Search

Raffinose family oligosaccharides (RFOs) have diverse structures and exhibit various biological activities. When using RFOs as prebiotics, their structures need to be identified. If we first knew whether an RFO was classical or non-classical, structural identification would become much easier. Here, we cloned and expressed an α-galactosidase (BF0224) from Bacteroides fragilis which showed strict specificity for hydrolyzing α-Gal-(1 → 6)-Gal linkages in RFOs. BF0224 efficiently distinguished classical from non-classical RFOs by identifying the resulting hydrolyzed oligo- and mono-saccharides with HPAEC-PAD-MS. Using this strategy, we identified a non-classical RFO from Pseudostellaria heterophylla (Miquel) Pax with DP6 (termed PHO-6), as well as a classical RFO from Lycopus lucidus Turcz. with DP7 (termed LTO-7). To characterize these RFO structures, we employed four other commercial or reported α-galactosidases in combination with NMR and methylation analysis. Using this approach, we elucidated the accurate chemical structure of PHO-6 and LTO-7. Our study provides an efficient analytical approach to structurally analyze RFOs. This enzyme-based strategy also can be applied to structural analysis of other glycans., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Soybean utilization is limited by the presence of raffinose oligosaccharides (RFO), which are not digested by humans and cause gastrointestinal discomfort. This study explores the potential of α-galactosidases from Penicillium griseoroseum for RFO hydrolysis in soymilk. Two distinct α-galactosidase enzymes, designated α-Gal1 and α-Gal2, were purified using a combination of ion-exchange chromatography and native polyacrylamide gel electrophoresis. Both enzymes exhibited characteristics of multimeric proteins and displayed similar biochemical properties. Optimal activity was observed at a pH range of 4.5-5.0 and a temperature range of 40-45 °C. Notably, α-Gal1 demonstrated high thermostability with a half-life of 16 h at 40 °C. The α-galactosidases displayed different substrate affinitiesfor the substrates ρ-NP-αGal, o-NP-αGal, rD-raffinose, d-stachyose, and mD-melibiose. The Michaelis-Menten constant (Km) values for α-Gal1 were 1.06, 1.31, 28.74, 19.88, and 4.77 mmol/L, respectively, while those for α-Gal2 were 0.8, 1.26, 30.46, 21.74 and 5.01 mmol/L, respectively. Both α-Gal1 and α-Gal2 were strongly inhibited by metal ions (Ag⁺, Cu 2 ⁺, Fe 2 ⁺, and Hg 2 ⁺) and moderately inhibited by d-melibiose. Importantly, both enzymes efficiently hydrolyzed RFOs, achieving complete d-stachyose elimination from soymilk after a 6-h incubation. These findings propose the promising application of these α-galactosidases in industrial soymilk production, potentially enhancing its nutritional value and alleviating gastrointestinal issues in consumers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Almeida, M. N. reports equipment, drugs, or supplies was provided by Federal University of São João del-Rei. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacterial contamination of raw diffusion juice poses unique challenges during the sugar extraction process. This study profiled bacterial communities by using full-length 16S rRNA amplicon sequencing and quantified the carbohydrate concentrations in raw diffusion juice samples received from sugar factory regions across the USA and Canada. Juice samples were collected at four time points during the 2021 and 2022 processing campaigns. Firmicutes was the dominant phylum from the raw diffusion juice samples collected during both campaigns and comprised 85.5% of total bacterial abundance. Lactic acid bacteria such as Leuconostoc and Lactobacillus were among the core genera which also dominated the bacterial community in raw diffusion juice. Positive correlations in the abundance of functionally and taxonomically related bacterial communities were identified. During the 2021 campaign, 44 bacterial genera were differentially abundant in raw diffusion juice extracted from sugarbeet roots in Periods 1 to 4. This number declined sixfold during the 2022 campaign to three genera. The concentration of raffinose in raw diffusion juice positively correlated to the relative abundance of Leuconostoc . Furthermore, an in vitro assay was performed to assess the growth dynamics of Leuconostoc mesenteroides in sucrose or raffinose-rich medium and observed the rapid consumption of both carbohydrates by this bacterium. This finding is important for deciphering microbial growth dynamics in raw diffusion juice that can be useful in minimizing sugar loss during the factory processing.IMPORTANCEFindings additionally provide baseline information that can be used to develop mitigation strategies that reduce losses due to microbial contamination of sucrose processing streams., Competing Interests: The authors declare no conflict of interest.

Deuterium labelling of the non-labile protium atoms in starch granules has been achieved for the first time, by growing genetically modified yeast on deuterated media. Mass spectrometry of the glucose monomers from digested starch showed 44 % average deuteration of the non-labile protium when grown on partially deuterated raffinose (with average deuteration 48 %); yielding starch with 26 % average overall deuteration. Non-labile deuteration was also demonstrated using D 2 O solvent in the culture medium. Solid-state NMR revealed that deuteration was not evenly distributed across the monomer, being highest at the C6 carbon and lowest at the C1 carbon. SANS revealed two structural features at q = 0.05 Å -1 and 0.4 Å -1 , the first corresponding to a lamellar repeat of approximately 12-13 nm while the latter is consistent with B-type crystalline polymer packing. Furthermore, solvent contrast variation SANS analysis yielded a contrast match point of 66 mol% D 2 O indicative of approximately 30-35 % average deuteration of the bulk granules, consistent with mass spectroscopy. When coupled with the more traditional process of exchange of labile protium in the hydroxyl groups by D 2 O solvent exchange, the biosynthesis of highly deuterated starch opens new opportunities for neutron scattering experiments involving multicomponent starch-based systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron . Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron . Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide., Importance: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource., Competing Interests: The authors declare no conflict of interest.

