO, KOG, and KEGG databases (Fig. 4A; Table S3). As a result, a total of 65,911 unigenes (67.39% of all unigenes) had at least one putative function from one of these databases. For NR annotation, a total of 61,803 (63.19%) unigenes were annotated. As shown in Fig. S2, 36,107 unigenes were assigned with a best score to Dendrobium catenatum (58.42%) and 14,862 ones were assigned to Phalaenopsis equestris (24.05%). For GO annotation, 45,555 unigenes were annotated into three GO categories, including cellular component (CC), molecular function (MF), and biological process (BP) (Fig. S3). The cellular component class contained cellular anatomical entity and intracellular and protein-containing complex. The most abundant molecular functions were binding and catalytic activity. Among the biological processes, cellular and metabolic processes were more abundant. Other entries such as biological regulation, localization and response to stimulus were relatively high. Unigenes among different groups were chiefly classified into carbohydrate transport and metabolism, function unknown, general function prediction only, posttranslational modification, protein turnover, chaperones, signal transduction mechanisms and transcription (Fig. 4B).

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

Functional annotation of differentially expressed genes

Differential expression analysis of gene expression at different stages of symbiotic germination

To identify the genes involved in symbiosis, we compared, as pairs, the transcriptomes of the four developmental periods of the symbiotic seeds. The pair SY1-CA, represented the symbiotic seeds at 6 days (fungal invasion) compared to 0 day (fungus-free seeds), while SY2-SY1 was the symbiotic material at 12 days (fungal colonization) compared to the stage of fungal invasion at day 6. The pair SY3-SY2 presented the transition to fungal degradation and protocorms with meristem from the fungal colonization at 12 days. A total of 15,382 genes showed significantly different expression levels (log2 FPKM≥2.0, Qvalue≤0.05) among all libraries (Fig. 4C). Among these differentially expressed genes (DEG), 5050 genes were up-regulated and 4818 ones were down-regulated in SY1-CA (Fig. 4D). In SY2-SY1, 5285 genes were up-regulated and 1893 ones were down-regulated in SY2-SY1. In SY3-SY2, 449 genes were up-regulated and 669 genes were down-regulated (Fig. 4D; Table S4). We found that the highest number of DEGs was shared between the early mycelium penetration stages (SY1-CA), followed by the early germination stage (SY2-SY1), and then the protocorm stage (SY3-SY2) (Fig. 4D).

GO enrichment analysis

To characterize the DEGs, we performed a GO enrichment analysis using Rage tool. Among the GO terms of DEGs, there were 51, 100 and 153 terms significantly overrepresented in SY1-CA, SY2-SY1 and SY3-SY2, respectively (Table S5). In total, 25 GO terms were over-represented in the three comparisons (Table 3). In particular, these terms included some significantly overrepresented GO terms related to oxidation reactions such as heme binding, monooxygenase activity, dioxygenase activity, peroxidase activity and iron ion binding. In addition, the terms of response to water, metal iron binding and endo-1,4-β-xylanase activity were annotated in SY1 versus CA. The terms of ATP binding, channel activity and cell wall macromolecule catabolic process were identified in SY2-SY1. However, xyloglucan and carbohydrate metabolic process were also found.

Table 3 Significantly overrepresented gene ontology terms during the symbiotic germination of C. appendiculata with C. disseminatus DJF-10

KEGG enrichment analysis

To investigate the differences in metabolic processes at different stages of symbiotic germination in more detail, a KEGG pathway enrichment analysis with the DEGs was conducted, based on the Qvalue≤0.05 (Table S6, S7). At SY1-CA, the categories flavonoid biosynthesis, plant hormone signal transduction, fatty acid degradation and phenylpropanoid biosynthesis were significantly enriched. The pathways of phenylpropanoid and flavonoid biosynthesis were also dramatically enriched in SY2-SY1 and SY3-SY2. Pathways for fatty acid degradation, carbon fixation in photosynthetic organisms, glycosaminoglycan degradation and carbon metabolism were enriched in SY2-SY1. Pathways of protein processing in endoplasmic reticulum, glycosaminoglycan degradation, plant signal transduction and other glycan degradation were significantly enriched between SY3-SY2. Taken together, these findings suggested that the fungus induced the expression of plant hormone signal transduction pathways which may break the seed dormancy. Moreover, it induced the expression of fatty acid degradation, carbon metabolism and endoplasmic reticulum processing, and hence improves the efficiency of utilization of stored nutrients.

Expression patterns of symbiosis-related genes at each stage

The lignified seed coat forms a hydrophobic barrier to water permeation during germination. The lignin content of the seeds was sharply reduced after symbiosis with the fungus C. disseminatus. Once the hydrophobic barrier was removed, the embryo was able to absorb water efficiently. In this study, we paid much attention to terms related to water absorption. Fifteen genes (including 9 CaDHN1 genes, 4 CaXero1 genes, 1 hypothetical protein, and 1 CaDHN4-like) related to the term response to water were down-regulated (Fig. 5; Table S8), and remained at low expression levels in SY2 and SY3. Among these genes, the expression of five CaDHN1 genes (CL1194.Contig2_All, CL1194.Contig3_All, CL1194.Contig5_All, Unigene16008_All and Unigene31280_All) decreased from 176.4, 241.7, 311.4, 166.1 and 38.2 in CA to 0.3, 0.6, 5.4, 4.1 and 0 in SY1, respectively. Two CaXero1 genes (CL1194.Contig6_All and CL1194.Contig7_All) showed high expression levels of 504.1 and 600.1, respectively at CA, but were down-regulated to 4.1 and 12.0, respectively in SY1. The expression level of a hypothetical protein (CL1194.Contig1_All) decreased from 229.7 in CA to 2.9 in SY1. The results suggested that these genes regulate the response to fungal stimulation during seed germination of C. appendiculata after symbiosis with the mycorrhizal fungus.

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

Indeed, plant hormones play a vital role in the process of symbiotic germination. The current study revealed that a large number of DEGs was annotated to signal transduction mechanisms. Carotenoid biosynthesis provides the precursors for the synthesis of ABA. In our study, zeaxanthin epoxidase ZEP (CL5276.Contig3_All), 9-cis-epoxycarotenoid dioxygenase NCED3 (CL6018.Contig1_All) and β-carotene hydroxylase (Unigene9557_All and Unigene9558_All) were significantly down-regulated in SY1-CA (Fig. 5; Table S8). Furthermore, most of the abscisic acid receptor pyrabactin resistance 1-like (PYLs) were up-regulated in SY1-CA. Seven genes of protein phosphatase 2C (PP2C), six of SNF1-related protein kinase 2 (SnRK2) and four basic region-leucine zipper (bZIP) transcription factors were down-regulated in this group (Fig. 5; Table S8).

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