BrBCAT1 mutation resulted in deficiency of epicuticular wax crystal in Chinese cabbage.

KEY MESSAGE: BrBCAT1 encoding a branched-chain amino acid aminotransferase was responsible for the glossy trait, which was verified by allelic mutants in Chinese cabbage. The glossy characteristic, thanks to the epicuticular wax crystal deficiency, is an excellent commodity character for leafy vegetables. Herein, two allelic glossy green mutants, wdm11 and wdm12, were isolated from an ethyl methane sulfonate (EMS)-mutagenized population of Chinese cabbage, and the mutant phenotype was recessive inherited. Cryo-SEM detected that epicuticular wax crystal in the mutant leaves was virtually absent. MutMap and Kompetitive allele-specific PCR analyses demonstrated that BraA06g006950.3C (BrBCAT1), homologous to AtBCAT1, encoding a branched-chain amino acid aminotransferase was the candidate gene. A SNP (G to A) on the fourth exon of BrBCAT1 in wdm11 caused the 233rd amino acid to change from glycine (G) to aspartic acid (D). A SNP (G to A) on the second exon of BrBCAT1 in wdm12 led to the 112th amino acid change from glycine (G) to arginine (R). Both of the allelic mutants had genetic structural variation in the candidate gene, which indicated that the mutant phenotype was triggered by the BrBCAT1 mutation. The expression levels of BrBCAT1 and genes related to fatty acid chain extension were decreased significantly in the mutant compared to the wild-type, which might result in epicuticular wax crystal deficiency in the mutants. Our findings proved that the mutation of BrBCAT1 induced the glossy phenotype and provided a valuable gene resource for commodity character improvement in Chinese cabbage.

Microencapsulation of Essential Oils Using Faba Bean Protein and Chia Seed Polysaccharides via Complex Coacervation Method.

The aim of this study was to develop microcapsules containing juniper or black pepper essential oils, using a combination of faba bean protein and chia seed polysaccharides (in ratios of 1:1, 1:2, 2:1). By synergizing these two polymers, our goal was to enhance the efficiency of essential oil microencapsulation, opening up various applications in the food industry. Additionally, we aimed to investigate the influence of different polymer mixing ratios on the properties of the resulting microcapsules and the course of the complex coacervation process. To dissolve the essential oils and limit their evaporation, soybean and rapeseed oils were used. The powders resulting from the freeze-drying of coacervates underwent testing to assess microencapsulation efficiency (65.64-87.85%), density, flowability, water content, solubility, and hygroscopicity. Additionally, FT-IR and DSC analyses were conducted. FT-IR analysis confirmed the interactions between the components of the microcapsules, and these interactions were reflected in their high thermal resistance, especially at a protein-to-polysaccharide ratio of 2:1 (177.2 °C). The water content in the obtained powders was low (3.72-7.65%), but it contributed to their hygroscopicity (40.40-76.98%).

Effect of dual modifications with ultrasonication and succinylation on Cicer arietinum protein-iron complexes: Characterization, digestibility, in-vitro cellular mineral uptake and preparation of fortified smoothie.

The research aimed to evaluate the effect of ultrasonication and succinylation on the functional, iron binding, physiochemical, and cellular mineral uptake efficacy of chickpea protein concentrate. Succinylation resulted in significant improvements in the water-holding capacity (WHC) (25.47 %), oil-holding capacity (OHC) (31.38 %), and solubility (5.80 %) of the chickpea protein-iron complex. Mineral bioavailability significantly increased by 4.41 %, and there was a significant increase in cellular mineral uptake (64.64 %), retention (36.68 %), and transport (27.96 %). The ferritin content of the succinylated chickpea protein-iron complex showed a substantial increase of 66.31%. Furthermore, the dual modification approach combining ultrasonication and succinylation reduced the particle size of the protein-iron complex with a substantial reduction of 83.25 %. It also resulted in a significant enhancement of 51.5 % in the SH (sulfhydryl) content and 48.92 % in the surface hydrophobicity. Mineral bioavailability and cellular mineral uptake, retention, and transport were further enhanced through dual modification. In terms of application, the addition of single and dual-modified chickpea protein-iron complex to a fruit-based smoothie demonstrated positive acceptance in sensory attributes. Overall, the combined approach of succinylation and ultrasonication to the chickpea protein-iron complex shows a promising strategy for enhancing the physiochemical and techno-functional characteristics, cellular mineral uptake, and the development of vegan food products.

