Raffinose Family Oligosaccharides Content Research Articles

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.

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.

Transcription Factor VvDREB2A from Vitis vinifera Improves Cold Tolerance.

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 H2S, 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.

Spatio-temporal expression pattern of Raffinose Synthase genes determine the levels of Raffinose Family Oligosaccharides in peanut (Arachis hypogaea L.) seed

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.

Changes in the raffinose family oligosaccharides content in the lentil and common bean seeds during malting and mashing processes

Raffinose family oligosaccharides (RFOs) are sugars, which are considered anti-nutritional substances, which are not digestible by human gastric enzymes and can lead to flatulence. Legume seeds are often rich in these compounds, which can be cumbersome for many people, such as vegetarians or the population of developing countries, whose diets consists of large amounts of these food products. In this study, simple procedures used around the world in the brewing industry (malting and mashing) were used to determine, whether these processes could be applied to popular legume seeds (lentil and bean) to reduce the RFOs content. Acquired malts and worts were characterised by radically decreased concentration (up to 90%) of most ubiquitous RFOs, such as raffinose and stachyose.

Raffinose family oligosaccharides (RFOs): role in seed vigor and longevity

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.

Galactinol synthase 1 improves cucumber performance under cold stress by enhancing assimilate translocation.

Cucumber (Cucumis sativus L.) predominantly translocates raffinose family oligosaccharides (RFOs) in the phloem and accumulates RFOs in leaves. Galactinol synthase (GolS) catalyzes the critical step of RFO biosynthesis, and determining the functional diversity of multiple GolS isoforms in cucumber is of scientific significance. In this study, we found that all four isoforms of CsGolS in the cucumber genome were upregulated by different abiotic stresses. β-Glucuronidase staining and tissue separation experiments suggested that CsGolS1 is expressed in vascular tissues, whereas the other three CsGolSs are located in mesophyll cells. Further investigation indicates that CsGolS1 plays double roles in both assimilate loading and stress response in minor veins, which could increase the RFO concentration in the phloem sap and then improve assimilate transport under adverse conditions. Cold-induced minor vein-specific overexpression of CsGolS1 enhanced the assimilate translocation efficiency and accelerated the growth rates of sink leaves, fruits, and whole plants under cold stress. Finally, our results demonstrate a previously unknown response to adverse environments and provide a potential biotechnological strategy to improve the stress resistance of cucumber.

Monitoring of early‐stage water uptake by hyperspectral imaging and evaluation of nutritional and technological functionality of germinated faba bean (Vicia faba L.) var. minor and var. major as food ingredients

AbstractFaba bean (Vicia faba L.) is a potential, sustainable protein alternative. Germination behavior of Vicia faba L. var. minor and Vicia faba L. var. major needs further studies in order to enable larger scale bioprocessing. In this study, early‐stage water uptake of two distinct faba bean varieties was assessed by hyperspectral imaging. Nutritional and technological functionality of germinated faba bean ingredients as such and in combination with fermentation were evaluated. Hyperspectral imaging revealed that early‐stage water uptake in faba beans occurred evenly from the different sides of the beans. Germination on petri dishes and in pilot‐scale showed that smaller faba beans moistened and germinated significantly (p < 0.05) faster and retained water better through germination than larger faba beans. Germinated faba flour of minor‐type variety resulted in 72% higher dextran production in Weissella confusa VTT E‐143403 fermentation than respective native faba flour. With both types of faba bean varieties, germination as such, and with minor‐type variety, germination as combined with fermentation decreased notably the content of raffinose family oligosaccharides. These bioprocessing methods also improved functionality of faba bean ingredients by increasing protein separation efficiency in air classification, protein solubility, foaming capacity, and foam stability. Based on this study, minor‐ and major‐type or small‐ and large‐seeded faba bean varieties set different requirements to the germination process. In addition, germination alone or as combined with fermentation was proved to improve the nutritional and technological quality of faba bean material promoting its use in several food applications including also gluten‐free products.

Sequential expression of raffinose synthase and stachyose synthase corresponds to successive accumulation of raffinose, stachyose and verbascose in developing seeds of Lens culinaris Medik.

