Raffinose Family Oligosaccharides Research Articles

Purification, characterisation and immobilisation of a protease‐resistant α‐galactosidase from Monascus purpureus and its application in removal of raffinose family oligosaccharides

SummaryIn this study, an α‐galactosidase (MPG) was produced by submerged fermentation from Monascus purpureus, a food‐grade fungus used for making red yeast rice. MPG was purified from intracellular extract which showed a subunit molecular mass of 95 kDa. MPG was optimally active at pH 4.0 and 50 °C and was stable between pH 3.5–8.0 and at temperatures up to 50 °C. The enzyme retained its activity in the presence of most of the metal ions except Cu2+ and Fe2+. MPG exhibited remarkable resistance to the proteases, pepsin, trypsin and proteinase K as well as to the sugars, galactose and sucrose. Moreover, it retained complete activity under simulated gastric conditions. The enzyme displayed specificity for the substrates, melibiose, raffinose and stachyose and was able to hydrolyze these oligosaccharides. It was effective in the removal of non‐digestible raffinose family oligosaccharides (RFOs) from soybeans, chickpeas and kidney beans. MPG was immobilised onto chitosan and the immobilised enzyme (iMPG) displayed higher optimal temperature (60 °C), better thermostability and substrate specificity as compared to MPG. iMPG was also capable of hydrolyzing RFOs in chickpeas and could be efficiently reused six times. In view of their superior properties, MPG and iMPG may have great prospects in the food and feed industries for the removal of RFOs from soy and other legumes.

SlGolS2 influences the metabolism of chlorophyll, carotenoid, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyzes the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR-Cas9 gene editing technology to create the slgols2 mutation showing uniformly green fruits without the dark green shoulder, and promoting fruit ripening. Further analysis revealed that the galactinol was undetected in slgols2 ovaries and fruits. Additionally, the mutant suppressed the accumulation of chlorophyll (Chl) and chloroplast development in tomato fruits. RNA sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development, such as SlPORB, SlGLK2, and SlCABs were downregulated in slgols2 fruits. Moreover, slgols2 lines prompted early color transformation and ethylene release by regulating the expression of genes such as SlPSY1, SlCRTISO, SlACS2, SlACS4, SlE4, SlE8, SlRIN, SlNOR and SlAP2a. Overall, our study provides evidence for the involvement of SlGolS2 in the pigment and ethylene metabolism of tomato fruits.

Variation of sugar compounds in Phoebe chekiangensis seeds during natural desiccation.

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.

CsHSFA1d Promotes Drought Stress Tolerance by Increasing the Content of Raffinose Family Oligosaccharides and Scavenging Accumulated Reactive Oxygen Species in Cucumber.

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.

More than a meat- or synthetic nitrogen fertiliser-substitute: a review of legume phytochemicals as drivers of 'One Health' via their influence on the functional diversity of soil- and gut-microbes.

Legumes are essential to healthy agroecosystems, with a rich phytochemical content that impacts overall human and animal well-being and environmental sustainability. While these phytochemicals can have both positive and negative effects, legumes have traditionally been bred to produce genotypes with lower levels of certain plant phytochemicals, specifically those commonly termed as 'antifeedants' including phenolic compounds, saponins, alkaloids, tannins, and raffinose family oligosaccharides (RFOs). However, when incorporated into a balanced diet, such legume phytochemicals can offer health benefits for both humans and animals. They can positively influence the human gut microbiome by promoting the growth of beneficial bacteria, contributing to gut health, and demonstrating anti-inflammatory and antioxidant properties. Beyond their nutritional value, legume phytochemicals also play a vital role in soil health. The phytochemical containing residues from their shoots and roots usually remain in-field to positively affect soil nutrient status and microbiome diversity, so enhancing soil functions and benefiting performance and yield of following crops. This review explores the role of legume phytochemicals from a 'one health' perspective, examining their on soil- and gut-microbial ecology, bridging the gap between human nutrition and agroecological science.

The Pea Oligosaccharides Could Stimulate the In Vitro Proliferation of Beneficial Bacteria and Enhance Anti-Inflammatory Effects via the NF-κB Pathway.

