Adequate water supply enhances seedling growth and metabolism in Festuca kryloviana: insights from physiological and transcriptomic analys.

BACKGROUND: Festuca kryloviana is a significant native grass species in the Qinghai Lake region, and its low emergence rate is a primary factor limiting the successful establishment of cultivated grasslands. The region's arid and low-rainfall climate characteristics result in reduced soil moisture content at the surface. Despite the recognized impact of water availability on plant growth, the specific role of moisture in seedling development remains not fully elucidated. This study aims to investigate the germination rate and seedling growth velocity of F. kryloviana seeds under varying moisture conditions, and to integrate physiological and transcriptomic analyses of seedlings under these conditions to reveal the mechanisms by which water influences seedling development. RESULTS: The emergence rate of F. kryloviana seedlings exhibited an initial increase followed by a decrease with increasing moisture content. The highest emergence rate, reaching 75%, was observed under 20% soil moisture conditions. By the eighth day of the experiment, the lengths of the plumules and radicles under the optimal emergence rate (full water, FW) were 21.82% and 10.87% longer, respectively, than those under closely matching the soil moisture content during the background survey (stress water, SW). The differential development of seedlings under varying moisture regimes is attributed to sugar metabolism within the seeds and the accumulation of abscisic acid (ABA). At FW conditions, enhanced sugar metabolism, which generates more energy for seedling development, is facilitated by higher activities of α-amylase, sucrose synthase, and trehalose-6-phosphate synthase compared to SW conditions. This is reflected at the transcriptomic level with upregulated expression of the α-amylase (AMY2) gene and trehalose-6-phosphate synthase (TPS6), while genes associated with ABA signaling and transduction are downregulated. Additionally, under FW conditions, the expression of genes related to the chloroplast thylakoid photosystems, such as photosystem II (PSII) and photosystem I (PSI), is upregulated, enhancing the seedlings' light-capturing ability and photosynthetic efficiency, thereby improving their autotrophic capacity. Furthermore, FW treatment enhances the expression of the non-enzymatic antioxidant system, promoting metabolism within the seeds. In contrast, SW treatment increases the activity of the enzymatic antioxidant system, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), to cope with water stress. CONCLUSIONS: Our experiment systematically evaluated the impact of moisture conditions on the growth and development of F. kryloviana seedlings. Physiological and transcriptomic data collectively indicate that adequate water (20%) supply enhances seedling growth and development by reducing ABA levels and increasing α-amylase activity within seeds, thereby boosting sugar metabolism and promoting the growth of seedling, which in turn leads to an improved emergence rate. Considering water management in future cultivation practices may be a crucial strategy for enhancing the successful establishment of F. kryloviana in grassland ecosystems.

'Emerging into the World' - the regulation and control of dormancy and sprouting in geophytes.

Geophytic plants synchronize growth and quiescence with the external environment to survive and thrive under changing seasons. Besides seasonal growth adaptation, dormancy and sprouting are critical factors determining crop yield and market supply as various geophytes also serve as major food, floriculture, and ornamental crops. Dormancy in such crops decides crop availability in the market, as most of such crops are consumed during the dormant stage. On the other hand, uniform/maximal sprouting is crucial for maximum yield. Thus, dormancy and sprouting regulation have great economic importance. Dormancy-sprouting cycles in geophytes are regulated by genetic, exogenous (environmental), and endogenous (genetic, metabolic and hormonal, etc.) factors. Comparatively, the temperature is more dominant in regulating dormancy and sprouting in geophytes, unlike aboveground tissues, where both photoperiod and temperature control are involved. Despite huge economic importance, studies concerning the regulation of dormancy and sprouting are scarce in the majority of geophytes. To date, only a few molecular factors involved in the process have been suggested. Recently, omics studies on molecular and metabolic factors involved in dormancy and growth regulations of underground vegetative tissues have provided more insight into the mechanism. Here, we discuss current knowledge of the environmental and molecular regulation and control of dormancy and sprouting in geophytes and discuss challenges/questions that need to be addressed in the future for crop improvement.

