Transcriptome analysis provides insights into the non-methylated lignin synthesis in Paphiopedilum armeniacum seed

doi:10.21203/rs.2.13152/v1

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Research article

Transcriptome analysis provides insights into the non-methylated lignin synthesis in Paphiopedilum armeniacum seed

https://doi.org/10.21203/rs.2.13152/v1

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published 29 Jul, 2020

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Abstract

Backgrounds Paphiopedilum is an important genus of orchid family (Orchidaceae) with high horticultural value. The wild populations are under the threat of extinction because of over collection and habitat destruction. Mature seeds of most Paphiopedilum species are difficult to germinate, which severely restricts the germplasm resources protection and commercial production. The germination inhibition factors are largely unknown. Results In this study, we found large amounts of non-methylated lignin were accumulated during seed maturation of Paphiopedilum armeniacum (P. armeniacum), which negatively correlates with the germination rate. We then further compared the transcriptome profiles of P. armeniacum seed at different development stages to explore molecular clues for the non-methylated lignin synthesis. KEGG enrichment analysis showed that a large number of genes associated with phenylpropanoid biosynthesis and phenylalanine metabolism as the seed maturation were differentially expressed. Several key genes in the lignin biosynthetic pathways displayed different expression patterns during the lignification process. PAL, 4CL, HCT and CSE were up-regulated to accelerate the C and H lignin accumulation. The expression of CCoAOMT, F5H and COMT were maintained at a low level or down-regulated to inhibit the conversion to the typical G and S lignin. Quantitative real-time RT-PCR analysis confirmed the altered expression levels of these genes among seeds and vegetative tissues. Conclusions This work demonstrated the plasticity of natural lignin polymer assembly in seed, and provided a better understanding of the molecular mechanism of seed-specific lignification process.

Epigenetics & Genomics

Paphiopedilum armeniacum

lignin

transcriptome analysis

germination

Paphiopedilum Pftzer (Orchidacease) are commonly known as Lady’s slipper orchids because of their slipper-shaped pouch. Members of this genus have high ornamental value because the unique flowers are available in a wide variety of colors, sizes and shapes. Besides the horticultural value, wild populations of Paphiopedilum are under the threat of extinction due to over-collection and habitat destruction, and all species are listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix I and their trade is prohibited¹.

Many terrestrial orchid seeds including Paphiopedilum are tiny and contain no endosperm, in contrast to well-developed embryos in most angiosperm seeds. In the natural habitat, Paphiopedilum seeds are generally unable to germinate on their own. They need to engage in a mycorrhizal relationship with fungus that help to feed the emerging seedling^2,3. Even offering enough nutrient supply in asymbiotic germination, mature seeds of most Paphiopedilum species are still difficult to germinate, which severely restricts their conservation and large-scale production. Paphiopedilum seeds developed heavily lignified secondary cell walls to reinforce mechanic support and to make it impermeable to water and nutrients^4,5. In the natural habitat, this feature will enhance the survival of orchid seeds in harsh conditions. However, several studies have implied that the accumulation of lignin contributed to the germination inhibition during aseptic germination ^6,7.

