Dendrobium nobile was grown on the imitation wild cultivation base at Wanglong in Chishui City, Guizhou province. Based on the growth cycle of D. nobile, the suitable harvest time of D. nobile stem was between September and October in Guizhou. Three-year-old mature stems of D. nobile were collected in October 2020. Immediately after harvesting, the samples were put in dry ice for transportation, and stored in a freezer at -80°C until used.

The solvents, including LC/MS-grade water, acetonitrile, methanol, formic acid, acetic acid, and trifluoroacetic acid, were bought from Sigma-Aldrich (Saint Louis, MO, USA). 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 2‐mercaptobenzothiazole (2-MBT) were purchased from J&K Scientific (Beijing, China). Dendrobine standard was purchased from Yuanye Shengwu (Shanghai, China).

The fresh stem was crushed manually on ice. The crushed sample (1g) was extracted with 10 mL of 80% methanol (v/v) followed by an ultrasonic bath for 30 min at room temperature and then centrifugation (6000 r/min) for 15 min using a Cence H1850 centrifuge (Changsha, China). The supernatant was transferred, and the extraction was repeated twice. The combined supernatant was evaporated under nitrogen flow at room temperature to obtain extracts and kept at -20 °C. Prior to UPLC-QTOF-MS analysis, the extract was dissolved in 80% methanol (v/v) to a concentration of 1 mg/mL and then subjected to centrifugation (15000 r/min). The supernatant (100 μL) was added to the sample vial with a 250 μL insert pipe for analysis.

The preparation of D. nobile stems sections were based on previously reported methods (He et al., 2019) with few modification. Briefly, fresh stems were stored at -80 °C until use; the stems were cryo-sectioned into 20-μm-thick slices at -20 °C on a freezing microtome (Leica CM1860, Germany) and then were immediately adhered to indium tin oxide (ITO) glass microscope slides. A matrix solution containing 2-MBT (12 mg/mL) in acetonitrile/ddH₂O/TFA (80:20:0.2, v/v/v) was prepared and sprayed to the surfaces of stem sections by air-brush sprayer via five cycles (5 s spraying, 60 s drying). After air-drying in a fume hood, an extra matrix spray was conducted on the same stem sections for 40 cycles. The optical images of the stem section were obtained using a Photo Scanner (Epson Perfection V550). The standard histological optical images were gained by Hematoxylin and eosin (H&E) staining following a previous procedure (Casadonte and Caprioli, 2011).

The UPLC-QTOF-MS analysis was executed with an ACQUITY UPLC coupled to Xevo G2 QTOF mass spectrometer system (Waters, Milford, MA, USA). The UPLC system was equipped with ACQUITY UPLC BEH C18 column (2.1×50 mm, I.D. 1.7 μm), autosampler, binary pump, and column compartment. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient elution procedure was as follows: 0-1.0 min, 3-13% B; 1.0-2.5 min, 13-25% B; 2.5-4.0 min, 25-40% B; 4.0-8.0 min, 40-60% B; 8.0-8.5 min, 60-97% B; 8.5-11.0 min, 97% B; 11.0-13.5 min, 97-3% B, 13.5-15.0 min, 3% B. The column temperature was 40°C, the flow rate was 0.5 mL/min, and the injection volume was 1 μL.

Mass spectrometry was obtained from both positive and negative ionization modes with scan time (1 s) and scan mass range (50-1500 Da). The capillary voltages were 3.0 kV (positive mode) and 2.5 kV (negative mode), and the sample cone voltage was 30 V. The desolvation temperature and ion source temperature were 400°C and 110°C, respectively. Nitrogen was used as the carrier gas with an 800 L/h flow rate for desolvation gas and a 50 L/h flow rate for sample cone gas. The low collision energy was 6 eV, and the high collision energy was changed repeatedly from 20 eV to 60 eV. Leucine-enkephalin (1 μg/mL) was used as Lock-Mass solution during data collection. The UPLC-QTOF-MS system was controlled by MassLynx 4.2 software (Waters, Milford, MA, USA).

