Integrative transcriptome and metabolome analysis reveals the discrepancy in the accumulation of active ingredients between Lycium barbarum cultivars.

MAIN CONCLUSION: The combined analysis of transcriptome and metabolome provided molecular insight into the dynamics of multiple active ingredients biosynthesis and accumulation across different cultivars of Lycium barbarum. Lycium barbarum L. has a high concentration of active ingredients and is well known in traditional Chinese herbal medicine for its therapeutic properties. However, there are many Lycium barbarum cultivars, and the content of active components varies, resulting in inconsistent quality between Lycium barbarum cultivars. At present, few research has been conducted to reveal the difference in active ingredient content among different cultivars of Lycium barbarum at the molecular level. Therefore, the transcriptome of 'Ningqi No.1' and 'Qixin No.1' during the three development stages (G, T, and M) was constructed in this study. A total of 797,570,278 clean reads were obtained. Between the two types of wolfberries, a total of 469, 2394, and 1531 differentially expressed genes (DEGs) were obtained in the 'G1 vs. G10,' 'T1 vs. T10,' and 'M1 vs. M10,' respectively, and were annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. Using these transcriptome data, most DEGs related to the metabolism of the active ingredients in 'Ningqi No.1' and 'Qixin No.1' were identified. Moreover, a widely targeted metabolome analysis of the metabolites of 'Ningqi 1' and 'Qixin 1' fruits at the maturity stage revealed 1,135 differentially expressed metabolites (DEMs) in 'M1 vs. M10,' and many DEMs were associated with active ingredients such as flavonoids, alkaloids, terpenoids, and so on. We further quantified the flavonoid, lignin, and carotenoid contents of the two Lycium barbarum cultivars during the three developmental stages. The present outcome provided molecular insight into the dynamics of multiple active ingredients biosynthesis and accumulation across different cultivars of Lycium barbarum, which would provide the basic data for the formation of Lycium barbarum fruit quality and the breeding of outstanding strains.

LbCOPT1 is a copper transporter induced in Lycium barbarum mycorrhizal roots, which allows tobacco with improved growth and nutrient uptake.

KEY MESSAGE: Overexpressing the copper transporter LbCOPT1 leads to a notable increase in the abundance of mycorrhizal arbuscules that suggests the potential application of LbCOPT1 in breeding programs aimed at enhancing symbiotic nutrient uptake in Lycium barbarum L.

Development of a pre-column derivatization ultra-high-performance liquid chromatography method for polysaccharide and monosaccharide quantification in Lycium barbarum.

Given the limited specificity and accuracy observed in the current official colorimetric quantification of polysaccharide in Lycium barbarum, our study aims to establish a novel, specific, accurate, and economic pre-column derivatization ultra-high-performance liquid chromatography (UHPLC) method for determining the monosaccharide and polysaccharide content in L. barbarum. The optimization of extraction, hydrolysis, and derivatization (using 1-phenyl-3-methyl-5-pyrazolone) processes for polysaccharide from L. barbarum was conducted initially, followed by separation of nine monosaccharides within 20 min using UHPLC with a C18 column. Subsequently, a novel method known as quantitative analysis of multiple components by single marker was developed, utilizing either additive 2-deoxy-D-ribose or any monosaccharide present in the sample as a single reference standard to simultaneously detect the contents of polysaccharide and nine monosaccharides in L. barbarum. To validate the accuracy of the established method, the quantitative results of our approach were compared to both external and internal standard method methods. The minimal relative errors in the quantitative determination of monosaccharides among the three methods confirmed the dependability of the method. By analyzing 20 batches of L. barbarum samples, D-galacturonic acid exhibited the highest content and the polysaccharide levels ranged from 3.02 to 13.04 mg/g. All data implied the specificity and accuracy of the method.

Transcriptional deciphering of the metabolic pathways associated with the bioactive ingredients of wolfberry species with different quality characteristics.