Mixed-phase weak anion-exchange/reversed-phase LC-MS/MS for analysis of nucleotide sugars in human fibroblasts.

Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-ß-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond.

Transcriptomic and metabolomic profiling provide insight into the role of sugars and hormones in leaf senescence of Pinellia ternata.

KEY MESSAGE: The interaction network and pathway map uncover the potential crosstalk between sugar and hormone metabolisms as a possible reason for leaf senescence in P. ternata. Pinellia ternata, an environmentally sensitive medicinal plant, undergoes leaf senescence twice a year, affecting its development and yield. Understanding the potential mechanism that delays leaf senescence could theoretically decrease yield losses. In this study, a typical senescent population model was constructed, and an integrated analysis of transcriptomic and metabolomic profiles of P. ternata was conducted using two early leaf senescence populations and two stay-green populations. The result showed that two key gene modules were associated with leaf senescence which were mainly enriched in sugar and hormone signaling pathways, respectively. A network constructed by unigenes and metabolisms related to the obtained two pathways revealed that several compounds such as D-arabitol and 2MeScZR have a higher significance ranking. In addition, a total of 130 hub genes in this network were categorized into 3 classes based on connectivity. Among them, 34 hub genes were further analyzed through a pathway map, the potential crosstalk between sugar and hormone metabolisms might be an underlying reason of leaf senescence in P. ternata. These findings address the knowledge gap regarding leaf senescence in P. ternata, providing candidate germplasms for molecular breeding and laying theoretical basis for the realization of finely regulated cultivation in future.

Role of sugars in the apical hook development of Arabidopsis etiolated seedlings.

KEY MESSAGE: The sugar supply in the medium affects the apical hook development of Arabidopsis etiolated seedlings. In addition, we provided the mechanism insights of this process. Dicotyledonous plants form an apical hook structure to shield their young cotyledons from mechanical damage as they emerge from the rough soil. Our findings indicate that sugar molecules, such as sucrose and glucose, are crucial for apical hook development. The presence of sucrose and glucose allows the apical hooks to be maintained for a longer period compared to those grown in sugar-free conditions, and this effect is dose-dependent. Key roles in apical hook development are played by several sugar metabolism pathways, including oxidative phosphorylation and glycolysis. RNA-seq data revealed an up-regulation of genes involved in starch and sucrose metabolism in plants grown in sugar-free conditions, while genes associated with phenylpropanoid metabolism were down-regulated. This study underscores the significant role of sugar metabolism in the apical hook development of etiolated Arabidopsis seedlings.

Frequent Acquisition of Glycoside Hydrolase Family 32 (GH32) Genes from Bacteria via Horizontal Gene Transfer Drives Adaptation of Invertebrates to Diverse Sources of Food and Living Habitats.

Glycoside hydrolases (GHs, also called glycosidases) catalyze the hydrolysis of glycosidic bonds in polysaccharides. Numerous GH genes have been identified from various organisms and are classified into 188 families, abbreviated GH1 to GH188. Enzymes in the GH32 family hydrolyze fructans, which are present in approximately 15% of flowering plants and are widespread across microorganisms. GH32 genes are rarely found in animals, as fructans are not a typical carbohydrate source utilized in animals. Here, we report the discovery of 242 GH32 genes identified in 84 animal species, ranging from nematodes to crabs. Genetic analyses of these genes indicated that the GH32 genes in various animals were derived from different bacteria via multiple, independent horizontal gene transfer events. The GH32 genes in animals appear functional based on the highly conserved catalytic blades and triads in the active center despite the overall low (35-60%) sequence similarities among the predicted proteins. The acquisition of GH32 genes by animals may have a profound impact on sugar metabolism for the recipient organisms. Our results together with previous reports suggest that the acquired GH32 enzymes may not only serve as digestive enzymes, but also may serve as effectors for manipulating host plants, and as metabolic enzymes in the non-digestive tissues of certain animals. Our results provide a foundation for future studies on the significance of horizontally transferred GH32 genes in animals. The information reported here enriches our knowledge of horizontal gene transfer, GH32 functions, and animal-plant interactions, which may result in practical applications. For example, developing crops via targeted engineering that inhibits GH32 enzymes could aid in the plant's resistance to animal pests.

