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Sestavení a zprovoznûní aparatury pro mûfiení
fluorescenãních spekter jednotliv˘ch molekul
TOMÁ· FESSL
Aparatura pro mûfiení fluorescenãních spekter jednotliv˘ch molekul byla sestavena, zprovoznûna a testována metodick˘mi experimenty na jednotlivé molekule DIOC, chlorofylu
a jednotlivém komplexu fotosystému I. Aktivnû jsem se podílel na sestavení a zprovoznûní
aparatury pro mûfiení spekter jednotliv˘ch molekul. Provedl jsem metodické experimenty za
úãelem ovûfiení funkãnosti aparatury. Aparatura je schopna mûfiení signálÛ na úrovni jednotliv˘ch molekul. Pfiíãinou potíÏí pfii mûfiení spekter chlorofylu je zpÛsob pfiípravy vzorkÛ. Není tedy zpÛsobena nízkou citlivostí aparatury. Tato potíÏ je podle mû pfiekonatelná a nijak nebrání v dal‰ím pouÏití aparatury. Mûfiení spekter chlorofylu nebylo hlavním cílem, jednalo se
pouze o mezikrok v cestû k biologick˘m aplikacím. Aparatura není primárnû urãena pouze
k mûfiení fluorescenãních spekter jednotliv˘ch molekul.Velmi zajímavá je také dynamika
fluorescenãních dûjÛ, sledování ãasové závislosti fluorescence umoÏÀuje fluorescenãní
korelaãní spektroskopie. Právû ta bude tedy dal‰ím krokem ve v˘stavbû aparatury.
Diplomová práce
Autor: Mgr. TomበFessl
Vedoucí práce:
Doc. RNDr. Franti‰ek Vácha, Ph.D.
Místo a datum obhajoby:
BF JU â. Budûjovice, kvûten, 2006
Kontakt: [email protected]
Denní zmûny fotosyntetické aktivity rozsivky
Thalassiosira weissflogii: vliv dynamického svûtelného
reÏimu a limitace dusíkem.
VOJTùCH KASALICK¯
V této magisterské práci jsem sledoval denní zmûny
fotosyntetické aktivity v promûnném svûtelném reÏimu u mofiské rozsivky Thalassiosira weissflogii pûstované v ustálenû rostoucí kontinuální kultufie. Podafiilo se mi zjistit pravidelné denní rytmy v koncentraci
pigmentÛ v buÀce, aktivitû fotosystému II, maximální
fotochemické úãinnosti, kinetice reoxidace primárního akceptoru QA ve fotosystému II, maximální rychlosti fixace CO2 fotoaklimaci buÀek a dal‰ích parametrÛ mûfien˘ch u buÀek rostoucích v kompletním
médiu i u buÀek limitovan˘ch dusíkem. Mnohé
z tûchto parametrÛ byly patrny i po zmûnû svûtelného reÏimu na konstantní. Jako jeden
z prvních jsem pozoroval v˘razné denní rytmy intenzitû thermoluminescence z fotosystému II, i ve tvaru thermoluminescenãní kfiivky.
Pro nov˘ pfiístroj od firmy P.S.I. jsem zavedl metodiku mûfiení thermoluminescence z fotosyntetick˘ch mikroorganismÛ. Tato jednoduchá metodika je vyuÏitelná pfii sledování
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denních zmûn v aktivitû fotosystému II, charakterizování fotosyntetick˘ch mutantÛ
i aktivity chlororespiraãních procesÛ. Poslední z tûchto moÏností lze sledovat pomocí
specifick˘ch inhibitirÛ oxidoreduktáz ãi infraãerven˘m záfiením. Dále jsem se zab˘val
metodickou otázkou rozdílu maximálního fluorescenãního v˘tûÏku získaného excitací
jedním krátk˘m zábleskem a celou sérií tûchto zábleskÛ („multiple turnover“) u izolovan˘ch thylakoidních membrán hrachu (Pea sativa).Fitováním kinetiky reoxydace primárního akceptoru lze urãit druh procesorÛ a jejich pfiíspûvek k nemûfienému fluorescenãnímu v˘tûÏku.
Diplomová práce
Autor: Mgr. Vojtûch Kasalick˘
Vedoucí práce: Doc. Ondfiej Prá‰il, Ph.D.