MALDI-MSI was carried out on Bruker AutoFlex Speed MALDI TOF mass spectrometer (Bruker Daltonics, Germany) equipped with a 2000 Hz solid-state Smartbeam Nd: YAG UV laser (355 nm, Azura Laser AG). The detected mass ranges from 120 Da to 500 Da in positive-ion mode. Profiling data of MALDI-MS were obtained from 5000 laser shots accumulation, and each scan accumulated 500 laser shots. The images were acquired using Bruker’s FlexImaging v. 4.1. Moreover, serine ([M + H]⁺, m/z 106.0498), L-glutamic ([M + H]⁺, m/z 148.0604), and proline ([M + H]⁺, m/z 116.0706) were used for external mass calibration. 2-MBT ([M + H]⁺, m/z 167.994) was used for internal mass calibration. The calibration was performed with cubic enhanced mode.

The raw mass spectrum data with .raw format obtained in centroid mode were converted by Reifycs ABF converter software (https://www.reifycs.com/AbfConverter/) to get.abf format files, and furtherly imported into MS-DIAL software (Ver.4.7) for peak alignment, peak picking, normalization, deconvolution, and compound identification (Tsugawa et al., 2019). The main parameter settings were as follows: retention time range, 0-15 min; MS1 tolerance, 0.015 Da; MS2 tolerance, 0.02 Da; mass range, 50-1500 Da; smoothing level, 3 scans; minimum peak width, 5 scans; retention time tolerance, 0.05 min; identification score cut off, 80%. Adduct types as [M+H]⁺, [M+Na]⁺, [M+H-H₂O]⁺, [2M+H]⁺, and [2M +Na]⁺ in positive-ion mode, and [M-H]^-, [M-H-H₂O]^-, [M+FA-H]^-, [2M-H]^-, and [2M+FA-H]^- in negative-ion mode. Identification was performed by comparing MS, MS/MS, and retention index with MS/MS-Public-Pos/Neg database in MS-DIAL. The metabolites were regarded as potential identifications when the matching degree with the spectrum database was higher than 80%. The .txt file results, including sample name, metabolites, peak area, retention time, and quantitative quality, were exported after MS-DAIL process. The fragment ions of exported compounds were further confirmed by its original MS and MSe data in MassLynx with mass error<10 ppm and compared with mass data reported in related databases (SciFinder, PubMed, Mzcloud) and literatures.

Heatmap analysis were carried out using HeatMap illustrator tool in TBtool v1.01 software. The ion maps of detected compounds were reconstructed using Bruker FlexImaging 4.1 software. The general histogram analysis was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) and was calculated on the base of the peak intensity of identified compounds. The data were presented as mean ± standard deviation (SD).

Based on the established UPLC-QTOF-MS methods, the prepared extracts of D. nobile stems were analyzed and acquired the mass spectral data in both positive and negative ionization modes. The MS-DAIL platform and MassLynx software were used to identify and characterize the metabolites. By comparing the multi-level ion fragment information, mass spectrometry database, and related literature data, a total of 34 compounds were tentatively identified, including 11 alkaloids, 10 sesquiterpenes, 5 amino acids, 1 lignan, 1 steroid, and 6 others. Detailed information on the identifications, including compound type, name, formula, and fragments were performed in Table S1 . The results showed alkaloids and sesquiterpenes were the main constituents in the mature D. nobile stem, which was accordant to the previous report that alkaloids and sesquiterpenes were the primary active constituents in the stems of D. nobile, especially alkaloids were representatives of the earliest identified classification of compounds from Dendrobium species (Xu et al., 2013; Mou et al., 2021). Based on the structural features of identified alkaloids and sesquiterpenes, we classified alkaloids as dendrobine-type alkaloids (dendrobine, dendrobine-N-oxide, dendramine, mubironine B, N-methyl-dendrobinium, and N-isopentenyl-dendrobinium) ( Figure 1A ), and dendroxine-type alkaloids (dendroxine, 4-hydroxy-dendroxine/6-hydroxy-dendroxine, N-isopentenyl-6-hydroxydendroxinium, N-isopentenyl-dendroxinium, and nobilonine) ( Figure 1B ). Moreover, the identified sesquiterpenes were also classified into two types, the one was dendrobine-type sesquiterpenes (findlayanin, endroterpene C, nobilomethylene, dendroside G, dendroside F, and dendronobilin F) ( Figure 1C ), and the remaining were listed as other-type ( Figure 1D ).