Formation mechanism of quinoa protein hydrolysate-EGCG complexes at different pH conditions and its effect on the protein hydrolysate-lipid co-oxidation in emulsions.

This study aimed to investigate the interaction, structure, antioxidant, and emulsification properties of quinoa protein hydrolysate (QPH) complexes formed with (-)-epigallocatechin gallate (EGCG) at pH 3.0 and 7.0. Additionally, the effect of pH conditions and EGCG complexation on protein hydrolysate-lipid co-oxidation in QPH emulsions was explored. The results indicated that QPH primarily interacted with EGCG through hydrophobic interactions and hydrogen bonds. This interaction led to alterations in the secondary structure of QPH, as well as a decrease in surface hydrophobicity and free SH content. Notably, the binding affinity between QPH and EGCG was observed to be higher at pH 7.0 compared to pH 3.0. Consequently, QPH-EGCG complexes exhibited more significant enhancement in antioxidant and emulsification properties at pH 7.0 than pH 3.0. The pH level also influenced the droplet size, ζ-potential, and interfacial composition of emulsions formed by QPH and QPH-EGCG complexes. Compared to QPH stabilized emulsions, QPH-EGCG stabilized emulsions were more capable of mitigating destabilization during storage and displayed fewer lipid oxidation products, carbonyl generation, and sulfhydryl groups and fluorescence loss, which implied better oxidative stability of the emulsions. Furthermore, the QPH-EGCG complexes formed at pH 7.0 exhibited better inhibition of protein hydrolysate-lipid co-oxidation. Overall, these findings provide valuable insights into the potential application of QPH and its complexes with EGCG in food processing systems.

Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Fabrication of an Antifouling Surface Plasmon Resonance Sensor with Stratified Zwitterionic Peptides for Highly Efficient Detection of Peanut Allergens in Biscuits.

Peanut allergen monitoring is currently an effective strategy to avoid allergic diseases, while food matrix interference is a critical challenge during detection. Here, we developed an antifouling surface plasmon resonance sensor (SPR) with stratified zwitterionic peptides, which provides both excellent antifouling and sensing properties. The antifouling performance was measured by the SPR, which showed that stratified peptide coatings showed much better protein resistance, reaching ultralow adsorption levels (<5 ng/cm2). Atomic force microscopy was used to further analyze the antifouling mechanism from a mechanical perspective, which demonstrated lower adsorption forces on hybrid peptide coatings, confirming the better antifouling performance of stratified surfaces. Moreover, the recognition of peanut allergens in biscuits was performed using an SPR with high efficiency and appropriate recovery results (98.2-112%), which verified the feasibility of this assay. Therefore, the fabrication of antifouling sensors with stratified zwitterionic peptides provides an efficient strategy for food safety inspection.

Design, Synthesis, and Biological Evaluation of Pyridazinone-Containing Derivatives As Novel Protoporphyrinogen IX Oxidase Inhibitor.