Místo a datum obhajoby:
Biologické centrum AV,
â. Budûjovice, kvûten 2006
Kontakt: [email protected]
The effect of diffusibility of atmospheric gas
components on stomata, leaf development and gas
exchange betwwen leaf and atmosphere
JANA TOM·ÍâKOVÁ
The aim of thesis was to find out whether diffusibility of gas component in external
atmosphere plays a role in development of leaves (especially on stomata). Plant grown
in He10x (mixture of He 79%, O2 21% and CO2 and H2O added) where diffusivity was
2.3 times higher, in comparison to plant grown in standard ambient atmosphere
(N2 79%, O2 21% and CO2 and H2O) showed changes in stomatal patterning.
The hypothesis was that the main effect causing the changes was concentration of CO2
in ambient air and/or relative humidity of the air. This hypothesis was further tested.
Diplomová práce
Autor: Mgr. Jana Tom‰íãková
Vedoucí práce: Doc. Ing. Jifií ·antrÛãek, CSc.
Místo a datum obhajoby:
MBÚ, TfieboÀ, kvûten, 2006
Kontakt: [email protected]
Heterogeneity in photosynthetic performance as
a result of metabolic, physiological and genetic
regulation of photosynthesis
NAILA FERIMAZOVA
Three following projects were major components of the thesis. (1) Photosynthetic
oscillations in individual cells of leaves and isolated leaf cell protoplast of four
Brassicaceae species were studied and characteristics of frequency spectrum and
dumping of oscillations under different actinic light irradiances and gas composition
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have been analyzed. (2) Interactions between photosynthesis and nitrogen fixation in
the non-heterocystous marine cyanobacterium Trichodesmium IMS101 have been
investigated. (3) Regulation of photosynthesis during heterocysts differentation in
Anabaena PCC 7120 has been compared between three different sets of the step-down
experiment. All three projects were performed using two-dimensional (imaging)
technique and Fluorescence Kinetic Microscope that allowed the measurements of
chlorophyll fluorescence kinetics at a single-cell level.
Doktorská disertaãní práce
Autor: Naila Ferimazova
Vedoucí práce: RNDr. Ivan ·etlík, CSc.
Místo a datum obhajoby:
MBÚ, TfieboÀ, ãerven,2006
Kontakt: [email protected]
Electrochemical biosensor for herbicide detection
based on the Photosystem II complex.
JAN MAL¯
Environmental monitoring is important for rapid assessment of the status of ecosystems.
Practical monitoring methods require rapid, simple, and low-cost screening procedures
for the detection of harmful chemicals in aquatic and soil environments. The
development of photosynthetic biosensors represents a valuable step towards the
improvement of pollutant monitoring in ecosystems.
Till now, biosensors have not yet achieved the prominent commercial position that was
initially predicted – due to the strict criteria which a successful biosensor has to meet in
order to become a commercial application. The demands of accurate response,
precision, reproducibility and linear response over the necessary analytical range, as
well as a long life for its biological component, are often not guaranteed. The key point,
usually influencing the overall performance of the biosensor, is the method used for the
immobilisation of the bioactive part. It determines the effectiveness of the interaction
between the biochemical recognition moiety and the electrochemical transducer.
The purpose of the present work has been to study various immobilisation methods in
order to optimize the photosystem II-based biosensor for the detection of herbicides.
The application of several approaches based on: (i) immobilisation of PSII by grosslinking in gel; (ii) immobilisation of recombinant PSII in the form of monolayers with
mediated signal transduction; and (iii) mediator-less electron transport using PSII
immobilised. Moreover, a novel immobilisation method suitable for the
electrochemically-driven and spatially-distinguished immobilisation of biomolecules in
a chip-array has been developed and presented.
Doktorská disertaãní práce
Autor: Jan Mal˘
Vedoucí práce: Doc. RNDr. Jifií Masojídek, CSc.
Místo a datum obhajoby:
Západoãeská universita, PlzeÀ, listopad, 2005
Kontakt: [email protected]
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The PsbH protein: A small membrane protein of the
Photosystem II as a model for structural and functional
studies
ZBYNùK HALBHUBER
Interaction of the PsbH protein of the photosystem II was analyzed by combination of CD,
NMR spectroscopy, and computer modelling. CD and NMR spectroscopy indicated that
interactions of the PsbH protein to all bilayer-forming lipids, including those extracted
directly from cyanobacterial thylakoid membranes, interacted with the PsbH protein nonspecifically. The molecular dynamic calculation of the PsbH-bilayer forming lipid
interactions indicates that both salt-bridge formation and hydrogen bond formation in the
lipid-water interface are feasible and stabilize a fraction of at least 10 lipid molecules in
the neighbourhood of the protein. This may lead to formation of lipid domains which
were hypothesized to be involved in assembly of membrane protein complexes.
