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Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association.

ABSTRACT: Dendrobium is one of the largest genera in the Orchidaceae, and D. officinale is used in traditional medicine, particularly in China. D. officinale seeds are minute and contain limited energy reserves, and colonization by a compatible fungus is essential for germination under natural conditions. When the orchid mycorrhizal fungi (OMF) initiates symbiotic interactions with germination-driven orchid seeds, phytohormones from the orchid or the fungus play key roles, but the details of the possible biochemical pathways are still poorly understood. In the present study, we established a symbiotic system between D. officinale and Tulasnella sp. for seed germination. RNA-Seq was used to construct libraries of symbiotic-germinated seeds (DoTc), asymbiotic-germinated seeds (Do), and free-living OMF (Tc) to investigate the expression profiles of biosynthesis and metabolism pathway genes for three classes of endogenous hormones: JA (jasmonic acid), ABA (abscisic acid) and SLs (strigolactones), in D. officinale seeds and OMF under symbiotic and asymbiotic conditions. Low concentrations of endogenous JA, ABA, or SLs were detected in the D. officinale-Tulasnella symbiont compared with the asymbiotic tissues. Gene annotation results suggest that the expression of DEGs (differentially expressed genes) related to JA and ABA biosynthesis from D. officinale were down-regulated, while most of the key DEGs related to SL biosynthesis from D. officinale were up-regulated in the symbiotic germinated seeds compared with the asymbiotic germinated seeds. Moreover, in the OMF, we found a significantly up-regulated differential expression of the JA and ABA biosynthesis-related genes in the symbiotic interaction, with the opposite expression trends to those found in Dendrobium. This indicates that Dendrobium seed symbiotic germination may be stimulated by the apparent involvement of the OMF in the production of hormones, and relatively low concentrations of endogenous JA, ABA, or SLs might be maintained to promote the growth of the D. officinale-Tulasnella symbiotic protocorm-like body. These results will increase our understanding of the possible roles played by endogenous hormones in the regulation of the orchid-fungus symbiosis.

SUBMITTER: Wang T

PROVIDER: S-EPMC6274778 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association.

Wang Tao T Song Zheng Z Wang Xiaojing X Xu Lijun L Sun Qiwu Q Li Lubin L

International journal of molecular sciences 20181106 11

Dendrobium is one of the largest genera in the Orchidaceae, and D. officinale is used in traditional medicine, particularly in China. D. officinale seeds are minute and contain limited energy reserves, and colonization by a compatible fungus is essential for germination under natural conditions. When the orchid mycorrhizal fungi (OMF) initiates symbiotic interactions with germination-driven orchid seeds, phytohormones from the orchid or the fungus play key roles, but the det ...[more]

PMID: 30404159

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Project description:BackgroundOrchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination.ResultsThe two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation.ConclusionsCompatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

Project description:Hydroxyproline-rich glycoproteins (HRGPs) are abundant cell wall components involved in mycorrhizal symbiosis, but little is known about their function in orchid mycorrhizal association. To gain further insight into the role of HRGPs in orchid symbiosis, the location and function of HRGPs were investigated during symbiotic germination of Dendrobium officinale. The presence of JIM11 epitope in developing protocorms was determined using immunodot blots and immunohistochemical staining procedures. Real-time PCR was also employed to verify the expression patterns of genes coding for extensin-like genes selected from the transcriptomic database. The importance of HRGPs in symbiotic germination was further investigated using 3,4-dehydro-L-proline (3,4-DHP), an inhibitor of HRGP biosynthesis. In symbiotic cultures, immunodot blots of JIM11 signals were moderate in mature seeds, and the signals became stronger in swollen embryos. After germination, signal intensities decreased in developing protocorms. In contrast, in asymbiotic cultures, JIM11 signals were much lower as compared with those stages in symbiotic cultures. Immunofluorescence staining enabled the visualization of JIM11 epitope in mature embryo and protocorm cells. Positive signals were initially localized in the larger cells near the basal (suspensor) end of uninfected embryos, marking the future colonization site of fungal hyphae. After 1 week of inoculation, the basal end of embryos had been colonized, and a strong signal was detected mostly at the mid- and basal regions of the enlarging protocorm. As protocorm development progressed, the signal was concentrated in the colonized cells at the basal end. In colonized cells, signals were present in the walls and intracellularly associated with hyphae and the pelotons. The precise localization of JIM11 epitope is further examined by immunogold labeling. In the colonized cells, gold particles were found mainly in the cell wall and the interfacial matrix near the fungal cell wall. Four extensin-like genes were verified to be highly up-regulated in symbiotically germinated protocorms as compared to asymbiotically germinated ones. The 3,4-DHP treatment inhibited the accumulation of HRGPs and symbiotic seed germination. In these protocorms, fungal hyphae could be found throughout the protocorms. Our results indicate that HRGPs play an important role in symbiotic germination. They can serve as markers for fungal colonization, establishing a symbiotic compartment and constraining fungal colonization inside the basal cells of protocorms.

Project description:Introduction: Numerous studies have demonstrated that the stems of D. officinale have the effect of lowering blood glucose, but the leaves of D. officinale have seldom been investigated. In this study, we mainly studied the hypoglycemic effect and mechanism of D. officinale leaves. Methods: Initially in vivo, male C57BL/6 mice were administered either standard feed (10 kcal% fat) or high-fat feed (60 kcal% fat) along with either normal drinking water or drinking water containing 5 g/L water extract of D. officinale leaves (EDL) for 16 weeks, and changes in body weight, food intake, blood glucose, etc., were monitored weekly. Next in vitro, C2C12 myofiber precursor cells which were induced to differentiate into myofibroblasts and cultured with EDL to detect the expression of insulin signaling pathway related proteins. HEPA cells were also cultured with EDL to detect the expression of hepatic gluconeogenesis or hepatic glycogen synthesis related proteins. Eventually after separating the components from EDL by ethanol and 3 kDa ultrafiltration centrifuge tube, we conducted animal experiments using the ethanol-soluble fraction of EDL (ESFE), ethanol-insoluble fraction of EDL (EIFE), ESFE with a molecular weight of >3 kDa (>3 kDa ESFE), and ESFE with a molecular weight of <3 kDa (<3 kDa ESFE) for intensive study. Results: The results in vivo revealed that the mice fed the high-fat diet exhibited significantly decreased blood glucose levels and significantly increased glucose tolerance after the EDL treatment, whereas the mice fed the low-fat diet did not. The results in vitro showed that EDL activated the expression of protein kinase B (AKT), the phosphorylation of AKT, and the expression of downstream GSK3β in the insulin signaling pathway. EDL treatment of HEPA cells confirmed that EDL did not affect hepatic gluconeogenesis or hepatic glycogen synthesis. In the experiment of studying the composition of EDL, we found that the >3 kDa ESFE displayed the effect of lowering blood glucose. In summary, the effect of EDL in lowering blood glucose may bethanole achieved by activating the insulin signaling pathway to increase insulin sensitivity, and the main functional substance was contained within the >3 kDa ESFE. Discussion: The findings of this study represent a reference point for further exploration of the hypoglycemic effects of D. officinale leaves and may assist in both the identification of new molecular mechanisms to improve insulin sensitivity and the isolation of monomeric substances that lower blood glucose. Furthermore, the obtained results may provide a theoretical basis for the development of hypoglycemic drugs with D. officinale leaves as the main component.

Dataset Information

Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association.

Publications

Functional Insights into the Roles of Hormones in the <i>Dendrobium officinale-Tulasnella</i> sp. Germinated Seed Symbiotic Association.

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