Semi-Quantification of Lectins in Rice (Oryza sativa L.) Genotypes via Hemagglutination

Lectins are unique glycoproteins that react with specific sugar residues on cell surfaces resulting in agglutination. They offer enormous applications in therapeutics, diagnostics, medicine, and agriculture. Rice lectins are naturally expressed during biotic and abiotic stresses suggesting their importance in stress resistance physiology. The objective of this study was to determine the presence and relative concentration of lectins in different accessions of rice obtained from IABGR/NARC Islamabad mainly originated from Pakistan. About 210 rice accessions including 02 local varieties and 05 transgenic seeds were screened for seed lectins using a hemagglutination (HA) assay with 5% Californian bred rabbits’ erythrocytes. A protein concentration of 3–8 mg/100 mg of seed flour was measured for all the rice accessions; the highest was 8.03 mg for accession 7600, while the lowest noted was 3.05 mg for accession 7753. Out of 210 accessions, 106 showed the highest HA activity. These 106 genotypes were further screened for titer analysis and specific activity. The highest titer and specific activity were observed for accession 7271 as 1024 and 236 hemagglutination unit (HAU), respectively. The selected accessions’ relative affinity and HA capability were evaluated using blood from four different sources: human, broiler chicken, local rabbit, and Californian-breed rabbit. The highest HA activity was observed with Californian-breed rabbit RBCs. The lectin assay was stable for about 1–2 h. After the required investigations, the accessions with higher lectin concentration and HA capability could be used as a readily available source of lectins for further characterization and utilization in crop improvement programs.

All sugars ain't sweet: selection of particular mono-, di- and trisaccharides by western carpenter ants and European fire ants

Ants select sustained carbohydrate resources, such as aphid honeydew, based on many factors including sugar type, volume and concentration. We tested the hypotheses (H1–H3) that western carpenter ants, Camponotus modoc, seek honeydew excretions from Cinara splendens aphids based solely on the presence of sugar constituents (H1), prefer sugar solutions containing aphid-specific sugars (H2) and preferentially seek sugar solutions with higher sugar content (H3). We further tested the hypothesis (H4) that workers of both Ca. modoc and European fire ants, Myrmica rubra , selectively consume particular mono-, di- and trisaccharides. In choice bioassays with entire ant colonies, sugar constituents in honeydew (but not aphid-specific sugar) as well as sugar concentration affected foraging decisions by Ca. modoc . Both Ca. modoc and M. rubra foragers preferred fructose to other monosaccharides (xylose, glucose) and sucrose to other disaccharides (maltose, melibiose, trehalose). Conversely, when offered a choice between the aphid-specific trisaccharides raffinose and melezitose, Ca. modoc and M. rubra favoured raffinose and melezitose, respectively. Testing the favourite mono-, di- and trisaccharide head-to-head, both ant species favoured sucrose. While both sugar type and sugar concentration are the ultimate cause for consumption by foraging ants, strong recruitment of nest-mates to superior sources is probably the major proximate cause.

The Holocene lake-evaporation history of the afro-alpine Lake Garba Guracha in the Bale Mountains, Ethiopia, based on δ18O records of sugar biomarker and diatoms

In eastern Africa, there are few long, high-quality records of environmental change at high altitudes, inhibiting a broader understanding of regional climate change. We investigated a Holocene lacustrine sediment archive from Lake Garba Guracha, Bale Mountains, Ethiopia, (3,950 m asl), and reconstructed high-altitude lake evaporation history using δ18O records derived from the analysis of compound-specific sugar biomarkers and diatoms. The δ18Odiatom and δ18Ofuc records are clearly correlated and reveal similar ranges (7.9‰ and 7.1‰, respectively). The lowest δ18O values occurred between 10–7 cal ka BP and were followed by a continuous shift towards more positive δ18O values. Due to the aquatic origin of the sugar biomarker and similar trends of δ18Odiatom, we suggest that our lacustrine δ18Ofuc record reflects δ18Olake water. Therefore, without completely excluding the influence of the ‘amount-effect’ and the ‘source-effect’, we interpret our record to reflect primarily the precipitation-to-evaporation ratio (P/E). We conclude that precipitation increased at the beginning of the Holocene, leading to an overflowing lake between ca. 10 and ca. 8 cal ka BP, indicated by low δ18Olake water values, which are interpreted as reduced evaporative enrichment. This is followed by a continuous trend towards drier conditions, indicating at least a seasonally closed lake system.

Optimal Control Applied to Oenological Management of Red Wine Fermentative Macerations

The management of wineries for industrial red winemaking is limited by the capacity and availability of fermentation tanks over the harvest season. The winemakers aim to optimize the wine quality, the fermentative maceration length, and the fermentation tank’s productive cycle simultaneously. Maceration in varietal wine production is carried out until a specific sugar content (digging-out point) is attained, finishing before alcoholic fermentation. Winemakers have found that by trial and error handling of the digging-out point, they can improve the winery capacity and production cost. In this work, we develop an optimal control problem for managing the digging-out point considering two objectives associated with process efficiency and costs. A good compromise between these objectives was found by applying multi-criteria decision-making (MCDM) techniques and the knee point. Two control strategies were compared: free nutrition and traditional nutrition. TOPSIS and LINMAP algorithms were used to choose the most suitable strategy that coincided with the knee point. The preferred option was nitrogen addition only at the beginning of fermentation (6.6–10.6 g/hL of DAP) and a high fermentation temperature (30 °C), yielding the desired digging-out point with a small error (6–9 g/L).