BACKGROUND: Wolfberry is rich in carotenoids, flavonoids, vitamins, alkaloids, betaines and other bioactive ingredients. For over 2,000 years, wolfberry has been used in China as a medicinal and edible plant resource. Nevertheless, the content of bioactive ingredients varies by cultivars, resulting in uneven quality across wolfberry cultivars and species. To date, research has revealed little about the underlying molecular mechanism of the metabolism of flavonoids, carotenoids, and other bioactive ingredients in wolfberry. RESULTS: In this context, the transcriptomes of the Lycium barbarum L. cultivar 'Ningqi No. 1' and Lycium chinense Miller were compared during the fruit maturity stage using the Illumina NovaSeq 6000 sequencing platform, and subsequently, the changes of the gene expression profiles in two types of wolfberries were analysed. In total, 256,228,924 clean reads were obtained, and 8817 differentially expressed genes (DEGs) were identified, then assembled by Basic Local Alignment Search Tool (BLAST) similarity searches and annotated using Gene Ontology (GO), Clusters of Orthologous Groups of proteins (KOG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). By combining these transcriptome data with data from the PubMed database, 36 DEGs related to the metabolism of bioactive ingredients and implicated in the metabolic pathway of carotenoids, flavonoids, terpenoids, alkaloids, vitamins, etc., were identified. In addition, among the 9 differentially expressed transcription factors, LbAPL, LbPHL11 and LbKAN4 have raised concerns. The protein physicochemical properties, structure prediction and phylogenetic analysis indicated that LbAPL and LbPHL11 may be good candidate genes involved in regulating the flavonoid metabolism pathway in wolfberry. CONCLUSIONS: This study provides preliminary evidence for the differences in bioactive ingredient content at the transcription level among different wolfberry species, as well as a research and theoretical basis for the screening, cloning and functional analysis of key genes involved in the metabolism of bioactive ingredients in wolfberry.

Transcriptome analysis reveals genes related to the synthesis and metabolism of cell wall polysaccharides in goji berry (Lycium barbarum L.) from various regions.

BACKGROUND: In goji berries (Lycium barbarum L.), the cell wall properties and ripening environment affect fruit quality and their economic benefits. However, the mechanism underlying the cell wall remains to be fully elucidated. RESULTS: The results showed that total sugar content was higher in Qinghai berries (13.87%, P < 0.01), whereas cellulose content peaked in Zhongning berries (28%, P < 0.05). Arabinose, galactose, and galacturonic acid were the principal components of the cell wall polysaccharides in goji berries. Among them, the content of galactose in Zhongning was significantly the highest (P < 0.05). Interestingly, we found that highly expressed ß-glucosidase and lowly expressed endoglucanase led to cellulose accumulation by RNA-sequencing analysis. The expression analysis results suggested that pectate lyase and pectinesterase enzymes could be major factors related to higher galactose and galacturonic acid contents in Zhongning compared to in Qinghai and Gansu. The starch and sucrose metabolism pathway, pentose and glucuronate interconversions pathway, and galactose metabolism pathway played a significant role in cell wall polysaccharide synthesis and metabolism. CONCLUSION: In the present study, we aimed to provide some insights into the cell wall on polysaccharide composition, structural features, and gene analysis in goji berries from Zhongning, Qinghai, and Gansu in China. These results might help to clarify the molecular function of the major genes in the cell wall polysaccharides of goji berries and provide a solid foundation for further study. © 2023 Society of Chemical Industry.

Diversity and spatiotemporal dynamics of fungal communities in the rhizosphere soil of Lycium barbarum L.: a new insight into the mechanism of geoherb formation.

Lycium barbarum L. is a well-known traditional geoherb in Ningxia, China. The fruits of L. barbarum contain several dietary constituents, and thus, they exert many beneficial effects on human health. However, a few studies have been conducted on the geoherb L. barbarum and its rhizosphere soil fungal community. In this study, we determined the physicochemical properties and fungal community structure of rhizosphere soil of L. barbarum from three regions of China, namely Ningxia (NX), Qinghai (QH), and Xinjiang (XJ), during three development stages of L. barbarum. Soil pH varied between 7.56 and 8.60 across the three regions, indicating that alkaline soil is conducive to the growth of L. barbarum. The majority of soil properties in NX, an authentic geoherb-producing area, were substantially inferior to those in XJ and QH during all three developmental stages. Total sugar, polysaccharide (LBP), and flavonoid contents were the highest in wolfberry fruits from NX. High-throughput sequencing showed that the abundance of the soil fungal population in NX was higher than that in QH and XJ during the flowering and fruiting stage and summer dormant stage. Moreover, the soil fungal diversity increased with the development of wolfberry. Ascomycota and Mortierellomycota were the predominant phyla in the rhizosphere fungal communities in all samples. Redundancy analysis showed a significant correlation of the soil-available phosphorus and LBP of wolfberry fruits with the fungal community composition. The characteristics of rhizosphere fungal communities determined in the present study provide insights into the mechanism of geoherb formation in NX wolfberry.