Allotetraploid white clover (Trifolium repens) formed during the last glaciation through hybridisation of two European diploid progenitors from restricted niches: one coastal, the other alpine. Here, we examine which hybridisation-derived molecular events may have underpinned white clover's postglacial niche expansion. We compared the transcriptomic frost responses of white clovers (an inbred line and an alpine-adapted ecotype), extant descendants of its progenitor species and a resynthesised white clover neopolyploid to identify genes that were exclusively frost-induced in the alpine progenitor and its derived subgenomes. From these analyses we identified galactinol synthase, the rate-limiting enzyme in biosynthesis of the cryoprotectant raffinose, and found that the extant descendants of the alpine progenitor as well as the neopolyploid white clover rapidly accumulated significantly more galactinol and raffinose than the coastal progenitor under cold stress. The frost-induced galactinol synthase expression and rapid raffinose accumulation derived from the alpine progenitor likely provided an advantage during early postglacial colonisation for white clover compared to its coastal progenitor., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Raffinose mitigates plant heat, drought, and cold stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize raffinose synthase mutant zmrafs-1 had seedlings that lack raffinose, generated fewer and shorter adventitious roots, and were more sensitive to waterlogging stress, while overexpression of the raffinose synthase gene, ZmRAFS, increased raffinose content, stimulated adventitious root formation, and enhanced waterlogging tolerance of maize seedlings. Transcriptome analysis of null segregant seedlings compared with zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were up-regulated by raffinose. Additionally, indole-3-acetic acid content was significantly decreased in zmrafs-1 seedlings and increased in ZmRAFS-overexpressing seedlings. Inhibition of the hydrolysis of raffinose by 1-deoxygalactonojirimycin decreased the waterlogging tolerance of maize seedlings, the expression of genes encoding proteins related to auxin transport-related genes, and the indole-3-acetic acid level in the seedlings, indicating that the hydrolysis of raffinose is necessary for maize waterlogging tolerance. These data demonstrate that raffinose catabolism stimulates adventitious root formation via the auxin signaling pathway to enhance maize waterlogging tolerance., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub-Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow- and white-fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo-inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow-fleshed cultivars, in comparison to their white-fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The study investigates the efficacy of an enzymatic preparation primarily with α-galactosidase activity for improving the quality of white sugar from poor-quality sugar beets. Focused on overcoming raffinose accumulation challenges in sugar beets, especially those harvested prematurely or stored for extended periods, an innovative exploration of enzymatic application in an industrial setting for the first time was conducted. By integrating theoretical calculations and experimental data, the findings reveal that α-galactosidase preparation notably diminishes raffinose content in beet juice, thus enhancing the sucrose yield and overall sugar quality. A reliable method to process lower-quality beets, promising enhanced efficiency in sugar production, was presented. The study also highlights the economic benefits of incorporating enzyme preparation into the production process, demonstrating a notable return on investment and underscoring the potential of enzymatic treatments to address industry challenges.

Drought is the most severe form of stress experienced by plants worldwide. Cucumber is a vegetable crop that requires a large amount of water throughout the growth period. In our previous study, we identified that overexpression of CsHSFA1d could improve cold tolerance and the content of endogenous jasmonic acid in cucumber seedlings. To explore the functional diversities of CsHSFA1d, we treat the transgenic plants under drought conditions. In this study, we found that the heat shock transcription factor HSFA1d (CsHSFA1d) could improve drought stress tolerance in cucumber. CsHSFA1d overexpression increased the expression levels of galactinol synthase (CsGolS3) and raffinose synthase (CsRS) genes, encoding the key enzymes for raffinose family oligosaccharide (RFO) biosynthesis. Furthermore, the lines overexpressing CsHSFA1d showed higher enzymatic activity of GolS and raffinose synthase to increase the content of RFO. Moreover, the CsHSFA1d-overexpression lines showed lower reactive oxygen species (ROS) accumulation and higher ROS-scavenging enzyme activity after drought treatment. The expressions of antioxidant genes CsPOD2, CsAPX1 and CsSOD1 were also upregulated in CsHSFA1d-overexpression lines. The expression levels of stress-responsive genes such as CsRD29A, CsLEA3 and CsP5CS1 were increased in CsHSFA1d-overexpression lines after drought treatment. We conclude that CsHSFA1d directly targets and regulates the expression of CsGolS3 and CsRS to promote the enzymatic activity and accumulation of RFO to increase the tolerance to drought stress. CsHSFA1d also improves ROS-scavenging enzyme activity and gene expression indirectly to reduce drought-induced ROS overaccumulation. This study therefore offers a new gene target to improve drought stress tolerance in cucumber and revealed the underlying mechanism by which CsHSFA1d functions in the drought stress by increasing the content of RFOs and scavenging the excessive accumulation of ROS., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Mitogen-activated protein kinase (MAPK) cascades and raffinose have been observed to increase in plants exposed to cold. However, it remains elusive whether and how MAPK regulates raffinose synthesis under cold stress. Here, overexpression of SlMAPK3 promoted the accumulation of galactinol and raffinose under cold stress, while CRISPR/Cas9-mediated mutants showed the opposite results. Moreover, SlMAPK3 promoted the expression of SlWRKY46 at low temperatures and interacted with SlWRKY46 protein. Overexpression of SlWRKY46 enhanced cold resistance. Furthermore, SlWRKY46 directly bound to the promoter of SlGols1 to enhance its expression and promoted the accumulation of raffinose. Virus-induced gene-silencing (VIGS)-mediated knockdown of SlGols1 remarkably elevated cold sensitivity and reduced raffinose content. Meanwhile, exogenous supplementation of raffinose could improve the cold tolerance of tomato plants. Thus, our data indicates that SlMAPK3 modulates cold resistance by regulating raffinose content and SlWRKY46 expression. SlWRKY46 also promotes the accumulation of raffinose by inducing the expression of SlGols1.

