The major medicinal constituents of Dendrobium include alkaloids, flavonoids, polysaccharides, polyphenols, etc. (Lu et al., 2014). Among these compounds, alkaloids are the most important medicinal components and the first category extracted and characterized from Dendrobium plants. Thus far, five types of structurally confirmed alkaloids including sesquiterpene alkaloids, imidazole alkaloids, phthalide alkaloids, pyrrolidine alkaloids, and indolizidine alkaloids have been clarified (Ng et al., 2012). Despite the complex composition of alkaloids, sesquiterpene alkaloid dendrobine has been regarded as the quality standard for many Dendrobium plants (Li R. et al., 2017). Evidences from modern pharmacology have demonstrated that Dendrobium alkaloids have remarkable antihypertensive, anticancer, antipyretic, eye-benefiting, neuroprotective, and immune regulatory effects in preclinical studies (Wang J.H. et al., 2010; Li et al., 2011). Currently, alkaloids are primarily obtained through extraction and chemical synthesis (Kreis and Carreira, 2012). However, neither of these approaches is efficient enough because of extremely low accumulation levels in the Dendrobium plants and technical problems in total synthesis. Due to the high market demand, overexploitation, and deterioration of natural habitats, wild Dendrobium resources have been increasingly depleted. Therefore, the biotechnology-based strategy is promising for stably producing large quantities of alkaloids to meet the market demand and protect the wild resources.

One of the main purposes of transcriptome research in medicinal Dendrobium plants is to analyze the biosynthetic pathway and identify the key enzyme genes involved in specialized metabolite production. Several transcriptome studies have focused on the biosynthetic pathway of alkaloids in Dendrobium species. For instance, a previous study (Yuan et al., 2018) revealed that the alkaloids in the Dendrobium genus are mostly sesquiterpenoid alkaloids or terpenoid indole alkaloids (TIA). Functional analysis based on KEGG terms revealed 25 genes associated with alkaloid backbone construction that belonged to the TIA class by using the Roche 454 GS FLX Titanium platform (Guo et al., 2013; Figure 1). The upstream of the TIA pathway, which can be further divided into terpenoid-forming and indole pathway, is conserved among alkaloid-producing plants and initiated from the shikimate, mevolonate (MVA), or the methylerythritol phosphate (MEP) pathway (Figure 2). In the terpenoid-forming pathway, 10-hydroxylase (G10H) catalyzes geraniol to produce 10-hydroxygenraniol. After a series of enzymatic reactions, 10-hydroxygenraniol is converted to loganin, which is then further catalyzed by secologanin synthase (SCS) to generate secologanin (Wang C.T. et al., 2010). From the indole pathway, tryptamine is synthesized. These two intermediates, secologanin and tryptamine, then combined with each other by strictosidine synthase (STR) to form strictosidine (Zhu et al., 2014), a common precursor for all TIA biosynthesis.

The upstream of alkaloid biosynthesis is mainly through three pathways: the shikimate pathway, the MEP pathway, and the MVA pathway (Figure 2). The shikimate pathway in plants is essential for a variety of second metabolite synthesis (Tzin et al., 2012). A series of key enzymes involved in shikimate pathway have been identified. Among them, 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a key enzyme involved in the formation of enolpyruvylshikimate 3-phosphate (Klee et al., 1987). The stem-specific expression of EPSP in D. huoshanense enhanced the accumulation of tryptamine, which is a precursor for strictosidine biosynthesis (Yuan et al., 2018). Seventeen unigenes associated with six enzymes were revealed by transcriptome analysis in D. officinale and were mapped to the shikimate pathway (Shen et al., 2017), including 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DHS), 3-dehydroquinate synthase (DHQS), 3-dehydroquinate acid dehydratase (DHD), shikimate dehydrogenase (SKDH), 5-enolpyruvylshikimate-3-phosphate synthase (SHKG), and farnesyl diphosphate synthase (FPS). In the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) is the first key enzyme, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the second and rate-limiting enzyme catalyzing a branched isovaleric precursor to form a straight chain pentose sugar (Ramak et al., 2013). Overexpression of DXS in Spike lavender or DXR in Salvia miltiorrhiza resulted in a significant increase in terpenoid accumulation (Munoz-Bertomeu et al., 2006). In the MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) is one of the key enzymes involved in terpenoid biosynthesis. It is capable of catalyzing 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to form MVA. Overexpression of HMGR1 in ginseng enhanced the steroid and triterpene production (Kim et al., 2014), suggesting the promoting role of HMGR1 in ginsenoside biosynthesis.