The oligosaccharides extracted from the seeds of peas, specifically consisting of raffinose, stachyose, and verbascose, fall under the category of raffinose family oligosaccharides (RFOs). The effect of RFOs on intestinal microflora and the anti-inflammatory mechanism were investigated by in vitro fermentation and cell experiments. Firstly, mouse feces were fermented in vitro and different doses of RFOs (0~2%) were added to determine the changes in the representative bacterial community, PH, and short-chain fatty acids in the fermentation solution during the fermentation period. The probiotic index was used to evaluate the probiotic proliferation effect of RFOs and the optimal group was selected for 16S rRNA assay with blank group. Then, the effects of RFOs on the inflammatory response of macrophage RAW264.7 induced by LPS were studied. The activity of cells, the levels of NO, ROS, inflammatory factors, and the expression of NF-κB, p65, and iNOS proteins in related pathways were measured. The results demonstrated that RFOs exerted a stimulatory effect on the proliferation of beneficial bacteria while concurrently inhibiting the growth of harmful bacteria. Moreover, RFOs significantly enhanced the diversity of intestinal flora and reduced the ratio of Firmicutes-to-Bacteroides (F/B). Importantly, it was observed that RFOs effectively suppressed NO and ROS levels, as well as inflammatory cytokine release and expression of NF-κB, p65, and iNOS proteins. These findings highlight the potential of RFOs in promoting intestinal health and ameliorating intestinal inflammation.

Novel Soybean Variety Lacking Raffinose Synthase 2 Activity.

Variation in the raffinose family oligosaccharide (RFO) content in soybean is advantageous for livestock farming and health science. In this study, a soybean variety (GmJMC172) with a significantly low stachyose content in its seeds was identified in the NARO Genebank core collection. The results of the single-nucleotide polymorphism (SNP) analysis suggested that this phenomenon was related to a single-base deletion, inducing a frameshift mutation in raffinose synthase 2 (RS2), rather than the polymorphisms in the RS3, RS4, and stachyose synthase (STS) sequences. Differences in the enzymatic properties between the native RS2 and truncated RS2 were examined by using a three-dimensional model predicted using Alphafold2. In addition to revealing the missing active pocket in truncated RS2, the modeled structure explained the catalytic role of W331* and suggested a sufficient space to bind both sucrose and raffinose in the ligand-binding pocket. The soybean line, with seeds available from the NARO Genebank, could serve as breeding materials for manipulating the RFO content.

Caracterização do germoplasma de soja com aptidão ao consumo humano no Uruguai

Abstract Soybean (Glycine max (L.) Merrill) is a crop of world economic importance; and its grain constitutes a significant source of protein and carbohydrates for human consumption. This work aimed to characterize soybean genotypes available in Uruguay for human consumption regarding protein quantity and quality, carbohydrate composition and oil content in relation to yield, both in genetically modified and conventional genotypes. In this study, 13 genotypes grown in three different environments (two locations, two years), a conventional set (22 genotypes) and a genetically modified set (36 genotypes), grown in a single environment were subject to study. The traits measured were yield, total protein, oil content, soluble protein, glycinin (11S), conglycinin (7S), the 11S/7S ratio, soluble carbohydrates, sucrose and total raffinose family oligosaccharides (RFOs). A significant environmental and genotype effect was detected for most of the parameters. The interaction between genotype and environment was significant for total protein, oil content and sucrose. Soluble protein and the 11S fraction were only affected by environment; soluble carbohydrates and the 7S fraction were only affected by genotype. It was possible to identify genotypes with good characteristics for human consumption (high total protein, sucrose, ratio 11S/7S and low oligosaccharides) across environments, both genetically modified and conventional genotypes. Variability was found among the genotypes available in Uruguay in the parameters studied.

The Sink-Source Relationship in Cucumber (Cucumis sativus L.) Is Modulated by DNA Methylation.

The optimization of the sink-source relationship is of great importance for crop yield regulation. Cucumber is a typical raffinose family oligosaccharide (RFO)-transporting crop. DNA methylation is a common epigenetic modification in plants, but its role in sink-source regulation has not been demonstrated in RFO-translocating species. Here, whole-genome bisulfite sequencing (WGBS-seq) was conducted to compare the nonfruiting-node leaves (NFNLs) and leaves of fruit setting (FNLs) at the 12th node by removing all female flowers in other nodes of the two treatments. We found considerable differentially methylated genes enriched in photosynthesis and carbohydrate metabolic processes. Comparative transcriptome analysis between FNLs and NFNLs indicated that many differentially expressed genes (DEGs) with differentially methylated regions were involved in auxin, ethylene and brassinolide metabolism; sucrose metabolism; and RFO synthesis pathways related to sink-source regulation. Moreover, DNA methylation levels of six sink-source-related genes in the pathways mentioned above decreased in leaves after 5-aza-dC-2'-deoxycytidine (5-Aza-dC, a DNA methyltransferase inhibitor) treatment on FNLs, and stachyose synthase (CsSTS) gene expression, enzyme activity and stachyose content in RFO synthesis pathway were upregulated, thereby increasing fruit length and dry weight. Taken together, our findings proposed an up-to-date inference for the potential role of DNA methylation in the sink-source relationship, which will provide important references for further exploring the molecular mechanism of DNA methylation in improving the yield of RFO transport plants.