LbAMT3-1, an ammonium transporter induced by arbuscular mycorrhizal in Lycium barbarum, confers tobacco with higher mycorrhizal levels and nutrient uptake.

KEY MESSAGE: An ammonium transporter LbAMT3-1 overexpression increases the arbuscular abundance of mycorrhizal that opens the possibility of using LbAMT3-1 in breeding programs to improve symbiotic nutrient uptake in Lycium barbarum. Nitrogen (N) is one of the most essential nutrients required by plants and limits net primary production much of the time in most terrestrial ecosystems. Arbuscular mycorrhizal (AM) fungi can enhance plant nutrient uptake and improve plant productivity in nutrient limit ecosystems. Here, we identified an ammonia transporter, LbAMT3-1, specifically induced by AM fungi in Lycium barbarum. To understand the expression characteristics and biological functions, LbAMT3-1 was cloned, characterized, and overexpressed in Nicotiana tabacum (tobacco). A BLAST search identified the coding sequence for LbAMT3-1 with an open-reading frame of 1473 bp. Reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that, besides mycorrhizal roots, LbAMT3-1 were barely detectable in other tissues, including stems and leaves. Promoter-GUS assay showed that GUS staining was detected in mycorrhizal roots, and GUS activity driven by the LbAMT3-1 promoter was exclusively confined to root cells containing arbuscules. LbAMT3-1 functionally complemented the yeast mutant efficiently, and yeast expressing LbAMT3-1 showed well growth on the agar medium with 0.02, 0.2, and 2 mM NH4+ supply. Moreover, overexpression of LbAMT3-1 in N. tabacum resulted a significant increase in arbuscular abundance and enhanced the nutrient acquisition capacity of mycorrhizal plants. Based on the results of our study, we propose that overexpression of LbAMT3-1 can promote P and N uptake of host plants through the mycorrhizal pathway, and increase the colonization intensity and arbuscular abundance, which opens the possibility of using LbAMT3-1 in breeding programs.

Efficient saccharification of Lycium barbarum leaf biomass by using enzyme cocktails produced by a novel fungus Aspergillus costaricensis LS18.

The utilization of agro-industrial residues is an interesting issue contributing to sustainable development and environmental protection. Lycium barbarum leaves (LBL) are agro-industrial residues of the L. barabrum berry cultivation and seriously underutilized, leading to resource waste and environmental pollution. In this study, we prepared cost-effective enzyme cocktails with high xylanase activity from a novel soil-derived fungal strain Aspergillus costaricensis LS18. The xylanase activity of these on-site produced enzyme cocktails was 3.49 ± 0.55 U/mL. Through the hydrolysis using the enzyme cocktails with 6% substrate loading at 45 °C for 12 h, 86.57 ± 1.81% of total reducing sugars (RS) from LBL was released. The concentration of RS in the hydrolysates reached 8.17 ± 0.33 mg/mL. In this study, LBL were added values by two mutually independent bioprocess ways. On the one hand, LBL were used as the only nutrients in the medium for the on-site production of enzyme cocktails by fermentation. On the other hand, through hydrolysis using this enzyme cocktail, LBL biomass was efficiently hydrolyzed and fermentable monosugars were gained. This study could benefit to the exploitation of LBL resources and provide the references for utilization of other agro-industrial residues.

Comparative Analysis of the Phenolic Profile of Lycium barbarum L. Fruits from Different Regions in China.

Lycium barbarum L. (LB) fruits have high nutritive values and therapeutic effects. The aim of this study was to comprehensively evaluate the differences in phenolic composition of LB fruits from different geographical regions. Different methods of characterization and statistical analysis of data showed that different geographic sources of China could be significantly separated from each other. The highest total phenolic compound (TPC) content was observed in LB fruits from Ningxia (LBN), followed by those from Gansu (LBG) and Qinghai (LBQ). The Fourier transform infrared (FTIR) spectra of LB fruits revealed that LBQ had a peak at 2972 cm-1 whereas there was no similar peak in LBG and LBQ. A new HPLC method was established for the simultaneous determination of 8 phenolic compounds by quantitative analysis of multiple components by a single marker (QAMS), including 4 phenolic acids (chlorogenic acid, caffeic acid, 4-hydroxycinnamic acid, and ferulic acid), 1 coumarin (scopoletin), and 3 flavonoids (kaempferol-3-O-rutinoside, rutin, and narcissoside). It was showed that rutin was the most dominant phenolic compound in LBQ, although the average content of 4 phenolic acids was also high in LBQ, and scopoletin was the richest in LBG. UHPLC-Q-TOF-MS was used to qualitatively analyze the phenolics, which showed LBN was abundant in phenolic acids, LBQ was rich in flavonoids, and coumarins were the most plentiful in LBG. In conclusion, this study can provide references for the quality control and evaluation of phenolics in LB fruits and their by-products.

