Raffinose Family Oligosaccharides Research Articles

Thermopriming reprograms metabolic homeostasis to confer heat tolerance

Heat stress threatens agriculture worldwide. Plants acquire heat stress tolerance through priming, which establishes stress memory during mild or severe transient heat stress. Such induced thermotolerance restructures metabolic networks and helps maintain metabolic homeostasis under heat stress. Here, we used an electrospray ionization mass spectrometry-based platform to explore the composition and dynamics of the metabolome of Arabidopsis thaliana under heat stress and identify metabolites involved in thermopriming. Primed plants performed better than non-primed plants under severe heat stress due to altered energy pathways and increased production of branched-chain amino acids, raffinose family oligosaccharides (RFOs), lipolysis products, and tocopherols. These metabolites serve as osmolytes, antioxidants and growth precursors to help plants recover from heat stress, while lipid metabolites help protect membranes against heat stress. The carbohydrate (e.g., sucrose and RFOs) and lipid superpathway metabolites showed the most significant increases. Under heat stress, there appears to be crosstalk between carbohydrate metabolism (i.e., the thermomemory metabolites stachyose, galactinol, and raffinose) and tyrosine metabolism towards the production of the thermomemory metabolite salidroside, a phenylethanoid glycoside. Crosstalk occurs between two glycerophospholipid pathways (the biosynthetic pathways of the thermomemory metabolite S-adenosyl-L-homocysteine and the terpenoid backbone) and the δ-tocopherol (chloroplast lipid) pathway, which favors the production of glycine betaine and other essential tocopherols, respectively, compounds which are essential for abiotic stress tolerance in plants. Therefore, metabolomic analysis can provide comprehensive insights into the metabolites involved in stress responses, which could facilitate plant breeding to maximize crop yields under adverse conditions.

Chemical Composition and Antioxidant Activity of Monguba (Pachira aquatica) Seeds

Monguba fruit has a seed with a chestnut-like flavor that can be consumed boiled, fried, and roasted. These nutritious seeds also have been used in popular medicine to treat several diseases. Nevertheless, the nutritional and functional potential of monguba seed is still underexploited. In this sense, we investigated the nutritional and functional components of monguba seeds. These seeds showed high total content of sugars, mainly sucrose, whereas the content of the raffinose family oligosaccharides was low. The mineral assay showed high amount of minerals, namely potassium, calcium, magnesium and zinc, which indicate that monguba seeds can be a new source of these minerals. UHPLC-ESI-MS/MS analysis showed caffeic, ferulic and 4-hydroxybenzoic acids as the main phenolic compounds, mainly in the esterified form, in these seeds. Monguba seed showed high lipid content, in which the main compounds were palmitic acid and γ-tocopherol. The soluble and insoluble phenolic fractions from monguba seeds showed high antioxidant activity measured by the oxygen radical absorption capacity (ORAC) and the trolox equivalent antioxidant capacity (TEAC) assays. Therefore, the monguba seeds have great potential to be explored by food, pharmaceutical and cosmetic industries due to their chemical composition.

Protective effects of α-galacto-oligosaccharides against a high-fat/western-style diet-induced metabolic abnormalities in mice.

In this study, we investigated the effects of a newly synthesized α-galacto-oligosaccharide mixture (α-GOSg), 0.5% in drinking water, on high-fat/western-style diet (HFWD)-induced metabolic abnormality in mice in a study of 13 weeks. Raffinose family oligosaccharides (RFOs) were included as a comparison. Mice treated with α-GOSg had significantly lower body weight and body fat (p < 0.05), while RFOs were less effective. Both α-GOSg and RFOs significantly reduced serum levels of total cholesterol and low-density lipoprotein cholesterol, alanine aminotransferase and liver lipids. However, only α-GOSg significantly decreased the histopathological score for liver steatosis and downregulated hepatic fatty acid synthesis gene acetyl CoA carboxylase-α. α-GOSg also significantly reduced the content of bile acids in the small intestine and significantly increased the abundance of gut Bifidobacterium and decreased the abundance of Clostridium leptum. These actions are proposed to be key mechanisms contributing to the beneficial health effects of α-GOSg.

