Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

Role of abscisic acid in modulating drought acclimation, agronomic characteristics and ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP) accumulation in grass pea (Lathyrus sativus L.).

BACKGROUND: ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is a physiological indicator in response to drying soil. However, how abscisic acid (ABA) modulates ß-ODAP accumulation and its related agronomic characteristics in drought stressed grass pea (Lathyrus sativus L.) continue to be unclear. The present study aimed to evaluate the effects of ABA addition on drought tolerance, agronomic characteristics and ß-ODAP content in grass pea under drought stress. RESULTS: Exogenous ABA significantly promoted ABA levels by 19.3% and 18.3% under moderate and severe drought stress, respectively, compared to CK (without ABA, used as control check treatment). ABA addition activated earlier trigger of non-hydraulic root-sourced signal at 69.1% field capacity (FC) (65.5% FC in CK) and accordingly prolonged its operation period to 45.6% FC (49.0% FC in CK). This phenomenon was mechanically associated with the physiological mediation of ABA, where its addition significantly promoted the activities of leaf superoxide dismutase, catalase and peroxidase enzymes and the biosynthesis of leaf proline, simultaneously lowering the accumulation of malondialdehyde and hydrogen peroxide under moderate and severe stresses. Interestingly, ABA application significantly increased seed ß-ODAP content by 21.7% and 21.3% under moderate and severe drought stress, but did not change leaf ß-ODAP content. Furthermore, ABA application produced similar shoot biomass and grain yield as control groups. CONCLUSION: Exogenous ABA improved the drought adaptability of grass pea and promoted the synthesis of ß-ODAP in seeds but not in leaves. Our findings provide novel insights into the agronomic role of ABA in relation to ß-ODAP enrichment in grass pea subjected to drought stress. © 2021 Society of Chemical Industry.

Field phenotyping and quality traits of grass pea genotypes in South Italy.

BACKGROUND: Grass pea (Laithyrus sativus L.) is a rustic plant whose seeds are rich in polyphenols and antioxidants, and it has been consumed as food by human beings since ancient times. This study was conducted in Italy between 2017and 2019 to evaluate, under field conditions, the stability of seed yield, biomass and 1000-seed weight (THS) and to assess the antioxidant composition and activity of 11 grass pea accessions. RESULTS: Analysis of variance revealed significant effects of the environment, accession and accession × environment (A × E) on the yield, above-ground biomass and THS. We found that the environment (year) and A × E explained 52.61% and 23.76% of the total seed yield variation, respectively. No relationship was observed between the yield and the total protein of seeds. Most grass pea accessions showed sensitivity to frost conditions that occurred in the third growing season. The total phenolic content ranged from 50.51 to 112.78 mg 100 g-1 seeds and antioxidant activity ranged from 0.576 to 0.898 mmol Trolox equivalents 100 g-1 seeds and from 0.91 to 1.6 mmol Fe2+  100 g-1 seeds in 2,20-azinobis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power, respectively. Among the accessions, the 'Campi Flegrei' and 'di Castelcività' showed the best performance with the highest yield and stability, phenolic content and superior antioxidant activity. CONCLUSION: The results showed that the yield of grass pea was mainly influenced by different climate conditions. This variability in yield, phenolic content and antioxidant activity among different accessions could help breeders and farmers select high-performance accessions for cultivation. © 2020 Society of Chemical Industry.

Development and physicochemical characterization of a new grass pea (Lathyrus sativus L.) miso.

BACKGROUND: Western consumers interest in Eastern fermented foods has been growing, due to their nutritional and healthy properties. In this study, new sweet misos and salty misos were produced using grass pea (Lathyrus sativus L.) - traditional Portuguese legume from local producers - to promote its consumption and preservation. The evolution of the new misos was evaluated in comparison to traditional miso (made from soybean), through analysis of the chemical composition, colour, texture and linear viscoelastic behaviour. RESULTS: Throughout the fermentation process, the ascorbic acid and phenolic compounds content - with important nutritional value - increased in all misos, mainly in misos produced using grass pea, besides, grass pea sweet miso presented the fastest evolution and darkest colour. The texture parameters (firmness and adhesiveness) of misos decreased over time: grass pea sweet miso showed the highest firmness reduction (51.63 N to 6.52 N) and soybean sweet miso the highest adhesiveness reduction (27.76 N to 3.11 N). Viscoelastic moduli also decreased, reflecting a reduction in the degree of internal structuring for all misos. However, grass pea misos presented more structured internal systems with faster maturation kinetics than soybean misos, for which stabilization started earlier. CONCLUSION: Two innovative misos were developed from grass pea. After 4 months, the texture parameters and viscoelastic moduli for grass pea misos, were similar to the control misos made from soybean, showing that grass pea can be used as a raw material to produce a sustainable miso with potentially healthy properties. © 2020 Society of Chemical Industry.

