Integrative Study Supports the Role of Trehalose in Carbon Transfer From Fungi to Mycotrophic Orchid

Abstract

Orchids rely on mycorrhizal symbiosis, especially in the stage of mycoheterotrophic protocorms, which depend on carbon and energy supply from fungi. The transfer of carbon from fungi to orchids is well-documented, but the identity of compounds ensuring this transfer remains elusive. Some evidence has been obtained for the role of amino acids, but there is also vague and neglected evidence for the role of soluble carbohydrates, probably trehalose, which is an abundant fungal carbohydrate. We therefore focused on the possible role of trehalose in carbon and energy transfer. We investigated the common marsh orchid (Dactylorhiza majalis) and its symbiotic fungus Ceratobasidium sp. using a combination of cultivation approaches, high-performance liquid chromatography, application of a specific inhibitor of the enzyme trehalase, and histochemical localization of trehalase activity. We found that axenically grown orchid protocorms possess an efficient, trehalase-dependent, metabolic pathway for utilizing exogenous trehalose, which can be as good a source of carbon and energy as their major endogenous soluble carbohydrates. This is in contrast to non-orchid plants that cannot utilize trehalose to such an extent. In symbiotically grown protocorms and roots of adult orchids, trehalase activity was tightly colocalized with mycorrhizal structures indicating its pronounced role in the mycorrhizal interface. Inhibition of trehalase activity arrested the growth of both symbiotically grown protocorms and trehalose-supported axenic protocorms. Since trehalose constitutes only an inconsiderable part of the endogenous saccharide spectrum of orchids, degradation of fungal trehalose likely takes place in orchid mycorrhiza. Our results strongly support the neglected view of the fungal trehalose, or the glucose produced by its cleavage as compounds transported from fungi to orchids to ensure carbon and energy flow. Therefore, we suggest that not only amino acids, but also soluble carbohydrates are transported. We may propose that the soluble carbohydrates would be a better source of energy for plant metabolism than amino acids, which is partially supported by our finding of the essential role of trehalase.

Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae)

“…Trehalose has been suggested as a fungi-derived carbon source for orchids; in Dactilorhiza majalis , the treatment by validamycin A (trehalase inhibitor) inhibited the growth of the protocorms infected with Ceratobasidium sp. [ 8 ]. In the Assembly 4, six contigs had high sequence similarity with the trehalase genes, and among them, read counts of three putative trehalase genes (evgLocus_101728.p1, evgLocus_593495.p1, and evgLocus_117844.p1) were highly detected in OM fungus-infected protocorms, but not in aseptically grown protocorms ( S2 Fig ).…”

“…In order to evaluate the validity of the contig set constructed in this study and to get any insights about genes important for the interaction between orchids and OM fungi, we estimated gene expression levels in our protocorm samples using Assembly 4 contig set as a reference. Nodulin-like genes, CSGs and SWEET genes have been shown to have roles in mutualistic plant–fungus interactions [ 51 – 56 ], and recently, the trehalase gene has been suggested to be involved in mycoheterotrophy in orchids [ 8 , 9 ]. When estimated expression levels of these genes, they were expected to be upregulated in the protocorms grown with OM fungi compared to those in the aseptic condition ( S2 Fig ).…”

Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons

“…Kristiansen et al, 2001;Selosse et al, 2004;Cameron et al, 2006;Chang & Chou, 2007;Hobbie & H€ ogberg, 2012;Bougoure et al, 2014;Kuga et al, 2014;Suetsugu et al, 2017;Favre-Godal et al, 2020). Aside from soluble carbohydrates (Cameron et al, 2006;Ponert et al, 2021), amino acids, playing a significant role in N transfer, have been recognized to participate in C transfer in orchid mycorrhiza (Cameron et al, 2006(Cameron et al, , 2008Dearnaley & Cameron, 2017;Fochi et al, 2017;Favre-Godal et al, 2020;Valadares et al, 2021). Notably, fungal proteins and amino acids are enriched in 15 N by c. 10& relative to fungal chitin, irrespective of the part of the fungus examined (Taylor et al, 1997;Hobbie & Colpaert, 2003).…”

At the core of the endomycorrhizal symbioses: intracellular fungal structures in orchid and arbuscular mycorrhiza

“…Early experiments with radioactive tracers (see Smith & Read, 2008) showed that carbon is transferred to the plant before the pelotons extensively collapsed inside OrM protocorms, with early peaks of labeled trehalose and saccharose (Smith, 1967). Intriguingly, several orchid species can grow in vitro on trehalose as the sole sugar source, and Ponert et al (2021) reported in Dactylorhiza majalis an efficient trehalase-dependent metabolic pathway that enables orchids to utilize exogenous trehalose. Genomic data support this peculiar ability of orchids to utilize trehalose (Li et al, 2022), and transcriptomic data further suggest that trehalose may be transferred from the fungus to the plant because genes coding for plant trehalases are significantly upregulated in OrM protocorms (Ja z kalski et al, 2021;Chen et al, 2022) as well as in adult mycoheterotrophs (Li et al, 2022).…”