β-N-Oxalyl-L-α,β-diaminopropionic acid (β-ODAP), found in Lathyrus sativus at first, causes a neurological disease, lathyrism, when over ingested in an unbalanced diet. Our previous research suggested that β-ODAP biosynthesis is related to sulfur metabolism. In this study, β-cyanoalanine synthase (β-CAS) was confirmed to be responsible for β-ODAP biosynthesis via in vitro enzymatic analysis. LsCAS was found to be pyridoxal phosphate (PLP)-dependent via spectroscopic analysis and dual functional via enzymatic activity analysis. Generation of a M135T/M235S/S239T triple mutant of LsCAS, which are the key sites to control the ratio of CAS/cysteine synthase (CS) activity, switches reaction chemistry to that of a CS. LsCAS interactions were further screened and verified via Y2H, BiFC and pull-down assay. It was suggested that LsSAT2 interacts and forms a cysteine regulatory complex (CRC) with LsCAS in mitochondria, which improves LsSAT while reduces LsCAS activities to affect β-ODAP content positively. These results provide new insights into the molecular regulation of β-ODAP content in L. sativus.

Diversity of regional yeast can be influenced by geography, grape cultivars and the use of SO2, but at single vineyard scale in China, the impact of these factors on yeast population, particularly Saccharomyces cerevisiae, is not well studied. Here, we characterised yeast species and dynamics during spontaneous fermentations with/without SO2 using eight typical grape cultivars from Yuma vineyard in Ningxia wine region of China. Results show that distribution and abundance of yeast species varied by grape varieties, fermentation stage and SO2 treatment. A number of 290 S. cerevisiae isolates were further classified into 33 genotypes by Interdelta fingerprinting. A prevailing role of grape varieties in shaping the genetic divergence of S. cerevisiae in Yuma vineyard was observed, as compared to the impacts of fermentation stage and SO2 treatment. Pre-selected S. cerevisiae strains were subjected to vinification with Cabernet Sauvignon and Chardonnay. All strains completed fermentations but the physiochemical parameters and volatile profiles of wines were strain-specific. Some indigenous S. cerevisiae yielded more desirable aroma compounds compared to the commercial strains, among which NX16 and NX18 outcompeted others, therefore having potential for use as starters. This study provides comprehensive analysis on yeast diversity at vineyard scale in Ningxia. Information on the vinification using indigenous S. cerevisiae is of great value for improving Ningxia wine regionality.

No abstract

Grass pea, a protein-rich, high-yielding, and drought-tolerant legume, is used as food and livestock feed in several tropical and subtropical regions of the world. The abundant seed proteins of grass pea are salt-soluble globulins, which can be separated into vicilins and legumins. In many other legumes, the members of vicilin seed proteins have been identified as major allergens. However, very little information is available on the allergens of grass pea. In this study, we have identified an abundant 47 kDa protein from grass pea, which was recognized by immunoglobulin E (IgE) antibodies from sera drawn from several peanut-allergic patients. The IgE-binding 47 kDa protein was partially purified by affinity chromatography on a Con-A sepharose column. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of the 47 kDa grass pea protein revealed sequence homology to 47 kDa vicilin from pea and Len c 1 from lentil. Interestingly the grass pea vicilin was found to be susceptible to pepsin digestion in vitro. We have also isolated a cDNA encoding the grass pea 47 kDa vicilin (β-lathyrin), and the deduced amino acid sequence revealed extensive homology to several known allergens, including those from peanut and soybean. A homology model structure of the grass pea β-lathyrin, generated using the X-ray crystal structure of the soybean β-conglycinin β subunit as a template, revealed potential IgE-binding epitopes located on the surface of the molecule. The similarity in the three-dimensional structure and the conservation of the antigenic epitopes on the molecular surface of vicilin allergens explains the IgE-binding cross-reactivity.

LsSAT2 (serine acetyltransferase in Lathyrus sativus) is the rate-limiting enzyme in biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (β-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 β-CAS (β-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 β-ODAP in L. sativus.