Recovery and recycling of solvent of counter-current chromatography: The sample of isolation of zeaxanthin in the Lycium barbarum L. fruits.

An efficient method of recovering and recycling solvent for counter-current chromatography was established by which zeaxanthin was separated from Lycium barbarum L. fruits. A column with activated carbon combined with high performance counter-current chromatography formed the recovering and recycling solvent system. Using the solvent system of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v) from the references, five injections were performed with an almost unchanged purity of zeaxanthin (80.9, 81.2, 81.5, 81.3, and 80.2% respectively) in counter-current chromatography separation. Meanwhile, the mobile phase reduced by half than conventional counter-current chromatography. By this present method, an effective improvement of counter-current chromatography solvent utilization was achieved.

Integrative Analysis of Transcriptome and Metabolome Reveals Salt Stress Orchestrating the Accumulation of Specialized Metabolites in Lycium barbarum L. Fruit.

Salt stress seriously affects yield and quality of crops. The fruit of Lycium barbarum (LBF) is extensively used as functional food due to its rich nutrient components. It remains unclear how salt stress influences the quality of LBF. In this study, we identified 71 differentially accumulated metabolites (DAMs) and 1396 differentially expressed genes (DEGs) among ripe LBF with and without 300 mM of NaCl treatment. Pearson correlation analysis indicated that the metabolomic changes caused by salt stress were strongly related to oxidoreductases; hydrolases; and modifying enzymes, in particular, acyltransferases, methyltransferases and glycosyltransferases. Further analysis revealed that salt stress facilitated flavonoid glycosylation and carotenoid esterification by boosting the expression of structural genes in the biosynthetic pathways. These results suggested that salt stress prompts the modification of flavonoids and carotenoids to alleviate ROS damage, which in turn improves the quality of LBF. Our results lay a solid foundation for uncovering the underlying molecular mechanism of salt stress orchestrating LBF quality, and the candidate genes identified will be a valuable gene resource for genetic improvement of L. barbarum.

Determination of Biogenic Amines in Different Parts of Lycium barbarum L. by HPLC with Precolumn Dansylation.

The aim of this work was to characterize biogenic amines (BAs) in different parts of Lycium barbarum L. using HPLC with dansyl chloride derivatization, and jointly, to provide referential data for further exploration and utilization of Lycium barbarum L. The linear correlation coefficients for all BAs were above 0.9989. The limits of detection and quantification were 0.015-0.075 and 0.05-0.25 µg/mL, respectively. The relative standard deviations for the intra-day and inter-day precision were 0.66-2.69% and 0.91-4.38%. The described method has good repeatability and intermediate precision for the quantitative determination of BAs in different parts of Lycium barbarum L. Satisfactory recovery for all amines was obtained (79.3-110.3%). The result showed that there were four kinds of BAs. The highest putrescine content (20.9 ± 3.2 mg/kg) was found in the flower. The highest histamine content (102.7 ± 5.8 mg/kg) was detected in the bark, and the highest spermidine (13.3 ± 1.6 mg/kg) and spermine (23.7 ± 2.0 mg/kg) contents were detected in the young leaves. The high histamine (HIS) content in the bark may be one of the reasons why all of the parts of Lycium barbarum L., except the bark, are used for medicine or food in China. Meanwhile, the issue of the high concentration of HIS should be considered when exploiting or utilizing the bark of Lycium barbarum L.

Carotenoid Contents of Lycium barbarum: A Novel QAMS Analyses, Geographical Origins Discriminant Evaluation, and Storage Stability Assessment.