ZjHXK5 and ZjHXK6 negatively regulate the sugar metabolism of Ziziphus jujuba Mill.

Hexokinase (HXK) plays a crucial role in plants, catalyzing the phosphorylation of hexose substances, which is one of the key steps in sugar metabolism and energy production. While HXK genes have been well-studied in model plants, the evolutionary and functional characteristics of HXK gene family in jujube is unknow. In this study, the HXK gene family members were identified by bioinformatics methods, the key members regulating glucose metabolism were identified by transcriptome data, and finally the function of the key genes was verified by instantaneous and stable genetic transformation. Our results showed that seven HXK genes were identified in the jujube genome, all of which were predict located in the chloroplast and contain Hexokinase-1 (PF00349) and Hexokinase-2 (PF03727) conserved domains. Most of HXK proteins were transmembrane protein with stable, lipid-soluble, hydrophilic. The secondary structure of ZjHXK proteins main α-helix, and contains two distinct tertiary structure. All ZjHXK genes contain nine exons and eight introns. Predictions of cis-regulatory elements indicate that the promoter region of ZjHXK contains a large number of MeJA responsive elements. Finally, combined with the analysis of the relationship between the expression and glucose metabolism, found that ZjHXK5 and ZjHXK6 may the key genes regulating sugar metabolism. Transient overexpression of ZjHXK5 and ZjHXK6 on jujube, or allogeneic overexpression of ZjHXK5 and ZjHXK6 on tomato would significantly reduce the content of total sugar and various sugar components. Transient silencing of ZjHXK5 and ZjHXK6 genes results in a significant increase in sucrose and total sugar content. Interestingly, the expression of ZjHXK5 and ZjHXK6 were also affected by methyl jasmonate.

The role of the universal sugar transport system components PtsI (EI) and PtsH (HPr) in Enterococcus faecium.

Vancomycin-resistant enterococci (VRE) pose a serious threat to public health because of their limited treatment options. Therefore, there is an increasing need to identify novel targets to develop new drugs. Here, we examined the roles of the universal PTS components, PtsI and PtsH, in Enterococcus faecium to determine their roles in carbon metabolism, biofilm formation, stress response, and the ability to compete in the gastrointestinal tract. Clean deletion of ptsHI resulted in a significant reduction in the ability to import and metabolize simple sugars, attenuated growth rate, reduced biofilm formation, and decreased competitive fitness both in vitro and in vivo. However, no significant difference in stress survival was observed when compared with the wild type. These results suggest that targeting universal or specific PTS may provide a novel treatment strategy by reducing the fitness of E. faecium.

Mechanisms of Litchi Response to Postharvest Energy Deficiency via Energy and Sugar Metabolisms.

In the post-harvest phase, fruit is inexorably subjected to extrinsic stressors that expedite energy expenditure and truncate the storage lifespan. The present study endeavors to elucidate the response strategies of litchi to the alterations of energy state caused by 2,4-Dinitrophenol (DNP) treatment through energy metabolism and sugar metabolism. It was observed that the DNP treatment reduced the energy state of the fruit, exacerbated membrane damage and triggered rapid browning in the pericarp after 24 h of storage. Furthermore, the expression of genes germane to energy metabolism (LcAtpB, LcAOX1, LcUCP1, LcAAC1, and, LcSnRK2) reached their peak within the initial 24 h of storage, accompanied by an elevation in the respiratory rate, which effectively suppressed the rise in browning index of litchi pericarp. The study also posits that, to cope with the decrease of energy levels and membrane damage, litchi may augment the concentrations of fructose, glucose, inositol, galactose, and sorbose, thus safeguarding the canonical metabolic functions of the fruit. Collectively, these findings suggest that litchi can modulate energy and sugar metabolism to cope with fruit senescence under conditions of energy deficiency. This study significantly advances the understanding of the physiological responses exhibited by litchi fruit to post-harvest external stressors.

