Lupin seeds storage protein composition and their interactions with native flavonoids.

BackgroundThe late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties.ResultsPrevious research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins.ConclusionsThere is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones, via a role in DNA-binding. In general, non-globular and low-complexity proteins, such as the LEA proteins, pose particular challenges in determining their functions and modes of action. Rather than masking off and ignoring low-complexity domains, novel tools and tool combinations are needed which are capable of analysing such proteins in their entirety.

In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4g H2O g DW–1. Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds.

Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by eleven-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase, from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of citrate synthase during desiccation, but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy (TFE). This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.

The oil samples were rich in linoleic acid (mean: 45.39%), and then, the most common fatty acids were oleic (mean: 36.79%), palmitic (mean: 8.27%), and stearic acids (mea:n 5.61%). Calcium 463.80–4065.50 mg/kg, boron 0.00–12.26 mg/kg, chromium 0.00–10.25 mg/kg, copper 3.92–44.48 mg/kg, iron 0.00–149.23 mg/kg, magnesium 34.07–2002.53 mg/kg, potassium 2,463.21–6779.90 mg/kg, and sodium 91.69–8532.82 mg/kg were determined in the chips samples. Glycemic index values of the chips made using whole lupine flour varied between 50.10 and 60.90. In addition to this, the glycemic index values of the whole lupine flour added chips were found lower than the glycemic index values of hulled lupine flour added chips. It was observed that the glycemic index values of the lupine flour added samples were lower than the control group (without lupine flour added). The lowest glycemic index value was found in the sample with the highest ratio of lupine flour. Practical applications Lupine flour is an ideal food component because it has a high content of protein, dietary fiber and bioactive compound, and low lipid and starch content. It has been clearly demonstrated that lupine has a high potential as a food component and has increased its importance in the food industry. Lupine is one of the new protein sources of recent time and is more economical than animal protein sources. The fat content of the lupine seed is generally 10% saturated and 90% polyunsaturated fatty acids. Lupine does not contain gluten and gluten-like proteins and has become an alternative raw material for gluten-free products used in the diets of celiac patients.

In late embryogenesis, the water content of living cell is reduced tremendously that leads to a state of dehydration and thus, might impose severe irreparable damage to cellular and macromolecular structures. However, the mature orthodox seeds can withstand severe desiccation due to role of osmoprotectants viz., reducing sugars, prolines, glycinebetaines or Late Embryogenesis Abundant (LEA) proteins. These operate on the virtue of intrinsic molecular mechanisms that alleviate multiple abiotic stresses in plants such as protein desiccation, membrane degradation, salt stress and cold and chilling stress. The LEA proteins are a group of versatile, adaptive, hydrophilic proteins considerably defined as ‘molecular shields’ for their anti-stress properties attributable to partial or complete structural randomness. On the basis of their amino acid composition and sequencing, LEA proteins have been clubbed into seven groups that are further sub divided into a number of protein sub families. Out of these, Group 2 LEA proteins called the ‘Dehydrins’ are of prime importance in the plant kingdom. The latent and unique stress remediating characteristics of this class of proteins has been further enhanced by transgenic studies, wherein the target LEA genes have been identified, sequenced to understand their molecular role in plants. Further investigations into the behavior of LEA proteins and mode of their regulation in stressed plants will facilitate in elucidating the function of LEA proteins. The present chapter reviews the versatility and role of LEA proteins in plant stress protection.

Group II late embryogenesis abundant (LEA) proteins are crucial phytomolecules which accumulate mainly in the late phases of seed development and also in the vegetative tissues in response to exogenous stress. In spite of considerable research, their mechanism of action to generate plant tolerance against abiotic stresses still remains obscure. The present review focuses on the varied structural aspects which ultimately dictate the multifarious functions of the Group II LEA proteins when the plants are exposed to intense desiccation. Currently, several reports have been documented regarding newer in silico approaches in predicting LEA protein structure and the corresponding cis-elements. Coupled to recent transgenic approaches, these reports need to be properly structured to further characterize the physico-chemical and functional importance of LEA proteins in regulating tolerance against multiple abiotic stresses.

Late embryogenesis abundant (LEA) proteins include eight multigene families that are expressed in response to water loss during seed maturation and in vegetative tissues of desiccation tolerant species. To elucidate LEA proteins evolution and diversification, we performed a comprehensive synteny and phylogenetic analyses of the eight gene families across 60 complete plant genomes. Our integrated comparative genomic approach revealed that synteny conservation and diversification contributed to LEA family expansion and functional diversification in plants. We provide examples that: 1) the genomic diversification of the Dehydrin family contributed to differential evolution of amino acid sequences, protein biochemical properties, and gene expression patterns, and led to the appearance of a novel functional motif in angiosperms; 2) ancient genomic diversification contributed to the evolution of distinct intrinsically disordered regions of LEA_1 proteins; 3) recurrent tandem-duplications contributed to the large expansion of LEA_2; and 4) dynamic synteny diversification played a role on the evolution of LEA_4 and its function on plant desiccation tolerance. Taken together, these results show that multiple evolutionary mechanisms have not only led to genomic diversification but also to structural and functional plasticity among LEA proteins which have jointly contributed to the adaptation of plants to water-limiting environments.