Protoporphyrinogen IX oxidase (PPO, E.C. 1.3.3.4) plays a pivotal role in chlorophyll biosynthesis in plants, making it a prime target for herbicide development. In this study, we conducted an investigation aimed at discovering PPO-inhibiting herbicides. Through this endeavor, we successfully identified a series of novel compounds based on the pyridazinone scaffold. Following structural optimization and biological assessment, compound 10ae, known as ethyl 3-((6-fluoro-5-(6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate, emerged as a standout performer. It exhibited robust activity against Nicotiana tabacum PPO (NtPPO) with an inhibition constant (Ki) value of 0.0338 µM. Concurrently, we employed molecular simulations to obtain further insight into the binding mechanism with NtPPO. Additionally, another compound, namely, ethyl 2-((6-fluoro-5-(5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1(6H)-yl)benzo[d]thiazol-2-yl)thio)propanoate (10bh), demonstrated broad-spectrum and highly effective herbicidal properties against all six tested weeds (Leaf mustard, Chickweed, Chenopodium serotinum, Alopecurus aequalis, Poa annua, and Polypogon fugax) at the dosage of 150 g a.i./ha through postemergence application in a greenhouse. This work identified a novel lead compound (10bh) that showed good activity in vitro and excellent herbicidal activity in vivo and had promising prospects as a new PPO-inhibiting herbicide lead.

The impact of extraction processes on the physicochemical, functional properties and structures of bamboo shoot protein.

This study aimed to extract bamboo shoot protein (BSP) using different extraction approaches and compare their functional and physicochemical properties with commercial protein ingredients, including whey protein and soy protein isolates. The extraction methods including alkali extraction (AE), salt extraction (SE), and phosphate-aided ethanol precipitation (PE) were used. An enhanced solvent extraction method was utilized in combination, resulting in a significant improvement in the protein purity, which reached 81.59 %, 87.36 %, and 67.08 % respectively. The extraction methods had significant effects on the amino acid composition, molecular weight distribution, and functional properties of the proteins. SE exhibited the best solubility and emulsification properties. Its solubility reached up to 93.38 % under alkaline conditions, and the emulsion stabilized by SE with enhanced solvent extraction retained 60.95 % stability after 120 min, which could be attributed to its higher protein content, higher surface hydrophobicity, and relative more stable and organized protein structure. All three BSP samples demonstrated better oil holding capacity, while the SE sample showed comparable functional properties to soy protein such as foaming and emulsifying properties. These findings indicate the potential of BSP as an alternative plant protein ingredient in the food industry.

Structural and Immunological Features of PR-10 Allergens: Focusing on the Major Alder Pollen Allergen Aln g 1.

Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.

Novel gurmarin-like peptides from Gymnema sylvestre and their interactions with the sweet taste receptor T1R2/T1R3.

Gymnema sylvestre (GS) is a traditional medicinal plant known for its hypoglycemic and hypolipidemic effects. Gurmarin (hereafter Gur-1) is the only known active peptide in GS. Gur-1 has a suppressive sweet taste effect in rodents but no or only a very weak effect in humans. Here, 8 gurmarin-like peptides (Gur-2 to Gur-9) and their isoforms are reported in the GS transcriptome. The molecular mechanism of sweet taste suppression by Gur-1 is still largely unknown. Therefore, the complete architecture of human and mouse sweet taste receptors T1R2/T1R3 and their interaction with Gur-1 to Gur-9 were predicted by AlphaFold-Multimer (AF-M) and validated. Only Gur-1 and Gur-2 interact with the T1R2/T1R3 receptor. Indeed, Gur-1 and Gur-2 bind to the region of the cysteine-rich domain (CRD) and the transmembrane domain (TMD) of the mouse T1R2 subunit. In contrast, only Gur-2 binds to the TMD of the human T1R2 subunit. This result suggests that Gur-2 may have a suppressive sweet taste effect in humans. Furthermore, AF-M predicted that Gα-gustducin, a protein involved in sweet taste transduction, interacts with the intracellular domain of the T1R2 subunit. These results highlight an unexpected diversity of gurmarin-like peptides in GS and provide the complete predicted architecture of the human and mouse sweet taste receptor with the putative binding sites of Gur-1, Gur-2, and Gα-gustducin. In addition, gurmarin-like peptides may serve as promising drug scaffolds for the development of antidiabetic molecules.