Lignin is a phenolic polymer formed by oxidative polymerization of the primary hydroxycinamyl alcohols (monolignols), ρ-coumaryl, caffeyl, coniferyl, sinapyl and 5-hydroxylcinnamyl alcohols, to give rise to ρ-hydroxylphenyl (H), catechyl (C), guaiacyl (G), syringyl (S) and 5-hydroxyguaiacyl (5H/5-OH-G) lignin. The five monolignols differ by their degree and position of methoxylation. The H and C units are non-methoxylated, whereas the G and 5H units are singly methylated on the 3-hydroxyl group and the S subunit is methylated on both the 3- and 5-hydroxyl moieties⁸. Lignin composition varies among plant species and tissue types. Lignins found in the vascular tissues are mainly composed of G and S units. While seed lignins have a significant variation in monolignol compositions. In addition to the typical H, G and S units, C unit is found in the seed of certain species belonging to Orchidaceae, Cactaceae, Cleomaceae, and Euphorbiaceae^9-11. The significance of this variation remains unknown. The non-methylated monolignols are incorporated into lignin polymers via benzodioxane bonds forming a linear structure without side chains^12,13. The linear lignin has less cross-linking with other cell wall components, and is capable of enhancing the hydrophobicity and stability of the plant tissue¹⁴. Lignin biosynthesis pathway is well established, involving functionalization of intermediated by O-methyltransferase (CCoAOMT and COMT) to generate monolignols differing in their degree of methylation⁸. Disruption or downregulation of those genes can lead to the lignin composition changed, and therefore change the cell wall integrity and mechanical properties of the tissue^15,16.

Here, we describe the presence of non-methoxylated H and C lignin in the seed of Paphiopedilum armeniacum (P. armeniacum). The deposition of the non-typical lignin started at the early stage of the seed development of P. armeniacum, and eventually made up approximately 39% of the total seed dry mass. Despite the unique structure and potentially important function, the biosynthesis of lignins in plant seeds has received little attention, relative to that in vascular tissues. In this study, a detailed gene expression profiling was performed at five key development stages of P. armeniacum seeds to help us understand the seed-specific regulation of lignin biosynthesis and potential regulator factors.

Lignin accumulation during seed development

Several studies suggested the heavily lignified seed coat contributed to the germination inhibition in several species of orchids^3,6,7. The germination was scored along with the lignin content during the seed development (Figure 1B). At the early stage of the seed development (45-95 DAP), the germination rate gradually increased as the embryo formation. At 95 DAP when the globular embryo has been fully developed, the germination rate reached 96.2%. During this stage, lignin was maintained at a relatively low level (5-9% of the dry mass). After that, even though the starch and lipid globules continue to accumulate as described in our previous study³, the germination rate dropped sharply from 96% to 5%. Meanwhile, the amounts of lignin started to increase rapidly, as the seed coat turning from white to brown (Figure 1A). Lignin accumulation reached a plateau (~39% of the dry mass) at 150 DAP, by which time the seed coats became dark and hard.

Identification of non-methylated lignin in seeds

To analyze the monolignol composition from various tissue types, the samples were subjected to pyrolysis-gas chromatography (Py-GC/MS). The identities and relative molar abundances of the released compounds are listed in Table 1. The results indicated the lignin in the seeds contain high levels of C units and H units without any trace of G and S units. In contrast, lignins present in other tissues (stems, leaves and pods residues after seed isolation) were composed mainly of G and S units with essentially no C units.

Previous work has established the assignments for C lignin and G/S lignin in FTIR and NMR spectra in orchid seeds^9-11 . Next, the composition of monolignol structure in seeds was further characterized by those two techniques. FTIR spectra from the seeds at five developmental stages indicated C lignin deposited in the early stage with distinct C lignin bands at 1154 cm^-1 and 823 cm^-1(Figure 2A). The rapid development of those two bands was observed at 108 DAP. In agreement with Py-GC analysis, no typical G/S lignin was detected in P. armeniacum seeds of all five developmental stages. Analysis of the aromatic region of the 2D NMR spectra of mature seeds confirmed the presence of H and C lignin (Figure 2B). The results indicated that the monolignol composition varied among tissue types in P. armeniacum, and the non-methylated C lignin and H lignin specifically deposited in the seed.

