Kobe University Repository : Kernel Comparison of green and albino individuals of the partially タイトル mycoheterotrophic orchid Epipactis helleborine on molecular identities Title of mycorrhizal fungi, nutritional modes and gene expression in mycorrhizal roots Suetsugu, Kenji / Yamato, Masahide / Miura, Chihiro / Yamaguchi, 著者 Katsushi / Takahashi, Kazuya / Ida, Yoshiko / Shigenobu, Shuji / Author(s) Kaminaka, Hironori 掲載誌・巻号・ページ Molecular Ecology,26(6):1652-1669 Citation 刊行日 2017-03 Issue date 資源タイプ Journal Article / 学術雑誌論文 Resource Type 版区分 author Resource Version © 2017 John Wiley & Sons This is the peer reviewed version of the following article: [Molecular Ecology, 26(6):1652-1669, 2017], which has 権利 been published in final form at http://dx.doi.org/10.1111/mec.14021. Rights This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. DOI 10.1111/mec.14021 JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/90004566 PDF issue: 2021-10-05 1 Comparison of green and albino individuals of the partially mycoheterotrophic 2 orchid Epipactis helleborine on molecular identities of mycorrhizal fungi, nutritional 3 modes, and gene expression in mycorrhizal roots 4 5 Kenji Suetsugu1,†,*, Masahide Yamato2,†, Chihiro Miura3,†, Katsushi Yamaguchi4, Kazuya 6 Takahashi2, Yoshiko Ida5, Shuji Shigenobu4, Hironori Kaminaka3* 7 8 1 Department of Biology, Graduate school of Science, Kobe University, Kobe, Japan 9 2 Faculty of Education, Chiba University, Chiba, Japan 10 3 Faculty of Agriculture, Tottori University, Tottori, Japan 11 4 Functional Genomics Facility, NIBB Core Research Facilities, National Institute for 12 Basic Biology, Okazaki, Japan. 13 5 Graduate School of Agriculture, Tottori University, Tottori, Japan 14 15 †These authors contributed equally to this work. 16 17 Corresponding author 18 19 Kenji Suetsugu 20 E-mail address: [email protected] 21 22 Hironori Kaminaka 23 E-mail address: [email protected] 24 Runing title: Comparison of green and albino Epipactis 1 25 Abstract 26 Some green orchids obtain carbon from their mycorrhizal fungi, as well as from 27 photosynthesis. These partially mycoheterotrophic orchids sometimes produce fully 28 achlorophyllous, leaf-bearing (albino) variants. Comparing green and albino individuals 29 of these orchids will help to uncover the molecular mechanisms associated with 30 mycoheterotrophy. We compared green and albino Epipactis helleborine by molecular 31 barcoding of mycorrhizal fungi, nutrient sources based on 15N and 13C abundances, and 32 gene expression in their mycorrhizae by RNA-seq and cDNA de novo assembly. 33 Molecular identification of mycorrhizal fungi showed that green and albino E. helleborine 34 harbored similar mycobionts, mainly Wilcoxina. Stable isotope analyses indicated that 35 albino E. helleborine plants were fully mycoheterotrophic, whereas green individuals 36 were partially mycoheterotrophic. Gene expression analyses showed that genes involved 37 in antioxidant metabolism were up-regulated in the albino variants, which indicates that 38 these plants experience greater oxidative stress than the green variants, possibly due to a 39 more frequent lysis of intracellular pelotons. It was also found that some genes involved 40 in the transport of some metabolites, including carbon sources from plant to fungus, is 41 higher in albino than in green variants. This result may indicate a bidirectional carbon 42 flow even in the mycoheterotrophic symbiosis. The genes related to mycorrhizal 43 symbiosis in autotrophic orchids and arbuscular mycorrhizal plants were also up- 44 regulated in the albino variants, indicating the existence of common molecular 45 mechanisms among the different mycorrhizal types. 46 47 Key words: Epipactis, mixotrophy, mycoheterotrophy, mycorrhizae, partial 48 mycoheterotrophy, RNA sequencing, stable isotope, transcriptome analysis 2 49 Introduction 50 51 The largest family in the plant kingdom, the Orchidaceae, comprises approximately 52 25,000 species (Chase et al. 2015). One of the distinctive features of this family is the 53 production of numerous minute seeds that depend on their associations with mycorrhizal 54 fungi for the supply of carbohydrates and other nutrients until they develop into 55 photosynthetic seedlings (Leake 1994). 