HY5-dependent light-mediated regulation of galactinol synthase gene, AtGolS1, modulates galactinol biosynthesis in Arabidopsis

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.

Changes in Polar Metabolites during Seed Germination and Early Seedling Development of Pea, Cucumber, and Wheat

Seed-to-seedling transition plays a crucial role in plant vegetation. However, changes in the metabolome of crop seedlings during seed germination and early seedling development are mostly unknown and require a deeper explanation. The present study attempted to compare qualitative and quantitative changes in polar metabolites during the seed germination and early development of seedlings of three different and important crop types: pea, cucumber, and wheat. The application of gas chromatography coupled with a flame ionization detector, as well as gas chromatography coupled with mass spectrometry, identified 51 polar metabolites. During seed imbibition/germination, the rapid degradation of raffinose family oligosaccharides (RFOs) preceded a dramatic increase in the concentrations of intermediates of glycolysis and the TCA cycle in embryonic axes (of pea and cucumber) or embryos (of wheat), confirming the important role of RFOs in the resumption of respiration and seed-to-seedling transition. After germination, the metabolic profiles of the growing roots, epicotyl/hypocotyl/coleoptile, and cotyledons/endosperm changed according to fluctuations in the concentrations of soluble carbohydrates, amino acids, and organic acids along the timeline of seedling growth. Moreover, the early increase in species-specific metabolites justified their role in seedling development owing to their participation in nitrogen metabolism (homoserine in pea), carbon translocation (galactinol, raffinose, and stachyose), and transitory carbon accumulation (1-kestose in wheat). The obtained metabolic profiles may constitute an important basis for further research on seedling reactions to stress conditions, including identification of metabolic markers of stress resistance.

Effects of climatic factors on carbohydrate composition of soybeans in Argentina

AbstractCarbohydrates are the second largest component of soybean seeds and include soluble sugars, a major source of energy. However, some oligosaccharides such as the raffinose family of oligosaccharides (RFOs) are considered antinutritional factors. In Argentina, the soybean crop area shows high climatic variation during the seed‐filling period (R5–R7), affecting seed composition. The objective of this work was to evaluate the effect of climatic traits on carbohydrate (CHO) seed composition of different cultivar types. Daily air temperature and solar radiation were measured during R5–R7 in two soybean varieties: (non‐transgenic) ALIM3.14 and a commercial. Precipitation and evapotranspiration during the crop season (R1–R7) were used to calculate a hydric balance proxy. ALIM 3.14 showed a better CHO profile than the commercial soybean. ALIM 3.14 had lower stachyose and total RFOs (25.1 and 28.9 g kg−1) than the commercial cultivar (37.3 and 42.3 g kg−1), respectively. Air temperature explained variations in CHO. A decrease in sucrose was observed with the increase in mean temperature (−2.65 g 100 g−1 per °C). Sucrose/total RFO and total sugar concentration also declined with increasing mean temperature −0.10 g 100 g−1 per °C and −0.35 g 100 g−1 per °C, respectively. Soybean with desired nutritional profile for specialties, such as high sucrose concentration and low raffinose, were obtained at cool temperatures (<19.4°C) during seed filling. Producers and breeders can use this information to identify climatic variable thresholds that maximize the concentration of sugars in seeds and select traits and environments useful to improve soybean under climate warming conditions.