RNAseq analysis, de novo assembly and functional annotation

To characterize the transcriptome dynamics of P. armeniacum seed development and to identify genes involved in the seed-specific lignification, we performed an RNA-seq analysis of P. armeniacum seeds at five developmental stages (66, 87, 108, 122 and 150 DAP). Three biological replicate sequencing libraries were prepared from each stage. A total of 104.12 Gb clean data were originated from each library after filtering out low-quality data. The transcriptome details for each sample are given in Table S1. Q30 of the raw data ranged from 92.10 to 94.11% indicating a high-quality reads worthy of further analysis. Since no reference genome is available for Paphiopedilum, we de novo assembled the total 347,075,856 reads into 433,854 transcripts with an N50 length of 1180 bp and 183,737 unigenes with an average length of 860 bp, respectively. The transcripts and unigenes length distribution are shown in Table S2

For annotation, 183,737 unigenes were subjected to BLASTX searches against the sequences in the NCBI non-redundant protein sequences (NR), Swissprot, Gene Ontology (GO), the Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. As a result, a total of 92,235 unigenes (50.20% of all unigenes) could be assigned at least one putative function from one of these databases (Table S3). A total of 89,285 unigenes were annotated to the NR database. In a further analysis of the matched sequences, P. armeniacum transcript was highly similar to those Dendrobium catenatum (29.86%), Apostasia shenzhenica (8.38%) and Phalaenopsis equestris (5.45%), as shown in Figure 3A. For GO annotation, a total of 29,301 unigenes from P. armeniacum seeds were annotated into three GO pathways, as shown in Figure 3B. The functions of unigenes in biological process classifications contained cellular process, metabolic process and biological regulation. Cell part, cell and organelle were the most abundant functions in terms of cellular component classifications. The most abundant biological process functions are metabolic process and cellular process. In the molecular function classification, binding and catalytic activity were more abundant. The main GO entries revealed that the cells divided frequently during seed development, and some catalytic, metabolic and binding activities were relatively high.

Differential gene expression and KEGG enrichment analysis

With the restrictive conditions of False Discovery Rate (FDR) < 0.05 and log2 ratio > = 1.0, unigenes that were differentially expressed in the seeds at five stages were identified. In total 8,722 differentially expressed genes (DEGs) were identified among all the libraries. The number of DEGs increased with seed development, and peaked among 122 DAP and 66 DAP seeds (Figure 4A). A total of 576 common DEGs were identified in 87 DAP, 108 DAP, 122 DAP and 150 DAP among all developmental stages, implying that these DEGs might be responsible for seed development (Figure 4B).

To explore more insight into the DEGs regulating the lignin deposition at the later stage of seed development, we further performed KEGG enrichment analysis with the DEGs in 87 DAP vs 66 DAP, 108 DAP vs 66 DAP, 122 DAP vs 66 DAP and 150 vs 66 DAP (Figure 5). The top-enriched KEGG pathways of these DEGs were secondary metabolite biosynthesis pathways, including flavonoid, phenylpropanoid and flavone and flavonol biosynthesis pathways, etc. Among them, flavone and flavonol biosynthesis (ko00944) and flavonoid biosynthesis (ko00941) are highly enriched throughout the five key development stages, implying those pathways are generally important for seed development such as pigment accumulation, but not specifically related to lignin deposition. Our premise is that genes involved in non-methylated lignin accumulation would be induced primarily during the lignin rapid accumulation stage (from 101 DAP to 150 DAP). The analysis showed that phenylpropanoid biosynthesis (ko00940) and phenylalanine metabolism (ko00360) are indeed highly enriched in 108 DAP vs 66DAP, 122DAP vs 66DAP and 150 vs 66DAP. Other highly enriched pathways were DNA replication (ko03030) in 87DAP vs 66DAP, starch and sucrose metabolism (ko00500) in 87DAP vs 66DAP and 108DAP vs 66DAP, plant hormone signal transduction (ko04075) in 150DAP vs 66DAP, etc.

Identification of genes potentially involved in non-methylated lignin biosynthesis

The monolignol biosynthesis pathway is well established, and a series of enzymatic reactions catalyzed by specific enzymes have been identified, including phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate: CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), 4-coumarate 3-hydroxylase (C3H), caffeoyl shikimate esterase (CSE), ferulic acid 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT), and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT)^8,13 (Figure 6). We used the annotated lignin pathway gene in KEGG database, and identified 80 Paphiopedilum genes that are homologous to genes potentially involved in monolignol synthesis (Table S5).