Raffinose, stachyose and verbascose form the three major members of the raffinose family oligosaccharides (RFO) accumulated during seed development. Raffinose synthase (RS; EC 2.4.1.82) and stachyose synthase (STS; EC 2.4.1.67) have been associated with raffinose and stachyose synthesis, but the precise mechanism for verbascose synthesis is not well understood. In this study, full-length RS (2.7 kb) and STS (2.6 kb) clones were isolated by screening a cDNA library prepared from developing lentil seeds (18, 20, 22 and 24 days after flowering [DAF]) to understand the roles of RS and STS in RFO accumulation in developing lentil seeds. The nucleotide sequences of RS and STS genes were similar to those reported for Pisum sativum. Patterns of transcript accumulation, enzyme activities and RFO concentrations were also comparable to P. sativum. However, during lentil seed development raffinose, stachyose and verbascose accumulation corresponded to transcript accumulation for RS and STS, with peak transcript abundance occurring at about 22–24 DAF, generally followed by a sequential increase in raffinose, stachyose and verbascose concentrations followed by a steady level thereafter. Enzyme activities for RS, STS and verbascose synthase (VS) also indicated a sudden increase at around 24–26 DAF, but with an abrupt decline again coinciding with the subsequent steady state increase in the RFO. Galactan:galactan galactosyl transferase (GGT), the galactinol-independent pathway enzyme, however, exhibited steady increase in activity from 24 DAF onwards before abruptly decreasing at 34 DAF. Although GGT activity was detected, isolation of a GGT sequence from the cDNA library was not successful.

Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max.

Raffinose family oligosaccharides (RFO) play an important role in plants but are also considered to be antinutritional factors. A profound understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds, without compromising normal plant development and functioning. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean. In common bean, three galactinol synthase genes, two raffinose synthase genes and one stachyose synthase gene were identified for the first time. To discover the expression patterns of these genes in different tissues, two expression atlases have been created through re-analysis of publicly available RNA-seq data. De novo expression analysis through an RNA-seq study during seed development of three varieties of common bean gave more insight into the expression patterns of these genes during the seed development. The results of the expression analysis suggest that different classes of galactinol- and RFO synthase genes have tissue-specific expression patterns in soybean and common bean. With the obtained knowledge, important galactinol- and RFO synthase genes that specifically play a key role in the accumulation of RFOs in the seeds are identified. These candidate genes may play a pivotal role in reducing the RFO content in the seeds of important legumes which could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption.

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

EVALUATION OF MONO-, DI- AND OLIGO-SACCHARIDES OF STAPLE INDIAN GRAIN FLOURS

In this study, investigation of fourteen most common and traditionally used Indian staple grain flours: wheat, white rice, kutki rice, maize, maida, besan, sattu, little millet, quinoa, soybean, jowar, bajra, sooji and ragi was undertaken for the assessment of mono- (namely glucose, fructose and galactose) and di- (namely sucrose) and oligo-saccharides (namely raffinose, stachyose and verbascose), with a special interest on the indigestible flatulence producing Raffinose Family Oligosaccharides (RFOs). The soluble saccharides were extracted with ethanol and evaluated by calorimetric and high-performance liquid chromatography (HPLC) methods. The results showed that among the investigated flours, total RFOs content on dry matter (DM) basis ranges from 0.07 (maize) to 12.61 mmol/100 g DM (chickpea). Within RFOs, raffinose was the major sugar (0.1 to 8.04 mmol/100 g DM), followed by stachyose (negligible to 6.3 mmol/100 g DM) and verbascose (negligible to 0.14 mmol/100 g DM), except for soybean wherein the major RFO was stachyose. The observed sucrose content ranges from 0.05 (maize) to 6.32 mmol/100 g DM (quinoa). Within mono-saccharides: glucose content ranges from negligible (chickpea, little millet, quinoa, pearl millet and sooji) to 3.64 mmol/100 g DM (soybean), galactose content ranges from negligible (white rice, maize, sattu, quinoa, white millet, sooji and finger millet) to 1.72 mmol/100 g DM (chickpea) and fructose content ranges from negligible (chickpea, sattu, little millet, quinoa, soybean, sooji and finger millet) to 0.99 mmol/100 g DM (white rice). From the obtained results, the investigated grains can be classified as per the content of water soluble saccharides and accordingly a glycemic index and anti-flatulence chart can be prepared as a ready reference for dietary guidelines.

Raffinose family oligosaccharides in seeds of common vetch (Vicia sativa L. ssp. sativa)

The composition and content of low-molecular weight soluble carbohydrates in seeds of 66 accessions of common vetch (Vicia sativa L. ssp. sativa) stored in the Polish and German gene banks, originating from Europe, Asia, Middle East and North Africa, were analyzed using a gas chromatography method. Genotypes showed differences in the composition of carbohydrates depending on the origin of seeds. Common features of accessions were: i) the predominance of raffinose family oligosaccharides (RFOs) in soluble carbohydrates, and ii) verbascose as the main oligosaccharide in RFOs. Several accessions with lowered RFOs content were identified. They may be suitable for breeding focused on diminishing RFOs, as antinutritional compounds in seeds.

