Biosynthesis of barley wax ß-diketones: a type-III polyketide synthase condensing two fatty acyl units.

The surface coatings of cereal plants are dominated by waxy ß-diketones crucial for drought resistance and, therefore, grain yield. Here, barley (Hordeum vulgare) wax analyses reveal ß-diketone and associated 2-alkanol ester profiles suggesting a common C16 3-ketoacid precursor. Isotope analysis further shows that the major (C31) diketone is synthesized from two plastidial C16 acyl units. Previous studies identified a gene cluster encoding enzymes responsible for ß-diketone formation in barley, but left their biochemical functions unknown. Various assays now characterize one of these enzymes as a thioesterase producing long-chain (mainly C16) 3-ketoacids, and another one as a polyketide synthase (PKS) condensing the 3-ketoacids with long-chain (mainly C16) acyl-CoAs into ß-diketones. The two enzymes are localized to the plastids and Endoplasmic Reticulum (ER), respectively, implying substrate transfer between these two sub-cellular compartments. Overall, our findings define a two-step pathway involving an unprecedented PKS reaction leading directly to the ß-diketone products.

Redundant CAMTA Transcription Factors Negatively Regulate the Biosynthesis of Salicylic Acid and N-Hydroxypipecolic Acid by Modulating the Expression of SARD1 and CBP60g.

Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. CALMODULIN-BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2, and CAMTA3 are known redundant negative regulators of plant immunity, but the underlying mechanism also remains largely unknown. In this study, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3, which also negatively regulates the expression of SARD1, presumably via an indirect effect. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two single mutants defective in NHP biosynthesis. Interestingly, a suppressor screen conducted in the camta1/2/3 triple mutant background yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response.

Detection of meat species adulteration using high-resolution mass spectrometry and a proteogenomics strategy.

Due to the internationalisation of food production and distribution, there has been a significant increase of food fraud in recent years. Food fraud can have serious health implications, and it occurs when food manufacturers implement unethical practices such as making false label claims as well as using additives and fillers within their products to increase profitability. This has been a serious concern. Meat adulteration was examined using a well-defined proteogenomic annotation, carefully selected surrogate tryptic peptides and high-resolution mass spectrometry. Selected mammalian meat samples were homogenised and the proteins extracted and digested with trypsin. Chromatography was achieved using a 30-min linear gradient along with a BioBasic C8 100 × 1 mm column at a flow rate of 75 µl min-1. The mass spectrometer was operated in full-scan high-resolution and accurate mass using resolving powers of 140,000 and 17,500 (FWHM) in full-scan MS and MS/MS respectively. Data independent acquisition (DIA) mode was used including 12 DIA MS/MS scans to cover the mass range 600-1200 m/z. Methodically in silico analyses of myoglobin, myosin-1, myosin-2 and ß-haemoglobin sequences allow for the identification of a species-specific tryptic peptide mass lists and theoretical MS/MS spectra. Following comprehensive MS, MS/MS or DIA analyses, the method was capable of the detection and identification of very specific tryptic peptides for all four targeted proteins for each animal species tested with observed m/z below 3 ppm compared with the theoretical m/z. The analyses were successfully performed with raw and cooked meat. Specifically, the method was capable of detecting 1% (w/w) of pork or horse meat in a mixture before and after cooking (71°C internal temperature).

Assessment of meat authenticity using bioinformatics, targeted peptide biomarkers and high-resolution mass spectrometry.

In recent years a significant increase of food fraud has been observed, ranging from false label claims to the use of additives and fillers to increase profitability. Recently in 2013 horse and pig DNAs were detected in beef products sold from several retailers. Mass spectrometry (MS) has become the workhorse in protein research, and the detection of marker proteins could serve for both animal species and tissue authentication. Meat species authenticity is performed in this paper using a well-defined proteogenomic annotation, carefully chosen surrogate tryptic peptides and analysis using a hybrid quadrupole-Orbitrap MS. Selected mammalian meat samples were homogenised and proteins were extracted and digested with trypsin. The samples were analysed using a high-resolution MS. Chromatography was achieved using a 30-min linear gradient along with a BioBasic C8 100 × 1 mm column at a flow rate of 75 µl min(-1). The MS was operated in full-scan high resolution and accurate mass. MS/MS spectra were collected for selected proteotypic peptides. Muscular proteins were methodically analysed in silico in order to generate tryptic peptide mass lists and theoretical MS/MS spectra. Following a comprehensive bottom-up proteomic analysis, we detected and identified a proteotypic myoglobin tryptic peptide (120-134) for each species with observed m/z below 1.3 ppm compared with theoretical values. Moreover, proteotypic peptides from myosin-1, myosin-2 and ß-haemoglobin were also identified. This targeted method allowed comprehensive meat speciation down to 1% (w/w) of undesired product.