Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Effect of Chitosan Nanoparticles as Edible Coating on the Storability and Quality of Apricot Fruits

Apricots are a fragile fruit that rots quickly after harvest. Therefore, they have a short shelf-life. The purpose of this work is to determine the effect of coatings containing chitosan (CH) as well as its nanoparticles (CHNPs) as thin films on the quality and shelf-life of apricots stored at room (25 ± 3 °C) and cold (5 ± 1 °C) temperatures. The physical, chemical, and sensorial changes that occurred during storage were assessed, and the shelf-life was estimated. Transmission electron microscopy was used to examine the size and shape of the nanoparticle. The nanoparticles had a spherical shape with an average diameter of 16.4 nm. During the storage of the apricots, those treated with CHNPs showed an obvious decrease in weight loss, decay percent, total soluble solids, and lipid peroxidation, whereas total acidity, ascorbic acid, and carotenoid content were higher than those in the fruits treated with CH and the untreated fruits (control). The findings of the sensory evaluation revealed a significant difference in the overall acceptability scores between the samples treated with CHNPs and the other samples. Finally, it was found that CHNP coatings improved the qualitative features of the apricots and extended their shelf-life for up to 9 days at room temperature storage and for 30 days in cold storage

Bee Pollen as a Functional Product – Chemical Constituents and Nutritional Properties

Nutritional supplements play a role in promoting human protein synthesis, fitness recovery, and mental health protection. Pollen is considered a natural food with excellent nutritional value. The use of bee-gathered pollen as a nutritional supplement is now widespread around the world, and it is appreciated for its healing qualities. However, depending on the floral species and the region of origin, pollen has a wide range of nutritional value. It was found that clover pollen had the highest protein content (31.4 g/100 g DM), while the lowest content was observed in maize pollen (21.3%). Eucalyptus pollen had the highest lipid amount (9.49 g/100 g DM), while clover pollen had the lowest content (7.46 g/100 g). Phytochemical analysis showed that eucalyptus pollen occupied the highest total antioxidant activity (67.02%), followed by clover (58.25%) then maize (52.18%), whereas clover pollen had the highest phenolic content (1165 mg GAE/100 gm), compared with the other pollen varieties (949.4 to 1073 mg-GAE/100 gm). The contents of branched-chain amino acids were 29, 33.3, and 38.4 mg/g for maize, eucalyptus, and clover respectively, representing 17.87, 18.44, and 16.53% of total amino acids. The contents of total essential amino acids (EAAs) were 61.8, 73.0, and 83.5 mg/g for maize, eucalyptus, and clover pollen, respectively. Only eucalyptus pollen ultimately met the minimum requirements of EAAs for adults. In contrast, the other two types of pollen contained at least 3–4 limiting amounts of the essential amino acids leucine, lysine, phenylalanine, and valine. In conclusion, pollen is a good, affordable source of nutrients that can be utilized as beneficial dietary supplements for human health