Assessment and Partial Characterization of Candidate Genes in Dihydrochalcone and Arbutin Biosynthesis in an Apple-Pear Hybrid by De Novo Transcriptome Assembly.

Apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) are valuable crops closely related within the Rosaceae family with reported nutraceutical properties derived from secondary metabolites including phloridzin and arbutin, which are distinctive phenolic metabolites characterizing apples and pears, respectively. Here, we generated a de novo transcriptome assembly of an intergeneric hybrid between apple and pear, accumulating intermediate levels of phloridzin and arbutin. Combining RNA-seq, in silico functional annotation prediction, targeted gene expression analysis, and expression-metabolite correlations, we identified candidate genes for functional characterization, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. Despite exhibiting an active arbutin synthase in vitro, the natural lack of arbutin in apples is reasoned by the absence of the substrate and broad substrate specificity. Altogether, our study serves as the basis for future assessment of potential physiological roles of identified genes by genome editing of hybrids and pears.

Analysis of Methylesterase Gene Family in Fragaria vesca Unveils Novel Insights into the Role of FvMES2 in Methyl Salicylate-Mediated Resistance against Strawberry Gray Mold.

Methylesterases (MESs) hydrolyze carboxylic ester and are important for plant metabolism and defense. However, the understanding of MES' role in strawberries against pathogens remains limited. This study identified 15 FvMESs with a conserved catalytic triad from the Fragaria vesca genome. Spatiotemporal expression data demonstrated the upregulated expression of FvMESs in roots and developing fruits, suggesting growth involvement. The FvMES promoter regions harbored numerous stress-related cis-acting elements and transcription factors associated with plant defense mechanisms. Moreover, FvMES2 exhibited a significant response to Botrytis cinerea stress and showed a remarkable correlation with the salicylic acid (SA) signaling pathway. Molecular docking showed an efficient binding potential between FvMES2 and methyl salicylate (MeSA). The role of FvMES2 in MeSA demethylation to produce SA was further confirmed through in vitro and in vivo assays. After MeSA was applied, the transient overexpression of FvMES2 in strawberries enhanced their resistance to B. cinerea compared to wild-type plants.

Enrichment and Quantitation of Dipeptidyl Peptidase IV Inhibitory Peptides in Quinoa upon Systematic Malting.

Food-derived peptides with an inhibitory effect on dipeptidyl peptidase IV (DPP-IV) can be used as an additive treatment for type 2 diabetes. The inhibitory potential of food depends on technological protein hydrolysis and gastrointestinal digestion, as the peptides only act after intestinal resorption. The effect of malting as a hydrolytic step on the availability of these peptides in grains has yet to be investigated. In this study, quinoa was malted under systematic temperature, moisture, and time variations. In the resulting malts, the DPP-IV inhibition reached a maximum of 45.02 (±10.28) %, whereas the highest overall concentration of literature-known inhibitory peptides was 4.07 µmol/L, depending on the malting parameters. After in vitro gastrointestinal digest, the inhibition of most malts, as well as the overall concentration of inhibitory peptides, could be increased significantly. Additionally, the digested malts showed higher values in both the inhibition and the peptide concentration than the unmalted quinoa. Concerning the malting parameters, germination time had the highest impact on the inhibition and the peptide concentration after digest. An analysis of the protein sizes before and after malting gave first hints toward the origin of these peptides, or their precursors, in quinoa.

Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Cloning, Expression Characteristics of Farnesyl Pyrophosphate Synthase Gene from Platycodon grandiflorus and Functional Identification in Triterpenoid Synthesis.