Poaceae Type II Galactinol Synthase 2 from Antarctic Flowering Plant Deschampsia antarctica and Rice Improves Cold and Drought Tolerance by Accumulation of Raffinose Family Oligosaccharides in Transgenic Rice Plants.

Deschampsia antarctica is a Poaceae grass that has adapted to and colonized Antarctica. When D. antarctica plants were subjected to cold and dehydration stress both in the Antarctic field and in laboratory experiments, galactinol, a precursor of raffinose family oligosaccharides (RFOs) and raffinose were highly accumulated, which was accompanied by upregulation of galactinol synthase (GolS). The Poaceae monocots have a small family of GolS genes, which are divided into two distinct groups called types I and II. Type II GolSs are highly expanded in cold-adapted monocot plants. Transgenic rice plants, in which type II D. antarctica GolS2 (DaGolS2) and rice GolS2 (OsGolS2) were constitutively expressed, were markedly tolerant to cold and drought stress as compared to the wild-type rice plants. The RFO contents and GolS enzyme activities were higher in the DaGolS2- and OsGolS2-overexpressing progeny than in the wild-type plants under both normal and stress conditions. DaGolS2 and OsGolS2 overexpressors contained reduced levels of reactive oxygen species (ROS) relative to the wild-type plants after cold and drought treatments. Overall, these results suggest that Poaceae type II GolS2s play a conserved role in D. antarctica and rice in response to drought and cold stress by inducing the accumulation of RFO and decreasing ROS levels.

Environmental Stability of Carbohydrate Profiles in Different Soybean Genotypes

Soybean [Glycine max (L.) Merr.] is an important annual crop. The raffinose family oligosaccharides (RFOs) raffinose and stachyose are antinutritional carbohydrates present in soybean seeds. Consumption of soybean seed products with low RFO reduced flatulence in humans and increased metabolizable energy efficiency in chickens, pigs, and dogs. The development of soybean cultivars producing high metabolizable energy with low antinutritional factors in the seed is one avenue to increase soybean value. The soybean line PI 200508 with low RFO content was previously characterized to have a reduction of seed raffinose synthase enzyme activity. A variant allele of the raffinose synthase 2 gene (RS2) with a 3‐bp deletion leading to the elimination of a single conserved amino acid residue (RS2, rs2W331−) in PI 200508 was associated with the seed RFO phenotype. Another missense allele of RS2 and variant alleles of the RS3 gene have been identified. The objective of this study was to determine the environmental stability of the carbohydrate profiles for soybean lines containing different allele combinations of two key raffinose synthase (RS2 and RS3) genes in two locations over 2 yr. Although this study was conducted with different genetic backgrounds, the results indicated that the carbohydrate profile in soybean seed is mostly determined by RS2 and RS3 genotype, and galactinol and sucrose content were mainly affected by environmental factors. Genotype was the major factor for the RFO content in soybean seeds, and soybean lines with the rs2W331− allele of RS2 were the most stable for sucrose and RFOs over four environments. A novel genetic combination of RS2 and RS3 alleles demonstrated the importance of multiple variant alleles contributing to seed raffinose synthase activity.

The GARP/MYB-related grape transcription factor AQUILO improves cold tolerance and promotes the accumulation of raffinose family oligosaccharides

Grapevine (Vitis vinifera L.) is a widely cultivated fruit crop whose growth and productivity are greatly affected by low temperatures. On the other hand, wild Vitis species represent valuable genetic resources of natural stress tolerance. We have isolated and characterized a MYB-like gene encoding a putative GARP-type transcription factor from Amur grape (V. amurensis) designated as VaAQUILO. AQUILO (AQ) is induced by cold in both V. amurensis and V. vinifera, and its overexpression results in significantly improved tolerance to cold both in transgenic Arabidopsis and in Amur grape calli. In Arabidopsis, the ectopic expression of VaAQ increased antioxidant enzyme activities and up-regulated reactive oxygen species- (ROS) scavenging-related genes. Comparative mRNA sequencing profiling of 35S:VaAQ Arabidopsis plants suggests that this transcription factor is related to phosphate homeostasis like their Arabidopsis closest homologues: AtHRS1 and AtHHO2. However, when a cold stress is imposed, AQ is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GoLS) and raffinose synthase genes. Gene co-expression network (GCN) and cis-regulatory element (CRE) analyses in grapevine indicated AQ as potentially regulating VvGoLS genes. Increased RFO content was confirmed in both transgenic Arabidopsis and Amur grape calli overexpressing VaAQ. Taken together, our results imply that AQ improves cold tolerance through promoting the accumulation of osmoprotectants.