The alkaloids from D. nobile shown numerous significant pharmacological effects, including neuroprotection, hepatic lipid regulation, anti-tumor, anti-diabetes, anti-inflammatory, and anti-virus (Linghu et al., 2021). Meanwhile, the sesquiterpenes from D. nobile also exhibited multiple bioactivities such as anti-microbial, anti-malarial, anti-inflammatory, anti-tumor, and immunomodulatory (Gong et al., 2021).

Heatmap and boxplot analysis were applied to compare the relative content of the identified different metabolites in the matured stems of D. nobile. As shown in Figure 2A , alkaloids were the most abundant metabolites in stems, especially dendrobine-type alkaloids. Sesquiterpenes presented a certain abundance, but different metabolites in sesquiterpenes were varied in relative content. Most of amino acids showed lower content in mature stems. The identified lignan (niranthin, NIA) and steroid (stigmasterol, STS) displayed higher content than most of the identified amino acids. Furtherly, the boxplot demonstrated the relative content of each identified metabolites distinctly ( Figures 2B-G ). Notably, dendrobine-type alkaloids ( Figure 2B ) had higher content than dendroxine-type alkaloids ( Figure 2C ), revealing that the alkaloid biosynthesis and accumulation were main dendrobine-type and few dendroxine-type alkaloids during the development of D. nobile stem. Dendrobine (DDB) has the highest accumulation than other dendrobine-type alkaloids, particularly the relative content higher than dendrobine-N-oxide (DNO) and dendramine (DDM) have ( Figure 2B ). Dendrobine was the characteristic and first found alkaloid from D. nobile, which has considered as the evaluation indicator (mass content>0.4%) for the quality control of this species by Chinese Pharmacopoeia (2020 edition) (Mou et al., 2021). The low accumulations of DDM, DNO, N-methyl-dendrobinium (NMD), and mubironine B (MBB) were gradually reduced, indicating these compounds may have little biosynthesis or may provide intermediates for the biosynthesis of DDB. The relative content of dendroxine-type alkaloids decreased in the trends following dendroxine (DDX), N-isopentenyl-dendroxinium (NID), nobilonine (NBN), N-isopentenyl-6-hydroxydendroxinium (NHD), and 4/6-hydroxy-dendroxine (4/6HD) ( Figure 2C ), revealing that NID, NBN, NHD, and 4/6HD may have little biosynthesis or decompose at mature D. nobile stems.

The different sesquiterpenes were also varied in the relative content of mature D. nobile stem ( Figures 2D-E ). For the accumulation of dendrobine-type sesquiterpenes, findlayanin (FLN) showed the highest content, followed by dendroterpene C (DTC), nobilomethylene (NBM), dendroside G (DSG), dendroside F (DSF), and dendronobilin F (DBF) ( Figure 2D ), indicating that DBF may provide the basic skeleton; and both of DSF and DSG with glycosyl group may support energy for the biosynthesis of the sesquiterpene with more complex structure (such as NBM, DTC, and FLN) during the D. nobile stem mature. Most dendrobine-type sesquiterpenes have been reported as intermediates for the biosynthesis of dendrobine (Gong et al., 2021). Besides, the different accumulated levels of the identified amino acid and others, including lignin (NIA) and steroid (STS), were presented in Figures 2F, G , respectively.

Many studies previously focused on the qualitative and quantitative of D. nobile metabolites, particularly in alkaloids and sesquiterpenes. However, the spatial distributions of these critical compounds in stem are still lacking, which has severely hampered the thorough understanding of D. nobile physiological activities and the biosynthesis of the crucial metabolites. Thus, we herein analyzed the spatial distribution of alkaloids and sesquiterpenes in D. nobile stem for the first time to understand the biosynthesis of metabolites.