Doktorská disertaãní práce
Autor: Zbynûk Halbhuber
Vedoucí práce: RNDr. Dalibor ·tys, CSc.
Místo a datum obhajoby:
ÚFB JU, Nové Hrady, fiíjen, 2006
Kontakt: [email protected]
Limitation of Photochemical and Non-photochemical
Processes of Photosynthesis in Lichens under Extreme
Light
HANA VRÁBLÍKOVÁ
The main aim of this thesis was to evaluate the response of photosynthetic apparatus
and photoprotective compounds (zeaxanthin and glutathione) of different lichen
species to short-term high-light (HL) exposure. Hydrated thalli of several lichen species
collected in maritime Antarctica (Usnea antarctica, Umbilicaria decussata, Umbilicaria
antarctica) and in the Czech Republic (Lasallia pustulata) were exposed under
laboratory conditions to short-term HL to evaluate the response of photosystem II (PSII),
zeaxanthin (Z) and glutathione (GSH) pool and its redox state. The response of PSII was
measured by means of modulated fluorometer using chlorophyll (Chl) fluorescence
induction curve supplemented with analysis of quenching mechanisms. Z (expressed as
de-epoxidation state of xanthophyll cycle pigments, DEPS) and GSH were determined
by means of HPLC analysis. All Chl fluorescence parameters and antioxidants levels
were evaluated before and several times after exposition to HL in order to characterise
the extent of photoinhibition and subsequent recovery.
Short-term HL exposure (1500–2000 mmol m–2 s–1 for 30 min, at 5 °C or at 20 °C) of
fully hydrated thalli led to a decrease in FV/FM and fII in all studied species.
Particularly higher degree of photoinhibiton was found in fruticose (Usnea antarctica)
than in foliose (Umbilicaria decussata) lichen species. Immediately after HL treatment,
U. decussata exhibited a lower HL-induced decrease of FV/FM than U. antarctica.
Values of FV/FM fully recovered in U. decussata after 3 h in dark, while they did not in
U. antarctica. The interspecific difference in the extent of photoinhibition was also
apparent in fII. It could be, therefore, suggested that both potential and actual
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photosynthetic processes might be more photoinhibited in the lichen with fruticose
rather than foliose thallus type when exposed to HL under full thallus hydration.
In all studied species, FV/FM and fII values fully recovered during subsequent recovery
lasting several hours. However, species-specific differences were apparent. Fast phase
of recovery was distinguished, especially in U. antarctica, as a steep rise of FV/FM
values within the first 40 min after the end of photoinhibitory treatment. Slow phase of
recovery was characterized by less pronounced FV/FM rise ongoing within following 4 h.
High values of non-photochemical quenching (NPQ) induced by HL recovered much
faster and in more extent in U. decussata than Usnea antarctica. Therefore, the capacity of
the photoprotective mechanisms involved into NPQ may be considered to be higher in the
former species. In Umbilicaria antarctica, NPQ was highly correlated to oxidized
glutathione (GSSG) and Z values, indicating the involvement of these antioxidants in the
resistance of U. antarctica to HL stress. However, the Z-related protection was not involved
equally in the two studied species. In L. pustulata, maximum level of Z was reached at
relatively low NPQ. This indicates that Z formation represented a major component of nonphotochemical quenching of L. pustulata. In U. antarctica, contrastingly, the maximum
level of Z was reached at very high NPQ. Also different slope of DEPS to NPQ relation may
suggest that in U. antarctica, other mechanism than Z formation played substantial role in
non-photochemical dissipation of absorbed light energy.
Exposure of Umbilicaria antarctica and Lasallia pustulata to HL led to a decrease of
total GSH and an increase in oxidized glutathione (GSSG). In the subsequent recovery,
total amount of GSH was resynthetised slowly. The content of total GSH differed
between Umbilicaria antarctica and Lasallia pustulata. Thalli of L. pustulata contained
nearly twice as much total GSH than U. antarctica, and the decrease of total GSH after
the HL exposure was lower in L. pustulata. Obtained data suggested that L. pustulata
could be considered generally more resistant to stress than U. antarctica.
In conclusion, the results suggest that in situ photoinhibition is likely in lichens. The
capacity of the photoprotective mechanisms in fully hydrated thalli, although different
in the studied species, was sufficient in coping with HL-induced stress in all the
species. This finding might be of great importance, suggesting that lichens may resist HL
doses in the field under those microclimatological conditions that make drying of the
thallus impossible.