56 The mycoheterotrophic nature of orchid protocorms makes them particularly 57 predisposed to the evolution of life-long mycoheterotrophy, retaining their 58 achlorophyllous status throughout their entire lifecycle (Leake 1994). Indeed, full 59 mycoheterotrophy is surprisingly common among orchids, with more than 1% of all 60 species having completely lost the ability to photosynthesize (Bidartondo 2005). This 61 strategy probably allows them to colonize the dark forest understory and to occupy niches 62 less accessible by photosynthetic plants (Bidartondo et al. 2004). Though the vast 63 majority of orchids develop photosynthetic pigmentation with putative full autotrophy at 64 adulthood (Cameron et al. 2006), several linages of orchids appear to adopt a partially 65 mycoheterotrophic strategy that allows them to obtain carbon from their mycorrhizal 66 fungi, as well as from their own photosynthesis (Gebauer & Meyer 2003). The trophic 67 status of a plant appears to be an important factor determining the type of mycorrhizae 68 they harbor. The majority of putatively autotrophic orchids form associations with fungi 69 belonging to the anamorphic-form genus Rhizoctonia in the Basidiomycota (Roberts 70 1999). This is in contrast to most fully mycoheterotrophic and partially 71 mycoheterotrophic species, which interact mainly with ectomycorrhiza-forming fungi, 72 such as the Sebacinales, Russulaceae, and Thelephoraceae, suggesting that their ultimate 3 73 source of carbon is the photosynthate produced by nearby trees (Selosse & Roy 2009 but 74 also see Ogura-Tsujita et al. 2009; Lee et al. 2015; Gebauer et al. 2016). 75 The partially mycoheterotrophic state is often considered to be an intermediate 76 evolutionary step toward full mycoheterotrophy because phylogenetic analyses have 77 shown that full mycoheterotrophy evolves after the establishment of partial 78 mycoheterotrophy, rather than through a direct shift from autotrophy to 79 mycoheterotrophy (Selosse & Roy 2009; Motomura et al. 2010). Some partially 80 mycoheterotrophic orchids, such as Epipactis and Cephalanthera species, produce fully 81 achlorophyllous leaf-bearing variants that can reach almost the same size as their green 82 counterparts (Selosse & Roy 2009). The genetic basis for this albinism remains unknown, 83 but such albinism could result from permanent mutations, as suggested by the stability of 84 this phenotype over a period of years (Tranchida-Lombardo et al. 2010; Roy et al. 2013). 85 Notably, these albinos exhibit a physiological ecology distinct from that of their green 86 counterparts but the same as that of fully mycoheterotrophic plants, having a highly 87 elevated 13C: 12C ratio (Abadie et al. 2004; Julou et al. 2005; Stöckel et al. 2011). 88 Therefore, examining mixed populations of green and albino variants in species 89 that usually adopt a partially mycoheterotrophic lifestyle provides a unique opportunity 90 to investigate the physiological differences between partial mycoheterotrophy and full 91 mycoheterotrophy (Roy et al. 2013). Evolutionary shifts from autotrophy to 92 mycoheterotrophy are usually accompanied by the coevolution of a broad range of other 93 phenotypes, including a reduction of vegetative structures, degeneration of the stomata, 94 changes in the pattern of dormancy, and the development of novel defenses against 95 herbivores (reviewed by Imhof et al. 2013; Hynson et al. 2013; Waterman et al. 2013). 96 However, albino mutants of partially mycoheterotrophic orchids do not possess some of 4 97 the characteristics associated with the usual mycoheterotrophic evolution because some 98 traits are inherited intact from photosynthetic individuals. This indicates that the transition 99 to full mycoheterotrophy requires the progressive coevolution of these additional traits, 100 while the sudden loss of photosynthesis (i.e., albinism) leads to the production of unfit 101 plants, even though they can parasitize their fungal partners throughout their entire 102 lifecycle (Roy et al. 2013). 103 Albino variants of partially mycoheterotrophic species undoubtedly show 104 increased dependence on their mycorrhizal fungi as a source of carbon. Therefore, green 105 and albino variants of partially mycoheterotrophic orchids provide a fascinating model 106 for comparing the physiological changes that occur in the mycorrhizae of partially 107 mycoheterotrophic and mycoheterotrophic orchids that share similar genetic backgrounds 108 (Selosse & Roy 2009). Although some studies have investigated the spectrum of green 109 and albino variants of mycorrhizal fungi using molecular identification (Selosse & Roy 110 2009), no previous studies have conducted a detailed assessment of the molecular 111 physiology of the two variants. Indeed, the molecular mechanisms underlying orchid 112 mycorrhizal symbiosis remain poorly understood (Selosse 2014; Zhao et al. 2014). Since 113 previous studies have only focused on putative autotrophic orchids that form associations 114 with fungi belonging to Rhizoctonia (Perotto et al. 2014; Valadares 2014; Zhao et al. 115 2014), further investigations are needed. 116 In the current study, we focused on the coastal orchid Epipactis