A convenient and robust protocol for preparation of ODAP-free Lathyrus sativus protein.

The present study aimed to develop a protocol for easy removal of ß-ODAP neurotoxin by converting it into its isomer α-ODAP (reported to be less toxic) followed by its separation from the protein fraction in pH dependent manner. Use of ß-mercaptoethanol prevented aggregate formation and increased solubility of the prepared Lathyrus sativus protein. Validation of ODAP removal by paper chromatography and mass spectrometry indicated the robustness of the protocol. Removal of ODAP and presence of high antioxidants and homoarginine content can enable Lathyrus sativus to be an alternate source of protein, as well as have other health benefits, including benefits for patients with cardiovascular diseases.

The role of Lathyrus sativus flower surface wax in short-range attraction and stimulant for nymph laying by an adult viviparous aphid.

Aphis craccivora Koch (Hemiptera: Aphididae) is an important pest of Lathyrus sativus L. plants, and causes retarded plant growth and loss of seed production. The insect sucks cell sap from flowers and lays nymphs on flowers. Hence, an attempt has been made to observe whether flower surface wax compounds (alkanes and free fatty acids) from two cultivars (BIO L 212 Ratan and Nirmal B-1) of L. sativus could act as short-range attractant and stimulant for nymph laying by adult viviparous females. The n-hexane extracts of flower surface waxes were analyzed by TLC, GC-MS and GC-FID analyses. Twenty one and 22 n-alkanes between n-C12 and n-C36 were detected in BIO L 212 Ratan and Nirmal B-1, respectively; whereas 12 free fatty acids between C12:0 and C22:0 were identified in both cultivars. Pentadecane and tridecanoic acid were predominant n-alkane and free fatty acid, respectively. One flower equivalent surface wax of both cultivars served as short-range attractant and stimulant for nymph laying through Y-tube choice experiments and I-tube viviparity assays, respectively, by adult viviparous females. A synthetic blend of nonacosane, tridecanoic acid and linoleic acid, and a synthetic blend of tetradecane, pentadecane, nonacosane, tridecanoic acid and linoleic acid resembling in amounts as present in one flower equivalent surface wax of BIO L 212 Ratan and Nirmal B-1, respectively, served as short-range attractant and stimulant for nymph laying by adult viviparous females. This study suggests that both these blends could be used in lures in the development of baited traps in pest management programmes.

Sensory properties of yellow pea and macadamia honeys from conventional and flow hive extraction methods.

BACKGROUND: The process of harvesting honey is time consuming and labor intensive. A new system, the Flow Frame, has drastically simplified the harvesting process, enabling honey to be extracted directly from the hive with minimal processing. The sensory profile of honey is influenced, first, by botanical origin and subsequently by processing and storage parameters. A reduction in harvest processing may thus influence the sensory profile of honey harvested from FFs compared to that of honey produced from conventional processing. To test this hypothesis, two monofloral honeys (macadamia and yellow pea) were harvested from FFs, or by conventional honey extraction. Sensory profiling using conventional descriptive analysis was carried out for each floral source with an experienced trained panel. RESULTS: The two monofloral honeys harvested using the FF system had significantly (p < 0.05) higher floral and cleaner aftertaste sensory scores than the honey extracted using commercial (C) methods that involve the use of heat and centrifugation. CONCLUSION: The flow system retains honey's natural sensory properties compared to harvesting methods that require heat and centrifugation. © 2019 Society of Chemical Industry.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low ß-L-ODAP grass pea lines.

BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.

An Improved HILIC HPLC-MS/MS Method for the Determination of ß-ODAP and Its α Isomer in Lathyrus sativus.