Previous reports revealed 34 alkaloids isolated from 14 Dendrobium species, 21 of which were dendrobine alkaloids with a sesquiterpene skeleton structure (Xu et al., 2017). A pharmacology study demonstrated that dendrobine restrains the growth of A549 lung cancer cells and acts as a promising agent for treating virus infection (Li R. et al., 2017). The upstream biosynthetic pathway of dendrobine is composed of the MVA and MEP pathway, which are conserved to provide basic skeleton for terpenoid alkaloids (Figure 2). Both pathways can produce isopentenyl diphosphate (IPP), which is the precursor of synthetic terpenes that can be exchanged on the plasma membrane. Key enzymes involved in the MEP pathway (Figure 3) including DXS and DXR, as well as enzymes functioning in the MVA pathway such as hydroxymethylglutaryl-CoA synthase (HMGS) and HMGR have been annotated in D. officinale (Fan et al., 2016; Chen et al., 2019). In D. nobile, large-scale transcriptome analysis has been generated in response to MF23 infection with increased dendrobine production (Li R. et al., 2017). From the datasets, 16 genes encoding acetyl CoA acetyltransferase (AACT), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), and terpene synthase 21 (TPS21), which are members related to the biosynthesis of the backbone of sesquiterpene alkaloid dendrobine, have been identified, suggesting the dominant role of the MVA pathway in this process. In addition, 11 genes encoding nine enzymes were mapped onto the MEP pathway (Figure 3), which serves as a supplemental provider of isoprene units for dendrobine biosynthesis.

Following the generation of strictosidine, the alkaloid biosynthesis in Dendrobium is characterized mainly by a set of post modifications (PTM), such as cytochrome P450s (CYP450s)-mediated oxidation and hydroxylation reactions (Guo et al., 2013). As a complex superfamily of monooxygenase, CYP450s play key roles in specialized metabolite biosynthesis, and some of them have been isolated and characterized (Seki et al., 2008). For instance, annotation of the 454 EST pool of D. officinale against the SwissProt database revealed 93 CYP450s transcripts belonging to 17 families (Guo et al., 2013). Among them, transcripts of the CYP71 (9.7%) were likely to be involved in hydroxylation steps of alkaloid biosynthesis. In D. huoshanense, 229 unigenes were identified as putative CYP450s, most of which were also the CYP71 family members (7.8%), followed by CYP3A and CYP4 family members (Yuan et al., 2018). Moreover, several post-modification enzymes involved in the biogenic pathway of dendrobine, such as CYP450s, aminotransferase, and methyltransferase (Figure 3), have been identified in D. nobile (Li Q. et al., 2017) and D. officinale (Chen et al., 2019). Among them, CYP1D10, METTL23, ATX4, and BCAT2 were significantly upregulated in response to MF23 infection (Li Q. et al., 2017), suggesting their positive roles in promoting dendrobine biosynthesis.

Transcription factor (TF) also plays a vital role in coordinating the expression of alkaloid biosynthesis genes, thus affecting the constituents’ composition in higher plants (Table 3). For example, the AP2/ERF family members ORCA2 and ORCA3 were capable of regulating a subset of alkaloid biosynthesis genes (Van Der Fits and Memelink, 2000). Additionally, a large number of TFs, including C3H, bHLH, bZIP, MYB, and WRKY (Yuan et al., 2018), have been previously reported to play central roles in the regulation of alkaloid biosynthesis in Dendrobium.