Platycodon grandiflorus is a medicinal plant whose main component is platycodins, which have a variety of pharmacological effects and nutritional values. The farnesyl pyrophosphate synthase (FPS) is a key enzyme in the isoprenoid biosynthesis pathway, which catalyzes the synthesis of farnesyl diphosphate (FPP). In this study, we cloned the FPS gene from P. grandiflorus (PgFPS) with an ORF of 1260 bp, encoding 419 amino acids with a deduced molecular weight and theoretical pI of 46,200.98 Da and 6.52, respectively. The squalene content of overexpressed PgFPS in tobacco leaves and yeast cells extract was 1.88-fold and 1.21-fold higher than that of the control group, respectively, and the total saponin content was also increased by 1.15 times in yeast cells extract, which verified the biological function of PgFPS in terpenoid synthesis. After 48 h of MeJA treatment and 6 h of ethephon treatment, the expression of the PgFPS gene in roots and stems reached its peak, showing a 3.125-fold and 3.236-fold increase compared to the untreated group, respectively. Interestingly, the expression of the PgFPS gene in leaves showed a decreasing trend after exogenous elicitors treatment. The discovery of this enzyme will provide a novel perspective for enhancing the efficient synthesis of platycodins.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 µM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Computational investigation of cis-1,4-polyisoprene binding to the latex-clearing protein LcpK30.

Latex clearing proteins (Lcps) catalyze the oxidative cleavage of the C = C bonds in cis-1,4-polyisoprene (natural rubber), producing oligomeric compounds that can be repurposed to other materials. The active catalytic site of Lcps is buried inside the protein structure, thus raising the question of how the large hydrophobic rubber chains can access the catalytic center. To improve our understanding of hydrophobic polymeric substrate binding to Lcps and subsequent catalysis, we investigated the interaction of a substrate model containing ten carbon-carbon double bonds with the structurally characterized LcpK30, using multiple computational tools. Prediction of the putative tunnels and cavities in the LcpK30 structure, using CAVER-Pymol plugin 3.0.3, fpocket and Molecular Dynamic (MD) simulations provided valuable insights on how substrate enters from the surface to the buried active site. Two dominant tunnels were discovered that provided feasible routes for substrate binding, and the presence of two hydrophobic pockets was predicted near the heme cofactor. The larger of these pockets is likely to accommodate the substrate and to determine the size distribution of the oligomers. Protein-ligand docking was carried out using GOLD software to predict the conformations and interactions of the substrate within the protein active site. Deeper insight into the protein-substrate interactions, including close-contacts, binding energies and potential cleavage sites in the cis-1,4-polyisoprene, were obtained from MD simulations. Our findings provide further justification that the protein-substrate complexation in LcpK30 is mainly driven by the hydrophobic interactions accompanied by mutual conformational changes of both molecules. Two potential binding modes were identified, with the substrate in either extended or folded conformations. Whilst binding in the extended conformation was most favorable, the folded conformation suggested a preference for cleavage of a central double bond, leading to a preference for oligomers with 5 to 6 C = C bonds. The results provide insight into further enzyme engineering studies to improve catalytic activity and diversify the substrate and product scope of Lcps.

Insights into wheat-starch biosynthesis from two-dimensional macromolecular structure.

Starch structure is often characterized by the chain-length distribution (CLD) of the linear molecules formed by breaking each branch-point. More information can be obtained by expanding into a second dimension: in the present case, the total undebranched-molecule size. This enables answers to questions unobtainable by considering only one variable. The questions considered here are: (i) are the events independent which control total size and CLD, and (ii) do ultra-long amylopectin (AP) chains exist (these chains cannot be distinguished from amylose chains using simple size separation). This was applied here to characterize the structures of one normal (RS01) wheat and two high-amylose (AM) mutant wheats (an SBEIIa knockout and an SBEIIa and SBEIIb knockout). Absolute ethanol was used to precipitate collected fractions, then size-exclusion chromatography for total molecular size and for the size of branches. The SBEIIa and SBEIIb mutations significantly increased AM and IC contents and chain length. The 2D plots indicated the presence of small but significant amounts of long-chain amylopectin, and the asymmetry of these plots shows that the corresponding mechanisms share some causal effects. These results could be used to develop plants producing improved starches, because different ranges of the chain-length distribution contribute independently to functional properties.

