Autofluorescence-Based Investigation of Spatial Distribution of Phenolic Compounds in Soybeans Using Confocal Laser Microscopy and a High-Resolution Mass Spectrometric Approach.

In this research, we present a detailed comparative analysis of the bioactive substances of soybean varieties k-11538 (Russia), k-11559 (Russia), k-569 (China), k-5367 (China), k-5373 (China), k-5586 (Sweden), and Primorskaya-86 (Russia) using an LSM 800 confocal laser microscope and an amaZon ion trap SL mass spectrometer. Laser microscopy made it possible to clarify in detail the spatial arrangement of the polyphenolic content of soybeans. Our results revealed that the phenolics of soybean are spatially located mainly in the seed coat and the outer layer of the cotyledon. High-performance liquid chromatography (HPLC) was used in combination with an amaZon SL BRUKER DALTONIKS ion trap (tandem mass spectrometry) to identify target analytes in soybean extracts. The results of initial studies revealed the presence of 63 compounds, and 45 of the target analytes were identified as polyphenolic compounds.

The Content of Anthocyanins in Cowpea (Vigna unguiculata (L.) Walp.) Seeds and Contribution of the MYB Gene Cluster to Their Coloration Pattern.

The intensively pigmented legumes belonging to Phaseolus and Vigna spp. are valued as an essential component of healthy nutrition due to their high content of flavonoids. In this context, we used the accessions of Vigna unguiculata with different colors of seed coats from the N.I. Vavilov All-Russian Institute of Plant Genetic Resources collection as the main object of this research. We applied confocal laser scanning microscopy, biochemical analysis, and wide in silico and molecular genetic analyses to study the main candidate genes for anthocyanin pigmentation within the MYB cluster on chromosome 5. We performed statistical data processing. The anthocyanin content ranged from 2.96 mg/100 g DW in reddish-brown-seeded cowpea accessions to 175.16 mg/100 g DW in black-seeded ones. Laser microscopy showed that the autofluorescence in cowpea seeds was mainly caused by phenolic compounds. The maximum fluorescence was observed in the seed coat, while its dark color, due to the highest level of red fluorescence, pointed to the presence of anthocyanins and anthocyanidins. Genes of the MYB cluster on chromosome 5 demonstrated a high homology and were segregated into a separate clade. However, amplification products were not obtained for all genes because of the truncation of some genes. Statistical analysis showed a clear correlation between the high content of anthocyanins in cowpea seeds and the presence of PCR products with primers Vigun05g0393-300-1.

Identification and Spatial Distribution of Bioactive Compounds in Seeds Vigna unguiculata (L.) Walp. by Laser Microscopy and Tandem Mass Spectrometry.

The research presents a comparative metabolomic study of extracts of Vigna unguiculata seed samples from the collection of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources. Analyzed samples related to different areas of use in agricultural production, belonging to different cultivar groups sesquipedalis (vegetable accessions) and unguiculata (grain accessions). Metabolome analysis was performed by liquid chromatography combined with ion trap mass spectrometry. Substances were localized in seeds using confocal and laser microscopy. As a result, 49 bioactive compounds were identified: flavonols, flavones, flavan-3-ols, anthocyanidin, phenolic acids, amino acids, monocarboxylic acids, aminobenzoic acids, fatty acids, lignans, carotenoid, sapogenins, steroids, etc. Steroidal alkaloids were identified in V. unguiculata seeds for the first time. The seed coat (palisade epidermis and parenchyma) is the richest in phenolic compounds. Comparison of seeds of varieties of different directions of use in terms of the number of bioactive substances identified revealed a significant superiority of vegetable accessions over grain ones in this indicator, 36 compounds were found in samples from cultivar group sesquipedalis, and 24 in unguiculata. The greatest variety of bioactive compounds was found in the vegetable accession k-640 from China.