Late embryogenesis abundant (LEA) proteins are small, hydrophilic, and largely unstructured proteins that are synthesized in seeds during late maturation and in vegetative tissues in response to abiotic stress. They have been associated with dehydration and desiccation tolerance in many plants, animal, and bacteria, and are documented to protect plants against desiccation and abiotic stress. In this study we characterize three Brassica napus LEA promoters; LEA 411 (Group1), ECP 66, and ECP248 from group 3. All three showed accumulation during seed development as well as in vegetative tissue in response to salt, drought, and cold stress. We show that the LEA proteins are being expressed as early as 10 days after flowering in seeds. The response to drought and salt stress occurred as quickly as 3 hours after stress treatment while that to cold took longer to detect. Promoter analysis revealed ABRE, DRE/CRT, DRE2, MYB and MYC motifs.

Late embryogenesis abundant (LEA) proteins are hydrophilic molecules that are believed to function in desiccation and low-temperature tolerance in some plants and plant propagules, certain prokaryotes, and several animal species. The brine shrimp Artemia franciscana can produce encysted embryos (cysts) that enter diapause and are resistant to severe desiccation. This ability is based on biochemical adaptations, one of which appears to be the accumulation of the LEA protein that is the focus of this study. The studies described herein characterize a 21 kDa protein in encysted Artemia embryos as a group 1 LEA protein. The amino acid sequence of this protein and its gene have been determined and entered into the NCBI database (no. EF656614). The LEA protein consists of 182 amino acids and it is extremely hydrophilic, with glycine (23%), glutamine (17%), and glutamic acid (12.6%) being the most abundant amino acids. This protein also consists of 8 tandem repeats of a 20 amino acid sequence, which is characteristic of group 1 LEA proteins from non-animal species. The LEA protein and its gene are expressed only in encysted embryos and not in larvae or adults. Evidence is presented to show that the LEA protein functions in the prevention of drying-induced protein aggregation, which supports its functional role in desiccation tolerance. This report describes, for the first time, the purification and characterization of a group 1 LEA protein from an animal species.

Late embryogenesis abundant (LEA) proteins are a family of hyper-hydrophilic proteins that accumulate in response to cellular dehydration. Originally identified as plant proteins associated with seed desiccation tolerance, LEA proteins have been identified in a wide range of organisms such as invertebrates and microorganisms. LEA proteins are thought to protect proteins and biomembranes under water-deficit conditions. Here, we characterized WCI16, a wheat (Triticum aestivum) protein that belongs to a class of plant proteins of unknown function, and provide evidence that WCI16 shares common features with LEA proteins. WCI16 was induced during cold acclimation in winter wheat. Based on its amino acid sequence, WCI16 is highly hydrophilic, like LEA proteins, despite having no significant sequence similarity to any of the known classes of LEA proteins. Recombinant WCI16 protein was soluble after boiling, and (1)H-nuclear magnetic resonance (NMR) spectroscopy revealed that the structure of WCI16 is random and has no hydrophobic regions. WCI16 exhibited in vitro cryoprotection of the freeze-labile enzyme l-lactate dehydrogenase as well as double-stranded DNA binding activity, suggesting that WCI16 may protect both proteins and DNA during environmental stresses. The biological relevance of these activities was supported by the subcellular localization of a green fluorescent protein (GFP)-fused WCI16 protein in the nucleus and cytoplasm. Heterologous expression of WCI16 in Arabidopsis (Arabidopsis thaliana) plants conferred enhanced freezing tolerance. Taken together, our results indicate that WCI16 represents a novel class of LEA proteins and is involved in freezing tolerance.

Functional synergy between disordered proteins and sugars in desiccation protection.

A comparative study on the bioavailability of phenolic compounds from organic and nonorganic red grapes

Late embryogenesis abundant (LEA) proteins comprise a diverse family whose members play a key role in abiotic stress tolerance. As intrinsically disordered proteins, LEA proteins are highly hydrophilic and inherently stress tolerant. They have been shown to stabilise multiple client proteins under a variety of stresses, but current hypotheses do not fully explain how such broad range stabilisation is achieved. Here, using neutron reflection and surface tension experiments, we examine in detail the mechanism by which model LEA proteins, AavLEA1 and ERD10, protect the enzyme citrate synthase (CS) from aggregation during freeze–thaw. We find that a major contributing factor to CS aggregation is the formation of air bubbles during the freeze–thaw process. This greatly increases the air–water interfacial area, which is known to be detrimental to folded protein stability. Both model LEA proteins preferentially adsorb to this interface and compete with CS, thereby reducing surface-induced aggregation. This novel surface activity provides a general mechanism by which diverse members of the LEA protein family might function to provide aggregation protection that is not specific to the client protein.

Late embryogenesis abundant (LEA) proteins are found in desiccation-tolerant species from all domains of life. Despite several decades of investigation, the molecular mechanisms by which LEA proteins confer desiccation tolerance are still unclear. In this study, dielectrophoresis (DEP) was used to determine the electrical properties of Drosophila melanogaster (Kc167) cells ectopically expressing LEA proteins from the anhydrobiotic brine shrimp, Artemia franciscana. Dielectrophoresis-based characterization data demonstrate that the expression of two different LEA proteins, AfrLEA3m and AfrLEA6, increases cytoplasmic conductivity of Kc167 cells to a similar extent above control values. The impact on cytoplasmic conductivity was surprising, given that the concentration of cytoplasmic ions is much higher than the concentrations of ectopically expressed proteins. The DEP data also supported previously reported data suggesting that AfrLEA3m can interact directly with membranes during water stress. This hypothesis was strengthened using scanning electron microscopy, where cells expressing AfrLEA3m were found to retain more circular morphology during desiccation, while control cells exhibited a larger variety of shapes in the desiccated state. These data demonstrate that DEP can be a powerful tool to investigate the role of LEA proteins in desiccation tolerance and may allow to characterize protein-membrane interactions in vivo, when direct observations are challenging.

The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli