Advances in mass spectrometry imaging for plant metabolomics-Expanding the analytical toolbox.

Mass spectrometry imaging (MSI) has become increasingly popular in plant science due to its ability to characterize complex chemical, spatial, and temporal aspects of plant metabolism. Over the past decade, as the emerging and unique features of various MSI techniques have continued to support new discoveries in studies of plant metabolism closely associated with various aspects of plant function and physiology, spatial metabolomics based on MSI techniques has positioned it at the forefront of plant metabolic studies, providing the opportunity for far higher resolution than was previously available. Despite these efforts, profound challenges at the levels of spatial resolution, sensitivity, quantitative ability, chemical confidence, isomer discrimination, and spatial multi-omics integration, undoubtedly remain. In this Perspective, we provide a contemporary overview of the emergent MSI techniques widely used in the plant sciences, with particular emphasis on recent advances in methodological breakthroughs. Having established the detailed context of MSI, we outline both the golden opportunities and key challenges currently facing plant metabolomics, presenting our vision as to how the enormous potential of MSI technologies will contribute to progress in plant science in the coming years.

Potential locus W and candidate gene McPRR2 associated with pericarp pigment accumulation in bitter gourd (Momordica charantia L.) revealed via BSA-seq analysis.

Pericarp color is a prominent agronomic trait that exerts a significant impact on consumer and breeder preferences. Genetic analysis has revealed that the pericarp color of bitter gourd is a quantitative trait. However, the underlying mechanism for this trait in bitter gourd remains largely unknown. In the present study, we employed bulked segregant analysis (BSA) to identify the candidate genes responsible for bitter gourd pericarp color (specifically, dark green versus white) within F2 segregation populations resulting from the crossing of B07 (dark green pericarp) and A06 (white pericarp). Through genomic variation, genetic mapping, and expression analysis, we identified a candidate gene named McPRR2, which was a homolog of Arabidopsis pseudo response regulator 2 (APRR2) encoded by LOC111023472. Sequence alignment of the candidate gene between the two parental lines revealed a 15-bp nucleotide insertion in the coding region of LOC111023472, leading to a premature stop codon and potentially causing a loss-of-function mutation. qRT-PCR analysis demonstrated that the expression of McPRR2 was significantly higher in B07 compared to A06, and it was primarily expressed in the immature fruit pericarp. Moreover, overexpression of McPRR2 in tomato could enhance the green color of immature fruit pericarp by increasing the chlorophyll content. Consequently, McPRR2 emerged as a strong candidate gene regulating the bitter gourd pericarp color by influencing chlorophyll accumulation. Finally, we developed a molecular marker linked to pericarp color, enabling the identification of genotypes in breeding populations. These findings provided valuable insights into the genetic improvement of bitter gourd pericarp color.

The light and hypoxia induced gene ZmPORB1 determines tocopherol content in the maize kernel.

Tocopherol is an important lipid-soluble antioxidant beneficial for both human health and plant growth. Here, we fine mapped a major QTL-qVE1 affecting γ-tocopherol content in maize kernel, positionally cloned and confirmed the underlying gene ZmPORB1 (por1), as a protochlorophyllide oxidoreductase. A 13.7 kb insertion reduced the tocopherol and chlorophyll content, and the photosynthetic activity by repressing ZmPORB1 expression in embryos of NIL-K22, but did not affect the levels of the tocopherol precursors HGA (homogentisic acid) and PMP (phytyl monophosphate). Furthermore, ZmPORB1 is inducible by low oxygen and light, thereby involved in the hypoxia response in developing embryos. Concurrent with natural hypoxia in embryos, the redox state has been changed with NO increasing and H2O2 decreasing, which lowered γ-tocopherol content via scavenging reactive nitrogen species. In conclusion, we proposed that the lower light-harvesting chlorophyll content weakened embryo photosynthesis, leading to fewer oxygen supplies and consequently diverse hypoxic responses including an elevated γ-tocopherol consumption. Our findings shed light on the mechanism for fine-tuning endogenous oxygen concentration in the maize embryo through a novel feedback pathway involving the light and low oxygen regulation of ZmPORB1 expression and chlorophyll content.

Reveal the kernel dehydration mechanisms in maize based on proteomic and metabolomic analysis.

BACKGROUND: Kernel dehydration is an important factor for the mechanized harvest in maize. Kernel moisture content (KMC) and kernel dehydration rate (KDR) are important indicators for kernel dehydration. Although quantitative trait loci and genes related to KMC have been identified, where most of them only focus on the KMC at harvest, these are still far from sufficient to explain all genetic variations, and the relevant regulatory mechanisms are still unclear. In this study, we tried to reveal the key proteins and metabolites related to kernel dehydration in proteome and metabolome levels. Moreover, we preliminarily explored the relevant metabolic pathways that affect kernel dehydration combined proteome and metabolome. These results could accelerate the development of further mechanized maize technologies. RESULTS: In this study, three maize inbred lines (KB182, KB207, and KB020) with different KMC and KDR were subjected to proteomic analysis 35, 42, and 49 days after pollination (DAP). In total, 8,358 proteins were quantified, and 2,779 of them were differentially expressed proteins in different inbred lines or at different stages. By comparative analysis, K-means cluster, and weighted gene co-expression network analysis based on the proteome data, some important proteins were identified, which are involved in carbohydrate metabolism, stress and defense response, lipid metabolism, and seed development. Through metabolomics analysis of KB182 and KB020 kernels at 42 DAP, 18 significantly different metabolites, including glucose, fructose, proline, and glycerol, were identified. CONCLUSIONS: In sum, we inferred that kernel dehydration could be regulated through carbohydrate metabolism, antioxidant systems, and late embryogenesis abundant protein and heat shock protein expression, all of which were considered as important regulatory factors during kernel dehydration process. These results shed light on kernel dehydration and provide new insights into developing cultivars with low moisture content.

Integrated transcriptomic and metabolomic analysis of chili pepper fruits provides new insight into the regulation of the branched chain esters and capsaicin biosynthesis.

Chili pepper (Capsicum spp.) is one of the world's most popular vegetables and spices. Aroma is an important quality indicator of pepper, but the nature of the related volatiles is still not clear. In this study, we investigated the fruit of two pepper varieties, one with strong fruity aroma 'CC' Capsicum chinense and one without 'TJ' Capsicum annuum at four different developmental stages using transcriptomic and metabolomic analysis. The results showed that the content of green leaf volatiles (GLVs) was higher in TJ than in CC and was higher in the young fruit stage in both varieties. GLVs content was positively correlated with the expression of 13-LOX1, 2, 5 and HPL. But the levels of branched-chain (BC) esters and capsaicin were higher in CC, and were positively correlated with the expression of IMPS4 and DADH1. Our findings shed light on the molecular mechanism of aroma biosynthesis in pepper and provide a theoretical basis for the molecular breeding of high-quality pepper fruits.

Integrated metabolomics and lipidomics analyses suggest the temperature-dependent lipid desaturation promotes aflatoxin biosynthesis in Aspergillus flavus.

Temperature is one of the main factors affecting aflatoxin (AF) biosynthesis in Aspergillus flavus. Previous studies showed that AF biosynthesis is elevated in A. flavus at temperatures between 28°C-30°C, while it is inhibited at temperatures above 30°C. However, little is known about the metabolic mechanism underlying temperature-regulated AF biosynthesis. In this study, we integrated metabolomic and lipidomic analyses to investigate the endogenous metabolism of A. flavus across 6 days of mycelia growth at 28°C (optimal AF production) and 37°C (no AF production). Results showed that both metabolite and lipid profiles were significantly altered at different temperatures. In particular, metabolites involved in carbohydrate and amino acid metabolism were up-regulated at 37°C on the second day but down-regulated from days three to six. Moreover, lipidomics and targeted fatty acids analyses of mycelia samples revealed a distinct pattern of lipid species and free fatty acids desaturation. High degrees of polyunsaturation of most lipid species at 28°C were positively correlated with AF production. These results provide new insights into the underlying metabolic changes in A. flavus under temperature stress.

Diel dynamics of multi-omics in elkhorn fern provide new insights into weak CAM photosynthesis.

