Original title:
Sekretorická dráha závislá na exocystu
Translated title:
Plant exocyst dependent secretory pathway and iron nutrition
Authors:
Batík, Adam ; Žárský, Viktor (advisor) ; Vaňková, Radomíra (referee) Document type: Master’s theses
Year:
2024
Language:
eng Abstract:
[eng][cze] Previous results from our lab suggested possible phenotypic deviation of a double T-DNA knock- out mutant of an EXOCYST subunit EXO70E2 and vacuolar transmembrane protein LAZ1H1 upon iron starvation. To confirm these preliminary results, single mutants were obtained, with EXO70E2 knock-out mutant being produced here using CRISPR/Cas9 technology. Result did not confirm the previous findings in neither of the mutants tested (exo70e2, laz1h1, exo70e2/laz1h1 and exo70h4). This discrepancy is attributed to improper iron starvation protocol and different storage age of seeds used in the previously conducted experiments. Coumarins are synthetized and secreted from roots during iron starvation; protocol for their visualization was adopted and WT compared with mutants. No phenotypic deviations were observed in mutants. Pharmacological treatments of iron starved WT plants using BFA, wortmannin and concanamycin A did not suggest that endomembrane vesicle trafficking affect accumulation or secretion of coumarins. Previously published susceptibility of T-DNA knock-out mutant of EXO70E2 to drought stress was tested using CRISPR/Cas9 mutant produced here, the reported susceptibility was not confirmed. Upregulation of EXO70E2 mRNA upon UV exposure reported in eFP browser was also tested and not confirmed under...Predošlé výsledky z nášho laboratória naznačovali možnú fenotypickú deviáciu dvojitého T-DNA mutanta podjednotky EXOCYST-u EXO70E2 a vakuolárneho transmembránového proteínu LAZ1H1 na médiu s nedostatkom železa. Jednoduchý mutanti oboch génov, EXO70E2 mutant bol vytvorený v tejto práci za pomoci CRISPR/Cas9 technológie, boli podobrení hladovaniu na železo aby sa potvrdili predošlé výsledky. Výsledky nepotvrdili žiadnu fenotypickú odchýlku v žiadnom z testovaných mutantov (exo70e2, laz1h1, exo70e2/laz1h1 a exo70h4) pestovaných na médiu bez železa. Nezrovnalosť s predošlými výsledkami bola pravdepodobne zapríčinená nesprávnym protokolom a použitím semien s rôznym vekom v predošlých pokusoch. Kumaríny sú syntetizované a vylučované koreňmi pri hladovaní na železo. Bol vytvorený protokol na ich pozorovanie a WT rastliny boli porovnané s mutantmi. Žiadne fenotypické odchýlky neboli pri mutantoch pozorované. Pozorovania hladujúcich WT rastlín ošetrenými inhibítormi endomembránového vezikulárneho transportu (BFA, wortmannin a concanamycin A) nenaznačujú že by tento transport ovplyvňoval akumuláciu alebo sekréciu kumarínov. V literatúre popisovaná citlivosť exo70e2 T-DNA mutanta na sucho bola testovaná CRISPR/Cas9 mutantom vyprodukovaným v tejto práci, citlivosť sa nepotvrdila. Zvýšená transkripcia EXO70E2...
Keywords:
endomembranes; exocyst; iron; mineral nutrition; secretory pathway; endomembrány; exocyst; minerální výživa; sekretorická dráha; železo
Institution: Charles University Faculties (theses)
(web)
Document availability information: Available in the Charles University Digital Repository. Original record: http://hdl.handle.net/20.500.11956/190423