To investigate the role of sugar metabolism in desiccation-sensitive seeds, we performed a natural desiccation treatment on Phoebe chekiangensis seeds in a room and systematically analyzed the changes in seed germination, sugar compounds, malondialdehyde, and relative electrical conductivity during the seed desiccation. The results revealed that the initial moisture content of P. chekiangensis seed was very high (37.06%) and the seed was sensitive to desiccation, the germination percentage of the seed decreased to 5.33% when the seed was desiccated to 22.04% of moisture content, therefore, the seeds were considered recalcitrant. Based on the logistic model, we know that the moisture content of the seeds is 29.05% when the germination percentage drops to 50% and that it is desirable to keep the seed moisture content above 31.74% during ambient transportation. During seed desiccation, sucrose and trehalose contents exhibited increasing trends, and raffinose also increased during the late stage of desiccation, however, low levels of the non-reducing sugar accumulations may not prevent the loss of seed viability caused by desiccation. Glucose and fructose predominated among sugar compounds, and they showed a slight increase followed by a significant decrease. Their depletion may have contributed to the accumulation of sucrose and raffinose family oligosaccharides. Correlation analysis revealed a significant relationship between the accumulation of sucrose, trehalose, and soluble sugars, and the reduction in seed viability. Sucrose showed a significant negative correlation with glucose and fructose. Trehalose also exhibited the same pattern of correlation. These results provided additional data and theoretical support for understanding the mechanism of sugar metabolism in seed desiccation sensitivity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Raffinose Family of Oligosaccharides (RFOs), including Galactinol, Raffinose, and Stachyose, are pivotal carbohydrates with significant roles in abiotic stress tolerance and growth within dynamic environments. Plant development is profoundly influenced by light, a major environmental signal. Despite this, the interconnections between the biosynthesis of secondary sugars and light signaling have remained unexplored. This study reveals that exposure to light induces the expression of Galactinol synthase (AtGolS1), a key enzyme in the RFO biosynthesis pathway. The light-inducible response of AtGolS1 operates downstream of ELONGATED HYPOCOTYL 5 (HY5), a central regulator in light signaling. Mutant seedlings with disrupted HY5 function (hy5-215) exhibit reduced AtGolS1 transcript accumulation compared to wild-type (WT) and HY5 overexpression seedlings. DNA-protein interaction studies demonstrate that HY5 directly binds to light-responsive cis-elements in the promoter region of AtGolS1, thereby mediating its light responsiveness. Quantification of galactinol revealed a diminished accumulation in the hy5-215 mutant compared to wild-type (WT) and HY5 overexpression seedlings. Consequently, these findings shed light on the intricate crosstalk between RFO biosynthesis and light signaling in Arabidopsis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Raffinose family oligosaccharides (RFOs) are synthesized from sucrose and subsequent addition of galactose moieties which was provided by galactinol. Galactinol is synthesized from UDP-galactose and myo-inositol. RFOs accumulate at late stage of seed development and play important roles in seed longevity. RFOs are major components in seeds of many plant species. Here, we document a methodology for extraction and quantitative analysis of raffinose metabolism-related soluble sugars or the derivative alcohols in plant seeds. This protocol, based on high-performance liquid chromatography (HPLC), achieves the efficient separation and accurate quantification of sucrose, myo-inositol, galactinol, and raffinose within 25 min of retention time., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Six oligosaccharides were discovered and isolated for the first time from Ziziphi Spinosae Semen. On the basis of spectroscopic analysis, their structures were determined to be verbascose (1), verbascotetraose (2), stachyose (3), manninotriose (4), raffinose (5), and melibiose (6). The prebiotic effect of the oligosaccharide fraction was assayed by eight gut bacterial growth in vitro, revealing a significant increase in cell density, up to 4-fold, for Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus johnsonii. The impact of six oligosaccharides with different degrees of polymerization (DPs) and structures on the growth of Lactobacillus acidophilus was evaluated. As a result, stachyose and raffinose demonstrated superior support for bacterial growth compared to the other oligosaccharides. This study explored the structure-activity relationship of raffinose family oligosaccharides (RFOs) and showed that the more the monosaccharide type, the more supportive the gut bacteria growth when oligosaccharides have the same molecular weight., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Frequent outbreaks of harmful cyanobacterial blooms and the microcystins (MCs) they produce seriously affect the survival of aquatic organisms. Interactions between gut microbiota and hosts often play crucial roles in driving the adaptation of aquatic organisms to environmental changes. In this study, we investigated the phenotypic indicators of the freshwater gastropod Bellamya aeruginosa, after uptake of Microcystis aeruginosa and explored its gut microbial composition and gut metabolites in response to toxic cyanobacterial stress. Results showed that the MCs concentration in the hepatopancreas of snails fed with toxic cyanobacteria decreased from 2.64 ± 0.14 μg·g -1 on day 7 to 1.16 ± 0.10 μg·g -1 on day 14. The compositions of the intestinal microbiota of snails fed with different algae significantly differed, and the relative abundance of gut microbes such as Lactobacillus and Sphingobium significantly increased after feeding toxic cyanobacteria. Significant differences also existed in intestinal metabolites, the relative abundance of the following metabolites significantly increased: l-proline, 5,6-DHET, stachyose, raffinose, and 3-isopropylmalate. Sankey network diagrams showing links between gut microbes and gut metabolites. The association of Lactobacillus and Sphingobium with amino acids may be related to host tolerance to toxicity, and the linkages of gut microbes with metabolites such as levan, imidazolepropionic acid, and eicosanoids may be associated with involvement in host immune responses. The association of microbes with stachyose and raffinose can help the host to regulate energy homeostasis. These results reveal the underlying mechanisms of gut microbes in the snail adaptation to toxic cyanobacterial stress. This study could be great important for gaining new insights into toxic cyanobacteria-induced changes in snail gut microbes and metabolites and their roles in snail adaptation to toxic cyanobacterial stress, and may provide important insights into the use of freshwater gastropods for the prevention and control of cyanobacterial blooms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cassava is an ideal food security crop in marginal and drought environment. However, the post-harvest storage of cassava is urgent problem to be resolved. In this study, the storage tolerant and non-tolerant cassava were screened by measuring the change of Peroxidase (POD), Superoxide dismutase (SOD), Catalase (CAT) and Malondialdehyde (MDA) in seven cultivars of cassava. Compared with other cultivars, the cultivar of SC14 showed the highest level of SOD, MDA and POD respectively at 0 day, 12 day and 9 day postharvest while exhibited lowest level of CAT at 0 day postharvest, indicating the strongest antioxidant capability and storage tolerance. In contrast, GR15231, termed as storage non-tolerance cultivars, showed lowest SOD and POD at 12 day and kept a relative high level of CAT at 12 day post-harvest. In addition, SC14 has higher level of starch and dry substance than GR15231. Mass spectrum was performed for SC14 and GR15231 to explore the key metabolites regulating the storage tolerance of cassava. The results showed that the expression of glutathione (reduced) and raffinose was significantly decreased at 12 day post-harvest both in tolerant SC14 and non-tolerant GR15231. Compared with GR15231, SC14 showed higher level of raffinose both at 0 and 12 day post-harvest, indicating that raffinose may be the potential metabolites protecting SC14 cultivar from deterioration post-harvest. Additionally, raffinose ratio of SC14a/SC14b was five times less than that of GR15231a/GR15231b, reflecting the slower degradation of raffinose in SC14 cultivar compared with GR15231 cultivar. In conclusion, the antioxidant microenvironment induced by reduced glutathione and higher level of raffinose in SC14 cultivar might be the promising metabolites to improve its antioxidant capacity and antibiosis and thus maintained the quality of Cassava root tubers., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Galactitol synthetase (GolS) as a key enzyme in the raffinose family oligosaccharides (RFOs) biosynthesis pathway, which is closely related to stress. At present, there are few studies on GolS in biological stress. The expression of MnGolS2 gene in mulberry was increased under Botrytis cinerea infection. The MnGolS2 gene was cloned and ectopically expressed in Arabidopsis . The content of MDA in leaves of transgenic plants was decreased and the content of CAT was increased after inoculation with B. cinerea . In this study, the role of MnGolS2 in biotic stress was demonstrated for the first time. In addition, it was found that MnGolS2 may increase the resistance of B. cinerea by interacting with other resistance genes. This study offers a crucial foundation for further research into the role of the GolS2 gene.

Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: "domesticated" strains with low genetic diversity, and "environmental" strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications. [ABSTRACT FROM AUTHOR]

The morphology of somatic embryos (SE) is not a sufficient criterion to determine the level of maturation and the optimal stage to transfer embryos for germination, unlike the biochemical components. This composition characterization in the laboratory is too restrictive to be considered at each maturation cycle, as would be necessary. It is, therefore, essential to consider alternative methods. The objectives of this work were to achieve a complete biochemical characterization of the embryos during their development, to serve as a reference and develop a characterization based on infrared spectrometry and chemometrics. During the precotyledonary stage (0-3 weeks of maturation), water content and glucose and fructose levels were high, which is consistent with SE development. After 4 weeks, the cotyledonary SE had a metabolism oriented towards the storage accumulation of lipids, proteins and starch, whereas raffinose only appeared from 8 weeks. Mid-infrared calibration models were developed for water, proteins, lipids, carbohydrates, glucose, fructose, inositols, raffinose, stachyose and starch contents with an r 2 average of 0.84. A model was also developed to discriminate the weeks of SE maturation. Different classes of age were discriminated with at least 72% of accuracy. Infrared analysis of the SE based on their full biochemical spectral fingerprint revealed a very slight variation in composition between 7 and 9 weeks, information that is very difficult to obtain by conventional analysis methods. These results provide novel insights into the maturation of conifer SE and indicate that mid-infrared spectrometry could be an easy and effective method for SE characterization., (© 2023 Scandinavian Plant Physiology Society.)

Lupines and faba beans are protein-rich legumes, which can be utilized as a plant-based substitute for animal proteins in human nutrition in general and in the beverage industry in particular. However, their application is hampered by low protein solubility in the acidic pH range and by antinutrients such as flatulence-causing "raffinose family oligosaccharides" (RFOs). Germination is known in the brewing industry for increasing enzymatic activities and mobilizing storage compounds. Therefore, germinations of lupines and faba beans were performed at different temperatures, and their impact on protein solubility, the concentration of free amino acids, and degradation of RFOs, alkaloids, and phytic acid was evaluated. In general, changes were comparable for both legumes but less pronounced in faba beans. Germination depleted the RFOs entirely in both legumes. The protein size distribution was found to be shifted toward smaller fractions, the concentrations of free amino acids multiplied, and protein solubility increased. No substantial reductions in the binding capacity of phytic acid toward iron ions were observed, but a release of free phosphate in lupines was detected. The results prove that germination is an applicable process for refining lupines and faba beans for use not only in refreshing beverages or milk alternatives but also in other food applications.

Low temperatures restrict the growth of the grapevine industry. The DREB transcription factors are involved in the abiotic stress response. Here, we isolated the VvDREB2A gene from Vitis vinifera cultivar 'Zuoyouhong' tissue culture seedlings. The full-length VvDREB2A cDNA was 1068 bp, encoding 355 amino acids, which contained an AP2 conserved domain belonging to the AP2 family. Using transient expression in leaves of tobacco, VvDREB2A was localized to the nucleus, and it potentiated transcriptional activity in yeasts. Expression analysis revealed that VvDREB2A was expressed in various grapevine tissues, with the highest expression in leaves. VvDREB2A was induced by cold and the stress-signaling molecules H 2 S, nitric oxide, and abscisic acid. Furthermore, VvDREB2A -overexpressing Arabidopsis was generated to analyze its function. Under cold stress, the Arabidopsis overexpressing lines exhibited better growth and higher survival rates than the wild type. The content of oxygen free radicals, hydrogen peroxide, and malondialdehyde decreased, and antioxidant enzyme activities were enhanced. The content of raffinose family oligosaccharides (RFO) also increased in the VvDREB2A -overexpressing lines. Moreover, the expression of cold stress-related genes ( COR15A , COR27 , COR6.6 , and RD29A ) was also enhanced. Taken together, as a transcription factor, VvDREB2A improves plants resistance to cold stress by scavenging reactive oxygen species, increasing the RFO amount, and inducing cold stress-related gene expression levels.

Based on factual scientific health claims, prebiotics have gained significant importance in ever-growing food and pharmaceutical industries. The diverse nature of distinct prebiotics influences the host differently in distinguishable patterns. Functional oligosaccharides are either plant-derived or commercially prepared. Raffinose, stachyose, and verbascose are the 3 types of raffinose family oligosaccharides (RFOs) that have been extensively used as medicine, cosmetic, and food additives. These dietary fiber fractions avert the adhesion and colonization by enteric pathogens and add nutrition metabolites for a healthy immune system. Enrichment of RFOs in healthy foods should be promoted as these oligosaccharides augment gut microecology by enhancing the health conferring microbes i.e. bifidobacteria and lactobacilli. RFOs influence the host's multiorgan systems due to their physiological and physicochemical properties. For example, the fermented microbial products of such carbohydrates affect neurological processes, including memory, mood, and behavior in humans. Raffinose-type sugar uptake is thought to be a ubiquitous property of bifidobacteria. This review paper summarizes the source of RFOs and their metabolizing entities, highlighting bifidobacterial carbohydrate utilization and health benefits., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Photosynthetic active radiation (PAR) refers to photons between 400 and 700 nm. These photons drive photosynthesis, providing carbohydrates for plant metabolism and development. Far-red radiation (FR, 701-750 nm) is excluded in this definition because no FR is absorbed by the plant photosynthetic pigments. However, including FR in the light spectrum provides substantial benefits for biomass production and resource-use efficiency. We investigated the effects of continuous FR addition and end-of-day additional FR to a broad white light spectrum (BW) on carbohydrate concentrations in the top and bottom leaves of sweet basil ( Ocimum basilicum L.), a species that produces the raffinose family oligosaccharides raffinose and stachyose and preferentially uses the latter as transport sugar. Glucose, fructose, sucrose, raffinose, and starch concentrations increased significantly in top and bottom leaves with the addition of FR light. The increased carbohydrate pools under FR light treatments are associated with more efficient stachyose production and potentially improved phloem loading through increased sucrose homeostasis in intermediary cells. The combination of a high biomass yield, increased resource-use efficiency, and increased carbohydrate concentration in leaves in response to the addition of FR light offers opportunities for commercial plant production in controlled growth environments.

Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis, cremoris, hordniae and tructae, but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: “domesticated” strains with low genetic diversity, and “environmental” strains that are the main contributors of the genetic diversity of the subsp. lactis. As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications.

Raffinose family oligosaccharides (RFOs) are known to have important physiological functions in plants. However, the presence of RFOs in legumes causes flatulence, hence are considered antinutrients. To reduce the RFOs content to a desirable limit without compromising normal plant development and functioning, the identification of important regulatory genes associated with the biosynthetic pathway is a prerequisite. In the present study, through comparative RNA sequencing in contrasting genotypes for seed RFOs content at different seed maturity stages, differentially expressed genes (DEGs) associated with the pathway were identified. The DEGs exhibited spatio-temporal expression patterns with high RFOs variety showing early induction of RFOs biosynthetic genes and low RFOs variety showing a late expression at seed maturity. Selective and seed-specific differential expression of raffinose synthase genes (AhRS14 and AhRS6) suggested their regulatory role in RFOs accumulation in peanut seeds, thereby serving as promising targets in low RFOs peanut breeding programs. Despite stachyose being the major seed RFOs fraction, differential expression of raffinose synthase genes indicated the complex metabolic regulation of this pathway. The transcriptomic resource and the genes identified in this study could be studied further to develop low RFOs varieties, thus improving the overall nutritional quality of peanuts., (© 2023. The Author(s).)

Raffinose synthetase (RS) is a key enzyme in the process of raffinose (Raf) synthesis and is involved in plant development and stress responses through regulating Raf content. As a sweetener, Raf makes an important contribution to the sweet taste of white tea. However, studies on the identification, analysis and transcriptional regulation of CsRSs ( Camellia sinensis RS genes) are still lacking. In this study, nine CsRSs were identified from the tea plant ( Camellia sinensis ) genome database. The CsRSs were classified into five groups in the phylogenetic tree. Expression level analysis showed that the CsRSs varied in different parts of the tea plant. Transcriptome data showed that CsRSs could respond to persistent drought and cold acclimation. Except for CsRS5 and CsRS9 , the expression pattern of all CsRSs increased at 12 h and decreased at 30 h during the withering process of white tea, consistent with the change trend of the Raf content. Furthermore, combining yeast one-hybrid assays with expression analysis, we found that CsDBB could potentially regulate the expression of CsRS8 . Our results provide a new perspective for further research into the characterization of CsRS genes and the formation of the white tea flavour.

Seed vigor and longevity are important agronomic attributes, as they are essentially associated with crop yield and thus the global economy. Seed longevity is a measure of seed viability and the most essential property in gene bank management since it affects regeneration of seed recycling. Reduced seed life or storability is a serious issue in seed storage since germplasm conservation and agricultural enhancement initiatives rely on it. The irreversible and ongoing process of seed deterioration comprises a complex gene regulatory network and altered metabolism that results in membrane damage, DNA integrity loss, mitochondrial dysregulation, protein damage, and disrupted antioxidative machinery. Carbohydrates and/or sugars, primarily raffinose family oligosaccharides (RFOs), have emerged as feasible components for boosting or increasing seed vigor and longevity in recent years. RFOs are known to perform diverse functions in plants, including abiotic and biotic stress tolerance, besides being involved in regulating seed germination, desiccation tolerance, vigor, and longevity. We emphasized and analyzed the potential impact of RFOs on seed vigor and longevity in this review. Here, we comprehensively reviewed the molecular mechanisms involved in seed longevity, RFO metabolism, and how RFO content is critical and linked with seed vigor and longevity. Further molecular basis, biotechnological approaches, and CRISPR/Cas applications have been discussed briefly for the improvement of seed attributes and ultimately crop production. Likewise, we suggest advancements, challenges, and future possibilities in this area., (© 2022 The Author(s).)

The C2H2 zinc finger proteins (ZFPs) play essential roles in regulating cold stress responses. Similarly, raffinose accumulation contributes to freezing stress tolerance. However, the relationship between C2H2 functions and raffinose synthesis in cold tolerance remains uncertain. Here, we report the characterization of the cold-induced C2H2-type zinc finger protein PhZFP1 in Petunia hybrida. PhZFP1 was found to be predominantly localized in the nucleus. Overexpression of PhZFP1 conferred enhanced cold tolerance in transgenic petunia lines. In contrast, RNAi mediated suppression of PhZFP1 led to increased cold susceptibility. PhZFP1 regulated the expression of a range of abiotic stress responsive-genes including genes encoding proteins involved in reactive oxygen species (ROS) scavenging and raffinose metabolism. The accumulation of galactinol and raffinose, and the levels of PhGolS1-1 transcripts, were significantly increased in PhZFP1-overexpressing plants and decreased in PhZFP1-RNAi plants under cold stress. Moreover, the galactinol synthase (GolS)-encoding gene PhGolS1-1 was identified as a direct target of PhZFP1. Taken together, these results demonstrate that PhZFP1 functions in cold stress tolerance by modulation of galactinol synthesis via regulation of PhGolS1-1. This study also provides new insights into the mechanisms underlying C2H2 zinc finger protein-mediated cold stress tolerance, and has identified a candidate gene for improving cold stress tolerance., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The reconfiguration of the primary metabolism is essential in plant-pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans ( Psl ) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P) + ] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo -inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phospho enol pyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.

Galactinol synthase (GolS) catalyzes the first and rate-limiting step in the synthesis of raffinose family of oligosaccharides (RFOs), which serve as storage and transport sugars, signal transducers, compatible solutes and antioxidants in higher plants. The present work aimed to assess the potential functions of citrus GolS in mechanisms of stress response and tolerance. By homology searches, eight GolS genes were found in the genomes of Citrus sinensis and C. clementina. Phylogenetic analysis showed that there is a GolS ortholog in C. clementina for each C. sinensis GolS, which have evolved differently from those of Arabidopsis thaliana. Transcriptional analysis indicated that most C. sinensis GolS (CsGolS) genes show a low-level tissue-specific and stress-inducible expression in response to drought and salt stress treatments, as well as to 'Candidatus Liberibacter asiaticus' infection. CsGolS6 overexpression resulted in improved tobacco tolerance to drought and salt stresses, contributing to an increased mesophyll cell expansion, photosynthesis and plant growth. Primary metabolite profiling revealed no significant changes in endogenous galactinol, but different extents of reduction of raffinose in the transgenic plants. On the other hand, a significant increase in the levels of metabolites with antioxidant properties, such as ascorbate, dehydroascorbate, alfa-tocopherol and spermidine, was observed in the transgenic plants. These results bring evidence that CsGolS6 is a potential candidate for improving stress tolerance in citrus and other plants., Competing Interests: The authors have declared that no competing interests exist.