Given the standard substances of zeaxanthin and its homologues obtained from Lycium barbarum L. (LB) are extremely scarce and unstable, a novel quantitative analysis of carotenoids by single marker method, named QAMS, was established. Four carotenoids including lutein, zeaxanthin, ß-carotene, and zeaxanthin dipalmitate were determined simultaneously by employing trans-ß-apo-8'-carotenal, a carotenoid component which did not exist in LB, as standard reference. Meanwhile, ß-carotene, another carotenoid constituent which existed in LB, was determined as contrast. The QAMS methods were fully verified and exhibited low standard method difference with the external standard method (ESM), evidenced by the contents of four carotenoids in 34 batches of LB samples determined using ESM and QAMS methods, respectively. HCA, PCA, and OPLS-DA analysis disclosed that LB samples could be clearly differentiated into two groups: one contained LB samples collected from Ningxia and Gansu; the other was from Qinghai, which was directly related to the different geographical location. Once exposed under high humidity (RH 75 ± 5%) at a high temperature (45 ± 5 °C) as compared with ambient temperature (25 ± 5 °C), from day 0 to day 28, zeaxanthin dipalmitate content was significantly decreased, and ultimately, all the decrease rates reached about 80%, regardless of the storage condition. Our results provide a good basis for improving the quality control of LB.

The major zeaxanthin dipalmitate derivatives from wolfberry.

Zeaxanthin dipalmitate (3) and two zeaxanthin dipalmitate derivatives, including one new compound (1), were obtained from wolfberry [the fruit of Lycium barbarum L. (Solanaceae)]. Their structures were unambiguously elucidated by spectroscopic analyses. Compound 2 is isolated from the genus Lycium for the first time, and its 1D/2D NMR data are firstly reported. All the compounds belong to carotenoids which are a kind of major bioactive constituents in wolfberry and are also responsible for wolfberry's red color.

Coupling Ultrafiltration-Based Processes to Concentrate Phenolic Compounds from Aqueous Goji Berry Extracts.

In this work, a membrane-based process for the purification and concentration of antioxidant compounds from aqueous Goji (Lycium barbarum L.) berry extracts was investigated. The aqueous extract was previously clarified with hollow fiber ultrafiltration (UF) membranes in order to remove suspended solids and ß-carotene and to produce a clarified extract enriched in phenolic compounds. Then, three UF flat sheet polyamide membranes with a molecular weight cut-off (MWCO) in the range 1000-3500 Da were tested to purify and concentrate phenolic compounds from the clarified extract. The effect of MWCO and transmembrane pressure (TMP) on the performance of selected membranes in terms of productivity and selectivity towards total dissolved solids (TDS), total phenolic compounds (TPC), total carbohydrates (TC) and total antioxidant activity (TAA) was evaluated. Experimental results indicated that the 2500 Da membrane exhibited a lower fouling index, higher cleaning efficiency, lower rejection towards carbohydrates (lower than 30%) and higher rejection towards phenolic compounds (higher than 50%) in comparison to the other investigated membranes. The inclusion of a diafiltration process in the treatment of the clarified extract with this membrane in a spiral-wound configuration improved the concentration of sugar compounds in the permeate stream and increased the purification of phenolic compounds in the retentate fraction.

Impact of Nitrogen Fertilizer Levels on Metabolite Profiling of the Lycium barbarum L. Fruit.

The yield and quality of goji (Lycium barbarum L.) fruit are heavily dependent on fertilizer, especially the availability of nitrogen, phosphorus, and potassium (N, P, and K, respectively). In this study, we performed a metabolomic analysis of the response of goji berry to nitrogen fertilizer levels using an Ultra Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS/MS) method. There was no significant difference in the fruit yield or the commodity grade between N0 (42.5 g/plant), N1 (85 g/plant), and N2 (127.5 g/plant). The primary nutrients of the goji berry changed with an increasing nitrogen fertilization. Comparative metabolomic profiling of three nitrogen levels resulted in the identification of 612 metabolites, including amino acids, flavonoids, carbohydrates, organic acids, and lipids/alcohols, among others, of which 53 metabolites (lipids, fatty acids, organic acids, and phenolamides) demonstrated significant changes. These results provide new insights into the molecular mechanisms of the relationship between yield and quality of goji berry and nitrogen fertilizer.

Lycium barbarum L. Polysaccharide (LBP) Reduces Glucose Uptake via Down-Regulation of SGLT-1 in Caco2 Cell.

Lycium barbarum L. polysaccharide (LBP) is prepared from Lycium barbarum L. (L. barbarum), which is a traditional Chinese medicine. LPB has been shown to have hypoglycemic effects. In order to gain some mechanistic insights on the hypoglycemic effects of LBP, we investigated the uptake of LBP and its effect on glucose absorption in the human intestinal epithelial cell line Caco2 cell. The uptake of LBP through Caco2 cell monolayer was time-dependent and was inhibited by phloridzin, a competitive inhibitor of SGLT-1. LPB decreased the absorption of glucose in Caco2 cell, and down-regulated the expression of SGLT-1. These results suggest that LBP might be transported across the human intestinal epithelium through SGLT-1 and it inhibits glucose uptake via down-regulating SGLT-1.

