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.

Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

U ovom radu predstavljena je ekspresija rekombinantne atipične aspartatne proteinaze heljde (Fagopyrum esculentum) bogate cisteinom, gde su testirana različita ekspresiona svojstva pet sojeva E. coli. Takođe je analiziran i uticaj fuzionih partnera (His6 i MBP) na efikasnost ekspresije. U slučaju His6-FeAPL1, dobijena je velika količina nerastvornog proteina, smeštenog u inkluzionim telima. S druge strane, MBP-FeAPL1 je bio lokalizovan i u citoplazmi i u inkluzionim telima u oba upotrebljena soja E. coli (BL21 i Rosetta-gami). Međutim, samo za rekombinantni protein proizveden u soju Rosetta-gami, dokazana je proteolitička aktivnost na supstratu BSA, pri pH 3,0. Rezultati su takođe ukazali da FeAPL1 sadrži PRO segment, čije je odstranjivanje neophodno za njegovu proteolitičku aktivnost. Aktivnost FeAPL1, pokazana samo u soju Rosetta-gami, gde je moguće formiranje disulfidnih veza, ukazuje na značaj 12 cisteina u uspostavljanju pravilne strukture koja omogućava funkcionalnost enzima.Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality

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)

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

A search of Brassica SI-involved orthologs in buckwheat leads to novel buckwheat sequence identification: MLPK possibly involved in SI response

Kod biljaka cvetnica postoje genetički određeni sistemi self-inkompatibilnosti (SI), koji sprečavaju samooprašivanje i ukrštanje u srodstvu održavajući genetičku raznovrsnost vrsta. SI se javlja u dva oblika, kao gametofitna i sporofitna SI, koje se razlikuju u načinu određivanja SI fenotipa polena - kod GSI je SI fenotip polena određen polenovim sopstvenim haploidnim genomom, dok je kod SSI određen dipolidnim genotipom majke biljke. SSI se javlja kao homomorfna (jedan tip cveta u biljaka jedne vrste) i heteromorfna (dva ili tri tipa cveta u biljaka jedne vrste). Heteromorfna SSI je u poređenju sa homomorfnom SSI i GSI izuzetno malo proučena i za sada je upoznavanje na molekularnom nivou tek započelo. Kod heljde je prisutna distilna heteromorfna SSI, o kojoj je sakupljeno dosta podataka na fiziološkom nivou, ali o kojoj za sada nema molekularnih podataka. Na osnovu fiziološke sličnosti SI odgovora biljaka rodova Brassica i Prunus sa tram i pin morfom heljde, respektivno, zatim na osnovu toga što postoje dokazi da slični biohemijski mehanizmi leže u osnovi različitih SI odgovora i na osnovu toga što i evolutivno udaljene SI vrste mogu posedovati iste ili slične predačke SI gene, mi smo odlučili da ispitamo prisustvo ortologih gena uključenih u SI odgovore Brassica i Prunus u genomu heljde. Upotrebom izrođenih prajmera dizajniranih na osnovu evolutivno očuvanih regiona SRK, SLG, SP11 i MLPK sekvenci Brassica rapa, kao i S-RNaza i SFB gena roda Prunus, dostupnih u NCBI bazi podataka, ispitano je prisustvo ortologa ovih gena u genomu heljde. Takođe je prisustvo S-RNaza ispitano u proteinskim izolatima neoprašenih i kompatibilno i inkompatibilno oprašenih tučkova heljde oba morfa. Rezultati su pokazali da nema ortologa SRK, SLG, SP11, kao ni S-RNaza i SFB u genomu heljde, ali da postoji MLPK ortolog kod heljde. Izvedena aminokiselinska sekvenca pokazala je 80 % sličnosti sa MLPKf2 sekvencom Brassica rapa i APK1A Arabidopsis thaliana, potvrđujući da su u pitanju ortolozi koji bi mogli da imaju i sličnu ulogu. Naš sledeći korak je dobijanje cele nukleotidne sekvence MLPK heljde uz is- pitivanje postojanja alternativnih mesta iskrajanja i određivanje nivoa ekspresije po tkivima, kao i ispitivanje moguće uloge u SI odgovoru heljde. Ovi odgovori omogućiće bolje upoznavanje heteromorfnih SSI sistema koji su još uvek u svojoj najranijoj fazi istraživanja i obezbediće podatke nužne za uvid u evoluciju SI sistema biljaka cvetnica. Najzad, rasvetljavanjem SSI sistema heljde, koja se koristi u ishrani, biće moguće genetički kontrolisati ukrštanje heljde i dobijanje linija sa željenim hranljivim i/ili fiziološkim osobinama.Self-incompatibility (SI) systems, gamethophytic (GSI) and sporophytic (SSI), prevent self-pollination in angiosperms. Buckwheat displays heteromorphic SSI, with pollination allowed only between different flower morphs - thrum and pin. The physiology of thrum and pin morph SI responses are entirely different, resembling homomorphic Brassica SSI and Prunus GSI responses, respectively. Considering angiosperm species may share ancestral SI genes, we examined the presence of Brassica and Prunus SI-involved gene orthologs in the buckwheat genome. We did not find evidence of SRK, SLG and SP11 Brassica or S-RNase and SFB Prunus orthologs in the buckwheat genome, but we found a Brassica MLPK ortholog. We report the partial nucleotide sequence of the buckwheat MLPK and discuss the possible implications of this finding

Two metallothionein gene family members in buckwheat: Expression analysis in flooding stress using Real Time RT-PCR technology

Metalotioneini (MT) pripadaju velikoj grupi proteina male molekulske težine bogatih cisteinom, izražene sposobnosti za vezivanje jona metala, uključenih u procese održavanja homeostaze metalnih jona i detoksifikacije od teških metala. U radu je analizirana struktura dva transkripta gena za MT tipa 3 poreklom iz semena heljde u razviću. Razlike su nađene pre svega u okviru 3’- UTR sekvenci. Nakon analiza sekvenci urađena je analiza ekspresije tokom hipoksije korišćenjem tehnologije Real Tme RT-PCR.Metallothioneins (MTs) are an extensive and diverse family of small cysteine-rich proteins with metal-binding ability that are involved in metal homeostasis and detoxification. Two cDNA clones of the MT3 type, differing in 3’ UTRs, were isolated from the developing buckwheat seed cDNA library. Following sequence analyses, expression profiles during flooding stress were monitored by Real Time RT PCR technology

Transient expression in tobacco Bright Yellow 2 cells and pollen grains: A fast, efficient and reliable system for functional promoter analysis of plant genes

Ekspresija gena je regulisana posredstvom promotora-DNK sekvenci uzvodno od kodirajućeg regiona. Promotori svojom sekvencom određuju mesta vezivanja transkripcionih faktora, regulatora i RNK polimeraze. Strukturna i funkcionalna analiza promotora neophodna je za razumevanje mehanizama genske ekspresije. Cilj ovog rada je razvijanje brzog, efikasnog i pouzdanog sistema za testiranje bazalne promotorske aktivnosti kao i otkrivanje sekvenci polen-specifičnih elemenata promotora. U ovom radu je testirana funkcionalnost promotora za metalotionein heljde, posredstvom histohemijskog GUS-eseja u dva tranzijentna sistema za ekspresiju: BY2 ćelijama i polenovim zrnima. U oba sistema je uočena izražena aktivnost ispitivanog promotora.Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems

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