Identification of Novel Loci Precisely Modulating Pre-Harvest Sprouting Resistance and Red Color Components of the Seed Coat in T. aestivum L.

The association between pre-harvest sprouting (PHS) and seed coat color has long been recognized. Red-grained wheats generally exhibit greater PHS resistance compared to white-grained wheat, although variability in PHS resistance exists within red-grained varieties. Here, we conducted a genome-wide association study on a panel consisting of red-grained wheat varieties, aimed at uncovering genes that modulate PHS resistance and red color components of seed coat using digital image processing. Twelve loci associated with PHS traits were identified, nine of which were described for the first time. Genetic loci marked by SNPs AX-95172164 (chromosome 1B) and AX-158544327 (chromosome 7D) explained approximately 25% of germination index variance, highlighting their value for breeding PHS-resistant varieties. The most promising candidate gene for PHS resistance was TraesCS6B02G147900, encoding a protein involved in aleurone layer morphogenesis. Twenty-six SNPs were significantly associated with grain color, independently of the known Tamyb10 gene. Most of them were related to multiple color characteristics. Prioritization of genes within the revealed loci identified TraesCS1D03G0758600 and TraesCS7B03G1296800, involved in the regulation of pigment biosynthesis and in controlling pigment accumulation. In conclusion, our study identifies new loci associated with grain color and germination index, providing insights into the genetic mechanisms underlying these traits.

QTL Analysis for Bread Wheat Seed Size, Shape and Color Characteristics Estimated by Digital Image Processing.

The size, shape, and color of wheat seeds are important traits that are associated with yield and flour quality (size, shape), nutritional value, and pre-harvest sprouting (coat color). These traits are under multigenic control, and to dissect their molecular and genetic basis, quantitative trait loci (QTL) analysis is used. We evaluated 114 recombinant inbred lines (RILs) in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative, ITMI/MP) grown in 2014 season. We used digital image analysis for seed phenotyping and obtained data for seven traits describing seed size and shape and 48 traits of seed coat color. We identified 212 additive and 34 pairs of epistatic QTLs on all the chromosomes of wheat genome except chromosomes 1A and 5D. Many QTLs were overlapping. We demonstrated that the overlap between QTL regions was low for seed size/shape traits and high for coat color traits. Using the literature and KEGG data, we identified sets of genes in Arabidopsis and rice from the networks controlling seed size and color. Further, we identified 29 and 14 candidate genes for seed size-related loci and for loci associated with seed coat color, respectively.

Characteristics and Stability Assessment of Therapeutic Methionine γ-lyase-Loaded Polyionic Vesicles.

Pyridoxal 5'-phosphate-dependent methionine γ-lyase from Citrobacter freundii (MGL, EC 4.4.1.11) is studied as an antitumor enzyme and in combination with substrates as an antibacterial agent in enzyme pro-drug therapy. For the possibility of in vivo trials, two mutant forms, C115H MGL and V358Y MGL, were encapsulated into polyionic vesicles (PICsomes). Five pairs of polymers with the number of polymer chain units 20, 50, 70, 120, and 160 were synthesized. The effect of polymer length-PEGylated poly-l-aspartic acid and poly-l-lysine-on the degree of MGL incorporation into PICsomes and their size was investigated. Encapsulation of proteins in PICsomes is a rather new technique. Our data demonstrated that the length of the polymers and, therefore, the ratio of the hydrophobic and hydrophilic fragments most likely should be selected individually for each protein to be encapsulated. The efficiency of encapsulation of MGL mutant forms into PICsomes was up to 11%. The hydrodynamic diameter and surface potential of hollow and MGL-loaded PICsomes were evaluated by the dynamic light scattering method. The size and morphology of the PICsomes were determined by atomic force microscopy. The most acceptable for further in vivo studies were PICsomes20 with a size of 57-64 nm, PICsomes70 of 50-90 nm, and PICsomes120 of 100-105 nm. The analysis of the steady-state parameters has demonstrated that both mutant forms retained their catalytic properties inside the nanoparticles. The release study of the enzymes from PICsomes revealed that about 50% of the enzymes remained encapsulated in PICsomes70 and PICsomes120 after 24 h. Based on the data obtained, the most promising for in vivo studies are PICsomes70 and PICsomes120.