Effect of temperature during flowering, pod set, and seed development on yield components and accumulation of protein, starch, and low molecular weight carbohydrates in two faba bean (Vicia faba L.) cultivars

AbstractThe aim was to explore the impact of temperature during seed development on yield performance and seed quality in faba bean when grown at cool temperatures representative for high latitude regions. Two varieties, an early and a medium late maturing, were grown in climate chambers with three temperature regimes (day/night temperatures of 14°C/12°C, 19°C/12°C, and 24°C/12°C) from onset of flowering to maturation. Yield components were recorded, and the accumulation of protein, starch, and low molecular weight carbohydrates including the raffinose family oligosaccharides was followed during the accumulation phase until physiological maturity. The lower temperature regimes strongly delayed pod and seed development compared with 24°C/12°C. Temperature affected the number of pods per plant for the upper node group. Plants grown at 19°C had the highest total dry seed weight compared with plants grown at 14°C and 24°C. Temperature per se did not influence the content of starch, protein, and low molecular weight carbohydrates, while their accumulation followed the moisture content in the seed, and thus the seed development stage. The content of raffinose family oligosaccharides increased sharply when the seed moisture dropped below 70% and leveled off at about 40% and 50% moisture for verbascose and stachyose, respectively, coinciding with physiological maturity. The results provide more knowledge about the seed maturation and accumulation in faba bean under low temperatures, important for cultivation under high latitude regions.

A horizontal view of primary metabolomes in vegetative desiccation tolerance.

Vegetative desiccation tolerance (VDT), the ability of such tissues to survive the near complete loss of cellular water, is a rare but polyphyletic phenotype. It is a complex multifactorial trait, typified by universal (core) factors but with many and varied adaptations due to plant architecture, biochemistry and biotic/abiotic dynamics of particular ecological niches. The ability to enter into a quiescent biophysically stable state is what ultimately determines desiccation tolerance. Thus, understanding the metabolomic complement of plants with VDT gives insight into the nature of survival as well as evolutionary aspects of VDT. In this study, we measured the soluble carbohydrate profiles and the polar, TMS-derivatisable metabolomes of 7 phylogenetically diverse species with VDT, in contrast with two desiccation sensitive (DS) species, under conditions of full hydration, severe water deficit stress, and desiccation. Our study confirmed the existence of core mechanisms of VDT systems associated with either constitutively abundant trehalose or the accumulation of raffinose family oligosaccharides and sucrose, with threshold ratios conditioned by other features of the metabolome. DS systems did not meet these ratios. Considerable chemical variations among VDT species suggest that co-occurring but distinct stresses (e.g., photooxidative stress) are dealt with using different chemical regimes. Furthermore, differences in the timing of metabolic shifts suggest there is not a single "desiccation programme" but that subprocesses are coordinated differently at different drying phases. There are likely to be constraints on the composition of a viable dry state and how different adaptive strategies interact with the biophysical constraints of VDT.

Fine-tuning CRISPR/Cas9 gene editing in common bean (Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models.

The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success. We initially tested three different transformation methods to induce hairy roots and selected a preferred method suitable for a variety of different common bean genotypes. This method involved inoculating a severed radicle with Rhizobium rhizogenes K599 and was fast, had a high transformation frequency of 42-48%, and resulted in numerous hairy roots. This method was further used for the transformation of explants using R. rhizogenes harboring different CRISPR/Cas9 constructs and evaluated the on-target activity of sgRNAs targeting raffinose family oligosaccharides biosynthetic genes and the impact of different promoters driving Cas9 on the gene editing efficiency. Additionally, we evaluated the reliability of the in silico tools, CRISPOR, CRISPR RGEN, and inDelphi to predict the sgRNA efficiencies and resulting mutations. Our results showed that the hairy root transformation system allows for rapid evaluation of multiple sgRNAs and promoters. We also identified several highly efficient sgRNAs that induced frameshift mutations at rates of up to 70% when a parsley ubiquitin promoter was driving Cas9 expression, providing valuable information for the selection of the most effective sgRNAs and promoters for future transformation experiments. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta results, the Lindel model proved to be the most reliable for P. vulgaris, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. Furthermore, the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants because they provide the possibility of predicting repair outcomes.

Quantitative Trait Loci and Candidate Genes That Control Seed Sugars Contents in the Soybean 'Forrest' by 'Williams 82' Recombinant Inbred Line Population.

