Transcriptome Analysis Provides Insights Into the Non-Methylated

Fang et al. BMC Genomics (2020) 21:524 https://doi.org/10.1186/s12864-020-06931-1 RESEARCH ARTICLE Open Access Transcriptome analysis provides insights into the non-methylated lignin synthesis in Paphiopedilum armeniacum seed Lin Fang1†, Xin Xu1,2†,JiLi1,2, Feng Zheng1, Mingzhi Li3, Jingwei Yan4, Yuan Li1, Xinhua Zhang1, Lin Li1, Guohua Ma1, Aying Zhang4, Fubing Lv5, Kunlin Wu1* and Songjun Zeng1,6* Abstract Backgrounds: Paphiopedilum is an important genus of the orchid family Orchidaceae and has high horticultural value. The wild populations are under threat of extinction because of overcollection and habitat destruction. Mature seeds of most Paphiopedilum species are difficult to germinate, which severely restricts their germplasm conservation and commercial production. The factors inhibiting germination are largely unknown. Results: In this study, large amounts of non-methylated lignin accumulated during seed maturation of Paphiopedilum armeniacum (P. armeniacum), which negatively correlates with the germination rate. The transcriptome profiles of P. armeniacum seed at different development stages were compared to explore the molecular clues for non-methylated lignin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that a large number of genes associated with phenylpropanoid biosynthesis and phenylalanine metabolism during seed maturation were differentially expressed. Several key genes in the lignin biosynthetic pathway displayed different expression patterns during the lignification process. PAL, 4CL, HCT, and CSE upregulation was associated with 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 in 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. Keywords: Paphiopedilum armeniacum, Lignin, Transcriptome analysis, Germination Background under threats of extinction due to overcollection and habi- Paphiopedilum Pftzer (Orchidacease) is commonly tat destruction. As a result, all species are listed in the known as Lady’s slipper orchid because of its slipper- Convention on International Trade in Endangered Species shaped pouch. Members of this genus have high ornamen- of Wild Fauna and Flora (CITES) Appendix I which also tal value because the flowers are uniquely shaped and prohibit their trade [1]. come in a wide variety of colors and sizes. Despite their Many orchid seeds including Paphiopedilum are tiny horticultural value, wild populations of Paphiopedilum are and contain no endosperm. In contrast, most angio- sperm seeds have well-developed embryos. Paphiopedi- * Correspondence: [email protected]; [email protected] lum † seeds are generally unable to germinate on their Lin Fang and Xin Xu contributed equally to this work. own. They form a mycorrhizal relationship that help 1Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China with the nourishment of emerging seedling [2, 3]. Even Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fang et al. BMC Genomics (2020) 21:524 Page 2 of 15 with the additional nutrients, mature seeds of most mass. Despite its unique structure and potentially im- Paphiopedilum species still have difficulty germinating, portant function, the biosynthesis of lignins in plant which severely restricts their conservation and large- seeds has received little attention relative to that of vas- scale production. However, the factors inhibiting ger- cular tissues. In this study, a detailed gene expression mination are still unknown. Paphiopedilum seeds profiling was performed at five key developmental stages develop heavily lignified secondary cell walls to provide of P. armeniacum seeds to help elucidate the seed- mechanical support and prevent permeability to water specific regulation of lignin biosynthesis. and nutrients [4, 5]. This feature enhances the survival of orchid seeds in harsh conditions. However, several Results studies imply that the accumulation of lignin contributes Embryo developmental features and lignin accumulation to the inhibition of germination during aseptic germin- during seed development ation [6, 7]. Immature orchid seeds with low degree of Zygotic embryos of P. armeniacum began to develop at lignification were shown to exhibit higher germination 45 days after pollination (DAP) as previously described rates than mature seed with higher lignification [2, 8]. [3]. At 66 DAP, the T-shaped pre-embryo with four cells Mature seeds subjected to lignin degradation resulted in was formed (Table 1). At 87 DAP, the globular embryo enhanced germination rates [2, 6, 9]. Based on these was formed. Around 108 DAP, there is a rapid lignifica- findings, it is possible that the high amounts of lignin tion of the seed coat, the inner testa disappears, and lipid act as germination inhibitors. globule and starch accumulate. At 122 DAP, the embryo Lignin is a phenolic polymer formed by oxidative is fully mature with a compact testa and no further ob- polymerization of the primary hydroxycinamyl alcohols served morphological changes. Five developmental (monolignols), ρ-coumaryl, caffeyl, coniferyl, sinapyl, and stages (66, 87, 108, 122, and 150 DAP) of P. armeniacum 5-hydroxylcinnamyl alcohols that give rise to ρ- embryos were selected for further lignin structure hydroxylphenyl (H), catechyl (C), guaiacyl (G), syringyl characterization and transcriptome profiles on the basis (S), and 5-hydroxyguaiacyl (5H/5-OH-G) lignin. The five of lignin accumulation pattern and embryo developmen- monolignols differ by their degree and position of meth- tal characteristics. oxylation. The H and C units are non-methoxylated, The germination was scored together with the lignin whereas the G and 5H units have a single methylation content during the seed development (Fig. 1a). During on the 3-hydroxyl group and the S subunit is methylated the early stages of seed development (45–95 DAP), the on both the 3- and 5-hydroxyl moieties [10]. Lignin germination rate gradually increased as the embryo for- composition varies among plant species and tissue types. mation. At 95 DAP, the globular embryo has been fully Lignins found in the vascular tissues are mainly composed developed, and the germination rate reached 96.2%. Dur- of G and S units. However, seed lignins have a significant ing this stage, lignin was maintained at a relatively low variation in monolignol compositions. Seed of certain spe- level (5–9% of the dry mass). After that, even though the cies belonging to Orchidaceae, Cactaceae, Cleomaceae, starch and lipid globules continue to accumulate as de- and Euphorbiaceae contain lignin with the typical H, G, scribed in a previous study [3], the germination rate and S units, as well as C unit [11–13]. The significance of dropped sharply from 96 to 5%. Meanwhile, the amounts these variations remain unknown. The non-methylated of lignin started to increase rapidly, with the seed coat monolignols are incorporated into lignin polymers via changing from white to brown (Fig. 1b). Lignin accumu- benzodioxane bonds, forming a linear structure without lation reached a plateau (~ 39% of the dry mass) at 150 side chains [14, 15]. The linear lignin has less cross- DAP, with the seed coats becoming dark and hard. linking with other cell wall components and is capable of enhancing the hydrophobicity and stability of the plant tis- Identification of non-methylated lignin in seeds sue [16]. The lignin biosynthesis pathway is well estab- To analyze the monolignol composition from various lished, involving O-methyltransferases (CCoAOMT and tissue types, the samples were subjected to pyrolysis-gas COMT) to generate monolignols differing in their degree chromatography/ mass spectrometry (Py-GC/MS). The of methylation [10]. Disruption or downregulation of identities and relative molar abundances of the released these genes can lead to changes in lignin composition, compounds are listed in Table 2. The results showed resulting in changes to cell wall integrity and mechanical that the lignin in the seeds contain high levels of C units properties

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