Relationship between the Characteristics of Bread Wheat Grains, Storage Time and Germination.

Seed storage is important to farmers, breeders and for germplasm preservation. During storage, seeds accumulate damage at the structural and metabolic level, which disrupt their function and reduce resistance to adverse external conditions. In this regard, issues related to seed aging prove to be relevant for maintaining the viability of genetic collections. We analyzed morphological characteristics of grains and their coat color for 44 recombinant inbred lines (RILs) of bread wheat grown in four different seasons, 2003, 2004, 2009 and 2014. Our investigations were performed in 2020. For 19 RILs from the same seasons germination was evaluated. Our results demonstrate that genotype significantly affects the variability of all seed traits, and the year of harvesting affects about 80% of them (including all the traits of shape and size). To identify the trend between changes in grain characteristics and harvesting year, we estimated correlation coefficients between them. No significant trend was detected for the grain shape/size traits, while 90% of the color traits demonstrated such a trend. The most significant negative correlations were found between the harvesting year and the traits of grain redness: the greater the storage time, the more intensive is red color component for the grains. At the same time, it was shown that grains of longer storage time (earlier harvesting year) have lighter coat. Analysis of linear correlations between germination of wheat seeds of different genotypes and harvesting years and their seed traits revealed a negative linear relationship between the red component of coat color and germination: the redder the grains, the lower their germination rate. The results obtained demonstrate manifestations of metabolic changes in the coat of grains associated with storage time and their relationship with a decrease of seed viability.

Encapsulated Methionine γ-Lyase: Application in Enzyme Prodrug Therapy of Pseudomonas aeruginosa Infection.

Lung disease caused by Pseudomonas aeruginosa is the leading reason for death in cystic fibrosis patients. Therapeutic efficacy of the pharmacological pairs, the naked/encapsulated mutant form of Citrobacter freundii methionine γ-lyase and the substrates, sulfoxides of S-substituted l-cysteine, generating thiosulfinates, was evaluated on the murine model of experimental sepsis caused by the multidrug-resistant P. aeruginosa 203-2 strain. The pairs containing the naked enzyme and substrates did not have antibacterial activity. The treatment of mice with the pair encapsulated enzyme and S-methyl-l-cysteine sulfoxide, generating dimethyl thiosulfinate, led to a complete recovery of the animals of the model, with the infecting dose equal to LD50. The pair generating diallyl thiosulfinate (allicin) proved to be less effective. So, the substituents, attached to the thiosulfinate moiety, affect the antibacterial activity of thiosulfinates against P. aeruginosa.

Methionine γ-lyase in enzyme prodrug therapy: An improvement of pharmacokinetic parameters of the enzyme.

Citrobacter freundii methionine γ-lyase (MGL), in addition to the physiological reaction, catalyzes the ß-elimination reaction of S-alk(en)yl-L-cysteine sulfoxides to yield thiosulfinates, which have antibacterial activity. We have obtained the mutant form C115H MGL, which cleaves S-alk(en)yl-L-cysteine sulfoxides more effectively than the wild type enzyme does. The binary system MGL/S-alk(en)yl-L-cysteine sulfoxides may be considered as a new pharmacological pair in enzyme prodrug therapy (EPT). Despite of the successful application of this pair in antibacterial studies in vitro, in vivo experiments may lead to several problems typical of therapeutic proteins including a relatively short-lasting biological activity. To circumvent these problems, we have investigated several approaches to improve safety and efficacy of the enzyme component of the pharmacological pair. This included covalent attachment of poly(ethylene glycol) to the enzyme, its encapsulation in liposomes and polymeric vesicles (PICsomes). The steady-state and pharmacokinetic parameters of modified/encapsulated enzyme were determined. It was demonstrated that the encapsulation in PICsomes prolongs in vivo stability of C115H MGL to over 42 h compared to PEGylated enzyme (3 h). Antibacterial activity of binary system ("pharmacological pair") modified/encapsulated enzyme/S-alk(en)yl-L-cysteine sulfoxides was tested and remained the same as for the naked enzyme. Thus, the usage of MGL-loaded PICsomes as enzymatic nanoreactors in ETP to produce antimicrobial thiosulfinates is promising.

