The role of arabinogalactan proteins in centaury (Centaurium erythraea Rafn.) morphogenesis in vitro

Kičica (Centaurium erythraea Rafn.) je biljna vrsta širokog spektra lekovitih dejstava, koja se odlikuje visokom regenerativnom sposobnošću u uslovima in vitro kulture. Osnovni cilj disertacije bio je izučavanje pojedinih biohemijskih i molekularnih aspekata in vitro morfogeneze kičice sa svrhom rasvetljavanja osnova razvojne plastičnosti ove biljne vrste koja se ispoljava u uslovima in vitro kulture. U ovom istraživanju je in vitro morfogeneza kičice indukovana iz odsečaka listova na svetlosti i u mraku. Na hranljivoj podlozi bez regulatora rastenja i na svetlosti odvijao se proces direktne organogeneze pupoljaka, a u mraku proces direktne rizogeneze. Regeneracija biljaka putem indirektne somatske embriogeneze (SE) je postignuta na hranljivim podlogama sa regulatorima rastenja 2,4-D i CPPU i na svetlosti i u mraku, dok je na istim podlogama, za regeneraciju pupoljaka indirektnom organogenezom, svetlost bila obligatorni faktor. Anatomska istraživanja su pokazala da je razvoj somatskih embriona bio asinhron, a na svetlosti se odvijao simultano sa razvojem adventivnih pupoljaka. U ovim istraživanjima je uspostavljen model-sistem pogodan za komparativnu analizu in vitro morfogenetskih procesa kod kičice na molekularnom i biohemijskom nivou. Ispitana je ukupna aktivnost antioksidativnih enzima, superoksid dismutaza (SOD), katalaza (CAT) i peroksidaza (POX), kao i zastupljenost i aktivnost pojedinih izoformi ispitivanih enzima tokom različitih morfogenetskih procesa. Analize su pokazale da su SOD i CAT uključene u antioksidativnu zaštitu listova kičice od oštećenja izazvanih povredom, do koje dolazi prilikom izolovanja eksplantata, dok je totalna aktivnost POX bila snažno indukovana tokom morfogeneze na svim ispitivanim hranljivim podlogama. Uloga arabinogalaktanskih proteina (AGP), proteina ćelijskog zida bogatih hidroksiprolinom, tokom različitih morfogenetskih procesa in vitro kičice istražena je primenom nekoliko pristupa. Dodavanje β-D-glukozil Yariv reagensa (βGlcY), koji specifično vezuje i precipitira AGP i na taj način sprečava AGP da obave svoju funkciju, u hranljive podloge dovelo je do smanjenja broja somatskih embriona i adventivnih pupoljaka po eksplantatu. Uporednom analizom je nađeno da se ispitivani morfogenetski procesi razlikuju po svojoj osetljivosti na βGlcY, te da se po rastućoj senzitivnosti mogu poređati kao: direktna rizogeneza (neosetljiva) < indirektna organogeneza pupoljaka < direktna organogeneza pupoljaka << indirektna SE (veoma osetljiva). Količina AGP, određena metodom radijalne difuzije, povećavala se tokom indukcije različitih morfogenetskih procesa. Metodom ukrštene elektroforeze je pokazano da su različiti morfogenetski procesi praćeni sintezom različitih setova AGP. Korišćenjem monoklonalnih antitela LM2, JIM4 i JIM13, specifičnih za ugljenohidratne delove AGP molekula, imunohistohemijski su lokalizovani specifični AGP epitopi u ćelijskom zidu i pokazana njihova razvojna regulacija tokom SE. Polazeći od sekvenciranog i rekonstruisanog transkriptoma korena i lista kičice, identifikovana su četiri AGP transkripta, označena kao CeAGP1 – 4. Sekvence ovih transkripata su potvrđene direktnim sekvenciranjem, dok je bioinformatičkom analizom pokazano da CeAGP1, CeAGP2 i CeAGP4 kodiraju AGP sa fasciklinskim domenima (FLA), dok je CeAGP3 AG-peptid sa konzervisanim DUF1070 domenom. Analizom ekspresije ovih gena qRT-PCR metodom je pokazano da se CeAGP1 specifično indukuje tokom indirektne SE, dok se CeAGP3 eksprimira tokom morfogeneze na svim hranljivim podlogama. U ovom radu je kičica po prvi put predložena kao odličan model-organizam u biologiji razvića biljaka, koji je pogodan za proučavanje biohemijskih i molekularnih aspekata in vitro morfogeneze. Dobijeni podaci o ulozi antioksidativnih enzima i AGP proteina tokom morfogeneze kičice in vitro predstavljaju