Domestication has altered gene expression and secondary metabolites in pea seed coat.

The mature seed in legumes consists of an embryo and seed coat. In contrast to knowledge about the embryo, we know relatively little about the seed coat. We analyzed the gene expression during seed development using a panel of cultivated and wild pea genotypes. Gene co-expression analysis identified gene modules related to seed development, dormancy, and domestication. Oxidoreductase genes were found to be important components of developmental and domestication processes. Proteomic and metabolomic analysis revealed that domestication favored proteins involved in photosynthesis and protein metabolism at the expense of seed defense. Seed coats of wild peas were rich in cell wall-bound metabolites and the protective compounds predominated in their seed coats. Altogether, we have shown that domestication altered pea seed development and modified (mostly reduced) the transcripts along with the protein and metabolite composition of the seed coat, especially the content of the compounds involved in defense. We investigated dynamic profiles of selected identified phenolic and flavonoid metabolites across seed development. These compounds usually deteriorated the palatability and processing of the seeds. Our findings further provide resources to study secondary metabolism and strategies for improving the quality of legume seeds which comprise an important part of the human protein diet.

Separation of Isomeric Tau Phosphopeptides from Alzheimer's Disease Brain by Cyclic Ion Mobility Mass Spectrometry.

Alzheimer's disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility-mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau-TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions (m/z 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at m/z 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.

The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication.

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy. We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas. The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity. Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.

Combination of electronically driven micromanipulation with laser desorption ionization mass spectrometry - The unique tool for analysis of seed coat layers and revealing the mystery of seed dormancy.

Electronically driven micromanipulation (EDM) with microscopic control was used as a novel tool for sample preparation prior to direct (matrix assisted) laser desorption/ionization mass spectrometric ((MA)LDI-MS) analysis of mature pea seed coat composition in defined layers. Microscissors were used for seed coat fragment shape adjustment, microtweezers for sample holding and "microjackhammer" Milling Pro for precise mechanical removing of cell layers in defined depths (2, 5 or 10 µm). These procedures circumvent the application of embedding media or enzymatic digestion of seed coat that would complicate mass spectra interpretation (presence of matrix signals, analyte signals enhancement or attenuation) and represent alternative for 3D metabolites profiling. In addition, microinjector was used to apply a solution on intact or micropeeled seed coat surface in nano-volumes, i.e. MALDI matrix and/or lithium salt, that provide improvement of signal of sugars. Utilization of EDM enabled optimization of matrix composition on a single small fragment of seed coat overcoming thus problems with biological (seed to seed) variability. LDI-MS data were studied by multivariate statistical analysis and significant metabolites in particular layers of seed coats were identified. Normalized intensities of signals (NS) of long-chain hydroxylated fatty acids (HLFA) on intact dormant pea genotype (JI64) seed coats were significantly higher than in their counterparts treated by micropeeling confirming HLFA accumulation in outermost layers (cutin). Fatty acids distribution differences between dormant and non-dormant genotypes were explored in detail. On the other hand, NS of sugar chains and particular polyphenols were significantly higher in micropeeled seed coats of studied dormant and non-dormant genotypes than in intact seed coats. Furthermore, combination of EDM with mass spectrometry imaging (MSI) allowed vertical profiling of metabolites in hilum (a place of former attachment of seed to maternal plant) and comparison of its composition with surrounding tissues. The obtained results contribute to the understanding of relations between seed coat chemical composition and physical seed dormancy.