Evolutionary diversification of galactinol synthases in Rosaceae: adaptive roles of galactinol and raffinose during apple bud dormancy.

Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply.

Carbohydrate Concentration in Lentils (Lens culinaris Medikus.): Genotypic and Environmental Effects

ABSTRACTLentils contain a range of prebiotic carbohydrates; however, genotypic and environmental implications of these components have not been well understood. The objective of this study was to determine the genetic and environmental variation of carbohydrate concentrations in three commonly grown lentil cultivars in South Dakota, USA at two locations over two growing seasons. Significant genetic and environmental variation was found for most of the constituents evaluated. Raffinose-family oligosaccharides (RFO) ranged from 3161 mg 100g−1 at Bison and 3670 mg 100g−1 at Wall and from 3217 mg 100g−1 to 3650 mg 100g−1 between cultivars. Moreover, RFO content was strongly correlated to a simple plant stress index (SI) in which higher estimated plant stress (e.g., low precipitation) produced higher levels of RFO. These results demonstrate important differences in prebiotic concentrations, which can be used for future lentil breeding efforts when matching cultivars to diverse growing climates with highly variable growing season precipitation.

Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana

Raffinose family oligosaccharides (RFOs) accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE (GOLS), RAFFINOSE SYNTHASE (RS), and STACHYOSE SYNTHASE (STS) are the enzymes responsible for RFO biosynthesis in plants. Interestingly, only raffinose is detected in maize seeds, and a unique maize RS gene (ZmRS) was identified. In this study, we found that two independent mutator (Mu)-interrupted zmrs lines, containing no raffinose but hyperaccumulating galactinol, have significantly reduced seed vigor, compared with null segregant controls. Unlike maize, Arabidopsis thaliana seeds contain several RFOs (raffinose, stachyose, and verbascose). Manipulation of A. thaliana RFO content by overexpressing ZmGOLS2, ZmRS, or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS, or overexpression of ZmGOLS2 alone, significantly increased the total content of RFOs and enhanced Arabidopsis seed vigor. Surprisingly, while overexpression of ZmRS increased seed raffinose content, its overexpression dramatically decreased seed vigor and reduced the seed amounts of galactinol, stachyose, and verbascose. In contrast, the atrs5 mutant seeds are similar to those of the wild type with regard to seed vigor and RFO content, except for stachyose, which accumulated in atrs5 seeds. Total RFOs, RFO/sucrose ratio, but not absolute individual RFO amounts, positively correlated with A. thaliana seed vigor, to which stachyose and verbascose contribute more than raffinose. Taken together, these results provide new insights into regulatory mechanisms of seed vigor and reveal distinct requirement for RFOs in modulating seed vigor in a monocot and a dicot.

Physiological and Biochemical Parameters of Lupin Seed Subjected to 29 Years of Storage

Physiological and biochemical changes resulting from long‐term storage of lupin (Lupinus luteus L.) seed were studied in a precisely controlled experiment to gain a better knowledge of the seed deterioration process. The seed was stored at different temperatures (0, −14, and +20°C) during a 29‐yr period. It was found that after long‐term storage at −14 and 0°C, the seed germinated well, whereas seed stored at +20°C did not germinate at all. The contents of albumin, biogenic amines, and soluble carbohydrates were determined. The content of albumin decreased in seed stored at +20°C. In two‐dimensional electrophoresis of seed proteins, 343 spots were obtained with seed stored at −14°C, whereas the extract of seed stored at +20°C gave 290 spots. Out of all detected spots, 27 displayed significant differences in intensity related to seed storage temperature (−14, 0, +20°C). The identified 27 proteins could be assigned to four major functional categories: storage proteins, proteins connected with defense mechanisms, proteins responsible for energy reactions, and proteins involved in growth and development. Spermine, spermidine, and putrescine were also analyzed in stored seed. The content of biogenic amines was higher in seed stored at +20°C. Significant changes occurred in the content of soluble carbohydrates during seed storage. In seed stored at +20°C, verbascose did not occur and the total content of raffinose family oligosaccharides decreased. The content of reducing monosaccharides and sucrose increased. The results show that lupin seed storage at −14 and 0°C resulted in very similar levels of physiological and biochemical changes, affecting the germination rate, proteome, biogenic amines, and soluble carbohydrates, whereas storage at +20°C is incomparably more detrimental.