Crassulacean acid metabolism (CAM) has high water-use efficiency (WUE) and is widely recognized to have evolved from C3 photosynthesis. Different plant lineages have convergently evolved CAM, but the molecular mechanism that underlies C3-to-CAM evolution remains to be clarified. Platycerium bifurcatum (elkhorn fern) provides an opportunity to study the molecular changes underlying the transition from C3 to CAM photosynthesis because both modes of photosynthesis occur in this species, with sporotrophophyll leaves (SLs) and cover leaves (CLs) performing C3 and weak CAM photosynthesis, respectively. Here, we report that the physiological and biochemical attributes of CAM in weak CAM-performing CLs differed from those in strong CAM species. We investigated the diel dynamics of the metabolome, proteome, and transcriptome in these dimorphic leaves within the same genetic background and under identical environmental conditions. We found that multi-omic diel dynamics in P. bifurcatum exhibit both tissue and diel effects. Our analysis revealed temporal rewiring of biochemistry relevant to the energy-producing pathway (TCA cycle), CAM pathway, and stomatal movement in CLs compared with SLs. We also confirmed that PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) exhibits convergence in gene expression among highly divergent CAM lineages. Gene regulatory network analysis identified candidate transcription factors regulating the CAM pathway and stomatal movement. Taken together, our results provide new insights into weak CAM photosynthesis and new avenues for CAM bioengineering.

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide, jasmonic acid, and phytoalexin metabolic pathways.

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H2 O2 ) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H2 O2 in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.

Metabolic profiling reveals genotype-associated alterations in carotenoid content during banana postharvest ripening.

Banana fruits have attracted considerable attention for health-promoting effects attributed to ubiquitous functional metabolites. However, genotype-dependent accumulation patterns of carotenoids in banana remain largely unclear. Here, we performed a systematic metabolomic investigation of 18 banana cultivars of the AAA, AAB, or ABB genome groups. Our results indicate that the levels of soluble sugars increase during postharvest ripening regardless of genotype, whereas amino acids (AAs) and tricarboxylic acid (TCA) cycle-derived organic acids display genotype-dependent patterns. The levels of AAs derived from the glycolytic pathway increased, whereas those derived from the TCA cycle significantly decreased during ripening. The carotenoid composition in banana pulp was genotype-specific, and the contents of α-carotene were the highest in AAA-genome bananas. Moreover, high α-carotene and ß-carotene contents in banana were correlated with elevated levels of TCA cycle-derived AAs and decreased levels of glycolysis-derived AAs. Taken together, these findings provide a comprehensive understanding of genotype-associated carotenoid accumulation, thereby facilitating the breeding of future high carotenoid banana cultivars.

Linolenic Acid-Derived Oxylipins Inhibit Aflatoxin Biosynthesis in Aspergillus flavus through Activation of Imizoquin Biosynthesis.

Oxylipins play important signaling roles in aflatoxin (AF) biosynthesis in Aspergillus flavus. We previously showed that exogenous supply of autoxidated linolenic acid (AL) inhibited AF biosynthesis in A. flavus via oxylipins, but the molecular mechanism is still unknown. Here, we performed multiomics analyses of A. flavus grown in media with or without AL. Targeted metabolite analyses and quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) showed that the imizoquin (IMQ) biosynthetic pathway was distinctly upregulated in the presence of AL. 13C-glucose labeling confirmed in parallel that the tricarboxylic acid cycle was also enhanced by AL, consistent with observed increases in mycelial growth. Moreover, we integrated thermal proteome profiling and molecular dynamics simulations to identify a potential receptor of AL; AL was found to interact with a transporter (ImqJ) located in the IMQ gene cluster, primarily through hydrophobic interactions. Further analyses of strains with an IMQ pathway transcription factor overexpressed or knocked out confirmed that this pathway was critical for AL-mediated inhibition of AF biosynthesis. Comparison of 22 assembled A. flavus and Aspergillus oryzae genomes showed that genes involved in the IMQ pathway were positively selected in A. oryzae. Taken together, the results of our study provide novel insights into oxylipin-mediated regulation of AF biosynthesis and suggest potential methods for preventing AF contamination of crops.