Soybean seed sugars are among the most abundant beneficial compounds for human and animal consumption in soybean seeds. Higher seed sugars such as sucrose are desirable as they contribute to taste and flavor in soy-based food. Therefore, the objectives of this study were to use the 'Forrest' by 'Williams 82' (F × W82) recombinant inbred line (RIL) soybean population (n = 309) to identify quantitative trait loci (QTLs) and candidate genes that control seed sugar (sucrose, stachyose, and raffinose) contents in two environments (North Carolina and Illinois) over two years (2018 and 2020). A total of 26 QTLs that control seed sugar contents were identified and mapped on 16 soybean chromosomes (chrs.). Interestingly, five QTL regions were identified in both locations, Illinois and North Carolina, in this study on chrs. 2, 5, 13, 17, and 20. Amongst 57 candidate genes identified in this study, 16 were located within 10 Megabase (MB) of the identified QTLs. Amongst them, a cluster of four genes involved in the sugars' pathway was collocated within 6 MB of two QTLs that were detected in this study on chr. 17. Further functional validation of the identified genes could be beneficial in breeding programs to produce soybean lines with high beneficial sucrose and low raffinose family oligosaccharides.

Mulberry MnGolS2 Mediates Resistance to Botrytis cinerea on Transgenic Plants.

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.

Environmental stability and genetic effect of soybeans differing in mutant allele combinations between rs and mips1 genes for soluble carbohydrate profiles

AbstractRaffinose and stachyose (raffinose family oligosaccharides [RFOs]) in soybeans [Glycine max (L.) Merr.] are anti‐nutritional carbohydrate components. Previous studies identified mutant alleles of raffinose synthase (rs) 2 and 3 and d‐myo‐inositol‐3‐phosphate synthase 1 (mips1) to increase sucrose and reduce RFOs content. Although earlier studies investigated the effects of rs2 and rs3 mutations, a mips1 mutation has not been tested yet. The objectives of this study were (1) to investigate the genetic effects and environmental stabilities of rs2, rs3, and mips1 mutations across 18 environments and (2) to evaluate germination rates of genotypes carrying these mutations. Sucrose was significantly affected by all environmental factors (year, location, and planting dates). However, raffinose was not affected by planting dates, and stachyose was only affected by location. The rs2 mutant allele (rs2W331‐) and mips1 showed the highest environmental stability for sucrose and raffinose, respectively. For stachyose, the wild‐type check showed the highest environmental stability. The lines with mips1 mutation showed significantly higher sucrose (11.1%) and lower raffinose (0.5%) and stachyose (0.6%) content than other genotypes across environments. The lines tested in field emergence tests showed no significant difference from the check. Lines with mips1 and rs2W331‐, respectively, showed significantly higher germination rates than the check in the accelerated aging test. In contrast, a line with mips1 showed a significantly higher germination rate than the check in the standard germination test. Desirable carbohydrate allele combinations will facilitate the development of new cultivars with modified carbohydrate profiles that are beneficial to human food and animal feed.

Isolation and structural elucidation of prebiotic oligosaccharides from Ziziphi Spinosae Semen

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.

Identification of raffinose family oligosaccharides in processed Rehmannia glutinosa Libosch using matrix-assisted laser desorption/ionization mass spectrometry image combined with machine learning.

Currently, research on oligosaccharides primarily focuses on the physiological activity and function, with a few studies elaborating on the spatial distribution characterization and variation in the processing of Rehmannia glutinosa Libosch. Thus, imaging the spatial distributions and dynamic changes in oligosaccharides during the steaming process is significant for characterizing the metabolic networks of R. glutinosa. It will be beneficial to characterize the impact of steaming on the active ingredients and distribution patterns in different parts of the plant. A highly sensitive matrix-assisted laser desorption/ionization mass spectrometry image (MALDI-MSI) method was used to visualize the spatial distribution of oligosaccharides in processed R. glutinosa. Furthermore, machine learning was used to distinguish the processed R. glutinosa samples obtained under different steaming conditions. Imaging results showed that the oligosaccharides in the fresh R. glutinosa were mainly distributed in the cortex and xylem. As steaming progressed, the tetra- and pentasaccharides were hydrolyzed and diffused gradually into the tissue section. MALDI-MS profiling combined with machine learning was used to identify the processed R. glutinosa samples accurately at different steaming intervals. Eight algorithms were used to build classification machine learning models, which were evaluated for accuracy, precision, recall, and F1 score. The linear discriminant analysis and random forest models performed the best, with prediction accuracies of 0.98 and 0.97, respectively, and thus can be considered for identifying the steaming durations of R. glutinosa. MALDI-MSI combined with machine learning can be used to visualize the distribution of oligosaccharides and identify the processed samples after steaming for different durations. This can enhance our understanding of the metabolic changes that occur during the steaming process of R. glutinosa; meanwhile, it is expected to provide a theoretical reference for the standardization and modernization of processing in the field of medicinal plants.