Identification of miRNAs Mediating Seed Storability of Maize during Germination Stage by High-Throughput Sequencing, Transcriptome and Degradome Sequencing.

Seed storability is an important trait for improving grain quality and germplasm conservation, but little is known about the regulatory mechanisms and gene networks involved. MicroRNAs (miRNAs) are small non-coding RNAs regulating the translation and accumulation of their target mRNAs by means of sequence complementarity and have recently emerged as critical regulators of seed germination. Here, we used the germinating embryos of two maize inbred lines with significant differences in seed storability to identify the miRNAs and target genes involved. We identified a total of 218 previously known and 448 novel miRNAs by miRNA sequencing and degradome analysis, of which 27 known and 11 newly predicted miRNAs are differentially expressed in two maize inbred lines, as measured by Gene Ontology (GO) enrichment analysis. We then combined transcriptome sequencing and real-time quantitative polymerase chain reaction (RT-PCR) to screen and confirm six pairs of differentially expressed miRNAs associated with seed storability, along with their negative regulatory target genes. The enrichment analysis suggested that the miRNAs/target gene mediation of seed storability occurs via the ethylene activation signaling pathway, hormone synthesis and signal transduction, as well as plant organ morphogenesis. Our results should help elucidate the mechanisms through which miRNAs are involved in seed storability in maize.

GWAS and RNA-seq analysis uncover candidate genes associated with alkaline stress tolerance in maize (Zea mays L.) seedlings.

Soil salt-alkalization is a common yet critical environmental stress factor for plant growth and development. Discovering and exploiting genes associated with alkaline tolerance in maize (Zea mays L.) is helpful for improving alkaline resistance. Here, an association panel consisting of 200 maize lines was used to identify the genetic loci responsible for alkaline tolerance-related traits in maize seedlings. A total of nine single-nucleotide polymorphisms (SNPs) and their associated candidate genes were found to be significantly associated with alkaline tolerance using a genome-wide association study (GWAS). An additional 200 genes were identified when the screen was extended to include a linkage disequilibrium (LD) decay distance of r2 ≥ 0.2 from the SNPs. RNA-sequencing (RNA-seq) analysis was then conducted to confirm the linkage between the candidate genes and alkali tolerance. From these data, a total of five differentially expressed genes (DEGs; |log2FC| ≥ 0.585, p < 0.05) were verified as the hub genes involved in alkaline tolerance. Subsequently, two candidate genes, Zm00001d038250 and Zm00001d001960, were verified to affect the alkaline tolerance of maize seedlings by qRT-PCR analysis. These genes were putatively involved protein binding and "flavonoid biosynthesis process," respectively, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. Gene promoter region contains elements related to stress and metabolism. The results of this study will help further elucidate the mechanisms of alkaline tolerance in maize, which will provide the groundwork for future breeding projects.

Fabrication and characterization of oil-in-water pickering emulsions stabilized by ZEIN-HTCC nanoparticles as a composite layer.

In this work, the ZEIN-HTCC nanoparticles formed by zein and N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCC) were used as stabilizers to prepare oil-in-water (O/W) Pickering emulsions. The preparation conditions including shearing time, volume fraction of corn oil, mass ratio of ZEIN:HTCC and total concentration of ZEIN-HTCC of emulsions were optimized by measuring the droplet size, zeta potential, PDI and surface tension of emulsions. The ZEIN-HTCC emulsions are stable at the pH range of 4-9 and in the low salt ion concentrations up to 0.2 mol L-1, and can keep stable up to 21 d during low temperature storage. Fourier transform infrared spectroscopy (FTIR), the confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM) were used to analyze the interaction between emulsion components, revealing that zein and HTCC form a composite layer by flocculation to adsorb on the surface of oil droplets, thus preventing emulsion droplets from aggregation. This novel, long-term stable, surfactant-free, and edible zein-based Pickering emulsion could be used as potential carriers for lipophilic nutrients delivery.

Complexation of ß-lactoglobulin with gum arabic: Effect of heat treatment and enhanced encapsulation efficiency.