Most lignin-related genes displayed a very similar transcript level between 66 DAP and 87 DAP, when the rapid accumulation of lignin has not started. Several monolignol synthesis related genes PAL, 4CL, HCT and CSE demonstrated increased expression levels from 87 DAP to 150 DAP. Despite the high expression level of these genes, most CCoAOMT, COMT and F5H were absent or down-regulated from developing seeds, and a few of them displayed a significant decrease in the expression level. CCoAOMT expression level is relatively high at 66 DAP. But the low expression level of F5H made the conversion of caffeoyl moieties to feruloyl moieties inefficient, which resulted in no G and S lignin production at the early stage of the seed development. Later, the decrease in the expression level of CCoAOMT and upregulation of upstream genes in monolignol pathway caused the accumulation of H and C lignins instead of G and C lignins.

qRT-PCR validation of candidate genes involved in lignin biosynthesis

To verify the accuracy and reproducibility of the RNA-seq results, we selected contigs corresponding to PAL, 4CL, HCT, C4H, CCoAOMT, F5H and COMT in five development stages of seed development for qRT-PCR validation. Methylated lignin rich tissue stems were also included to verify our predicted monolignol pathway. The sequences of the primers used are given in Table S6. In agreement with the transcriptomic data, PAL, 4CL and HCT showed increased expression level as the seed approach maturity (Figure 7). CCoAOMT displayed relatively high transcript levels at the early stage of the seed development, and low transcript level during the later stage. F5H and COMT were also down-regulated during seed development. The results confirm the reliability of the transcriptomic data. Stems displayed relatively high transcript levels of CCoAOMT, in contrast to the mature seeds, which suggested that down-regulation of the first lignin methylation gene CCoAOMT was crucial for non-methylated lignin production.

Transcription factors (TFs) potentially involved in lignin synthesis

A total of 944 unigenes that encode TFs associated with 54 families were identified from our assembled transcripts. 40, 188, 274 and 247 differentially expressed TFs were found in 87 DAP vs 66 DAP, 108 DAP vs 66 DAP, 122 DAP vs 66 DAP and 150 vs 66 DAP (Table S6). The number of DEGs peaked at 122DAP vs 66DAP. At this stage, the bHLH family contained the most DEGs (28), followed by the C2H2 (22), ERF (21), NAC (18), WRKY (17), GRAS (17), MYB (16) and MYB-related (12). Lignin deposition is mainly regulated by TFs, such as MYB and NAC^8,17,18. In our RNA-seq dataset, several differentially expressed MYB was screened out (Figure 8B). MYB2 and MYB308 displayed preferential expression in rapid lignin accumulation during seed development. These two TFs are positive regulators of most structure genes including 4CL, HCT and C3H in Ecalyptus¹⁹. MYB4 was down-regulated as the seed approach maturity. MYB4 is a positive regulator of CCoAOMT and a negative regulator of PAL, C4H, 4CL in Arabidopsis and Pinus tadeda^20,21. MYB21 is a negative regulator of CCoAOMT, the expression level of other structural genes remain unchanged in Poplar¹⁸. The expression of these TFs agreed with the expression of major structural genes involved in monolignol pathways, implying a potential role in lignin synthesis regulation.