Curcumin alleviates LPS-induced intestinal homeostatic imbalance through reshaping gut microbiota structure and regulating group 3 innate lymphoid cells in chickens.

Gastrointestinal dysfunction is associated with a disturbance of immune homeostasis, changes in the intestinal microbiome, alteration of the composition of the bile acid pool, and dynamic imbalance of group 3 innate lymphoid cells (ILC3s). Curcumin (CUR), a polyphenolic compound isolated from turmeric, has been known to attenuate intestinal inflammation in potential therapies for gastrointestinal diseases. It was hypothesized that CUR could target the gut microbiome to modulate bile acid (BA) metabolism and the function of ILC3s in ameliorating lipopolysaccharide (LPS)-induced imbalance of intestinal homeostasis in chickens. Seven hundred and twenty 1-day-old crossbred chickens were randomly divided into four treatments, namely CON_saline (basal diet + saline control), CUR_saline (basal diet + 300 mg kg-1 curcumin + saline), CON_LPS (basal diet + LPS), and CUR_LPS (basal diet + 300 mg kg-1 curcumin + LPS), each consisting of 6 replicates of 30 birds. On days 14, 17, and 21, the chickens in the CON_LPS and CUR_LPS treatments were intraperitoneally injected with LPS at 0.5 mg per kg BW. Dietary CUR supplementation significantly decreased LPS-induced suppression of growth performance and injury to the intestinal tight junctions and decreased the vulnerability to LPS-induced acute inflammatory response by inhibiting pro-inflammatory (interleukin-1ß and tumor necrosis factor-α) cytokines. CUR reshaped the cecal microbial community and BA metabolism, contributing to regulation of the intestinal mucosal immunity by promoting the anti-inflammatory (interleukin 10, IL-10) cytokines and enhancing the concentrations of primary and secondary BA metabolites (chenodexycholic acid, lithocholic acid). LPS decreased farnesoid X receptor (FXR) and G protein-coupled receptor class C group 5 member A synthesis, which was reversed by CUR administration, along with an increase in interleukin 22 (IL-22) production from ILC3s. Dietary supplementation of CUR increased the prevalence of Butyricicoccus and Enterococcus and enhanced the tricarboxylic acid cycle of intestinal epithelial cells. In addition, curcumin supplementation significantly increased sirtuin 1 and sirtuin 5 transcription and protein expression, which contributes to regulating mitochondrial metabolic and oxidative stress responses to alleviate LPS-induced enteritis. Our findings demonstrated that curcumin played a pivotal role in regulating the structure of the intestinal microbiome for health promotion and the treatment of intestinal dysbiosis. The beneficial effects of CUR may be attributed to the modulation of the BA-FXR pathway and inhibition of inflammation that induces IL-22 secretion by ILC3s in the intestinal lamina propria.

OsPP65 Negatively Regulates Osmotic and Salt Stress Responses Through Regulating Phytohormone and Raffinose Family Oligosaccharide Metabolic Pathways in Rice.

Although type 2C protein phosphatases (PP2Cs) have been demonstrated to play important roles in regulating plant development and various stress responses, their specific roles in rice abiotic stress tolerance are still largely unknown. In this study, the functions of OsPP65 in rice osmotic and salt stress tolerance were investigated. Here, we report that OsPP65 is responsive to multiple stresses and is remarkably induced by osmotic and salt stress treatments. OsPP65 was highly expressed in rice seedlings and leaves and localized in the nucleus and cytoplasm. OsPP65 knockout rice plants showed enhanced tolerance to osmotic and salt stresses. Significantly higher induction of genes involved in jasmonic acid (JA) and abscisic acid (ABA) biosynthesis or signaling, as well as higher contents of endogenous JA and ABA, were observed in the OsPP65 knockout plants compared with the wild-type plants after osmotic stress treatment. Further analysis indicated that JA and ABA function independently in osmotic stress tolerance conferred by loss of OsPP65. Moreover, metabolomics analysis revealed higher endogenous levels of galactose and galactinol but a lower content of raffinose in the OsPP65 knockout plants than in the wild-type plants after osmotic stress treatment. These results together suggest that OsPP65 negatively regulates osmotic and salt stress tolerance through regulation of the JA and ABA signaling pathways and modulation of the raffinose family oligosaccharide metabolism pathway in rice. OsPP65 is a promising target for improvement of rice stress tolerance using gene editing.