The raffinose synthetase ( RFS ) and galactinol synthase ( GolS ) are two critical enzymes for raffinose biosynthesis, which play an important role in modulating plant growth and in response to a variety of biotic or abiotic stresses. Here, we comprehensively analyzed the RFS and GolS gene families and their involvement in abiotic and biotic stresses responses at the genome-wide scale in kiwifruit. A total of 22 GolS and 24 RFS genes were identified in Actinidia chinensis and Actinidia eriantha genomes. Phylogenetic analysis showed that the GolS and RFS genes were clustered into four and six groups, respectively. Transcriptomic analysis revealed that abiotic stresses strongly induced some crucial genes members including AcGolS1/2/4/8 and AcRFS2/4/8/11 and their expression levels were further confirmed by qRT-PCR. The GUS staining of AcRFS4Pro ::GUS transgenic plants revealed that the transcriptionlevel of AcRFS4 was significantly increased by salt stress. Overexpression of AcRFS4 in Arabidopsis demonstrated that this gene enhanced the raffinose accumulation and the tolerance to salt stress. The co-expression networks analysis of hub transcription factors targeting key AcRFS4 genes indicated that there was a strong correlation between AcNAC30 and AcRFS4 expression under salt stress. Furthermore, the yeast one-hybrid assays showed that AcNAC30 could bind the AcRFS4 promoter directly. These results may provide insights into the evolutionary and functional mechanisms of GolS and RFS genes in kiwifruit.

Streptococcus mutans is a representative biofilm-forming bacterium that causes dental caries through glucosyltransferase (GTF) activity. Glucans are synthesized from sucrose by GTFs and provide binding sites for S. mutans to adhere tightly to the tooth enamel. Therefore, if a novel compound that interferes with GTF function is developed, biofilm formation control in S. mutans would be possible. We discovered that raffinose, an oligosaccharide from natural products, strongly inhibited biofilm formation, GTF-related gene expression, and glucan production. Furthermore, biofilm inhibition on saliva-coated hydroxyapatite discs through the reduction of bacterial adhesion indicated the applicability of raffinose in oral health. These effects of raffinose appear to be due to its ability to modulate GTF activity in S. mutans. Hence, raffinose may be considered an antibiofilm agent for use as a substance for oral supplies and dental materials to prevent dental caries. IMPORTANCE Dental caries is the most prevalent infectious disease and is expensive to manage. Dental biofilms can be eliminated via mechanical treatment or inhibited using antibiotics. However, bacteria that are not entirely removed or are resistant to antibiotics can still form biofilms. In this study, we found that raffinose inhibited biofilm formation by S. mutans, a causative agent of dental caries, possibly through binding to GtfC. Our findings support the notion that biofilm inhibition by raffinose can be exerted by interference with GTF function, compensating for the shortcomings of existing commercialized antibiofilm methods. Furthermore, raffinose is an ingredient derived from natural products and can be safely utilized in humans; it has no smell and tastes sweet. Therefore, raffinose, which can control S. mutans biofilm formation, has been suggested as a substance for oral supplies and dental materials to prevent dental caries.

Enterohepatic circulation of 12 α -hydroxylated (12 α OH) bile acid (BA) is enhanced depending on the energy intake in high-fat diet-fed rats. Such BA metabolism can be reproduced using a diet supplemented with cholic acid (CA), which also induces simple steatosis, without inflammation and fibrosis, accompanied by some other symptoms that are frequently observed in the condition of non-alcoholic fatty liver in rats. We investigated whether supplementation of the diet with raffinose (Raf) improves hepatic lipid accumulation induced by the CA-fed condition in rats. After acclimation to the AIN-93-based control diet, male Wistar rats were fed diets supplemented with a combination of Raf (30 g/kg diet) and/or CA (0·5 g/kg diet) for 4 weeks. Dietary Raf normalised hepatic TAG levels (two-way ANOVA P < 0·001 for CA, P = 0·02 for Raf and P = 0·004 for interaction) in the CA-supplemented diet-fed rats. Dietary Raf supplementation reduced hepatic 12 α OH BA concentration (two-way ANOVA P < 0·001 for CA, P = 0·003 for Raf and P = 0·03 for interaction). The concentration of 12 α OH BA was reduced in the aortic and portal plasma. Raf supplementation increased acetic acid concentration in the caecal contents (two-way ANOVA P = 0·001 as a main effect). Multiple regression analysis revealed that concentrations of aortic 12 α OH BA and caecal acetic acid could serve as predictors of hepatic TAG concentration ( R 2 = 0·55, P < 0·001). However, Raf did not decrease the secondary 12 α OH BA concentration in the caecal contents as well as the transaminase activity in the CA diet-fed rats. These results imply that dietary Raf normalises hepatic lipid accumulation via suppression of enterohepatic 12 α OH BA circulation.

Responding to change is a fundamental property of life, making time-series data invaluable in biology. For microbes, plate readers are a popular, convenient means to measure growth and also gene expression using fluorescent reporters. Nevertheless, the difficulties of analysing the resulting data can be a bottleneck, particularly when combining measurements from different wells and plates. Here we present omniplate, a Python module that corrects and normalises plate-reader data, estimates growth rates and fluorescence per cell as functions of time, calculates errors, exports in different formats, and enables meta-analysis of multiple plates. The software corrects for autofluorescence, the optical density's non-linear dependence on the number of cells, and the effects of the media. We use omniplate to measure the Monod relationship for the growth of budding yeast in raffinose, showing that raffinose is a convenient carbon source for controlling growth rates. Using fluorescent tagging, we study yeast's glucose transport. Our results are consistent with the regulation of the hexose transporter (HXT) genes being approximately bipartite: the medium and high affinity transporters are predominately regulated by both the high affinity glucose sensor Snf3 and the kinase complex SNF1 via the repressors Mth1, Mig1, and Mig2; the low affinity transporters are predominately regulated by the low affinity sensor Rgt2 via the co-repressor Std1. We thus demonstrate that omniplate is a powerful tool for exploiting the advantages offered by time-series data in revealing biological regulation., Competing Interests: The authors have declared that no competing interests exist.

