Proteinases from buckwheat (Fagopyrum esculentum moench) seeds: Purification and properties of the 47 kDa enzyme

Analizirane su aspartične proteinaze semena heljde. Upotrebom pepstatin A afinitetne hromatografije, iz zrelog semena izdvojene su tri forme aspartičnih proteinaza, od 47 kDa, 40 kDa i 28 kDa, dok je u ekstraktu nezrelog semena odsustvovala forma od 40 kDa. Protein od 47 kDa naknadno je razdvojen od ostalih formi kada je hromatografiji prethodila amonijum-sulfatna precipitacija. Pokazano je da tip proteolitičkog delovanja prečišćene forme enzima odgovara delovanju himozina, aspartične proteinaze animalnog porekla, čime bi se mogla objasniti njegova sposobnost da koaguliše mleko. Enzim je lokalizovan u membranskoj ćelijskoj frakciji.Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction

Proteinases from buckwheat (Fagopyrum esculentum moench) seeds: Purification and properties of the 47 kDa enzyme

Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction.

Seed-specific aspartic proteinase FeAP12 from buckwheat (Fagopyrum esculentum Moench)

Gen za aspartičnu proteinazu (FeAP12) je izolovan iz eDNA biblioteke semena heljde u razviću. Analiza izvedene amino kiselinske sekvence FeAP12 gena ukazuje na njenu visoku homologiju sa ostalim tipičnim biljnim aspartičnim proteinazama (AP) koje se odlikuju prisustvom biljno specifičnog inserta (plant specific insert PSI), jedinstvenog među AP. Pokazano je da gen FeAP12 nije eksprimiran u listu, korenu, stablu i cvetu, već da je iRNA za FeAP12 prisutna samo u semenu. Najveći nivo ekspresije ovog gena je uočen u ranim fazama razvića semena, što ukazuje na njegovu moguću ulogu u degradaciji nucelusa.Aspartic proteinase gene (FeAP12) has been isolated from the cDNA library of developing buckwheat seeds. Analysis of its deduced amino acid sequence showed that it resembled the structure and shared high homology with typical plant aspartic proteinases (AP) characterized by the presence of a plant-specific insert (PSI), unique among APs. It was shown that FeAP12 mRNA was not present in the leaves, roots, steam and flowers, but was seed-specifically expressed. Moreover, the highest levels of FeAP12 expression were observed in the early stages of seed development, therefore suggesting its potential role in nucellar degradation

Isolation and structural analysis of a gene coding for a novel type of aspartic proteinase from buckwheat seed (Fagopyrum esculentum Moench)

Iz biblioteke cDNK semena heljde u srednjoj fazi razvića izolovan je gen koji kodira novi tip aspartične proteinaze. Analizom sekvence ove cDNK (Fe-APL1) uočeno je odsustvo domena karakterističnog samo za biljne aspartične proteinaze, a analizom odgovrajućeg genomskog fragmenta da se radi o genu koji ne sadrži introne. Bioinformatičkim analizama genoma arabidopsisa je pokazano da je većina potencijalnih gena za aspartične proteinaze upravo sa ovim osobinama, iako je to eksperimentalno dokazano samo kod malog broja gena. Rezultati ovog rada daju doprinos u analizi raznovrsnosti unutar familije biljnih aspartičnih proteinaza. .A novel type of aspartic proteinase gene was isolated from the cDNA library of developing buckwheat seeds. This cDNA, FeAPL1, encoded an AP-like protein lacking the plant-specific insert (PSI) domain characteristic of typical plant aspartic proteinases. In addition the corresponding genomic fragment was isolated. It is demonstrated that this gene does not contain introns. Since bioinformatics analysis of the Arabidopsis genome showed that most potential AP genes are intronless and PSI-less, it appears that "atypical" is an inappropriate word for that class of AP. Isolation of this specific buckwheat gene among the small group of those isolated from other plant species provides a new perspective on the diversity of AP family members in plants.

Seed-specific aspartic proteinase FeAP12 from buckwheat (Fagopyrum esculentum Moench)

Aspartic proteinase gene (FeAP12) has been isolated from the cDNA library of developing buckwheat seeds. Analysis of its deduced amino acid sequence showed that it resembled the structure and shared high homology with typical plant aspartic proteinases (AP) characterized by the presence of a plant-specific insert (PSI), unique among APs. It was shown that FeAP12 mRNA was not present in the leaves, roots, steam and flowers, but was seed-specifically expressed. Moreover, the highest levels of FeAP12 expression were observed in the early stages of seed development, therefore suggesting its potential role in nucellar degradation

Antioxidative enzymes in the response of buckwheat (Fagopyrum esculentum Moench) to complete submergence

Oxidative stress and antioxidative defense system activity were studied in buckwheat leaves after complete submergence and re-aeration. The levels of H2O2 and lipid peroxidation were found to be significantly higher in stressed than in untreated buckwheat leaves. Enzymes catalyzing the degradation of H2O2 and peroxides were shown to participate actively, whereas superoxide dismutase did not take part in the buckwheat leaf response to flooding stress. The most prominent increase in antioxidative enzyme activities was noticed upon return to air, when the strongest oxidative stress occurred and the need for antioxidative defense was the greatest

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate.

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nataša seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.This is a post-peer-review, pre-copyedit version of an article published in Protoplasma. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00709-018-1250-0

Biochemical and molecular changes in buckwheat leaves during exposure to salt stress

In spite of the great nutritive and pharmacological potentials of buckwheat, data about the abiotic stress tolerance of this plant species are very limited. The aim of this work was to analyze the biochemical and molecular response of buckwheat plants in the middle vegetative phase against short- and long-term salt stress. Changes in relative water content, level of lipid peroxidation, content and localization of H2O2 as well as changes in antioxidative enzyme activity and expression of ubiquitin and dehydrins, were investigated. Reasons for observed buckwheat salt stress sensitivity as well as possibilities for enhancing stress tolerance are discussed

Treatment of isolated pistils with protease inhibitors overcomes the self-incompatibility response in buckwheat

Isolated pistils of distylous buckwheat (Fagopyrum esculentum Moench) were treated with protease inhibitors (PMSF, pepstatin A, and antipain). Pistils were cross- or self- pollinated, and growth of pollen tubes was observed under a fluorescence microscope. Treatments with all inhibitors suppressed inhibition of self-pollen tube growth, suggesting that activity of proteases is involved in rejection of self-pollen during the SI response

DOI:10.2298/ABS0701045M TREATMENT OF ISOLATED PISTILS WITH PROTEASE INHIBITORS OVERCOMES THE SELF-INCOMPATIBILITY RESPONSE IN BUCKWHEAT

Abstract – Isolated pistils of distylous buckwheat (Fagopyrum esculentum Moench) were treated with protease inhibitors (PMSF, pepstatin A, and antipain). Pistils were cross- or self- pollinated, and growth of pollen tubes was observed under a fluorescence microscope. Treatments with all inhibitors suppressed inhibition of self-pollen tube growth, suggesting that activity of proteases is involved in rejection of self-pollen during the SI response