ß-N-Oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is a non-protein amino acid present in Lathyrus sativus (grass pea) and other Lathyrus species, in parallel with its nontoxic isomer, α-ODAP. When consuming grass pea for several months as staple food, ß-ODAP may cause neurolathyrism, a motor neuron degeneration syndrome. Therefore, the independent quantification of both ODAP isomers instead of only the total amount in grass pea allows the identification of less toxic varieties and the development of tools to support breeding for improving grass pea quality. In this work, a simple and fast HPLC-MS/MS method was developed without sample derivatization, using a hydrophilic interaction chromatography (HILIC) column and an isocratic gradient of eluents for 18 min, which allowed the determination of both α- and ß-ODAP. The proposed method was fully validated and applied to the determination of α- and ß-ODAP contents in a diverse collection of 107 grass pea accessions representative of the main grass pea-growing geographical regions in the world, with the prompt identification of contrasting accessions. ß-ODAP content in the analyzed grass pea samples ranged from 0.45 ± 0.02 to 6.04 ± 0.45 mg g-1. The moderate correlation found between α- and ß-ODAP contents (0.65) in this collection reinforces the importance of the independent quantification of both ODAP isomers.

Reducing anti-nutritional factor and enhancing yield with advancing time of planting and zinc application in grasspea in Ethiopia.

BACKGROUND: Grasspea (Lathyrus sativus L.) is an important pulse crop for food, feed and sustainable crop production systems in Ethiopia. Despite its advantages in nutrition and adaptability to harsh climate and low fertile soil, it contains a neurotoxin, ß-N-oxalyl-α,ß-diamiono propionic acid (ß-ODAP), which paralyses the lower limbs and is affected by genotypic and agronomic factors. To determine the effect of zinc application and planting date on yield and ß-ODAP content of two genotypes, experiments were conducted in two regions of Ethiopia. RESULTS: The main effects of variety, sowing date and zinc and their interactions were significant (P < 0.001) for ß-ODAP and seed yield, which had a linear relationship with zinc. For the improved grasspea variety, an application of 20 kg ha-1 zinc showed a reduction of ß-ODAP from 0.15% to 0.088% at Debre Zeit and 0.14% to 0.08% at Sheno and increased its yield from 841 kg ha-1 to 2260 kg ha-1 at Debre Zeit and from 715 to 1835 kg ha-1 at Sheno. Early sowing showed a reduction in ODAP content in relation to the late sowing. CONCLUSION: An application of Zn beyond even 20 kg ha-1 with an early sowing is recommended for the improved variety. © 2017 Society of Chemical Industry.

ß-N-Oxalyl-l-α,ß-diaminopropionic Acid (ß-ODAP) Content in Lathyrus sativus: The Integration of Nitrogen and Sulfur Metabolism through ß-Cyanoalanine Synthase.

Grass pea (Lathyrus sativus L.) is an important legume crop grown mainly in South Asia and Sub-Saharan Africa. This underutilized legume can withstand harsh environmental conditions including drought and flooding. During drought-induced famines, this protein-rich legume serves as a food source for poor farmers when other crops fail under harsh environmental conditions; however, its use is limited because of the presence of an endogenous neurotoxic nonprotein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP). Long-term consumption of Lathyrus and ß-ODAP is linked to lathyrism, which is a degenerative motor neuron syndrome. Pharmacological studies indicate that nutritional deficiencies in methionine and cysteine may aggravate the neurotoxicity of ß-ODAP. The biosynthetic pathway leading to the production of ß-ODAP is poorly understood, but is linked to sulfur metabolism. To date, only a limited number of studies have been conducted in grass pea on the sulfur assimilatory enzymes and how these enzymes regulate the biosynthesis of ß-ODAP. Here, we review the current knowledge on the role of sulfur metabolism in grass pea and its contribution to ß-ODAP biosynthesis. Unraveling the fundamental steps and regulation of ß-ODAP biosynthesis in grass pea will be vital for the development of improved varieties of this underutilized legume.

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase.

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, ß-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme.

Attraction of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) to four varieties of Lathyrus sativus L. seed volatiles.

Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) is an important stored grain pest of Lathyrus sativus L. (Leguminosae), commonly known as khesari, in India, Bangladesh and Ethiopia. Volatiles were collected from four varieties, i.e., Bio L 212 Ratan, Nirmal B-1, WBK-14-7 and WBK-13-1 of uninfested khesari seeds, and subsequently identified and quantified by gas chromatography mass spectrometry and gas chromatography flame ionization detector analyses, respectively. A total of 23 volatiles were identified in the four varieties of khesari seeds. In Bio L 212 Ratan and WBK-13-1 seeds, nonanal was the most abundant followed by farnesyl acetone; whereas farnesyl acetone was predominant followed by nonanal in Nirmal B-1 and WBK-14-7 khesari seeds. The olfactory responses of female C. maculatus toward volatile blends from four varieties of khesari seeds, and individual synthetic compounds and their combinations were examined through Y-shaped glass tube olfactometer bioassays. Callosobruchus maculatus showed significant preference for the whole volatile blends from Bio L 212 Ratan seeds compared to whole volatile blends from other three varieties. The insect exhibited attraction to five individual synthetic compounds, 3-octanone, 3-octanol, linalool oxide, 1-octanol and nonanal. A synthetic blend of 448, 390, 1182, 659 and 8114 ng/20 µl methylene chloride of 3-octanone, 3-octanol, linalool oxide, 1-octanol and nonanal, respectively, was most attractive to C. maculatus, and this combination might be used for insect pest management program such as baited traps.

Determination of ß-N-oxalyl-L-α,ß-diaminopropionic acid and homoarginine in Lathyrus sativus and Lathyrus cicera by capillary zone electrophoresis.

BACKGROUND: Lathyrus species as legumes represent an alternative protein source for human and animal nutrition. Heavy consumption of these species can lead to lathyrism, caused by the non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP). Currently, there is no well-defined level below which ß-ODAP is considered non-toxic. In this work, the ß-ODAP content was determined in L. sativus and L. cicera samples to assess their potential toxicity. Homoarginine is another non-protein amino acid found in Lathyrus spp. with interesting implications for human and animal nutrition. RESULTS: The level of ß-ODAP found in these two species ranged from 0.79 to 5.05 mg g(-1). The homoarginine content of the samples ranged from 7.49 to 12.44 mg g(-1). CONCLUSION: This paper describes an accurate, fast and sensitive method of simultaneous detection and quantification of ß-ODAP and homoarginine by capillary zone electrophoresis in L. cicera and L. sativus seeds. Moreover, several methods of extraction were compared to determine the highest performance.

Valorization of traditional foods: nutritional and bioactive properties of Cicer arietinum L. and Lathyrus sativus L. pulses.

BACKGROUND: The use of traditional foods can enrich our diet, perpetuating important elements of local knowledge and cultural inheritance. Raw, soaked and cooked samples of two Fabaceae species (Cicer arietinum L. and Lathyrus sativus L.) were characterized regarding nutritional and bioactive properties. RESULTS: L. sativus gave the highest carbohydrate, protein, ash, saturated fatty acid and polyunsaturated fatty acid content, and lowest fat and energy value. Furthermore, it also showed the highest concentration of flavonoids and antioxidant activity. Cicer arietinum gave the highest concentration of sugars, organic acids and tocopherols. The soaking process did not significantly affect macronutrients, but cooking (boiling) decreased protein, ash, sugars and organic acids, and increased carbohydrates, fat, tocopherols, bioactive compounds and antioxidant activity. No differences were obtained for fatty acid composition. CONCLUSION: The present study highlights the nutritional profile and bioactive properties of these agricultural varieties of C. arietinum and L. sativus pulses, and valorizes their traditional consumption and the use in modern diets.

Effect of different levels of raw and heated grass pea seed (Lathyrus sativus) on nutrient digestibility, intestinal villus morphology and growth performance of broiler chicks.

This study aimed to investigate chemical composition and effect of different levels (0%, 10% and 20%) of raw grass pea (RGP) and heat-treated (120 °C for 30 min) grass pea seed (HGP) on nutrient digestibility, dressing percentage, relative internal organ weights, intestinal villous morphology and broiler chicks' performance. A total number of 200 day-old male chicks were raised under similar condition for 10 days. On day 11, chicks were randomly assigned to five dietary treatments and four replicates of 10 birds each. The result of chemical analysis indicated that Iranian grass pea seed has low levels of total and condensed tannin, and it may be considered as a good source of protein (36.1%) and energy (17.09 kJ GE/g). Heat treatment reduced (p < 0.05) the total and condensed tannin to 21% and 78% respectively. Grass peas seed had higher levels of nitrogen-free extract, P, Na, Mg and Zn than soya bean meal. The apparent digestibility of gross energy and lipid was affected (p < 0.01) by the treatment diets, and it was the lowest after feeding 20% of HGP (p < 0.05). The relative weight of breast and pancreas (p < 0.05) was affected by treatments. Percentage weight of breast and pancreas increased (p < 0.05) after feeding high levels (20%) of RGP and HGP. Substitution of 20% of RGP and HGP increased the duodenal crypt depth (p < 0.05); however, it had no suppressive effect on villus height as the absorptive surface of intestine. The feed conversion ratio was not affected by the treatments in the total experimental period. This study showed that, although the high level of grass pea seed caused a remarkable increase in the relative weight of pancreas and decreased the apparent digestibility of gross energy and lipid, it had beneficial effect on breast relative weight. It seems that heat processing is not effective method for improving quality of Iranian grass pea seed.