Effect of in ovo administration of different synbiotics on carcass and meat quality traits in broiler chickens

The aim of this study was to examine the effect of in ovo synbiotic administration on slaughter performance and meat quality traits of broiler chickens. On day 12 of incubation, 5,850 eggs (Cobb 500 FF) were randomly divided into 3 experimental groups and automatically injected in ovo with physiological saline (control, C) and 2 different synbiotic formulations (SYN1: Lactobacillus salivarius + galactooligosaccharides; SYN2: Lactobacillus plantarum + raffinose family oligosaccharides). After hatching, 240 males were randomly chosen (80 chicks per group) and split into 8 replicate pens (10 birds per pen). At 42 d of age, 15 birds per treatment were weighed and slaughtered. In ovo synbiotic administration had a low effect on investigated traits, but depends on the kind of synbiotic administered. Both synbiotic formulations did not affect final BW, weight, and yield of carcass and pectoral muscle (PM); likewise, physicochemical properties (pH, color, water holding capacity), intramuscular collagen properties, and cholesterol content of PM were not affected by treatment. Synbiotic administration reduced (P = 0.061) the lipid content compared with C group, markedly (P < 0.05) with synbiotic SYN2. Meat from SYN1 birds displayed a higher (P < 0.01) content of saturated fatty acids (SFA), lower monounsaturated fatty acids (P < 0.05 compared only to SYN2), and lower (P < 0.01 and P < 0.05) polyunsaturated fatty acids (PUFA), n-6 PUFA and n-3 PUFA compared to C and SYN2 groups. The ratio of n-6 to n-3 PUFA was affected by the synbiotic administration (P = 0.039). Meat from C and SYN2 groups displayed a higher (P < 0.01) ratio of PUFA to SFA and lower (P < 0.01) atherogenic and thrombogenic indices compared to SYN1. In conclusion, this study has shown that in ovo administration of synbiotics did not negatively affect slaughter performance and physicochemical properties of meat. However, meat from C and SYN2 birds showed a preferable fatty acid profile, with a positive effect on nutritional properties of chicken meat.

Abiotic stress regulates expression of galactinol synthase genes post-transcriptionally through intron retention in rice.

Expression of the Galactinol synthase genes in rice is regulated through post-transcriptional intron retention in response to abiotic stress and may be linked to Raffinose Family Oligosaccharide synthesis in osmotic perturbation. Galactinol synthase (GolS) is the first committed enzyme in raffinose family oligosaccharide (RFO) synthesis pathway and synthesizes galactinol from UDP-galactose and inositol. Expression of GolS genes has long been implicated in abiotic stress, especially drought and salinity. A non-canonical regulation mechanism controlling the splicing and maturation of rice GolS genes was identified in rice photosynthetic tissue. We found that the two isoforms of Oryza sativa GolS (OsGolS) gene, located in chromosomes 3(OsGolS1) and 7(OsGolS2) are interspersed by conserved introns harboring characteristic premature termination codons (PTC). During abiotic stress, the premature and mature transcripts of both isoforms were found to accumulate in a rhythmic manner for very small time-windows interrupted by phases of complete absence. Reporter gene assay using GolS promoters under abiotic stress does not reflect this accumulation profile, suggesting that this regulation occurs post-transcriptionally. We suggest that this may be due to a surveillance mechanism triggering the degradation of the premature transcript preventing its accumulation in the cell. The suggested mechanism fits the paradigm of PTC-induced Nonsense-Mediated Decay (NMD). In support of our hypothesis, when we pharmacologically blocked NMD, the full-length pre-mRNAs were increasingly accumulated in cell. To this end, our work suggests that a combined transcriptional and post transcriptional control exists in rice to regulate GolS expression under stress. Concurrent detection and processing of prematurely terminating transcripts coupled to repressed splicing can be described as a form of Regulated Unproductive Splicing and Translation (RUST) and may be linked to the stress adaptation of the plant, which is an interesting future research possibility.

