Identifikacija AGP gena kičice (Centaurium erythraea, Gentianaceae) i praćenje njihove ekspresije u odgovoru na mehaničke povrede biljnog tkiva gajenog in vitro

Abstract

Arabinogalaktanski proteini (AGP) su ekstenzivno glikozilovani proteini ćelijskog zida i predstavljaju podklasu O-glikozilovanih glikoproteina bogatih hidroksiprolinom (HRGP). Odlikuje ih visoka raznolikost primarne strukture, velika familija gena koja ih kodira, kao i širok spektar uloga tokom rastenja i razvića biljaka. AGP su proteini sa neuređenom strukturom što otežava njihovu identifikaciju na osnovu homologije sekvenci. Sa ciljem poboljšanja metodologije za identifikaciju i analizu HRGP sekvenci u sklopu ove doktorske disertacije razvijen je pristup zasnovan na mašinskom učenju. Ovaj pristup koristi glavnu odliku HRGP, a to je prisustvo nekarakteristične aminokiseline hidroksiprolina. Model za predviđanje verovatnoće hidroksilacije prolina na osnovu lokalne sekvence proteina inkorporiran je u ragp R paket uz brojne druge alate koji omogućavaju analize proteinskih sekvenci kao što su: klasifikacija HRGP sekvenci, fleksibilna pretraga karakterističnih motiva, efikasna komunikacija sa serverima za predviđanje N-terminalne signalne sekvence, mesta dodavanja glikozil-fosfatidilinozitolnog sidra, određivanje položaja neuređenih regiona i anotaciju domena u sekvencama. Navedena metodologija omogućava prilagodljivu identifikaciju sekvenci pri kojoj izbor nekoliko parametara utiče na rigoroznost procesa, a time i broj identifikovanih sekvenci. Navedeni softver omogućava efikasnu identifikaciju HRGP sekvenci u celim biljnim proteomima što predstavlja preduslov za njihovo dalje ispitivanje. Kičicu (Centaurium erythraea) karakteriše velika razvojna plastičnost i snažan morfogenetski potencijal, što je čini pogodnim model organizmom za istraživanja u razvojnoj biologiji. AGP su identifikovani kao značajan biohemijski marker somatske embriogeneze i organogeneze kičice i kao takvi mogu biti značajni za proces regeneracije kako biljaka gajenih u uslovima in vitro, tako i onih u prirodi. Povrede predstavljaju sastavni deo manipulacije biljnim tkivom in vitro i mogu indukovati morfogenetske procese. Odgovor biljke indukovan Yariv reagensom, koji specifično precipituje AGP, najsličniji je odgovoru indukovanom mehaničkim povredama biljnog tkiva. Može se pretpostaviti da postoji veza između procesa koji se odigravaju u i na ćeliji, a koji su inicirani mehaničkim povredama ili primenom Yariv reagensa kao i tokom indukcije somatske embriogeneze i organogeneze kičice. Nedavno sekvenciran transkriptom kičice uz razvoj ragp R paketa omogućio je identifikaciju velikog broja HRGP i AGP sekvenci kičice. Od identifikovanih sekvenci odabrano je osamnaest predstavnika za ispitivanje ekspresije u različitim uslovima. Ekspresija odabranih gena praćena je: a) 48 h nakon mehaničke povrede lista i korena biljaka kičice gajenih u uslovima in vitro b) u eksplantatima lista i korena kičice gajenih na različitim koncentracijama Yariv reagensa c) u različitim uzorcima biljaka gajenih in vitro, biljaka iz prirode i iz različitih morfogenetskih procesa kičice (somatske embriogeneze i organogeneze). Od odabranih osamnaest gena, CeAGp6, koji kodira kratak AG peptid, bio je izraženo indukovan nakon mehaničke povrede lista, a reprimiran nakon povrede korena. Moguće je da ovaj AG peptid uključen u određen tip prenosa signala nakon povrede biljnog tkiva. Pored njega CeFLA1, koji je pokazao povećanje ekspresije tokom 48 h nakon povrede lista, a indukovan je i u embriogenom kalusu, mogao bi biti deo mreže koja povezuje povrede i somatsku embriogenezu.

Arabinogalactan proteins (AGPs) are extensively glycosylated cell wall proteins belonging to the super family of O-glycosylated hydroxyproline-rich glycoproteins (HRGPs). They are characterized by a high diversity of the primary structure, they are encoded by a large gene family and are associated with a wide array of physiological roles in plant growth and development. AGPs are intrinsically disordered proteins which hinders their homology-based identification. In order to improve the methodology for identification and analysis of HRGP sequences, a new machine learning based approach was developed as part of this thesis. This approach exploits the main feature of HRGPs, the presence of the uncharacteristic amino acid hydroxyproline. A model for predicting proline hydroxylation probability based on local protein sequence has been incorporated into the ragp R package along with a number of diverse tools that allow the analysis of protein sequences such as: classification of HRGPs sequences, flexible scan for characteristic motifs, efficient communication with web servers for prediction of N-terminal signal peptides, glycosylphosphatidylinositol modification sites, disordered regions and domain annotation. The implemented pipeline enables adaptable identification of sequences where the choice of several parameters affects the stringency of the process and thus the number of sequences identified. This software allows efficient identification of HRGPs sequences in whole plant proteomes, which is a prerequisite for their further study. Common centaury (Centaurium erythraea) is characterized by an exceptional developmental plasticity and strong morphogenetic potential, which makes it a suitable model organism for developmental biology studies. AGPs have been identified as a significant factor during somatic embryogenesis and organogenesis of common centaury, and as such can be important for the regeneration process in both plants grown in vitro and plants from nature. Mechanical wounding is an integral part of plant tissue manipulation in vitro and can induce morphogenetic processes. The plant response induced by Yariv reagent, which specifically precipitates AGPs, is most similar to the response induced by mechanical wounding of plant tissue. It can be assumed that there is a connection between the processes that take place in and on the cell, which are initiated by mechanical wounding, application of Yariv reagents and during the induction of somatic embryogenesis and organogenesis. The recently sequenced C. erythraea transcriptome combined with the development of ragp R package allowed the identification of a large number of C. erythraea HRGP and AGP sequences. Eighteen representatives were selected from the identified sequences for expression analyses in different experimental conditions. The expression of selected genes was recorded: a) 48h following leaf and root mechanical wounding in vitro b) in leaf and root explants cultivated on different concentrations of Yariv reagent in medium c) in samples from plants cultivated in vitro, plants from nature and from different morphogenetic processes (somatic embryogenesis i organogenesis). Among the selected eighteen genes, CeAGp6, which encodes a short AG peptide, was significantly induced after mechanical wounding of leaf, and down-regulated after mechanical wounding of root. It is possible that this AG peptide is involved in some type of signal transduction after plant tissue wounding. CeFLA1 which showed a clear trend of increasing expression over time after leaf wounding, and is induced in embryogenic callus, could be part of a network linking wounding with somatic embryogenesis.