In contrast to well-developed embryo and endosperm present in most angiosperm seeds, the structure of orchid seeds is quite simple with a balloon like seed coat and a globular embryo. There is no endosperm. Orchids typically have tiny wind dispersed seeds, often called “dust seeds” ²². Most species are difficult to germinate on their own under natural conditions. P. armeniacum is one of the most difficult species for propagation, and the difficulty in germination severely restricted its large-scale production. The factors that inhibit germination are still unknown. Seed germination of Paphiopedilum and other orchid species is significantly affected by the degree of seed maturity ^7,23. Immature orchid seeds with a low degree of lignification, exhibit higher germination percentages than mature seed ^2,24. Enhance germination of mature seeds was documented after seed lignin degradation^2,6,25. Based on these findings, some researchers have proposed that the high amounts of lignin accumulation contributed to the germination inhibition. In this study, we first measured the lignin content during the seed development process to check whether the lignin content correlates with the germination rate. During the early stage of the development, the germination rate gradually increased as the embryo became fully developed with lignin maintained at a low level. The germination rate abruptly dropped when the seed lignin started rapid accumulating around 95 DAP. The results implied that the level of lignin negatively correlated with the germination rate. The lignin-rich seed coat provided mechanical restraint by preventing water uptake or interfere with diluting germination inhibitors such as abscisic acid⁴.

Besides the content, the structure and composition of lignin have been shown to impact the cell wall mechanical properties²⁶. Here, we discovered that lignins in the Paphiopedilum seeds are composed of H and C units, as clearly evident from Py-GC, two-dimensional NMR and FTIR analysis (Table 1and Figure 2). While the lignins in other parts of the Paphiopedilum plant are composed of typical G and S units. This discovery raised an important question: what’s the mechanism for regulating the seed specific deposition of non-methylated lignin. For non-model species lacking a well-studied genetic background, transcriptome analysis is a powerful approach to seeking answers for this question. Based on the dynamics of lignin accumulation and key anatomical features in embryo development, we selected five key stages for transcriptome analysis. A total of 104.12 Gb clean data were obtained from15 RNA-seq libraries of 45, 66, 87, 108, 122 and 150 DAP. The assembled data had an average N50 length of 1180 bp, which is similar to that in other orchid species, such as Dendrobium catenatum and Apostasia shenzhenica ^27,28.

Phenylpropanoid biosynthesis and phenylalanine metabolism are highly enriched during lignin rapid accumulation stages. Further DEGs analysis of phenylpropanoid biosynthesis pathway showed that major structural genes were upregulated with the exception of CCoAOMT, F5H and COMT. Both CCoAOMT and COMT catalyze the O-methylation of the C3 and C5 hydroxyl groups of lignin precursors during G and S lignin formation. Earlier studies demonstrated that low O-methyltransferase (both CCoAOMT and COMT) enzyme activities in the C-lignin containing seed of Cleome hasslerianan (C. hasslerianan) ¹⁵. CCoAOMT is the first OMT in monolignol pathway. Caffeyl alcohol would be formed if CCoAOMT became depleted or lost its function. Down regulation of CCoAOMT in Pinus radiata introduced caffeyl alcohol into lignification, resulting in low levels of C units present in the G lignin dominant tissues¹⁶. In contrast to pine, suppression of CCoAOMT in several angiosperm species such as Arabidopsis, alfalfa, poplar and tobacco did not result in the incorporation of caffeyl alcohol ^29-31. This suggested other factors might also determine the lignin composition, such as transcriptional regulation or availability of the specific monolignol transporters. NAC and MYB families have been identified as regulators of lignin deopsotion^8,17,18. Instead of the master switches, we are interested in the TFs that can specifically regulate CCoAOMT, since these TFs can control the non-methylated lignin accumulation. We identified a few MYBs that are regulators of CCoAOMT, such as MYB4 and MYB21. These TFs are good candidates for regulating different monolignol biosynthesis. Overall, the low expression level of CCoAOMT, COMT and F5H is likely the biochemical basis for the accumulation of non-methylated lignin in seeds. The detailed mechanism towards this fine coordination of monolignol biosynthetic genes needs further exploration.