Few studies have focused on the capacity of lactic acid bacteria in utilizing soybean carbohydrates by genetic and phenotypic approaches. In the current study, genetic and phenotypic characteristics of soybean carbohydrate metabolism in Leuconostoc mesenteroides , Lactococcus lactis and Streptococcus thermophilus were investigated. The results indicated that all the 3 species have the potential capacity to utilize sucrose with a sucrose phosphoenolpyruvate-dependent phosphotransferase system (PTS) pathway mainly consisted of scrA , sacA , scrK and scrR. Among the 15 strains, only 1 L. lactis strain possessed both sucrose permease and PTS pathway. On the other hand, only Leu. mesenteroides among the 3 species have the genes related to α-galactosidases for potential capacity to metabolize raffinose-family oligosaccharides (RFO) in soybean. Four Leu. mesenteroides strains, DQHXN_Q03M16, FSDLZ60M2, DSCAB2M6 and DQHXN_Q38M5, possessed the complete α-galactosidases gene clusters of lacS-galA-galK-galT and high α-galactosidases activity, which contributed to the capacity of these strains to utilize RFO. These findings provide an approach to investigate the genetic and the phenotypic characteristics of Leu. mesenteroides , L. lactis and S. thermophilus in soybean carbohydrate metabolism for suitable strain selection in soybean fermentation. • The complete sucrose PTS pathway was present in Leuconostoc mesenteroides , Lactococcus lactis and Streptococcus thermophilus. • Among the 15 strains, only 1 L. lacti s strain possessed both sucrose permease and PTS pathway. • Among the 3 species, only Leu. mesenteroides possessed the capacity to metabolize raffinose. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infected mice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Agnew, Brazel, Tikhomirova, van der Linden, McLean, Paton and Trappetti.)

The current gold standard of Static Cold Storage (SCS), which is static cold storage on ice (about + 4 °C) in a specialized media such as the University of Wisconsin solution (UW), limits storage to few hours for vascular and metabolically active tissues such as the liver and the heart. The liver is arguably the pinnacle of metabolism in human body and therefore metabolic pathway analysis immediately becomes very relevant. In this article, a Nash Equilibrium (NE) approach, which is a first principles approach, is used to model and simulate the static cold storage and warm ischemia of a proposed model of liver cells. Simulations of energy depletion in the liver in static cold storage measured by ATP content and energy charge are presented along with comparisons to experimental data. In addition, conversion of Nash Equilibrium iterations to time are described along with an uncertainty analysis for the parameters in the model. Results in this work show that the Nash Equilibrium approach provides a good match to experimental data for energy depletion and that the uncertainty in model parameters is very small with percent variances less than 0.1%., (© 2022. The Author(s).)

In agriculture, plant growth promoting bacteria (PGPB) are increasingly used for reducing environmental stress-related crop losses through mutualistic actions of these microorganisms, activating physiological and biochemical responses, building tolerances within their hosts. Here we report the use of radioactive carbon-11 (t ½ 20.4 min) to examine the metabolic and physiological responses of Zea mays to Azospirillum brasilense (HM053) inoculation while plants were subjected to salinity and low nitrogen stresses. Host metabolism of "new" carbon resources (as 11 C) and physiology including [ 11 C]-photosynthate translocation were measured in response to imposed growth conditions. Salinity stress caused shortened, dense root growth with a 6-fold increase in foliar [ 11 C]-raffinose, a potent osmolyte. ICP-MS analyses revealed increased foliar Na + levels at the expense of K + . HM053 inoculation relieved these effects, reinstating normal root growth, lowering [ 11 C]-raffinose levels while increasing [ 11 C]-sucrose and its translocation to the roots. Na + levels remained elevated with inoculation, but K + levels were boosted slightly. Low nitrogen stress yielded longer roots possessing high levels of anthocyanins. Metabolic analysis revealed significant shifts in "new" carbon partitioning into the amino acid pool under low nitrogen stress, with significant increases in foliar [ 11 C]-glutamate, [ 11 C]-aspartate, and [ 11 C]-asparagine, a noted osmoprotectant. 11 CO 2 fixation and [ 11 C]-photosynthate translocation also decreased, limiting carbon supply to roots. However, starch levels in roots were reduced under nitrogen limitation, suggesting that carbon repartitioning could be a compensatory action to support root growth. Finally, inoculation with HM053 re-instated normal root growth, reduced anthocyanin, boosted root starch, and returned 11 C-allocation levels back to those of unstressed plants., (© 2022 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals , stachyose was highly accumulated in RNAi- CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi- CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi- CsAGA1 and RNAi- CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi- CsAGA1 and RNAi- CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Calmodulin-like proteins (CMLs) are one of the Ca 2+ sensors in plants, but the functions of most CMLs remain unknown. The regulation of cold tolerance and flowering time by MtCML42 in Medicago truncatula and the underlying mechanisms were investigated using MtCML42-overexpressing plants and cml42 Medicago mutants with a Tnt1 retrotransposon insertion. Compared with the wild type (WT), MtCML42-overexpressing lines had increased cold tolerance, whereas cml42 mutants showed decreased cold tolerance. The impaired cold tolerance in cml42 could b complemented by MtCML42 expression. The transcript levels of MtCBF1, MtCBF4, MtCOR413, MtCAS15, MtLTI6A, MtGolS1 and MtGolS2 and the concentrations of raffinose and sucrose were increased in response to cold treatment, whereas higher levels were observed in MtCML42-overexpressing lines and lower levels were observed in cml42 mutants. In addition, early flowering with upregulated MtFTa1 and downregulated MtABI5 transcripts was observed in MtCML42-overexpressing lines, whereas delayed flowering with downregulated MtFTa1 and upregulated MtABI5 was observed in cml42. MtABI5 expression could complement the flowering phenotype in the Arabidopsis mutant abi5. Our results suggest that MtCML42 positively regulates MtCBF1 and MtCBF4 expression, which in turn upregulates the expression of some COR genes, MtGolS1 and MtGolS2, which leads to raffinose accumulation and increased cold tolerance. MtCML42 regulates flowering time through sequentially downregulating MtABI5 and upregulating MtFTa1 expression., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Nutritional dependencies, especially those regarding nitrogen sources, govern numerous microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary, organoleptic, and health properties of most fermented products, such positive interactions have previously been studied between yogurt bacteria. However, they have never been exploited to create artificial cocultures of LAB that would not necessarily coexist naturally, i.e., from different origins. The objective of this study was to promote LAB positive interactions, based on nitrogen dependencies in cocultures, and to investigate how these interactions affect some functional outputs, e.g., acidification rates, carbohydrate consumption, and volatile-compound production. The strategy was to exploit both proteolytic activities and amino acid auxotrophies of LAB. A chemically defined medium was thus developed to specifically allow the growth of six strains used, three proteolytic and three nonproteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was cocultured with each one of the nonproteolytic LAB strains, L. lactis NCDO2111 and Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and cocultures. Acidification, carbohydrate consumption, and volatile-compound production were evaluated in direct cocultures. Each proteolytic strain induced different types of interactions: strongly positive interactions, weakly positive interactions, and no interactions were seen with E. faecalis CIRM-BIA2412, L. lactis NCDO2125, and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations of tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to higher acidification rates, lower pH, higher raffinose utilization, and higher concentrations of five volatile compounds. IMPORTANCE Interactions of lactic acid bacteria (LAB) are often studied in association with yeasts or propionibacteria in various fermented food products, and the mechanisms underlying their interactions are being quite well characterized. Concerning interactions between LAB, they have mainly been investigated to test antagonistic interactions. Understanding how they can positively interact could be useful in multiple food-related fields: production of fermented food products with enhanced functional properties or fermentation of new food matrices. This study investigated the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and nonproteolytic strains. The results suggest that proteolytic LAB do not equally stimulate nonproteolytic LAB and that the stronger the interactions between LAB are, the more functional outputs we can expect. Thus, this study gives insight into how to create new associations of LAB strains and to guarantee their positive interactions.