A novel growth-promoting dark septate endophytic fungus improved drought tolerance in blueberries by modulating phytohormones and non-structural carbohydrates.

Drought is a significant global issue affecting agricultural production, and the utilization of beneficial rhizosphere microorganisms is one of the effective ways to increase the productivity of crops and forest under drought. In this study, we characterized a novel growth-promoting dark septate endophytes (DSE) fungus R16 derived from the blueberry roots. Hyphae or microsclerotia were visible within the epidermal or cortical cells of R16-colonized blueberry roots, which was consistent with the typical characteristics of DSE fungi. Inoculation with R16 promoted the growth of blueberry seedlings and the advantage over the control group was more significant under PEG-induced drought. Comparison of physiological indicators related to drought resistance between the inoculated and control groups was performed on the potted blueberry plants, including the chlorophyll content, net photosynthetic rate, root activities, MDA and H2O2 content, which indicated that R16 colonization mitigated drought injury in blueberry plants. We further analyzed the effects of R16 on phytohormones and non-structural carbohydrates (NSCs) to explore the mechanism of increased drought tolerance by R16 in blueberry seedlings. The results showed that except for the GA content, IAA, ZT and ABA varied significantly between the inoculated and control groups. SPS and S6PDH activities in mature leaves, the key enzymes responsible for sucrose and sorbitol synthesis, respectively, as well as SDH, SuSy, CWINV, HXK and FRK in roots, the key enzymes involved in the NSCs metabolism, showed significant differences between the inoculated and control groups before and after drought treatment. These results suggested that the positive effects of R16 colonization on the drought tolerance of blueberry seedlings are partially attributable to the regulation of phytohormone and sugar metabolism. This study provided valuable information for the research on the interaction between DSE fungi and host plants as well as the application of DSE preparations in agriculture.

Drought Protective Effects of Exogenous ABA and Kinetin on Lettuce: Sugar Content, Antioxidant Enzyme Activity, and Productivity.

Drought is an environmental stressor that significantly impacts plant growth and development. Comprehending the complexity of drought stress and water utilization in the context of plant growth and development holds significant importance for sustainable agriculture. The aim of this study was to evaluate the effect of exogenously applied phytohormones on lettuce (Lactuca sativa L.) sugar content profiles and antioxidant enzyme activity and productivity. Lettuce plants were grown under normal and drought conditions in a growth chamber with a photoperiod of 14/10 h (day/night). Kinetin and abscisic acid were applied separately and in combinations when the second leaf was fully expanded. The results showed that sugar accumulation and productivity of the pretreated plants under drought were significantly higher than the controls. The perspective offered by this work showed that growth-related and stress-related phytohormones significantly influenced plant sugar metabolism, metabolic profiles, and productivity, thus enabling the control of yield and quality.

Integrated transcriptomics and metabolomics provides insights into the Nicotiana tabacum response to heat stress.

Heat stress is a prevalent factor that significantly damages crops, especially with the ongoing global warming and increasing frequency of extreme weather events. Tobacco is particularly sensitive to temperature fluctuations, experiencing reduced yield and quality under high temperatures. However, the underlying molecular mechanisms of heat resistance in tobacco remain poorly understood. This study comprehensively analyzed biochemical, transcriptomic, and metabolomic responses to heat stress on the root and shoot of the tobacco cultivar K326 compared to control conditions. Heat stress significantly increased the activities of antioxidant enzymes (CAT, POD, and SOD) and levels of osmotic mediators (soluble sugars, sucrose, and proline) in the shoot. Furthermore, transcriptome analysis identified 13,176 differentially expressed genes (DEGs) in the root (6,129 up-regulated and 7,047 down-regulated) and 12,283 DEGs (6,621 up-regulated and 5,662 down-regulated) in the shoot. The root had 24 enriched KEGG pathways, including phenylpropanoid metabolism, while the shoot had 32 significant pathways, such as galactose metabolism and MAPK signaling. The metabolomic data identified 647 metabolites in the root and 932 in the shoot, with carbohydrates and amino acids being the main categories. The root had 116 differentially abundant metabolites (DAMs) (107 up-regulated and 9 down-regulated), and the shoot contained 256 DAMs (251 up-regulated and 5 down-regulated). Joint transcriptome and metabolome analysis showed that galactose metabolism and starch and sucrose metabolism were co-enriched in both tissues. In contrast, amino sugar and nucleotide sugar metabolism was enriched in the root, and purine metabolism in the shoot. The purine metabolic pathway in the shoot can modulate the expression of MYB transcription factors by influencing ABA synthesis and signaling, thereby controlling the accumulation of HSPs, raffinose, sucrose, and trehalose to enhance heat tolerance. Furthermore, NtMYB78, an MYB transcription factor, enhances tolerance for heat stress in tobacco. This research offers a foundational framework for investigating and implementing heat-resistant genes and metabolic pathways in the root and shoot of tobacco seedlings.

