Content determination and microscopic observation proved that dendrobine accumulation in the stem of Dendrobium nobile Lindl. increased after infection with mycorrhizal fungus MF23 (Mycena sp.). Large-scale transcriptome sequencing of symbiotic and asymbiotic D. nobile revealed that 30 unigenes encoding proteins were possibly related to the biosynthesis of dendrobine sesquiterpene backbone. A qRT-PCR experiment of 16 unigenes, selected randomly, proved that there were significant changes in the expression levels of AACT, MVD, PMK and TPS21 at 9 weeks after inoculation. These results implied that MF23 might stimulate dendrobine biosynthesis by regulating the expressions of genes involved in the mevalonate (MVA) pathway. The biogenetic pathway of dendrobine was suggested systematically according to the structural features of dendrobine alkaloids and their sesquiterpene precursors, which implied that post-modification enzymes might play a major role in dendrobine biosynthesis. Thus, genes encoding post-modification enzymes, including cytochrome P450, aminotransferase and methyltransferase, were screened for their possible involvement in dendrobine biosynthesis. This study provides a good example of endophytes promoting the formation of bioactive compounds in their host and paves the way for further investigation of the dendrobine biosynthetic pathway.

Polysaccharides are among the most important active compounds of Dendrobium nobile Lindl., and they improve immunity, strengthen memory, and prevent and fight tumours. Evidence suggests that the mycorrhizal fungus Mycena sp. may increase the polysaccharide content of D. nobile. To understand the regulatory mechanism by which Mycena sp. promotes polysaccharide accumulation in D. nobile, a model including MF23 (Mycena sp.) and D. nobile was developed. Content measurements and electron microscopy showed that the change in polysaccharides was associated with the level of colonization. Large-scale transcriptome sequencing analyses of D. nobile infected with MF23 and of wild D. nobile revealed that 30 unigenes encoding key enzymes were potentially associated with polysaccharide accumulation. A qRT-PCR experiment involving 13 randomly chosen unigenes suggested that the combined effects of the carbohydrate biosynthesis and consumption pathways resulted in changes in polysaccharide levels. MF23 might increase polysaccharide level by inducing genes involved in photosynthesis (petF), starch and sucrose metabolism (SUS, bcsA, glgA) and fructose and mannose metabolism (PMM, GPMM) and by repressing genes involved in glycolysis (PGK, PFK) and the citrate cycle (PDHB, CS, SDHA). This study provides a good example of an endophyte promoting the synthesis of bioactive compounds in its host and paves the way for further investigations of the associated regulatory mechanism using molecular methods.

Dendrobium nobile Lindl. has been used as a traditional Chinese medicine for a long time, in which the most important compound is dendrobine functioning in a variety of pharmacological activities. Farnesyl diphosphate synthase (FPPS) is one of the key enzymes in the biosynthetic pathway of dendrobine. In this work, we found the expression profiles of DnFPPS were correlated with the contents of dendrobine under the methyl jasmonate (MeJA) treatments at different time. Then, the cloning and functional identification of a novel FPPS from D. nobile. The full length of DnFPPS is 1231 bp with an open reading frame of 1047 bp encoding 348 amino acids. The sequence similarity analysis demonstrated that DnFPPS was in the high homology with Dendrobium huoshanense and Dendrobium catenatum and contained four conserved domains. Phylogenetic analysis showed that DnFPPS was the close to the DhFPPS. Then, DnFPPS was induced to express in Escherichia coli, purified, and identified by SDS-PAGE electrophoresis. Gas chromatography-mass spectrometry analysis indicated that DnFPPS could catalyze dimethylallyl pyrophosphate and isopentenyl pyrophosphate to produce farnesyl diphosphate. Taken together, a novel DnFPPS was cloned and functionally identified, which supplied a candidate gene for the biosynthetic pathway of dendrobine.

Two new indolizidine alkaloids crepidatumines A (1) and B (2) together with the stereoisomer of dendrocrepidine B (3) and known analog dendrocrepine (4) were isolated from D. crepidatum. Their structures were determined by HR-ESI-MS, NMR, and Electronic Circular Dichroism (ECD) experiments together with comparison of analogues. Compound (1) possess a (5/6/6/5) tetra-hetero-cyclic ring, whereas compound (2) contains a tricyclic system with an unusual bridged ring, which are the first report in Nature. The biological evaluation revealed that dendrocrepine (4) displayed a potent hypoglycemic effect in vitro.

The quality of Dendrobium nobile Lindl. is related to its endophytic fungi. It has been reported that the mycorrhizal fungus MF23 helps to increase the content of dendrobine in Dendrobium, but few studies have explained the mechanism underlying this phenomenon. In a previous study, we verified the mechanism of symbiosis between MF23 and D. nobile on agar medium. The research carried out in this study on bark medium, similar to the natural environment, is of great importance because of its benefits for wide application. We found a significant effect, especially in the later period of cultivation, in which the highest dendrobine content in the experimental group was 0.147%, which is equivalent to 2.88 times that of the control group, and suggesting that MF23 promoted D. nobile in the natural environment, which verifies the application of the technique in field conditions. This result also implied that post-modification enzyme genes might play an important role in stimulating the biosynthesis of dendrobine.