Uloga arabinogalaktanskih proteina u procesu morfogeneze in vitro kod kičice (Centaurium erythraea Rafn.)
The role of arabinogalactan proteins in centaury (Centaurium erythraea Rafn.) morphogenesis in vitro
Abstract:
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.
Keywords:
Centaurium erythraea Rafn.; In vitro culture; Organogenesis; Somatic embryogenesis; Antioxidative enzymes; Arabinogalactan proteins; Β-D- glucosyl Yariv (βglcy) reagent; Immunohistochemistry; FLA; DUF1070Source:
University of Belgrade, Faculty of Biology, 2015, 1-181Funding / projects:
- Physiological, chemical and molecular analysis of the diversity of selected rare and endangered plant species and application of biotechnology for ex situ conservation and production of biologically active compounds (RS-MESTD-Basic Research (BR or ON)-173024)
URI
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http://nardus.mpn.gov.rs/123456789/4174
https://radar.ibiss.bg.ac.rs/handle/123456789/2812