Low-temperature headspace-trap gas chromatography with mass spectrometry for the determination of trace volatile compounds from the fruit of Lycium barbarum L.

The total saccharides content of Lycium barbarum L. is very high, and a high temperature would result in saccharide decomposition and the emergence of a large amount of water. Moreover, the volatile compounds from the fruit of L. barbarum L. are rather low in concentration. Hence, it is difficult for a conventional headspace method to study the volatile compounds from the fruit of L. barbarum L. Since headspace-trap gas chromatography with mass spectrometry is an excellent method for trace analysis, a headspace-trap gas chromatography with mass spectrometry method based on low-temperature (30°C) enrichment and multiple headspace extraction was developed to explore the volatile compounds from the fruit of L. barbarum L. The headspace of the sample was extracted in 17 cycles at 30°C. Each time, the compounds extracted were concentrated in the trap (Tenax TA and Tenax GR, 1:1). Finally, all the volatile compounds were delivered into the gas chromatograph after thermal desorption. With the method described above, a total of 57 compounds were identified. The identification was completed by mass spectral search, retention index, and accurate mass measurement.

Antioxidant, Antimicrobial Effects and Phenolic Profile of Lycium barbarum L. Flowers.

L. barbarum L. is a widely-accepted nutraceutical presenting highly advantageous nutritive and antioxidant properties. Its flowers have been previously described as a source of diosgenin, ß-sitosterol and lanosterol that can be further pharmaceutically developed, but no other data regarding their composition is available. The purpose of this work was to investigate the chemical constituents, antioxidant and antimicrobial activities of L. barbarum flowers, as an alternative resource of naturally-occurring antioxidant compounds. The free radical scavenging activity of the ethanolic extract was tested by TEAC, two enzymatic assays with more physiological relevance and EPR spectroscopy. The presence of several phenolic compounds, such as chlorogenic, p-coumaric and ferulic acids, but also isoquercitrin, rutin and quercitrin, was assessed by an HPLC/MS method. The antioxidant assays revealed that the extract exhibited a moderate antioxidant potential. The antimicrobial activity was mild against Gram-positive bacteria and lacking against Escherichia coli. These findings complete the scarce existing data and offer new perspectives for further pharmaceutical valorization of L. barbarum flowers.

iTRAQ-based proteomics identifies proteins associated with betaine accumulation in Lycium barbarum L.

In order to better understand the mechanism of betaine accumulation in Lycium barbarum L. (LBL), we used iTRAQ (Isotope relative and absolute quantitative labeling) proteomics to screen and identify differentially abundant proteins (DAPs) at five stages (S1-young fruit stage, S2-green fruit stage, S3-early yellowing stage, S4-late yellowing stage, S5-ripening stage). A total of 1799 DAPs and 171 betaine-related DAPs were identified, and phosphatidylethanolamine N-methyltransferase (NMT), choline monooxygenase (CMO), and betaine aldehyde dehydrogenase (BADH) were found to be the key enzymes related to betaine metabolism. These proteins are mainly involved in carbohydrates, amino acids and their derivatives, fatty acids, carboxylic acids, photosynthesis and photoprotection, isoquinoline alkaloid biosynthesis, peroxisomes, and glycine, serine, and threonine metabolism. Three of the key enzymes were also up- and down-regulated to different degrees at the mRNA level. The study provide new insights into the of mechanism of betaine accumulation in LBL. SIGNIFICANCE: Betaine, a class of naturally occurring, water-soluble alkaloids, has been found to be widespread in animals, higher plants, and microbes. In addition to being an osmotic agent, betaine has biological functions such as hepatoprotection, neuroprotection, and antioxidant activity. Betaine metabolism (synthesis and catabolism) is complexly regulated by developmental and environmental signals throughout the life cycle of plant fruit maturation. As a betaine-accumulating plant, little has been reported about the regulatory mechanisms of betaine metabolism during the growth and development of Lycium barbarum L. (LBL) fruit. Therefore, this study used iTRAQ quantitative proteomics technology to investigate the abundance changes of betaine-related proteins in LBL fruit, screen and analyze the differential abundance proteins related to betaine metabolism, and provide theoretical references for the in-depth study of the mechanism of betaine metabolism in LBL fruit.