Many Dendrobium plants are precious medicinal herbs partially because of their abundant polysaccharides found in stems, leaves, and flowers. In recent years, many soluble polysaccharides were extracted from various Dendrobium species, and the structure, composition, and bioactivity were determined (He et al., 2015). The major polysaccharides in Dendrobium stems were non-starch mannan polysaccharides, to a lesser extent glucose, and a small amount of galactose (Ng et al., 2012). Semithin and ultrathin sections demonstrated that polysaccharides that formed granules were stored in the plastids of stems (He et al., 2017a), similar to starch grains.

Polysaccharides are one of the major medicinal components in Dendrobium plants with antitumor, antioxidant, antiaging, antibacterial, antiviral, antiradiation, and anticoagulant activities (Liu et al., 2011; Wang et al., 2014). Thus far, a large number of carbohydrate-related genes (Li Q. et al., 2017) encoding glycosyltransferase (GT), glucosyltransferase, and mannosyltransferase, most of which were expressed more highly in stems than in leaves and roots, have been identified and might play vital roles in polysaccharide synthesis (Figure 2). Enzymes directly involved in polysaccharide metabolism, such as GDP-mannose pyrophosphorylase (GMP) in Dendrobium, have also been characterized (He et al., 2017c). Moreover, it has been confirmed that sucrose synthase (Susy) was positively correlated with the polysaccharide content in D. officinale and D. huoshanense and that two unique gene families, the galacturonosyltransferase and β-galactosidase gene families, were related to the richness of polysaccharides in D. officinale (Yan et al., 2015). GT is a group of carbohydrate-active enzymes that catalyzes the glycosidic bond formation in glycan and glycoside biosynthesis by transfer of sugar moieties from active donors to acceptor molecules (Shao et al., 2005; Lao et al., 2014). A recent transcriptome analysis in D. officinale (Shen et al., 2017) identified 280 GTs, including genes encoding glucosyltransferase (236), fucosyltransferase (11), mannosyltransferase (16), and xylosyltransferase (17) using BLASTX methods.

Mannan polysaccharides are the major component of polysaccharides from most of the Dendrobium species, accounting for as much as 58.3% of the dry weight of the crude polysaccharide fraction in D. officinale (Xing et al., 2015), and also promising bioactive ingredients for use in drugs. It has beneficial effects on human health with increased cytokine production and antioxidant and anticancer activities (Xing et al., 2015). Mannan polysaccharides can be further classified into four subfamilies (Buckeridge, 2010): pure mannan, glucomannan (GM), galactomannan (GGM), and galactoglucomannan. The biosynthesis of mannan polysaccharides is mediated by mannan synthases using GDP-D-mannose or GDP-D-glucose as substrates (Hassid, 1969). Moreover, the cellulose synthase A (CesA) superfamily genes have also been demonstrated to be involved in the biosynthesis of mannan polysaccharides (Lerouxel et al., 2006). The CesA superfamily can be subdivided into one cellulose synthase (CesA) family and nine cellulose synthase-like (Csl) families, CslA to CslJ (Suzuki et al., 2006). In D. officinale, eight CslA genes (DoCslA1 to DoCslA8) were identified and analyzed to provide genetic evidences for their roles in mannan polysaccharide biosynthesis (He et al., 2017a). Overexpression of DoCslA6 increased mannose content in Arabidopsis thaliana (He et al., 2017a). In addition, GDP-mannose transporter (GMT), which translocates GDP-mannose into the Golgi lumen, is indispensable for mannan polysaccharide biosynthesis. Three GMT genes, DoGMT1 to DoGMT3 (Yu et al., 2018), have been identified in D. officinale with the highest transcript levels in stems.