In summary, the presence of H and C lignin in Paphiopedilum seeds exemplifies the flexibility in the lignin monomers assembly in nature. Transcriptome profiling revealed the lignin related candidate genes expression pattern, which clarified the seed specific monolignol pathways. However, the seed monolignol composition is not consistent even within the same families of Cypripedium, Euphorbiaceae and Cleomaceae plants, suggesting the feature is not conservative. The difference in the degree of methylation and molecular structure of C and H lignin strongly suggests a functional difference compared to typical G and S lignin. Future work should focus on linking these unique non-methylated lignin structure to the mechanistic role for seed development and germination.

Plant materials and Growth Conditions: P. armeniacum S. C. Chen et F. Y. Liu plants were maintained in a glass greenhouse in the South China Botanical Garden, Guangzhou, China. The plants were potted in a substrate of Zhijing stone for orchids under 800 μ mol m⁻²s⁻¹ natural light maintained by a sunshade net. The average temperature and relative humidity ranged from 10–32 °C and 70–98%, respectively. The flowers from three-year-old adult plants were labeled and artificially self-pollinated. Capsules of different developmental stages from 45 DAP to 180 DAP were collected at 7-day intervals from grown plants. Seeds were removed from capsules, and immediately frozen in liquid nitrogen.

Seed germination assay: Seed capsules were surface sterilized by the method described previously³². More than 100 seeds were sown on Hyponex N026 medium supplemented with 1.5 g/ l activated charcoal, 2 g/L peptone, 15g/L sucrose and 5% coconut water. Germination was scored after embryo swollen and testa rupture.

Lignin content and composition: Lignin content was estimated using the acetyl bromide method with slight modification³³. Five (5) mg of finely ground seed was thoroughly exacted with ethanol/toluene (1:1) until the extracts no longer absorbed UV light at 280 nm. The dried samples were placed into loosely capped glass tubes containing 1 mL of acetyl bromide/ acetic acid (1:3) and incubated at 70°C for 30 min. The sample was then cooled in an ice bath and then mixed with 0.9 mL of 2 M NaOH, 5 mL glacial acetic acid, and 0.1 mL 7.5 M hydroxylamine hydrochloride. The final volume was adjusted to 10 mL with glacial acetic acid and the absorbance measured at 280 nm with a microplate reader (Tecan Infinity). A standard curve was generated with alkali lignin (Sigma-Aldrich, 471003) for lignin content calculation.

The chemical composition of lignin was analyzed by pyrolysis-gas chromatography (GC)/ mass spectrometry (MS) using a previously described method with some modifications³⁴. The pyrolysis was performed on Pyroprobe 5000 (CDS Analytical Inc.) with direct connection to Shimadzu GCMS-QP2010A equipped with HP-5MS column (30 m×0.25 mm×0.25 mm). The pyrolysis was carried out at 550 °C. The chromatograph was programmed from 50 °C to 250 °C at a rate of 15 °C/min, and the final temperature was held for 10 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The mass spectrometer was operated in scan mode and the ion source was maintained at 300 °C. The compounds were identified by comparing their mass spectra with those of the NIST library and those previously reported^35,36. Peak molar areas were used to calculate the lignin degradation products, and the summed areas were normalized.

Fourier transform infrared spectroscopy (FTIR): The dried samples were embedded in KBr pellets in the concentration of about 1 mg/100 mg KBr. FTIR spectra in the range of 4000–400 cm⁻¹ were recorded by using a Shimadzu IRAffinity-1S FTIR spectrophotometer. The spectra were recorded in the absorption mode at 64 scans per sample with a resolution of 4 cm ⁻¹.

2D ¹³C-¹H heteronuclear single-quantum coherence (HSQC) NMR spectroscopy: Seeds from 122 DAP was extracted and ball-milled as previously described³⁷. The gels were formed using DMSO-d₆/pyridine-d₅ (4:1) and sonicated until homogenous. The homogeneous solutions were transferred to NMR tubes. HSQC spectra were acquired at 25 °C using a Bruker Avance-500 MHz instrument. The detailed running conditions and assignment of the spectra was described elsewhere¹¹.