The results of the thesis were summarized in the bellow-listed publications
[I] BARTÁK M, VRÁBLÍKOVÁ H & HÁJEK J (2003) Sensitivity of photosystem 2 of Antarctic
lichens to high irradiance stress: A fluorometric study of fruticose (Usnea antarctica)
and foliose (Umbilicaria decussata) species. Photosynthetica 41: 497-504.
[II] BARTÁK M, HÁJEK J, VRÁBLÍKOVÁ H & DUBOVÁ J (2004) High-light stress and
photoprotection in Umbilicaria antarctica monitored by chlorophyll fluorescence
imaging and changes in zeaxanthin and glutathione. Plant Biology 6: 333-341.
[III] VRÁBLÍKOVÁ H, BARTÁK M & WÖNISCH A (2005) Changes in glutathione and
xanthophyll cycle pigments in high light-stressed lichens Umbilicaria antarctica and
Lasallia pustulata. Journal of Photochemistry and Photobiology, B: Biology 79: 35-41.
Ph.D. thesis
Author: Mgr. Hana Vráblíková
Head of Ph.D. thesis: Doc. Milo‰ Barták, CSc.
Place and time of defence:
Department of Plant Physiology and Anatomy
Faculty of Science, Masaryk University,
Brno 2005
Kontakt: [email protected]
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Fyziologické vlastnosti li‰ajníkov˘ch fotobiontov
rodu Trebouxia
PETER VÁCZI
Dizertaãná práca sa zaoberala ‰túdiom fyziologick˘ch vlastností li‰ajníkov˘ch symbiotick˘ch rias rodu Trebouxia. Cieºom práce prispieÈ k prehæbeniu poznatkov o vplyve teploty, svetla, toxicity ÈaÏk˘ch kovov a osmotického stresu na rast a procesy primárnej
fotosyntetickej produkcie u li‰ajníkov˘ch fotobiontov. Táto problematika bola rie‰ená
v sérii experimentov. V experimentoch s toleranciou fotobiontov voãi medi boli v sérii
testov porovnávané odpovede postupnou selekciou (postupne sa zvy‰ujúcim selekãn˘m
tlakom obsahu iónov Cu2+ v médiu) získaného „tolerantného“ kmeÀa a „divokého“ kmeÀa li‰ajníkovej symbiotickej riasy Trebouxia erici. Anal˘zou rastu oboch kultúr i anal˘zou obsahu chlorofylov boli medzi kmeÀmi dokázané signifikantné rozdiely a tolerantn˘ kmeÀ vykazoval niekoºkonásobne lep‰ie charakteristiky rastu (pestovanie na
celulózo-acetátov˘ch diskoch) a produkcie (anal˘zy obsahu chlorofylov) i na médiách
s obsahom medi aÏ 4 mmol.l–1 ako kmeÀ divok˘. V˘ledky poukázali na rozdiely v toxicite medi medzi kultúrami pestovan˘mi na pevnom (obsah Cu2+ 4 mmol.l–1) a v tekutom
médiu (obsah Cu2+ 0,5 mmol.l–1).