Lentil protein isolate (Lens culinaris) subjected to ultrasound treatment combined or not with heat-treatment: structural characterization and ability to stabilize high internal phase emulsions.

This study evaluated the effect of ultrasound treatment combined or not with heat treatment applied to lentil protein isolate (LPI) aiming to enhance its ability to stabilize high internal phase emulsions (HIPE). LPI dispersion (2%, w/w) was ultrasound-treated at 60% (UA) and 70% (UB) amplitude for 7 min; these samples were subjected to and then heat treatments at 70 °C (UAT70 and UBT70, respectively) or 80 °C (UAT80 and UBT80, respectively) for 20 min. HIPEs were produced with 25% untreated and treated LPI dispersions and 75% soybean oil using a rotor-stator (15,500 rpm/1 min). The LPI dispersions were evaluated for particle size, solubility, differential scanning calorimetry, electrophoresis, secondary structure estimation (circular dichroism and FT-IR), intrinsic fluorescence, surface hydrophobicity, and free sulfhydryl groups content. The HIPEs were evaluated for droplet size, morphology, rheology, centrifugal stability, and the Turbiscan test. Ultrasound treatment decreased LPI dispersions' particle size (∼80%) and increased solubility (∼90%). Intrinsic fluorescence and surface hydrophobicity confirmed LPI modification due to the exposure to hydrophobic patches. The combination of ultrasound and heat treatments resulted in a reduction in the free sulfhydryl group content of LPI. HIPEs produced with ultrasound-heat-treated LPI had a lower droplet size distribution mode, greater oil retention values in the HIPE structure (> 98%), lower Turbiscan stability index (< 2), and a firmer and more homogeneous appearance compared to HIPE produced with untreated LPI, indicating higher stability for the HIPEs stabilized by treated LPI. Therefore, combining ultrasound and heat treatments could be an effective method for the functional modification of lentil proteins, allowing their application as HIPE emulsifiers.

Lineage-specific gene duplication and expansion of DUF1216 gene family in Brassicaceae.

Proteins containing domain of unknown function (DUF) are prevalent in eukaryotic genome. The DUF1216 proteins possess a conserved DUF1216 domain resembling to the mediator protein of Arabidopsis RNA polymerase II transcriptional subunit-like protein. The DUF1216 family are specifically existed in Brassicaceae, however, no comprehensive evolutionary analysis of DUF1216 genes have been performed. We performed a first comprehensive genome-wide analysis of DUF1216 proteins in Brassicaceae. Totally 284 DUF1216 genes were identified in 27 Brassicaceae species and classified into four subfamilies on the basis of phylogenetic analysis. The analysis of gene structure and conserved motifs revealed that DUF1216 genes within the same subfamily exhibited similar intron/exon patterns and motif composition. The majority members of DUF1216 genes contain a signal peptide in the N-terminal, and the ninth position of the signal peptide in most DUF1216 is cysteine. Synteny analysis revealed that segmental duplication is a major mechanism for expanding of DUF1216 genes in Brassica oleracea, Brassica juncea, Brassica napus, Lepidium meyneii, and Brassica carinata, while in Arabidopsis thaliana and Capsella rubella, tandem duplication plays a major role in the expansion of the DUF1216 gene family. The analysis of Ka/Ks (non-synonymous substitution rate/synonymous substitution rate) ratios for DUF1216 paralogous indicated that most of gene pairs underwent purifying selection. DUF1216 genes displayed a specifically high expression in reproductive tissues in most Brassicaceae species, while its expression in Brassica juncea was specifically high in root. Our studies offered new insights into the phylogenetic relationships, gene structures and expressional patterns of DUF1216 members in Brassicaceae, which provides a foundation for future functional analysis.