prve rezultate istraživanja morfogeneze na biohemijskom i molekularnom nivou kod ove vrste.Common centaury (Centaurium erythraea Rafn.) is a plant species with a broad spectrum of medicinal effects, characterized with high regenerative potential under conditions of in vitro culture. The main objective of the current dissertation was the study of some biochemical and molecular aspects of in vitro morphogenesis of centaury, with an aim to elucidate the basis of its developmental plasticity that is manifested in in vitro culture. In the current study the in vitro morphogenesis was induced on light and in darkness. Direct shoot organogenesis was induced on growth regulator-free culture media on light, whereas on the same media in darkness, direct rhizogenesis occurred. Plant regeneration via indirect somatic embryogenesis (SE) was accomplished on growth media with growth regulators 2,4-D and CPPU on both light regimes, while for shoot regeneration by indirect organogenesis, light was an obligatory factor. Anatomical studies revealed that the development of somatic embryos was asynchronous and proceeded simultaneously with the development of adventitious buds on light. Thus, this work established a model-system for comparative analysis of in vitro morphogenetic processes in centaury on molecular and biochemical level. Total activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), as well as the presence and activities of their isoforms during different morphogenetic processes, were also determined. It was shown that SOD and CAT were involved in antioxidative protection of centaury leaves wounded during explant isolation, whereas total POX activity was strongly induced during morphogenesis on all growth media. The role of arabinogalactan proteins (AGPs), plant cell-wall hydroxyproline-rich proteins, during different morphogenetic processes in vitro was investigated using several approaches. The addition of β-D- glucosyl Yariv reagent (βGlcY) that specifically binds to, precipitates and thus inactivates AGPs, to the culture media, caused a reduction of the number of somatic embryos and adventitious buds formed per explant. Comparative analysis revealed that the investigated morphogenetic processes differ in sensitivity to βGlcY, so that they can be arranged in order of increasing sensitivity to bGlcY as: direct rhizogenesis (insensitive) < indirect shoot organogenesis < direct shoot organogenesis << indirect SE (very sensitive). The AGP content, as determined by radial diffusion method, increased during induction of different morphogenetic processes. Using crossed electrophoresis method it was shown that different morphogenetic processes are accompanied by synthesis of different AGP sets. Using monoclonal antibodies LM2, JIM4 and JIM13, specific for carbohydrate moieties of AGP molecules, the specific AGP epitopes were immunohistochemically localized to the cell wall and their developmental regulation during SE was demonstrated. Starting from sequenced and assembled leaf and root centaury transcriptome, four AGP genes were identified and named CeAGP1 through 4. The transcript sequences were verified by direct sequencing, and the bioinformatics analysis revealed that CeAGP1, CeAGP2 and CeAGP4 encode AGPs with fasciclin (FAS) domain, whereas CeAGP3 is an AG peptide with a conserved DUF1070 domain. The expression analysis of these genes by qRT-PCR amplification showed that CeAGP1 is specifically induced during indirect SE, while CeAGP3 is expressed during morphogenesis on all growth media. In the current work, centaury is for the first time proposed as an excellent model organism in developmental plant biology, suitable for study of biochemical and molecular aspects of in vitro morphogenesis. The data obtained on the role of antioxidative enzymes and AGPs during in vitro morphogenesis in centaury represent the first study of morphogenesis in this species on biochemical and molecular level