Non-stereoselective decomposition of (±)-S-alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates catalyzed by C115H mutant methionine γ-lyase from Citrobacter freundii.

S-Alk(en)yl-l-cysteine sulfoxides, initially found in plants of the genus Allium, are converted to antimicrobial thiosulfinates by pyridoxal 5'-phosphate(PLP)-dependent alliinase (EC 4.4.1.4). It was found that methionine γ-lyase (MGL, EC 4.4.1.11) catalyzes the ß-elimination reaction of (±)-S-alk(en)yl-l-cysteine sulfoxides to yield thiosulfinates. The efficient catalyst for the production of thiosulfinates, C115H mutant MGL, developed in our previous work, cleaves S-alk(en)yl-l-cysteine sulfoxides more effectively than the wild type enzyme. Thiosulfinates generated by the C115H MGL/sulfoxide system have demonstrated growth inhibition of Gram-positive, Gram-negative bacteria and clinical isolates of pathogenic bacteria from mice. In search of a more effective system for production of antibacterial thiosulfinates we synthesized S-substituted analogues of l-cysteine sulfoxide with a longer side chains - (±)-S-propyl-l-cysteine sulfoxide ((±)-propiin) and (±)-S-n-butyl-l-cysteine sulfoxide ((±)-butiin) and determined catalytic parameters of the ß-elimination reaction of two sulfoxides. It was found that C115H MGL cleaves (±)-propiin with the highest rate, as compared to other (±)-S-alk(en)yl-l-cysteine sulfoxides. Studies on interaction of the enzyme with (+)- or (-)-S-alk(en)yl-l-cysteine sulfoxides revealed that C115H MGL can decompose both diastereomers equally. The antibacterial activity of the mixture of the mutant MGL with (±)-propiin is comparable with those of the mixtures with S-allyl-l-cysteine sulfoxide (alliin) and S-methyl-l-cysteine sulfoxide (methiin). The results make MGL/sulfoxide system more advantageous in preparing antibacterial thiosulfinates as compared to alliinase-based system, which preferably cleaves naturally occurring (+)-sulfoxides.

Origin of Infrared Light Modulation in Reflectance-Mode Photoplethysmography.

We recently pointed out the important role of dermis deformation by pulsating arterial pressure in the formation of a photoplethysmographic signal at green light. The aim of this study was to explore the role of this novel finding in near-infrared (NIR) light. A light-emitting diode (LED)-based imaging photoplethysmography (IPPG) system was used to detect spatial distribution of blood pulsations under frame-to-frame switching green and NIR illumination in the palms of 34 healthy individuals. We observed a significant increase of light-intensity modulation at the heartbeat frequency for both illuminating wavelengths after a palm was contacted with a glass plate. Strong positive correlation between data measured at green and NIR light was found, suggesting that the same signal was read independently from the depth of penetration. Analysis of the data shows that an essential part of remitted NIR light is modulated in time as a result of elastic deformations of dermis caused by variable blood pressure in the arteries. Our observations suggest that in contrast with the classical model, photoplethysmographic waveform originates from the modulation of the density of capillaries caused by the variable pressure applied to the skin from large blood vessels. Particularly, beat-to-beat transmural pressure in arteries compresses/decompresses the dermis and deforms its connective-tissue components, thus affecting the distance between the capillaries, which results in the modulation of absorption and scattering coefficients of both green and NIR light. These findings are important for the correct interpretation of this widely used medical technique, which may have novel applications in diagnosis and treatment monitoring of aging and skin diseases.

Influence of a skin status on the light interaction with dermis.

We present experimental evidence that the parameters of green light remitted from a human tissue in-vivo strongly depend on skin contact status. In case when the skin is free of any contact, simultaneous recording of imaging photoplethysmogram (iPPG) and electrocardiogram revealed that contactless iPPG fails in correct estimates of the heart rate in almost half of the cases. Meanwhile, the number of successful correlations between ECG and iPPG is significantly increased when the skin is in contact with a glass plate. These observations are in line with the recently proposed model in which pulsatile arteries deform the connective-tissue components of the dermis thus resulting in temporal modulation of the capillary density interacting with slightly penetrating light.