Dendrobine, belonging to sesquiterpene alkaloids, accounted for 92.6% of D. nobile alkaloids (Xu et al., 2017). Moreover, dendrobine was also the first isolated bioactive alkaloid from D. nobile (Chen and Chen, 1935), and has been considered as the indicator ingredient for the quality evaluation of D. nobile stem attributing to its many important pharmacological effects (Li R et al., 2017; Song et al., 2019). Given this, the synthesis of dendrobine has attracted lots of researchers’ interest. So far, total chemical synthesis of dendrobine has been available, but the yield and purity of dendrobine still meet the challenge (Gong et al., 2021). Therefore, biosynthesis of dendrobine was prospect and primary investigation, which promoted some enzymes and genes of the biosynthetic pathway of dendrobine have been found, such as cytochrome P450 oxidase (CYP450), farnesyl diphosphate synthase (FPPS), sesquiterpene synthase (SES), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGR), and mevalonate diphosphate decarboxylase (MVD) (Li Q et al., 2017; Gong et al., 2021; Gong et al., 2022). However, due to the enormous genome of D. nobile and the complex structure of dendrobine, the biosynthesis of dendrobine was still unclear.

This study combined the superiority of both UPLC-QTOF-MS and MALDI-MSI, high accuracy and visualizable information, to analyze the biosynthesis and accumulation of dendrobine in D. nobile stem. Dendrobine was a sesquiterpene alkaloid, thus, the sesquiterpenes were proposed as precursors and intermediates in dendrobine biosynthesis, which pathway was also like that of sesquiterpene due to the sesquiterpene skeleton of dendrobine. Our metabolic profiling by UPLC-QTOF-MS has revealed that, a wide variety of sesquiterpene metabolites were identified in D. nobile stem, whereas the abundance was low. This result corresponded to the fact that sesquiterpene was consumed for dendrobine biosynthesis during the maturation of D. nobile stem. Figure 7 displayed information referring to the main compounds of the proposed dendrobine biosynthesis pathway in stems of D. nobile based on the data of UPLC-QTOF-MS and MALDI-MS, and previous speculation (Gong et al., 2021).

As shown in Figure 7 , farnesyl diphosphate (FPP) was first obtained from acetyl-CoA in the mevalonate (MVA) pathway, that is, the proposed biosynthetic pathway of dendrobine was started by the catalysis of FPPS. Then FPP can form dendronobilin G through intramolecular cyclization under the action of FPPS. Dendronobilin G putatively produces intermediate A under the effects of P450 oxidoreductase. Subsequently, the intermediate A undergoes molecule rearrangement, cyclization, and Michael addition to form dendronobilin K and (+)-(1R,5R,6S,8R,9R)-8,12-dihydroxy-copacamphan-3-en-2-one. These compounds were oxidized to produce B skeleton with opened-ring and hydrolyzation, and produce C- and D-type compounds, like dendronobiloside A and dendronobiloside B. D-type intermediates undergo redox reactions furtherly to form E, and E subjected to esterification to produce intermediate F. Thus far, the skeleton of picrotoxane-type sesquiterpene (E and F) was present through the intramolecular esterification of intermediate D. We have identified and visualized five picrotoxane-type sesquiterpenes in D. nobile stem. These picrotoxane-type sesquiterpenes may on the one hand form dendronobilin C by cyclization, dendronobilin C was aminated to form intermediate G, and G was finally methylated to produce dendrobine. On the other hand, the sesquiterpenes may be aminated to form nobilonine, nobilonine then transfer to dendrobine by cyclization and decarboxylation. Gong et al. (2021) have speculated a similar biosynthetic pathway of dendrobine, while our work found the most of sesquiterpenes in the pathway, including their relative contents and spatial distribution, which further confirmed the biosynthetic pathway of dendrobine.