Genome-Wide Identification and Expression Profiling Analysis of the Galactinol Synthase Gene Family in Cassava (Manihot esculenta Crantz)

Galactinol synthases (GolSs) are the key enzymes that participate in raffinose family oligosaccharides (RFO) biosynthesis, which perform a big role in modulating plant growth and response to biotic or abiotic stresses. To date, no systematic study of this gene family has been conducted in cassava (Manihot esculenta Crantz). Here, eight MeGolS genes are isolated from the cassava genome. Based on phylogenetic background, the MeGolSs are clustered into four groups. Through predicting the cis-elements in their promoters, it was discovered that all MeGolS members act as hormone-, stress-, and tissue-specific related elements to different degrees. MeGolS genes exhibit incongruous expression patterns in various tissues, indicating that different MeGolS proteins might have diverse functions. MeGolS1 and MeGolS3–6 are highly expressed in leaves and midveins. MeGolS3–6 are highly expressed in fibrous roots. Quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis indicates that several MeGolSs, including MeGolS1, 2, 5, 6, and 7, are induced by abiotic stresses. microRNA prediction analysis indicates that several abiotic stress-related miRNAs target the MeGolS genes, such as mes-miR156, 159, and 169, which also respond to abiotic stresses. The current study is the first systematic research of GolS genes in cassava, and the results of this study provide a basis for further exploration the functional mechanism of GolS genes in cassava.

Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds

Raffinose family oligosaccharides (RFOs) accumulate during seed development, and have been thought to be associated with the acquisition of desiccation tolerance (DT) by seeds. Here, comprehensive approaches were adopted to evaluate the changes of DT in developing Arabidopsis seeds of wild type, overexpression (OX-AtGS1/GS2/RS5), and mutant lines by manipulating the expression levels of the GALACTINOL SYNTHASE (GS) and RAFFINOSE SYNTHASE (RS) genes. Our results indicate that seeds of the double mutant (gs1, gs2) and rs5 delayed the timing of DT acquisition as compared to wild type. Subsequent detection confirmed that seeds from OX-AtGS1/GS2 plants with high levels of galactinol, raffinose, and stachyose, and OX-AtRS5 plants possess more raffinose and stachyose but less galactinol compared to wild type. These lines all showed greater germination percentage and shorter time to 50% germination after desiccation treatment at 11 and 15 days after flower (DAF). Further analysis revealed that the role of RFOs is time limited and mainly affects the middle stage (9–16 DAF) of seed development by enhancing seed viability and the ratio of GSH to GSSH in cells, but there is no significant difference in DT of mature seeds. In addition, RFOs could reduce damage to seeds caused by oxidative stress. We conclude that GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE play important roles in DT acquisition during Arabidopsis seed development, and that galactinol and RFOs are crucial protective compounds in the response of seeds to desiccation stress.

Phloem Unloading Strategies and Mechanisms in Crop Fruits

Carbohydrate produced by photosynthesis is loaded into phloem via collection phloem, translocated via the transport phloem, and unloaded by release phloem into sink organs. Phloem unloading strategy is an important physiological process and plays a key role in regulating the distribution of photoassimilate and the growth and development of crops. Here, the detailed unloading strategies and mechanisms in three types of sugar transporting-plants, sucrose, sugar alcohol, and Raffinose family of oligosaccharides (RFOs), are summarized and discussed. Symplastic and apoplastic unloading are the main phloem unloading strategies, and these strategies could shift from symplastic to apoplastic (such as grape), or otherwise (such as potato); some crops shift more than one time (jujube and cotton) during fruit development, depending on the type, structure, sugar component, and developmental stages of fruit and a series of genes involved in unloading process, such as transporters, α-galactosidase, invertase, and sucrose synthase. The diversity of the unloading strategies results in the difference in size, yield, quality, and flavor of crop fruit. Why are there a variety of phloem unloading strategies in crop fruit? What is the mechanism of the unloading strategy shift? These issues are summarized, discussed, and put forward a research agenda for the near future.

