The role of DREB2A transcription factor and small RNAsmiR398a/b and miR408 in dehidratation response of pea (Pisum sativum L.)

Jedan od glavnih faktora abiotičkog stresa kod biljaka je dehidratacija. U ovom radu ispitivan je uticaj dehidratacije na ekspresiju gena za transkripcioni faktor DREB2A i malih RNK miR398 i miR408 graška, u cilju sagledavanja značaja ovih molekula u odgovoru graška na dehidrataciju i rehidrataciju. Parcijalna cDNK (PsDREB2A) koja kodira za DREB2A transkripcioni faktor graška je dobijena primenom metode PCR sa prajmerima dizajniranim na osnovu sekvenci DREB2A gena iz model leguminoze Medicago truncatula. Dobijena sekvenca (495 bp) deponovana je u GeneBank (HM229349). PoreĎenje nukletidnih sekvenci pokazalo je da je dobijena sekvenca najsličnija sa sekvencom za DREB2A iz M. truncatula, kao i sa EREBP (ethylene responsive element binding protein) iz iste vrste. Analiza izvedene aminokiselinske sekvence pokazala je da PsDREB2A ima strukturu karakterističnu za DREB2A protein - prisustvo konzervisanog AP2 domena (17-55 ak), kao i da ima najveću homologiju sa DREB2A i sa ERBP proteinom iz M. truncatula. Na osnovu kompjuterske predikcije posttranslacionih modifikacija proteina utvrĎeno je prisustvo jednog potencijalnog mesta za sumoilaciju proteina, i to na poziciji Lys157 (MKQE), kao i prisustvo nekoliko potencijalnih mesta za fosforilaciju, najverovatnije protein kinazom A. U cilju ispitivanja ekspresije gena za DREB2A transkripcioni factor graška vršene su analize iRNK korena i nadzemnog dela graška u normalnom fiziološkom stanju, kao i u uslovima dehidratacije u trajanju 7 i 10 dana, i rehidratacije. Primenjena je metoda Real-time PCR uz upotrebu proba specifičnih za PsDREB2A. Pokazano je da se profil ekspresije razlikuje u korenu i nadzemnom delu biljaka. Kod korena maksimalni nivo ekspresije PsDREB2A utvrĎen je nakon 10 dana tretmana, dok je kod nadzemnog dela biljaka maksimum dostignut nakon 7 dana dehidratacije...Adverse environmental stresses, such as drought, low temperature and soil salinity have a strong influence on agricultural production and sustainability. A major limitation to yield and quality in many crop species is water availability throughout or at critical times during the growing season. We investigated the effect of dehydration on expression of DREB2A transcription factor in pea, as well as on the expression of conserved miRNAs-miR398a/b and miR408. Partial cDNA (PsDREB2A), which encodes a DREB2A transcription factor in pea, was obtained by PCR with primers designed according to DREB2A gene from the model legume Medicago truncatula. The resulting sequence (495 bp) has been deposited in Gene Bank (HM229349). Comparison of nucleotide sequences showed that the obtained sequence is most similar to the sequence of DREB2A from M. truncatula, and with EREBP (ethylene responsive element binding protein) from the same species. Analysis of deduced amino acid sequence showed that PsDREB2A has a structure characteristic of the DREB2A protein - presence of AP2 domains (17-55 a.a.), and has the highest homology with DREB2A and ERBP protein from M. truncatula. Computer prediction of post-translational modification of protein revealed the presence of a potential site for protein sumoylation, at the position of Lys157 (MKQE), and the presence of several potential sites for phosphorylation. To study the expression of PsDREB2A we performed the analysis of mRNA level in pea root and shoot in a normal physiological state, as well as during dehydration for 7 and 10 days, and rehydration. Real-time PCR using a probe specific for PsDREB2A has been applied. It has been shown that the expression profile was different in roots and aerial parts of the plants. In the roots the maximum level of expression PsDREB2A was determined after 10 days of treatment, while in the above-ground parts of plants the maximum was rached after 7 days of dehydration..

A simple and efficient DNA isolation method for Ornithogalum L. species (Hyacinthaceae, Asparagales)

We report an efficient, simple and cost-effective protocol for the isolation of genomic DNA from Ornithogalum species. Our modification of the standard CTAB protocol includes two polyphenol adsorbents (insoluble PVPP and activated charcoal), high NaCl concentrations (4 M) for removing polysaccharides, and addition of phenol to remove proteins and other contaminants. DNA yield obtained with our protocol was 223 and 312 μg DNA g-1 of dry leaf tissue. The absorbance ratio 260/280 nm was 1.879 (O. refractum) and 1.753 (O. sibthorpii), and the absorbance ratio 260/230 nm was 1.779 (O. refractum) and 1.545 (O. sibthorpii), revealing lack of contamination. PCR amplifications of one nuclear marker (26S rDNA) indicated that this DNA isolation protocol may be used for Ornithogalum plants containing many interfering compounds for further analyses in population genetics and phylogeographic studies

Synthesis and antioxidant activity of 1,3,4-oxadiazoles and their diacylhydrazine precursors derived from phenolic acids