RNA extraction, library construction and RNA sequencing: The five different development stages analyzed were 66, 87, 108, 122 and 150 days after pollination (DAP). The total RNA was extracted using Column Plant RNAout2.0 (Tiandz Inc., Beijing, China) according to the manufacturer’s protocol, which was specifically designed for materials rich in polysaccharides and polyphenolics. Extracted RNA was treated with DNase (Tiandz Inc., Beijing, China) to remove genomic DNA. The RNA quality was validated using agarose gel electrophoresis, Nanodrop One (Nanodrop Technologies Inc., DE, USA), and Agilent 2100 (Agilent Technologies Inc., CA, USA) to confirm the purity, concentration, and integrity, respectively. Library construction and sequencing were performed using Illumina HiSeq4000 platform (Illumina Inc., CA, USA) by Genepioneer Technologies Corporation (Nanjing, China).

De novo assembly, functional annotation of unigenes and DEGs analysis: The clean data was generated by removing adaptor sequences, ambiguous reads (‘N’ > 10%), and low-quality reads (that is, where more than 50% of bases in a read had a quality value Q ≤ 5) using perl script. All sequence data was uploaded into the BioProject databased hosted by the National Center for Biotechnology Information (NCBI) under the BioProject PRJNA550294. The transcripts were assembled using Trinity v2.4.0 program with default parameters³⁸, and gene expression was estimated by applying the fragments per kilobase per million mapped reads (FPKM). Functional annotations were performed using the public databases, including the NR ³⁹, Swiss-Prot⁴⁰, COG ⁴¹ and GO⁴². Differentially expressed genes (DEGs) between libraries were identified as those with the fold change (FC) of the expression level (FC≥ 2 or FC≤0.5 under P-value ≤0.05), FDR ≤0.05). GO enrichment and KEGG enrichment were performed using the obtained DEGs. To identify genes encoding for TFs, all DEGs were compared with protein sequences downloaded from a plant TF database, PlantTFDB with the E-values threshold of 10⁻¹⁰⁴³.

Verification of gene expression using qRT-PCR: Samples of RNA-seq were reversed transcribed into cDNA for real-time qPCR validation using GoScript Reverse Transcription System (Promega, CA, USA). The qRT-PCR reactions were performed on ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA) and the SYBR Premix ExTaq Kit (Takara, Dalian, China). Primer sequences are listed in Table S7. The expression level was calculated as 2^-ΔΔCt, and normalized to the Ct values of P. armeniacum Actin (TRINITY_DN40678_c2_g1).

P. armeniacum: Paphiopedilum armeniacum; Py-GC/MS: pyrolysis-gas chromatography; DAP: Days after pollination; FTIR: Fourier transform infrared spectroscopy; DEG: Differentially expressed genes; NR: non-redundant protein sequences; GO: Gene Ontology; COG: the Clusters of Orthologous Groups; KEGG: Kyoto Encyclopedia of Genes and Genomes; FDR: False Discovery Rate; PAL: Phenylalanine ammonia lyase; COMT: Caffeic acid O-methyltransferase; CCoAOMT: Caffeoyl-CoA 3-O-methyltransferase; TFs: Transcription factors.

Acknowledgements

The authors would like to thank Yunfei Yuan for assistance with NMR experiments, Yuting Yin, Chuangxue Mao and Sha Deng for assistance with plant growth and pollination.

Consent for publication

Not Applicable.

Ethics approval and consent to participate

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and the supplementary information files. The sequence data was deposited at NCBI database

under the BioProject PRJNA550294.

Funding

This work was funded by National Key Research and Development Program of China (2016YFC0503104), Natural Science Foundation of Guangdong Province (2018A030313686), Guangdong Key Technology Research and Development Program (2018B020202001, 2017A020208036, 2017B090901050), and Guangdong Modern Agricultural Industry Technology System Program (2018LM2176, 2018LM1142), Biological Resources Program, Chinese Academy of Sciences (KFJ-BRP-007), Guangzhou Science and Technology Program (201904010305),Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences (AB2018002, AB2018026). The Project was also supported by Program from Core Botanical Gardens, Chinese Academy of Sciences.