Polysaccharide synthesis and sucrose metabolism are closely linked because many monosaccharides, the basic building blocks for polysaccharide synthesis, are produced from sucrose hydrolysis or hydrolysis derivatives. In general, sucrose metabolism involves two distinct processes, sucrose synthesis and sucrose breakdown, which are mainly catalyzed by sucrose phosphate synthase (SPS) and Susy, respectively (Huber and Huber, 1996). A previous study revealed that the levels of polysaccharides in D. officinale were closely related to the concentrations of the reduced sugar and soluble sugar, which were directly affected by sucrose invertase and SPS activities (Yan et al., 2015). Yan Liang and colleagues (Yan et al., 2015) analyzed the genome sequence of D. officinale and identified 10 SPS and 15 Susy genes, which have undergone marked expansion through tandem duplication. Likewise, in D. huoshanense, 13 SPS and 18 Susy genes have been isolated (Yuan et al., 2018). Alkaline/neutral invertase (NI) is responsible for sucrose hydrolysis to produce glucose or fructose in the cytoplasm. An NI gene, DoNI, was cloned in D. officinale by the rapid amplification of cDNA ends (RASE) method. The expression of DoNI was associated with its activities in different tissues and, more importantly, the polysaccharide accumulation (Gao et al., 2016). Uridine diphosphate glucose (UDPG) is an important direct or indirect glycosyl donor for synthesis of polysaccharides. The UDPG pyrophosphorylase (UDPase) is a key enzyme for reversibly catalyzing UDPG into glucose-1-phosphate (Glc-P), which is then utilized in synthesis of polysaccharides by GTs. A novel UGPase gene, DoUGP, was identified from D. officinale (Wan et al., 2017). It was highly expressed in stems in comparison to other organs and positively correlated with the highest polysaccharide content there (Wan et al., 2017). Sucrose feeding significantly increased DoUGP expression and enhanced polysaccharide production accordingly in both protocorm of D. officinale and protocorm-like bodies of D. huoshanense in suspension cultures (Wan et al., 2017). Thus, DoUGP is probably involved in polysaccharide synthesis and might serve as a potential target for quality breeding of Dendrobium orchids.

Tropine is an alkaloid derived from tropinone, which can be reduced by tropinone reductase (TRs) using the NADPH as coenzyme (Nakajima et al., 1998). TRs can be further divided into two subgroups, TRI and TRII, based on the stereospecificity of reduction product. TRI is responsible for tropine production, whereas TRII is mainly involved in the generation of pseudotropine (Nakajima et al., 1998). Most of TR homologous genes found in other plant species do not have tropinone reduction activity, except for plants belonging to or closely related to the Solanaceae family (Drager, 2006) and CoTR from Cochlearia officinalis (Brock et al., 2008). Recently, DnTR1 and DnTR2 that encode peptides with similarity to known TRs were cloned from D. nobile. Catalytic activity assay revealed that both DnTR1 and DnTR2 were able to reduce 3-quinuclidinone hydrochloride and 4-methylcyclohexanone using NADPH as coenzyme (Chen et al., 2013). Moreover, DnTR1 could reduce tropinone, whereas DnTR2 couldn’t (Cheng et al., 2013), implying their tremendous variation in substrate specificity.

Flavonoids are the second most common compounds in D. officinale, exhibiting diverse medicinal functions including antioxidant and protective effects on cell toxicity and treatment of various degenerative and age-related diseases (Prochazkova et al., 2011; Wang et al., 2017). Most flavonoids in Dendrobium are C-glycosides with basic skeletons including vitexin, quercetin, luteolin, apigenin, etc. (Lei et al., 2018). The biosynthesis of most flavonoids begins in the phenylpropanoid pathway using malonyl-CoA and p-coumaroyl-CoA as precursors (Liu et al., 2013). The whole processes are regulated by many key enzymes, transcription factors, UDP-GT, and CYP450s (Liu et al., 2013). Transcriptome analysis (Lei et al., 2018) revealed that 31 unigenes encoding 14 enzymes were involved in the biosynthesis of flavonoids in D. catenatum. Synthesis of the three basic flavonoid glycoside skeletons are regulated by flavonol synthase (FLS), CYP75A, and flavonoid 3′-monooxygenase. Specifically, FLS is involved in the transformation of dihydroquercetin to quercetin, and dihydrokaempferol to kaempferol, which can be subsequently catalyzed into quercetin by CYP75A and flavonoid 3′-monooxygenase (Lei et al., 2018), two enzymes also regulating the synthesis of luteolin from apigenin.