# New Insights into the Symbiotic Relationship between Orchids and Fungi
## 4 Molecular Studies on Orchid Mycorrhizal Symbiosis by Next-Generation Sequencing and Proteomics
The 88 differentially expressed proteins identified at different protocorm development stages (achlorophilous or green) by the 2D-LC-MS/MS coupled to isobaric tagging are involved in molecular signaling, cell rescue and defense, energy and secondary metabolism (Table 1) [65]. Within these proteins, the fructose-bisphosphate aldolase and enolase in the glycolytic pathway and the isocitrate lyase in the Krebs cycle were accumulated in the achlorophyllus protocorms, suggesting the catabolism of organic C from the fungus may be induced. In contrast, the RuBisCO and Photosystem I P700 chlorophyll a apoprotein A1 were most abundant in the green protocorms. The result may imply the transition of metabolism from fully myco-heterotrophic to partially myco-heterotrophic or fully autotrophic. In the interaction between _O. sphauclatum_ [PERSON] and _Thanatephorus_ sp., eleven orchid proteins related to cell cycle, energy, purine metabolism, ribosome biogenesis and vesicle secretion were identified, and the fungal proteins involved in protein-protein interaction, stress response and protein and saccharides biosynthesis were found under the symbiosis condition (Table 1) [66]. To better understand orchid seed germination and protocorm development, [PERSON] et al. (2017) performed RNA-seq and proteomic analysis using iTRAQ (isobaric tags for relative and absolute quantification) at three different stages (germination, protocorm and seedling) to identify the differentially expressed genes/proteins involved in these processes in _Dendrobium officinal_[61]. For both symbiotic and asymbiotic germination, 308 out of the 2256 identified proteins were differentially expressed across the three stages. The results indicated that symbiotic and asymbiotic germination may share a common signaling pathway during the early stage. In addition, 229 proteins were identified to be differentially expressed in only symbiotic germination. Among them, 32 candidates were upregulated in both transcriptomic and proteomic experiments including those encode for symbiotic signal transduction, defense reaction, storage protein utilization and metabolisms of N, carbohydrates and lipids (Table 1). Interestingly, several genes or proteins identified in this study, such as acyl-CoA-binding proteins, acetyl-CoA carboxylase, \(\beta\)-oxidation multifunctional protein, may promote the utilization of the stored reserves in the embryo cells due to their key roles in lipid metabolism. By using isotopolog profiling, [PERSON] et al. (2017) demonstrated that lipids can be directly transferred from host plants to fungal cells in arbuscular mycorrhizal symbioses [27]. It would be also interesting to know whether the stored lipid in orchid embryos can be transported to the fungal partners for establishing orchid-fungus symbioses in further studies. Taken together, transcriptomic and proteomic analyses lead to a comprehensive understanding of the molecular processes activated in the orchid mycorrhizas. In addition to nutrient transporter genes, a set of genes regulating plant defense responses and secondary metabolism is also induced by symbiosis with mycorrhizal fungi. To fully understand the molecular mechanisms of the orchid-fungus symbiosis, further characterization of the differentially expressed genes is required.