Heat treatment is widely used in food industry. Proteins and polysaccharides as important natural polymers in food, under heat treatment, the interactions between them could mediate the conformation and functional properties of proteins. Thermally induced ß-lactoglobulin-gum arabic complexes (ß-Lg-GA) were fabricated, and the effect of heat treatment on physicochemical properties of the complexes was systematically investigated. The average particle size of ß-Lg-GA complexes decreased with temperature increased, at 85°C, a smaller size of 273 nm was obtained. A saturated adsorption of GA was found when mass ratio of ß-Lg/GA was <1:2. At pH = 4.2-7.0, electrostatic attraction between ß-Lg and GA was low and a fairly constant turbidity was observed, the formed composite particles had good stability to the pH value. Through UV, fluorescence, and FTIR spectroscopy, it was found that formation of the nanoparticles relied on thermal denaturation and aggregation of protein, the electrostatic, hydrophobic, and hydrogen bonding interactions between ß-Lg and GA were also important. Scanning electron microscope further indicated ß-Lg and GA had good compatibility, and the complexes had a spherical core-shell structure at molecular level. In addition, these prepared natural nanoparticles by heat treatment show significantly higher encapsulation efficiency for (-)-epigallocatechin-3-gallate (EGCG) than that of unheated, thus could be used as a promising carrier for biologically active substances.

Preparation of ß-lactoglobulin/gum arabic complex nanoparticles for encapsulation and controlled release of EGCG in simulated gastrointestinal digestion model.

In this work, the ß-lactoglobulin/gum arabic (ß-Lg-GA) complexes were prepared to encapsulate epigallocatechin gallate (EGCG), forming ß-Lg-GA-EGCG complex nanoparticles with an average particle size of 133 nm. The ß-Lg-GA complexes exhibited excellent encapsulation efficiency (84.5%), and the antioxidant performance of EGCG in vitro was improved after encapsulation. It was recorded that 86% of EGCG could be released in simulated intestinal fluid after 3 h of digestion, much faster than that in simulated gastric fluid, indicating that the ß-Lg-GA complexes were effective in enhancing EGCG stability, which was confirmed using SDS-PAGE and SEM. Further spectrum results demonstrated that various intramolecular interactions including electrostatic, hydrophobic and hydrogen bonding interactions contribute to the formation of ß-Lg-GA-EGCG complex nanoparticles. Also, XRDexperiments indicated that EGCG was successfully encapsulated by ß-Lg-GA complexes. Therefore, the ß-Lg-GA complexes hold great potentials in the protective delivery of sensitive bioactives.

Formation and Characterization of ß-Lactoglobulin and Gum Arabic Complexes: the Role of pH.

Protein-polysaccharide complexes have received increasing attention as delivery systems to improve the stability and bioavailability of multiple bioactive compounds. However, deep and comprehensive understanding of the interactions between proteins and polysaccharides is still required for enhancing their loading efficiency and facilitating targeted delivery. In this study, we fabricated a type of protein-polysaccharide complexes using food-grade materials of ß-lactoglobulin (ß-Lg) and gum arabic (GA). The formation and characteristics of ß-Lg-GA complexes were investigated by determining the influence of pH and other factors on their turbidity, zeta-potential, particle size and rheology. Results demonstrated that the ß-Lg and GA suspension experienced four regimes including co-soluble polymers, soluble complexes, insoluble complexes and co-soluble polymers when the pH ranged from 1.2 to 7 and that ß-Lg-GA complexes formed in large quantities at pH 4.2. An increased ratio of ß-Lg in the mixtures was found to promote the formation of ß-Lg and GA complexes, and the optimal ß-Lg/GA ratio was found to be 2:1. The electrostatic interactions between the NH3+ group in ß-Lg and the COO- group in GA were confirmed to be the main driving forces for the formation of ß-Lg/GA complexes. The formed structure also resulted in enhanced thermal stability and viscosity. These findings provide critical implications for the application of ß-lactoglobulin and gum arabic complexes in food research and industry.

Fabrication of lysozyme/κ-carrageenan complex nanoparticles as a novel carrier to enhance the stability and in vitro release of curcumin.

In this manuscript, lysozyme/κ-carrageenan (LYS-CRG) complexes were prepared and used to encapsulate curcumin. The LYS-CRG complexes demonstrate good encapsulation of curcumin (CUR), and the encapsulation efficiency (EE) and loading capacity (LC) reach 96.2% and 2.31%, respectively. The encapsulated CUR has high antioxidant activity, while the thermal stability and photostability of CUR are also increased. The LYS-CRG complexes could effectively improve the storage stability of CUR and increase its retention rate. In simulated gastric fluid, only 17.91% CUR in the CUR-LYS-CRG complex nanoparticles is released in 3 h, while in the simulated intestinal fluid, the CUR release rate quickly reaches 62.56% in 1.5 h. The release rate tends to be stable within 1.5 h to 3 h and the final release rate reaches 67.23%, suggesting that the formation of CUR-LYS-CRG complex nanoparticles does not affect CUR release in the simulated intestinal fluid.