Dal‰ia skupina experimentov studovala vplyv oÏiarenosti a sacharózou indukovaného
osmotického stresu na vzÈah primárnych fotochemck˘ch dejov na úrovni PS II a biochemick˘ch parametrov. U nestresovanej riasovej suspenzie, bol vzÈah medzi OER (r˘chlosÈ
v˘vina kyslíka) a fII (kvantov˘ v˘ÈaÏok elektrónového transporu fotosystémom II) lineárny. To svedãí o v˘raznej efektivite prenosu absorbovanej energie fotónu v rámci fotochemick˘ch a biochemick˘ch procesov fotosyntézy. V podmienkach osmotického stresu bol
sledovan˘ vzÈah OER a fII nelineárny (krivkov˘) a bola zistená v˘razne niωia OER
(v porovnaní s kontrolou), ão bylp pripísané zníÏenej fotosyntetickej fixácii CO2 u stresovanej suspenzie T. erici kvôli zv˘‰enej fotorespirácii. Zvlá‰È pri vysok˘ch PPFD
(500 mmol.m–2.s–1) za osmotického stresu dochádzalo u T. erici k váÏnemu zníÏeniu
OER [0,04 pg(O2).(1000 buniek)–1.s–1]. Bolo zistené, Ïe osmotick˘ stres inicioval nárast
nefotochemického zhá‰ania (NPQ), predov‰etk˘m komponenty qE. Z dosiahnut˘ch v˘sledkov vyplynulo, Ïe u osmoticky dehydratovan˘ch buniek riasového symbionta (T. erici) je práve qE hlavnou zloÏkou NPQ.Je moÏné zhrnúÈ, Ïe v protiklade s kontrolou,
osmoticky stresovaná T. erici vykazuje nelineárny vzÈah fII a OER. Zistenú nelinearitu je
dobré maÈ na zreteli v terénnych ekofyziologick˘ch ‰túdiách li‰ajníkov vyuÏívajúcich v˘hradne techniky Chl fluorescencie. V niektor˘ch prípadoch, predov‰etk˘m pri ãiastoãnej
dehydrácii, nemôÏe byÈ teda PN priamo odvodená od fII v dôsledku nepredvídateºného
vzÈahu medzi fII a PN. Preto, pre presné zhodnotenie fotosyntetick˘ch procesov u ãiastoãne dehydrovan˘ch li‰ajníkov je pouÏitie paralelného fluorometrického (fII) a gazometrického (PN) merania nevyhnutnosÈou.
Doktorská disertaãná práca
Author: Mgr. Peter Váczi
Vedoucí práce: Doc. Milo‰ Barták, CSc.
Místo a datum obhajoby:
Katedra fyziologie a anatomie rostlin
Pfiírodovûdecká fakulta, Masarykova Univerzita,
Brno 2005
Kontakt: [email protected]
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Mechanizmy smrti v bunûãn˘ch liniích tabáku
s geneticky modifikovanou citlivostí vÛãi PCD
indukujícím faktorÛm
OND¤EJ SMETANA
Cílem práce bylo ovûfiit platnost hypotézy o úloze cytokininÛ jako pfiirozen˘ch induktorÛ programované bunûãné smrti (programmed cell death – PCD) u rostlin. Jako modelov˘ materiál byly vyuÏity bunûãné linie tabáku – a to jednak pÛvodního „wild“ typu linie
BY-2, jednak novû pfiipravené linie transgenní. Jako potenciální PCD induktor slouÏil
6-benzylaminopurin (BAP). V˘sledky byly získány pomocí ‰irokého spektra molekulárních, bunûãn˘ch i biochemick˘ch anal˘z a lze je shrnout do následujících závûrÛ:
1) Pfiídavek BAP do kultivaãního media ovlivÀuje fenotyp bunûãné linie BY-2 v závislosti na aktuálním fyziologickém stavu kultury. Inokula nízké denzity ve fázi exponenciálního rÛstu jsou nejcitlivûj‰ím biotestem.
2) Nízké koncentrace BAP (cca do 20 mM) nevyvolávají smrt bunûk, pfii dlouhodobé
aplikaci mûní fenotyp kultur (porucha polarity bunûk i agregátÛ) a mohou vést k postupné selekci BAP – tolerantních sublinií.
3) Vysoké koncentrace BAP (cca nad 30 mM) velmi rychle sníÏily viabilitu kultury, indukovaly tedy bunûãnou smrt, detekovatelnou rÛzn˘mi technikami.
4) SniÏování viability kultur bylo doprovázeno specifickou fragmentací DNA, která byla
detekována jiÏ v brzk˘ch fázích úãinku vysok˘ch koncentracích BAP. Rychlost fragmentace DNA korelovala s dynamikou zmûn v architektufie bunûk.
5) Cytologická charakteristika kultur potvrdila v˘skyt nejménû dvou typÛ PCD vyvolané
vysok˘mi dávkami BAP. V prvním pfiípadû ‰lo zfiejmû o PCD autofagického typu, kdy
se po vakuolárním kolapsu vylily hydrolytické enzymy do cytosolu, coÏ bylo doprovázeno smr‰Èováním bunûãného obsahu. V druhém pfiípadû k autofagii nedocházelo.