Spatially resolved metabolomics reveals variety-specific metabolic changes in banana pulp during postharvest senescence.

Banana is one of most popular fruits globally due to health-promoting and disease-preventing effects, yet little is known about in situ metabolic changes across banana varieties. Here, we integrated gold nanoparticle (AuNP)-assisted laser desorption/ionization mass spectrometry imaging (LDI-MSI) and metabolomics to investigate the spatiotemporal distribution and levels of metabolites within Brazil and Dongguan banana pulps during postharvest senescence. Metabolomics results indicated that both postripening stages and banana varieties contribute to metabolite levels. Benefiting from improved ionization efficiency of small-molecule metabolites and less peak interference, we visualized the spatiotemporal distribution of sugars, amino acids (AAs) and monoamines within pulps using AuNP-assisted LDI-MSI for the first time, revealing that AAs and monoamines exclusively accumulated in the middle region near the seed zone. Monosaccharides and di/trisaccharides were generally distributed across entire pulps but exhibited different accumulation patterns. These findings provide a guide for breeding new varieties and improving extraction efficiency of bioactive compounds.

A reactive oxygen species burst causes haploid induction in maize.

Haploid induction (HI) is an important tool in crop breeding. Phospholipase A1 (ZmPLA1)/NOT LIKE DAD (NLD)/MATRILINEAL (MTL) is a key gene controlling HI in maize; however, the underlying molecular mechanism remains unclear. In this study, to dissect why loss of ZmPLA1 function could mediate HI we performed a comprehensive multiple omics analysis of zmpla1 mutant anthers by integrating transcriptome, metabolome, quantitative proteome, and protein modification data. Functional classes of significantly enriched or differentially abundant molecular entities were found to be associated with the oxidative stress response, suggesting that a reactive oxygen species (ROS) burst plays a critical role in HI. In support of this, we further discovered that a simple chemical treatment of pollen with ROS reagents could lead to HI. Moreover, we identified ZmPOD65, which encodes a sperm-specific peroxidase, as a new gene controlling HI. Taken together, our study revealed a likely mechanism of HI, discovered a new gene controlling HI, and created a new method for HI in maize, indicating the importance of ROS balance in maintaining normal reproduction and providing a potential route to accelerate crop breeding.

Dissecting the Protein Dynamics Coupled Ligand Binding with Kinetic Models and Single-Molecule FRET.

Protein-ligand interactions are crucial to many biological processes. Ligand binding and dissociation are the basic steps that allow proteins to function. Protein conformational dynamics have been shown to play important roles in ligand binding and dissociation. However, it is challenging to determine the ligand binding kinetics of dynamic proteins. Here, we undertook comprehensive single-molecule FRET (smFRET) measurements and kinetic model analysis to characterize the conformational dynamics coupled ligand binding of glutamine-binding protein (GlnBP). We showed that hinge and T118A mutations of GlnBP affect its conformational dynamics as well as the ligand binding affinity. Based on smFRET measurements, the kinetic model of ligand-GlnBP interactions was constructed. Using experimentally measured parameters, we solved the rate equations of the model and obtained the undetectable parameters of the model which allowed us to understand the ligand binding kinetics fully. Our results demonstrate that modulation of the conformational dynamics of GlnBP affects the ligand binding and dissociation rates. This study provides insights into the binding kinetics of ligands, which are related to the protein function itself.

Mechanism of carbendazim in treating pebrine disease of Bombyx mori based on GC/MS-based metabonomics.