Grass pea consumption & present scenario of neurolathyrism in Maharashtra State of India.

BACKGROUND & OBJECTIVES: Neurolathyrism is a non progressive motor neuron disorder engendered by the prolonged over-consumption of Lathyrus sativus (grass pea) seeds which contain a neurotoxic amino acid, ß-N oxalyl- L-α, ß-diaminopropionic acid (ß-ODAP). It is characterized by spastic paraparesis in the hind limbs. The present study was conducted in 105 households (HHs) of Gondia district in Maharashtra, India, where grass pea is cultivated and consumed to assess the health implication of its consumption. METHODS: Across-sectional survey was carried out in 105 HHS in five villages and grass pea samples were collected for ß-ODAP estimation. Amino acid analysis was also done, neurolathyrism cases were identified by snowball sampling method and neurological examination was carried out. RESULTS: The study revealed that 61 per cent of population was consuming this pulse as a part of diet. ß-ODAP concentration in grass pea was high in Bora village (1254.5 ± 528.21 mg %) and less in Malgaon village (413.6±415.79 mg %). The nutritional status of the people was within the normal range (BMI 18± 3.40 kg/m2) in the surveyed households. Consumption of grass pea was observed to be less than 25 g. CONCLUSIONS: The cases of neurolathyrism declined in all the studied villages due to reduced ß-ODAP exposure through Lathyrus sativus consumption, however, the grass pea was cultivated and consumed in Gondia district of Maharashtra State.

Effect of dehulling and germination on the functional properties of grass pea (Lathyrus sativus) flour.

The compositional, bioactive, functional, pasting, and thermal characteristics of native, dehulled, and germinated grass pea flour were examined. Germination significantly improved the protein content and bioactive properties while simultaneously reducing total carbohydrate and fat levels. However, dehulling increased the fat content, foaming, and emulsion properties. Dehulling and germination significantly increased (p < 0.05) the functional properties by improving flowability and cohesiveness. Although processing methods enhance functional properties, the pasting properties of dehulled and germinated flours differ significantly (p < 0.05) from the native flour. The X-ray diffraction patterns indicate a reduction in percentage crystallinity in germinated flours. Overall, the study suggests that the dehulling and germination processes enhanced the quality of grass peas by improving nutritive value and functional attributes.

Analysis of the glycosylation pattern of plant copper amine oxidases by MALDI-TOF/TOF MS coupled to a manual chromatographic separation of glycans and glycopeptides.

The N-glycosylation in pea seedling amine oxidase and lentil seedling amine oxidase was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of pea seedling amine oxidase by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and "trimmed" N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF MS. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a RP using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, e.g. in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.

Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities.

LsSAT2 (serine acetyltransferase in Lathyrus sativus) is the rate-limiting enzyme in biosynthesis of ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), a neuroactive metabolite distributed widely in several plant species including Panax notoginseng, Panax ginseng, and L. sativus. The enzymatic activity of LsSAT2 is post-translationally regulated by its involvement in the cysteine regulatory complex in mitochondria via interaction with ß-CAS (ß-cyanoalanine synthase). In this study, the binding sites of LsSAT2 with the substrate Ser were first determined as Glu290, Arg316, and His317 and the catalytic sites were determined as Asp267, Asp281, and His282 via site-directed/truncated mutagenesis, in vitro enzymatic activity assay, and functional complementation of the SAT-deficient Escherichia coli strain JM39. Furthermore, the C-terminal 10-residue peptide of LsSAT2 is confirmed to be critical to interact with LsCAS, and Ile336 in C10 peptide is the critical amino acid. These results will enhance our understanding of the regulation of LsSAT2 activities and the biosynthesis of ß-ODAP in L. sativus.