Role of pigeon pea (Cajanus cajan L.) in human nutrition and health: A review

Legumes and cereals are good, relatively inexpensive, sources of proteins and energy for third world countries, including India. India is a major pulse producing country, sharing 36 and 28% of total area and production of these crops. Pigeon pea (Cajanus cajan L.) is widely consumed in the form of dhal as an economical source of protein. It is a dense source of nutrients, but its nutritional elements are masked by some anti-nutritional factors decreased by different processing methods. The non-nutritive compounds of pigeon pea have been investigated for their role in the enhancement of the antioxidant and anti carcinogenic effects. Recent evidences suggest that pigeon pea bioactive compounds play a vital role in modulating the gut micribiota hence, can reduce inflammation. The Prebiotic potential of non digestible raffinose family oligosaccharides has also been investigated in animal models. Research on pigeon pea prebiotic oligosaccharide and health benefits is scanty and some research regarding this novel approach remains inconclusive and there is still much to be studied. This review article focuses on the nutritional, anti-nutritional factors bioactive compounds and other health benefits of pigeon pea.

Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs).

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.

A GENOME-WIDE ANALYSIS OF THE GALACTINOL SYNTHASEGENE FAMILY IN BANANA (Musa acuminata)

Galactinol synthase (GolS) is theenzyme that catalyzes the first step of the biosynthesis of the raffinose family oligosaccharides (RFOs), andisinvolved in manybiological processes in plants.In the present study, four putative GolSgenes were identified in the Musa acuminatagenome. We further characterized these MaGolSgenes in terms of protein length, molecular weight, theoretical isoelectric point and 3D protein structure. Genomic organization revealed that most MaGolSgenes have four exons. The conserved motifs were identified,demonstrating high group-specificityof all MaGolS proteins. Multiple sequence alignment showedthat theAPSAA typical domainispresent in all GolS proteins. Comparativephylogenetic analysis oftheMaGolS proteins revealed three distinct groups.These data provide insight to support new studies adressing the role of GolSgenes in this important fruit species.

Prebiotics offered to broiler chicken exert positive effect on meat quality traits irrespective of delivery route.

Elimination of antibiotic growth promoters from poultry production has encouraged intensive search for relevant alternatives. Prebiotics are proposed as efficient replacements to stimulate colonization/expansion of beneficial microflora in chickens. The aim of this study was to deepen the knowledge on the effect of prebiotic administration on slaughter performance and meat quality traits of broiler chickens by evaluating different routes of their delivery (in ovo vs. in-water vs. in ovo + in-water). At d 12 of incubation, 1,500 eggs (Ross 308) containing viable embryos were randomly allotted into 4 groups and injected in ovo with 0.2mL solution containing: 3.5mg/embryo BI (Bi2tos, trans-galactooligosaccharides); 0.88mg/embryo DN (DiNovo, extract of Laminaria spp.); 1.9mg/embryo RFO (raffinose family oligosaccharides) and 0.2mL physiological saline (C). All prebiotics increased final BW compared to C group (P < 0.01), irrespective of delivery route. The prebiotics injected in ovo (T1) or in ovo combined with in-water supplementation (T2) increased carcass weight as compared with in-water group (T3), while T3 had the lowest carcass yield compared to the other groups. All prebiotics increased breast muscle weight and yield (P < 0.01), as well as fiber diameter (P < 0.05). Ultimate meat pH was lower (P < 0.01) in T3 than in T2 group. Meat from chickens treated with prebiotics showed a lower redness index, while lightness and yellowness were not affected by the treatments. Saturated fatty acid (SFA), polyunsaturated fatty acid (PUFA) and n-3 fatty acids contents were higher (P < 0.01), and monounsaturated fatty acid (MUFA) level was lower (P < 0.01) in prebiotic groups compared with C group. Nutritional indexes (n-6/n-3, PUFA/SFA ratio and thrombogenic index) displayed favorable human health-promoting values in the meat of chickens which were treated with prebiotics, irrespective of delivery route. Muscle cholesterol content was not affected by prebiotics. In conclusion, this study has shown that prebiotics can exert positive effects on growth of broiler chickens, carcass and meat quality traits, irrespective of delivery route.

The occurrence and accumulation of d-pinitol in fenugreek (Trigonella foenum graecum L.)

This article presents changes in concentrations of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek (Trigonella foenumgraecum L.) during an entire plant growing period. d-Pinitol was the major cyclitol in all tested organs, representing 43–94% of total cyclitols and 2–77% of total soluble carbohydrates. The highest concentration of d-pinitol was found in pods (14–23 mg g−1 of dry weight, DW), lower in leaves and stems (5–20 and 9–10 mg g−1 DW, respectively), and the lowest in maturing seeds (2–5 mg g−1 DW). Although maturing seeds accumulate α-d-galactosides of d-pinitol (galactosyl pinitols, up to 6.6 mg g−1 DW), the major storage sugars were raffinose family oligosaccharides (RFOs, 65.37 mg g−1 DW). Both RFOs and galactosyl pinitols are hydrolyzed during seed germination, releasing sucrose and d-pinitol, respectively. Accumulation of free galactose was not detected. Owing to the high concentration of d-pinitol (up to 23.70 mg g−1 DW) and low concentration of soluble sugars, developing pods seem to be the best source of d-pinitol.

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in Jatropha curcas seedlings.

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12°C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation.The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.

Effect of synbiotic dietary supplementation on histological and histopathological parameters of Pectoralis major muscle of broiler chickens

Bioactive substances may constitute an alternative to antibiotics. Probiotics are mixtures of microorganisms that enhance the effectiveness and yield of nutrient absorption by competing for the substrate against pathogens that may cause intestinal infections. Prebiotics are organic substances which include complex carbohydrates and have an influence on the growth and activity of desirable intestinal microflora. Synbiotics are a combination of both of them. The aim of this study was to analyze the impact of synbiotics added to commercial feed on Pectoralis major muscle microstructure in broiler chickens. Research material consisted of 240 one-day-old Ross 308 female chicks. Birds were divided into 2 groups, 12 broilers each (replicated 10 times). The control group (C) was fed a commercial diet, and the SYN group was fed the same diet with added synbiotic: 0.8% of prebiotic RFO (raffinose family oligosaccharides) extracted from lupin seeds and 1% of probiotic Lavipan&lt;sup&gt;®&lt;/sup&gt;. Birds were slaughtered at day 42. Immediately after slaughter, samples of the Pectoralis major muscle for histological analysis were taken and frozen in liquid nitrogen. The staining procedures performed were: hematoxylin and eosin staining to evaluate fibre diameter and histopathological changes, oil red staining to determine intramuscular fat content, NADH-TR (tetrazolium reductase) activity to distinguish muscle fibre types and alkaline phosphatase reaction for counting the number of capillaries. The tendency to reduced muscle fibre diameter in chickens supplemented with synbiotics indicates an increase in muscle fibre density, which gives meat a more delicate structure. When assessing the occurrence of histopathological changes, significantly more fibre splitting was observed in the control group. Moreover, the positive effect of feed supplementation with synbiotics on muscle microvascularization could have an important practical meaning, because low physical activity of chickens during rearing may lead to ischemic muscles, increased changes in the structure of muscle fibres, and reduction of meat quality.

Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 μmol ml−1 min−1, and 0.413, 0.0026 s−1 mM−1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45–65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).

Comparison of properties of raw pulse flours with those of jet-cooked, drum-dried flours

The properties of whole bean flours of navy, pinto, and black beans and chickpeas were compared with those of flours that had been passed through a steam jet cooker and drum dried. Analysis of structure, particle size, color, solubility, pasting characteristics, dietary fiber, oligosaccharides, and protein digestibility revealed differences from raw flours with potential advantages for food applications, including increased soluble fiber on average from 53 to 62 g/kg. Hot water solubility increased from 10 to 37 g/100 g, while particle size, water absorption index, and viscosity after pasting were decreased. Color changes in the flours suggested solubilization and redistribution of seed coat pigments. The treatments increased extractability of raffinose family oligosaccharides from 34 to 37 mg g−1. Starch granules were dissolved during jet-cooking, and the dried product consisted of a uniform composite matrix. These results revealed changes in the physicochemical and functional properties of bean flours with the potential to increase the utilization of pulse flours in foods.

The functions of cucumber sucrose phosphate synthases 4 (CsSPS4) in carbon metabolism and transport in sucrose- and stachyose-transporting plants

Sucrose phosphate synthases (SPSs) are rate-limiting sucrose synthesis enzymes present in photosynthetic and non-photosynthetic tissues. The cucumber genome contains three SPSs that can be grouped into families A, B, and C. CsSPS1 and CsSPS2 are highly expressed in flowers and mature leaves, while the expression level of CsSPS4 increased gradually after leaf unfolding in our study and reached its peak after 20 days. In CsSPS4-overexpression tobacco plants, sucrose content and sucrose/starch ratio were increased significantly and resulted in improved leaf yield. By contrast, in CsSPS4-overexpression (CsSPS4-OE) cucumber lines, contents of sucrose and starch were unchanged, and raffinose was increased in transgenic cucumber leaves. The expression of cucumber raffinose family oligosaccharide (RFO)-synthesis-related genes increased obviously in cucumber CsSPS4-OE plants, and the sucrose, raffinose, and stachyose contents increased significantly in the petioles of CsSPS4-OE lines. In CsSPS4-antisense (CsSPS4-A) cucumber lines, decreases occurred in mRNA expression, enzyme activity, sucrose content, sucrose/starch ratio, and stachyose transport, but the RFO-synthesis-related genes were nearly unchanged. Together, these results suggest that overexpression of CsSPS4 can lead to carbon metabolism prioritizing sugar transport in cucumber, and suppression of CsSPS4 likely promotes carbon metabolism to accumulate starch, showing a more complicated carbon distribution model than in transgenic tobacco plants.

High-level expression of a novel protease-resistant α-galactosidase from Thielavia terrestris

Abstract A novel α-galactosidase gene from Thielavia terrestris (designated TtGal27A) was cloned and expressed at high level in Pichia pastoris. The enzyme shared highest identity (40%) with glycoside hydrolase family 27 α-galactosidases from Penicillium purpurogenum and Penicillium simplicissimum among the characterized proteins. Extracellular expression of TtGal27 A was up to 4402.1 U/mL. TtGal27A was purified and had a specific activity of 752.0 U/mg towards p-nitrophenyl-α-galactopyranoside. It showed optimal pH and temperature of 4.5 and 60 °C, respectively, and exhibited 36.0% of its maximum activity at pH 2.5. The enzyme was stable within pH 3.0–9.0 and at temperatures of up to 50 °C after 30 min incubation. TtGal27A hydrolyzed raffinose family oligosaccharides and various galactomannans. In addition, the enzyme showed good protease resistance (>80% of initial activity retained) towards α-chymotrypsin, papain, proteinase K and trypsin. These properties of TtGal27A make it potentially applicable in the food and feed industries.

The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum

The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose-containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.