Pebrine disease is caused by microporidia (Nosema bombycis) and is destructive to sericulture production. A carbendazim-based drug FangWeiLing (FWL) has a significant control effect on the disease, which is a successful example of drug treatment of microsporidia. In this study, the therapeutic effect and critical action time of FWL were investigated by silkworm rearing biological test. Besides, the hemolymph samples from silkworms in the control group, model group, and FWL group were analyzed by metabonomics based on gas chromatography-mass spectrometry (GC/MS). The results showed that FWL had a significant therapeutic effect on pebrine disease, and the critical action time was 24 ~ 48 h post inoculation. Forty-seven different metabolites related to pebrine disease were screened out, and correlated with starch and sucrose metabolism; aminoacyl-tRNA biosynthesis; arginine biosynthesis; glycine, serine, and threonine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. After pretreatment with FWL, the metabolites were all effectively regulated, indicating productive intervention. Principal component analysis (PCA) also showed that the overall metabolic profile of the FWL group tended toward the control group. Compared with the control group, 16 different metabolites were obtained from the hemolymph of B.mori in FWL group, mainly involving aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism. It indicated that FWL had some effect on silkworm metabolism, which might be related to the decrease in cocoon quality. In conclusion, combined with the life cycle of N. bombycis, the mechanism of carbendazim in the treatment of pebrine disease can be fully revealed. Carbendazim can effectively reduce the destruction of amino acid metabolism and carbohydrate metabolism by N. Bombycis infection by inhibiting the proliferation of the meronts in silkworms, thus maintaining the normal physiological state of B. mori and achieve therapeutic effects. GC/MS-based metabonomics is a valuable and promising strategy to understand the disease mechanism and drug treatment of pebrine disease.

Identification of Metabolomic Biomarkers of Seed Vigor and Aging in Hybrid Rice.

Seed deterioration during rice seed storage can lead to seed vigor loss, which adversely affects agricultural production, the long-term preservation of germplasm resources, and the conservation of species diversity. However, the mechanisms underlying seed vigor maintenance remain largely unknown. In this study, 16 hybrid rice combinations were created using four sterile lines and four restorer lines that have been widely planted in southern China. Following artificial aging and natural aging treatments, germination percentage and metabolomics analysis by gas chromatography-mass spectrometry was used to identify the metabolite markers that could accurately reflect the degree of aging of the hybrid rice seeds. Significant differences in the degree of seed deterioration were observed among the 16 hybrid rice combinations tested, with each hybrid combination having a different germination percentage after storage. The hybrid rice combination with the storage-resistant restorer line Guanghui122 exhibited the highest germination percentage under both natural and artificial storage. A total of 89 metabolic peaks and 56 metabolites were identified, most of which were related to primary metabolism. Interestingly, the content of galactose, gluconic acid, fructose and glycerol in the seeds increased significantly during the aging process. Absolute quantification indicated that galactose and gluconic acid were highly significantly negatively correlated with the germination percentage of the seeds under the different aging treatments. The galactose content was significantly positively correlated with gluconic acid content. Additionally, glycerol showed a significant negative correlation with the germination percentage in most hybrid combinations. Based on the metabolomics analysis, metabolite markers that could accurately reflect the aging degree of hybrid rice seeds were identified. Galactose and gluconic acid were highly significantly negatively correlated with the germination percentage of the seeds, which suggested that these metabolites could constitute potential metabolic markers of seed vigor and aging. These findings are of great significance for the rapid and accurate evaluation of seed aging degree, the determination of seed quality, and the development of molecular breeding approaches for high-vigor rice seeds.

Environmentally-driven metabolite and lipid variations correspond to altered bioactivities of black wolfberry fruit.

Black wolfberry is a commonly cultivated woody plant in China, and is rich in nutrients that are beneficial for human. To characterize the endogenous metabolite differences among black wolfberry fruits grown in different geographical regions, mass spectrometry-based metabolomic and lipidomic analyses were performed in black wolfberry grown in nine locations throughout five provinces in China, from which 204 primary and specialized metabolites, and 267 lipids were identified in their fruits. Three samples from Alxa Left Banner, Jinta, and Minqin showed dramatically altered metabolite profiles, displaying higher levels of phenolic acids, soluble sugars and flavonoids, but lower levels of tricarboxylic acid cycle intermediates and aromatic amino acids. Moreover, the lipid profile of the Alxa Left Banner sample was strikingly distinct from all other samples, with high levels of monogalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol, which are positively correlated with their anti-inflammatory capacities. These findings thus prompt for further studies on black wolfberry fruit for their health benefits.