Authors’ contributions

LF, KW and SZ conceived the research program and designed research. LF, XX, JL, FZ, JY and YL performed research, KW, ML, XZ, GM, FL and SZ analyzed the data. LF, XX, KW and SZ wrote the manuscript, AZ revised this manuscript.

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Table 1. Relative molar abundances (%) of the compounds released after pyro-GC/MS of seeds, stems, leaves and pods of P. armeniacum. Values in brackets are the standard deviations from three biological replicates.

Compound name	Origin	Formula	Molecular Mass	Main Mass fragments	Seeds (%)	Stems (%)	Leaves (%)	Pods (%)
Phenol	H	C₆H₆O	94	65,66,94	16.2 (0.1)	-	23.0 (6.9)	5.7 (1.2)
2-Methylphenol	H	C₇H₈O	108	77,107,108	18.7 (0.4)	-	-	-
3-Methylphenol	H	C₇H₈O	108	77,107,108	18.3 (0.4)	-	-	-
2,5-Dimethylphenol	H	C₈H₁₀O	122	77,107,122	7.2 (1.3)	-	-	-
Catechol	C	C₆H₆O₂	110	53,64,110	19.6 (2.7)	-	-	-
4-Methylcatechol	C	C₇H₈O₂	124	51,97, 125	13.4 (0.7)	-	-	-
4-Ethylcatechol	C	C₈H₁₀O₂	138	51,78,124	6.5 (0.1)	-	-	-
2-Methoxylphenol	G	C₇H₈O₂	124	81,109,124	-	14.9 (0.8)	16.1 (2.1)	19.0 (2.1)
2-Methoxy-5-methylphenol	G	C₈H₁₀O₂	138	95,123,138	-	5.6 (0.2)	-	13.4 (2.7)
4-Ethenyl-2-methoxyphenol	G	C₉H₁₀O₂	150	107,135,150	-	31.6 (4.6)	23.1 (3.2)	31.7 (3.2)
4-Hydroxy-3-methoxybenzaldehyde	G	C₈H₈O₃	152	109,151,152	-	4.5 (0.1)	-	5.5 (0.3)
2-Methoxy-4-propenylphenol	G	C₁₀H₁₂O₂	164	131,149,164	-	7.9 (0.1)	-	11.2 (0.6)
4-Hydroxy-3-methoxyphenyl acetone	G	C₁₀H₁₂O₃	180	122,137,180	-	4.1 (0.3)	18.1 (0.7)	-
2,6-Dimethoxyphenol	S	C₈H₁₀O₃	154	111,139,154	-	-	19.8 (4.7)	13.6 (2.6)
4-Hydroxy-3,5-dimethoxystyrene	S	C₁₀H₁₂O₃	180	137,165,180	-	31.6 (2.7)	-	-
% H units					60.5 (2.0)	-	23.0 (4.3)	5.7 (0.9)
% C units					39.5 (2.0)	-	-	-
% G units					-	68.4 (6.9)	57.2 (3.1)	80.7 (2.5)
% S units					-	31.6 (2.1)	19.8 (2.9)	13.6 (1.9)

Supplementary Table S1. Overview of transcriptome sequencing and de novo assembly results.

Supplementary Table S2. Overview of transcriptome assembly showing length distribution of transcript and unigene.

Supplementary Table S3. Summary of functional annotation of contigs from BLAST searches against public databases.

Supplementary Table S4. KEGG enrichment analysis list.

Supplementary Table S5. Lignin related genes with RPKM values.

Supplementary Table S6. Differentially expressed transcription factors (TFs) list.

Supplementary Table S7. Primers used for qRT-PCR.

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Transcriptome analysis provides insights into the non-methylated lignin synthesis in Paphiopedilum armeniacum seed

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