Organs from donors after controlled circulatory death (DCD III) exhibit a higher risk for graft dysfunction due to an initial period of warm ischemia. This procurement condition can also affect the yield of beta cells in islet isolates from donor pancreases, and hence their use for transplantation. The present study uses data collected and generated by our Beta Cell Bank to compare the number of beta cells in isolates from DCD III (n = 141) with that from donors after brain death (DBD, n = 609), before and after culture, and examines the influence of donor and procurement variables. Beta cell number per DCD III-organ was significantly lower (58 x 106 versus 84 x 106 beta cells per DBD-organ; p < 0.001) but their purity (24% insulin positive cells) and insulin content (17 μg / 106 beta cells in DCD III-organs versus 19 μg / 106 beta cells in DBD-organs) were similar. Beta cell number correlated negatively with duration of acirculatory warm ischemia time above 10 min; for shorter acirculatory warm ischemia time, DCD III-organs did not exhibit a lower beta cell yield (74 x 106 beta cells). Use of Institut Georges Lopez-1 cold preservation solution instead of University of Wisconsin solution or histidine-tryptophan-ketoglutarate also protected against the loss in beta cell yield from DCD III-organs (86 x 106 for IGL-1 versus 54 x 106 and 65 x 106 beta cells respectively, p = 0.042). Multivariate analysis indicates that both limitation of acirculatory warm ischemia time and use of IGL-1 prevent the reduced beta cell yield in islet cell isolates from DCD III-organs., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jacques Pirenne holds named chairs at the KU Leuven from the Institut Georges Lopez and from the “Centrale Afdeling voor Fractionering” (DGFCAF). The other authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

HL-1 is a cell line that shows a phenotype similar to adult cardiomyocytes. All major cardiac cell types release extracellular vesicles (EVs) that emerge as key mediators of intercellular communication. EVs can mediate intercellular cross-talk through the transfer of specific microRNAs (miRNAs). MiRNAs are known to play important regulatory roles during tissue differentiation and regeneration processes. Furthermore, miRNAs have recently been shown to be involved in the proliferation of adult cardiomyocytes. In this context, the purpose of this study was to analyze the transcriptomic profile of miRNAs expressed from HL-1 cardiac muscle cell-derived EVs, using next generation sequencing (NGS). Specifically, our transcriptomic analysis showed that the EVs derived from our HL-1 cells contained miRNAs that induce blood vessel formation and increase cell proliferation. Indeed, our bioinformatics analysis revealed 26 miRNAs expressed in EVs derived from our HL-1 that target genes related to cardiovascular development. In particular, their targets are enriched for the following biological processes related to cardiovascular development: heart morphogenesis, positive regulation of angiogenesis, artery development, ventricular septum development, cardiac atrium development, and myoblast differentiation. Consequently, EVs could become important in the field of regenerative medicine.

Dietary patterns and psychosocial factors, ubiquitous part of modern lifestyle, critically shape the gut microbiota and human health. However, it remains obscure how dietary and psychosocial inputs coordinately modulate the gut microbiota and host impact. Here, we show that dietary raffinose metabolism to fructose couples stress-induced gut microbial remodeling to intestinal stem cells (ISC) renewal and epithelial homeostasis. Chow diet (CD) and purified diet (PD) confer distinct vulnerability to gut epithelial injury, microbial alternation and ISC dysfunction in chronically restrained mice. CD preferably enriches Lactobacillus reuteri, and its colonization is sufficient to rescue stress-triggered epithelial injury. Mechanistically, dietary raffinose sustains Lactobacillus reuteri growth, which in turn metabolizes raffinose to fructose and thereby constituting a feedforward metabolic loop favoring ISC maintenance during stress. Fructose augments and engages glycolysis to fuel ISC proliferation. Our data reveal a diet-stress interplay that dictates microbial metabolism-shaped ISC turnover and is exploitable for alleviating gut disorders.

Alpha-galactosides or Raffinose Family Oligosaccharides (RFOs) are enriched in legumes and are considered as anti-nutritional factors responsible for inducing flatulence. Due to a lack of alpha-galactosidases in the stomachs of humans and other monogastric animals, these RFOs are not metabolized and are passed to the intestines to be processed by gut bacteria leading to distressing flatulence. In plants, alpha(α)-galactosides are involved in desiccation tolerance during seed maturation and act as a source of stored energy utilized by germinating seeds. The hydrolytic enzyme alpha-galactosidase (α-GAL) can break down RFOs into sucrose and galactose releasing the monosaccharide α-galactose back into the system. Through characterization of RFOs, sucrose, reducing sugars, and α-GAL activity in maturing and germinating chickpeas, we show that stored RFOs are likely required to maintain a steady-state level of reducing sugars. These reducing sugars can then be readily converted to generate energy required for the high energy-demanding germination process. Our observations indicate that RFO levels are lowest in imbibed seeds and rapidly increase post-imbibition. Both RFOs and the α-GAL activity are possibly required to maintain a steady-state level of the reducing monosaccharide sugars, starting from dry seeds all the way through post-germination, to provide the energy for increased germination vigor.