Eight 1,3,4-oxadiazole derivatives containing phenolic acid moieties (7a-h) and eight of their diacylhydrazine precursors (6a-h) were synthesized, characterized using spectroscopic methods and examined by scavenging of stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals. The most potent phenolic 1,3,4-oxadiazoles showed better DPPH scavenging activity in comparison with their corresponding diacylhydrazine precursors as a result of participation of both aromatic rings and a 1,3,4-oxadiazole moiety in resonance stabilization of the formed phenoxyl radical. Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxadiazoles (7d, 7e, 7g and 7h) with the best DPPH scavenging activity, were chosen for further evaluation of their antioxidant potential through various assays. The investigated compounds exerted pronounced ABTS radical scavenging capacity, moderate to good H2O2 scavenging properties and strong ferric ion reducing capacity. Further in vitro evaluation of the antioxidant properties of the most active compounds demonstrated their protective effects in normal lung fibroblasts MRC-5 against hydrogen peroxide induced oxidative stress. Diacylhydrazine 6h increased two times the activity of glutathione peroxidase in treated cells in comparison with a control sample and did not affect the superoxide dismutase activity.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2980

Antioxidant activity of pea protein hydrolysates produced by batch fermentation with lactic acid bacteria

Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO) activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW lt 10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity) and the recently developed direct current (DC) polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins

Identification of Seed Coat Phenolic Compounds from Differently Colored Pea Varieties and Characterization of Their Antioxidant Activity

The phenolic composition of seed coats in four differently colored pea varieties (Pisum sativum L.) was investigated using UHPLC-LTQ OrbiTrap MS. The obtained findings revealed that the seed coats of the examined pea genotypes possess a unique phenolic composition compared to previously studied European cultivars. In total, 41 phenolic compounds have been identified. The seed coats of the studied cultivars contained certain amounts of rosmarinic acid, rutin, galangin, morin, naringin, hesperetin and pinocembrin as well as ten flavonol glycosides that had not been reported previously. Additionally, the total phenolic content, antioxidant activity and metal chelating capacity of extracts was determined using Folin-Ciocalteu's method, 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric ion-reducing capacity and ferrous ion-chelating capacity assay, respectively. Dark colored genotypes MBK 168 and MBK 173 possessed the highest total phenolic contents as well the strongest antioxidant activities. On the other hand, bright colored genotypes MBK 88 and MBK 90 exhibited the strongest metal-chelating capacities. The examined pea seed coats may be considered as important potential contributors to human health due to the presence of bioactive phenolic constituents. In addition, our results could be used as a guideline for breeding new pea cultivars with high antioxidant activities applicable in the formulation of functional food products

Identification of seed coat phenolic compounds from differently colored pea varieties and characterization of their antioxidant activity

The phenolic composition of seed coats in four differently colored pea varieties (Pisum sativum L.) was investigated using UHPLC-LTQ OrbiTrap MS. The obtained findings revealed that the seed coats of the examined pea genotypes possess a unique phenolic composition compared to previously studied European cultivars. In total, 41 phenolic compounds have been identified. The seed coats of the studied cultivars contained certain amounts of rosmarinic acid, rutin, galangin, morin, naringin, hesperetin and pinocembrin as well as ten flavonol glycosides that had not been reported previously. Additionally, the total phenolic content, antioxidant activity and metal chelating capacity of extracts was determined using Folin-Ciocalteu's method, 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric ion-reducing capacity and ferrous ion-chelating capacity assay, respectively. Dark colored genotypes MBK 168 and MBK 173 possessed the highest total phenolic contents as well the strongest antioxidant activities. On the other hand, bright colored genotypes MBK 88 and MBK 90 exhibited the strongest metal-chelating capacities. The examined pea seed coats may be considered as important potential contributors to human health due to the presence of bioactive phenolic constituents. In addition, our results could be used as a guideline for breeding new pea cultivars with high antioxidant activities applicable in the formulation of functional food products

Supplementary material for the article: Stanisavljević, N. S.; Ilić, M. D.; Matić, I. Z.; Jovanović, Ž. S.; Čupić, T.; Dabić, D. Č.; Natić, M. M.; Tešić, Ž. L. Identification of Phenolic Compounds from Seed Coats of Differently Colored European Varieties of Pea (Pisum Sativum L.) and Characterization of Their Antioxidant and In Vitro Anticancer Activities. Nutrition and Cancer 2016, 68 (6), 988–1000. https://doi.org/10.1080/01635581.2016.1190019.

Supplementary material: [https://doi.org/10.1080/01635581.2016.1190019]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2291

Supplementary data for article: Mihailovic, N.; Marković, V.; Matić, I. Z.; Stanisavljević, N. S.; Jovanovic, Z. S.; Trifunović, S. S.; Joksović, L. Synthesis and Antioxidant Activity of 1,3,4-Oxadiazoles and Their Diacylhydrazine Precursors Derived from Phenolic Acids. RSC Advances 2017, 7 (14), 8550–8560. https://doi.org/10.1039/c6ra28787e

Supplementary material for: [https://doi.org/10.1039/c6ra28787e]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2398

Antioxidant Activity of Pea Protein Hydrolysates Produced by Batch Fermentation with Lactic Acid Bacteria

Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO) activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW lt 10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity) and the recently developed direct current (DC) polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins

Supplementary material for the article: Stanisavljević, N. S.; Ilić, M. D.; Matić, I. Z.; Jovanović, Ž. S.; Čupić, T.; Dabić, D. Č.; Natić, M. M.; Tešić, Ž. L. Identification of Phenolic Compounds from Seed Coats of Differently Colored European Varieties of Pea (Pisum Sativum L.) and Characterization of Their Antioxidant and In Vitro Anticancer Activities. Nutrition and Cancer 2016, 68 (6), 988–1000. https://doi.org/10.1080/01635581.2016.1190019.

Supplementary material: [https://doi.org/10.1080/01635581.2016.1190019]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2291