Can Plant Lectins Help to Elucidate Insect Lectin-Mediated Immune Response?

Lectins are carbohydrate-binding proteins that recognize and selectively bind to specific sugar structures. This group of proteins is widespread in plants, animals, and microorganisms, and exerts a broad range of functions. Many plant lectins were identified as exogenous stimuli of vertebrate immunity. Despite being the largest and most diverse taxon on earth, the study of lectins and their functions in insects is lagging behind. In insects, research on lectins and their biological importance has mainly focused on the C-type lectin (CTL) family, limiting our global understanding of the function of insect lectins and their role in insect immunity. In contrast, plant lectins have been well characterized and the immunomodulatory effects of several plant lectins have been documented extensively in vertebrates. This information could complement the missing knowledge on endogenous insect lectins and contribute to understanding of the processes and mechanisms by which lectins participate in insect immunity. This review summarizes existing studies of immune responses stimulated by endogenous or exogenous lectins. Understanding how lectins modulate insect immune responses can provide insight which, in turn, can help to elaborate novel ideas applicable for the protection of beneficial insects and the development of novel pest control strategies.

Cracking the “Sugar Code”: A Snapshot of N- and O-Glycosylation Pathways and Functions in Plants Cells

Glycosylation is a fundamental co-translational and/or post-translational modification process where an attachment of sugars onto either proteins or lipids can alter their biological function, subcellular location and modulate the development and physiology of an organism. Glycosylation is not a template driven process and as such produces a vastly larger array of glycan structures through combinatorial use of enzymes and of repeated common scaffolds and as a consequence it provides a huge expansion of both the proteome and lipidome. While the essential role of N- and O-glycan modifications on mammalian glycoproteins is already well documented, we are just starting to decode their biological functions in plants. Although significant advances have been made in plant glycobiology in the last decades, there are still key challenges impeding progress in the field and, as such, holistic modern high throughput approaches may help to address these conceptual gaps. In this snapshot, we present an update of the most common O- and N-glycan structures present on plant glycoproteins as well as (1) the plant glycosyltransferases (GTs) and glycosyl hydrolases (GHs) responsible for their biosynthesis; (2) a summary of microorganism-derived GHs characterized to cleave specific glycosidic linkages; (3) a summary of the available tools ranging from monoclonal antibodies (mAbs), lectins to chemical probes for the detection of specific sugar moieties within these complex macromolecules; (4) selected examples of N- and O-glycoproteins as well as in their related GTs to illustrate the complexity on their mode of action in plant cell growth and stress responses processes, and finally (5) we present the carbohydrate microarray approach that could revolutionize the way in which unknown plant GTs and GHs are identified and their specificities characterized.

Purification and Characterisation of Lectin Isolated from Nigeria Achatina achatina Snail.

Lectins are carbohydrate-binding proteins that are highly specific for sugar moieties of other molecules. They perform recognition on the cellular and molecular level and play numerous roles in biological recognition phenomena involving cells, carbohydrates, and proteins. Blood groups are inherited characters which give rise to antigen-antibody reaction. A total of 120 samples of local (Nigeria) Achatina achatina snail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used for all the actual tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination tests, Protein Assay and Specific Sugar determinations. The molecular weight was assessed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. The respective haemagglutination tests on the crude, partially and affinity purified lectin showed on standardisation, preferential agglutination with Blood group A type. The Protein contents of the lectin was deduced to be as follows: The crude extract contains 13.5mg/dl, Dialysed precipitate – 5.7mg/dl, Dialysed supernatant – 5.0mg/dl and the Affinity purified Lectin – 0.422mg/dl. Galactose N-acetyl amine (Gal NAc) residue was determined to be its specific sugar. The SDS-PAGE analysis showed the molecular weight of the lectin to be 250 KDaltons. This research has therefore succeeded in the Purification, Characterisation and illustration of the lectinic properties of the local Nigeria snail - Achatina achatina.

Stable Protein Sialylation in Physcomitrella

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

Stable Protein Sialylation in Physcomitrella

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrella (Physcomitrium patens, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, P. patens meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed P. patens. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of six mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for synthesis, activation, transport and transfer of sialic acid. Production of free and (CMP)-activated sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

Workflow for the Quantification of Soluble and Insoluble Carbohydrates in Soybean Seed

Soybean seed composition has a profound impact on its market value and commercial use as an important commodity. Increases in oil and protein content have been historically pursued by breeders and genetic engineers; consequently, rapid methods for their quantification are well established. The interest in complete carbohydrate profiles in mature seeds, on the other hand, has recently increased due to numerous attempts to redirect carbohydrates into oil and protein or to offer specialty seed with a specific sugar profile to meet animal nutritional requirements. In this work, a sequential protocol for quantifying reserve and structural carbohydrates in soybean seed was developed and validated. Through this procedure, the concentrations of soluble sugars, sugar alcohols, starch, hemicellulose, and crystalline cellulose can be determined in successive steps from the same starting material using colorimetric assays, LC–MS/MS, and GC–MS. The entire workflow was evaluated using internal standards to estimate the recovery efficiency. Finally, it was successfully applied to eight soybean genotypes harvested from two locations, and the resulting correlations of carbohydrate and oil or protein are presented. This methodology has the potential not only to guide soybean cultivar optimization processes but also to be expanded to other crops with only slight modifications.