Analysis of sugar components and identification of SPS genes in citrus fruit development.

Sugar is a primary determinant of citrus fruit flavour, but undergoes varied accumulation processes across different citrus varieties owing to high genetic variability. Sucrose phosphate synthase (SPS), a key enzyme in glucose metabolism, plays a crucial role in this context. Despite its significance, there is limited research on sugar component quality and the expression and regulatory prediction of SPS genes during citrus fruit development. Therefore, we analysed the sugar quality formation process in 'Kiyomi' and 'Succosa', two citrus varieties, and performed a comprehensive genome-wide analysis of citrus CsSPSs. We observed that the accumulation of sugar components significantly differs between the two varieties, with the identification of four CsSPSs in citrus. CsSPS sequences were highly conserved, featuring typical SPS protein domains. Expression analysis revealed a positive correlation between CsSPS expression and sugar accumulation in citrus fruits. However, CsSPS expression displays specificity to different citrus tissues and varieties. Transcriptome co-expression network analysis suggests the involvement of multiple transcription factors in shaping citrus fruit sugar quality through the regulation of CsSPSs. Notably, the expression levels of four CsWRKYs (CsWRKY2, CsWRKY20, CsWRKY28, CsWRKY32), were significantly positively correlated with CsSPSs and CsWRKY20 might can activate sugar accumulation in citrus fruit through CsSPS2. Collectively, we further emphasize the potential importance of CsWRKYs in citrus sugar metabolism, our findings serve as a reference for understanding sugar component formation and predicting CsSPS expression and regulation during citrus fruit development.

Gum arabic coating alleviates chilling injury of cold-stored peach by regulating reactive oxygen species, phenolic, and sugar metabolism.

In this study, gum arabic (GA) coating was employed to mitigate chilling injury in peach fruit, and it was observed that 10% GA coating exhibited the most favorable effect. GA coating significantly inhibited the decline of AsA content and enhanced antioxidant enzyme activity in peach fruit, thereby enhancing reactive oxygen species (ROS) scavenging rate while reducing its accumulation. Simultaneously, GA coating inhibited the activity of oxidative degradation enzymes for phenolics and enhanced synthase activity, thus maintaining higher levels of total phenolics and flavonoids in fruits. Additionally, compared to the control fruit, GA-coated fruits demonstrated higher concentrations of sucrose and sorbitol, accompanied more robust activity of sucrose synthase and sucrose phosphate synthase, as well as reduced activity of acid invertase and neutral invertase. Our study demonstrates that GA coating can effectively enhance the cold resistance of peach fruit by regulating ROS, phenolics, and sugar metabolism, maintaining high levels of phenolics and sucrose while enhancing antioxidant activity.

Advances in Engineering Nucleotide Sugar Metabolism for Natural Product Glycosylation in Saccharomyces cerevisiae.

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

Systems Metabolic Engineering to Elucidate and Enhance Intestinal Metabolic Activities of Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is one of the most widely used probiotics to treat gastrointestinal diseases. Recently, many studies have engineered EcN to release therapeutic proteins to treat specific diseases. However, because EcN exhibits intestinal metabolic activities, it is difficult to predict outcomes after administration. In silico and fermentation profiles revealed mucin metabolism of EcN. Multiomics revealed that fucose metabolism contributes to the intestinal colonization of EcN by enhancing the synthesis of flagella and nutrient uptake. The multiomics results also revealed that excessive intracellular trehalose synthesis in EcN, which is responsible for galactose metabolism, acts as a metabolic bottleneck, adversely affecting growth. To improve the ability of EcN to metabolize galactose, otsAB genes for trehalose synthesis were deleted, resulting in the ΔotsAB strain; the ΔotsAB strain exhibited a 1.47-fold increase in the growth rate and a 1.37-fold increase in the substrate consumption rate relative to wild-type EcN.

From acidity to sweetness: a comprehensive review of carbon accumulation in grape berries.

Most of the carbon found in fruits at harvest is imported by the phloem. Imported carbon provide the material needed for the accumulation of sugars, organic acids, secondary compounds, in addition to the material needed for the synthesis of cell walls. The accumulation of sugars during fruit development influences not only sweetness but also various parameters controlling fruit composition (fruit "quality"). The accumulation of organic acids and sugar in grape berry flesh cells is a key process for berry development and ripening. The present review presents an update of the research on grape berry development, anatomical structure, sugar and acid metabolism, sugar transporters, and regulatory factors.

Preharvest spraying of phenylalanine activates the sucrose and respiratory metabolism in muskmelon wounds during healing.

Phenylalanine (Phe) accelerates fruit wound healing by activating phenylpropanoid metabolism. However, whether Phe affects sucrose and respiratory metabolism in fruit during wound healing remains unknown. In this research, we found that preharvest Phe spray promoted sucrose degradation and increased glucose and fructose levels by activating acid invertase (AI), neutral invertase (NI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) on harvested muskmelons. The spray also activated hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G6PDH). In addition, the spray improved energy and reducing power levels in the fruit. Taken together, preharvest Phe spray can provide carbon skeleton, energy and reducing power for wound healing by activating the sucrose metabolism, Embden-Meyerhof-Parnas (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate (PPP) pathway in muskmelon wounds during healing, which is expected to be developed as a new strategy to accelerate fruit wound healing.

A comprehensive physiological and -Omic analysis of trypsin-mediated protection of green pepper fruits from chilling injury.

Chilling injury (CI) in green pepper fruits during low-temperature storage causes a significant decline in quality. The present study utilized physiological, transcriptomic, and metabolomic analyses to idneitfy the mechanisms by which trypsin mitigates CI in green peppers stored at 4 °C for 8 days, followed by 3 days of shelf life. Results indicated that the trypsin treatment significantly reduced electrolyte leakage and the CI index in peppers, effectively extending their shelf life and preserving postharvest quality. After 4 days of storage, comparative -omic analyses identified 2514 differentially expressed genes (DEGs) and 397 differentially abundant metabolites (DAMs) between trypsin-treated and control peppers. The trypsin treatment induced changes in sugar metabolism, modulating the expression of HK, SUS, INV, and GLGC, which affected the abundance of metabolites such as CDP-glucose and α-D-p-glucose. Trypsin also enhanced carotenoid metabolism, altering the abundance of rhodopinal glucoside, 1'-hydroxyl-γ-carotene glucoside, and farnesyl 1-PP, and influencing the expression of PDS, CRTH, CRTB, and LUT5. Notably, the trypsin treatment activated the mitogen-activated protein kinase (MAPK) pathway that plays an integral role in the signal transduction of abiotic stress. Differential expression of FLS2, ELF18, PTO, PR1, PTI5, WPKY, MEKK1, and MPK6 genes in the MAPK pathway was observed, which was correlated with CI mitigation in green peppers during cold storage. In conclusion, trypsin is an effective treatment for reducing CI in green peppers during cold storage. The present study provides valuable insights into its physiological and molecular impact on green pepper fruit.

Engineered Saccharomyces cerevisiae as a Biosynthetic Platform of Nucleotide Sugars.

Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.