6) Byly vytvofieny 4 transgenní linie exprimující geny s „funkãním vztahem“ k PCD – Ïivoãi‰né Bax (obecnû fieãeno, PCD v Ïivoãi‰n˘ch systémech podporující), Bcl-2 (PCD
potlaãující ) a pÛvodnû rostlinné, leã nikoliv tabákové Bax – inhibitorové BI-1/GFP
a BI-1 AS (antisense). Nûkteré z transgenních linií mûly sice transformací pozmûnûn˘
fenotyp (ztráta polarity), nicménû byly nadále pouÏitelné pro detailní cytologickou
anal˘zu. Vnitrobunûãné zmûny vyvolané BAP byly obecnû a kvalitativnû srovnatelné
s chováním pÛvodní linie BY-2, rozsah reakce v‰ak byl ovlivnûn transformací v souladu s pfiedpokládanou funkcí tûchto genÛ. PfiedbûÏné studie s liniemi BY-2/Bax1
a BY-2/Nt3 (s vnesen˘m Bax- inhibitorem v antisense formû) potvrdily oãekávanou
vy‰‰í senzitivitu k PCD indukujícímu efektu BAP. To mj. odpovídá pfiedstavû, Ïe
v PCD reakci na vysoké koncentrace BAP se úãastní systém apoptotick˘ch „caspase-like“ enzymÛ.
7) Byla vytvofiena transgenní linie BY-2/Y3, exprimující fúzní gen pro CFP a YFP a umoÏÀující tak sledovat „caspase-like“ proteázovou aktivitu technikou FRET anal˘zy na
úrovni jedné konkrétní buÀky. Díky této metodû, vÛbec poprvé pouÏité pro tyto anal˘zy u rostlin, byly rozli‰eny tfii typy bunûk. Typ 1 odpovídal buÀkám, které zfiejmû
pfiedãasnû ukonãily proces PCD. FRET anal˘za v dal‰ích dvou typech ukázala aktivitu proteáz bûhem exekuãní fáze PCD, v jednom pfiípadû (typ 3) se zfiejmû jedná o aktivaci metakaspáz a v druhém (typ 2) o aktivitu VPE. Tato pozorování jsou ve shodû
s v˘sledky uveden˘mi v bodû 3.
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Na základû obdrÏen˘ch v˘sledkÛ nebylo moÏné s urãitostí odpovûdût na otázku, nakolik je moÏno pozorovan˘ letální úãinek BAP povaÏovat za prÛkaz jedné z jejich standardních funkcí in vivo, ãi za pouh˘ toxick˘ úãinek nefyziologicky vysok˘ch koncentrací dané látky. K jejímu objasnûní je nezbytná dal‰í analytika, kupfi. definující dynamiku
skuteãn˘ch aktuálních hladin funkãních – tedy voln˘ch – cytokininÛ v situacích jimi fiízené cytodiferenciace resp. histogeneze, zahrnující i PCD.
Diplomová práce
Autor: Mgr. Ondfiej Smetana
Vedoucí diplomové práce:
Zdenûk Opatrn˘
Místo a datum obhajoby:
Katedra fyziologie rostlin PfiF UK,
Praha 2006
Somatická embryogeneze smrku ztepilého (Picea abies
(L.) Karst.): Ovlivnûní v˘tûÏkÛ a kvality embryí
zmûnami v dostupnosti sacharidÛ bûhem zrání
na tekut˘ch médiích
NIKOLA DRÁÎNÁ
Abstract:
Somatic embryogenesis of Norway spruce (Picea abies) presents both promising means
of propagation and valuable model system for investigating the structural, physiological
and molecular events occurring during embryo development. Somatic embryogenesis is
a process in which a bipolar structure, somatic embryo develops from non-zygotic cell
through series of developmental steps similar to zygotic embryogenesis. The efficiency
of every phase depends on the previous ones. Important changes in structure and
physiology of somatic embryo happen in maturation phase. Embryo accumulates
storage compounds (saccharides, lipids and proteins), that are necessary for postmaturation phases. Manipulation of the culture conditions during the maturation can
increase both quality and number of embryos produced in culture. Between factors that
influence somatic embryogenesis belong growth regulators, osmotic stress and
carbohydrates. Maturation is supported considerably by abscisic acid and nonpenetrating osmotica (e.g. PEG). The role of carbohydrates during maturation phase has
not been fully elucidated yet. Carbohydrates serve as source of energy and carbon,
osmotica, participate in protection of membrane and recently it is assumed that can be
signal molecules.
Presented study was made using the embryogenic line genotyp Picea abies AFO 541
(AFOCEL, France). Embryogenic culture was grown on Gupta and Durzan modified
liquid medium supplemented with ABA and different types of carbohydrates. To
contribute to elucidation of the role of carbohydrates during somatic embryo
maturation four variants of maturation of SE were conducted: 1) medium with 3%
sucrose and one week subcultivation interval, 2) medium with 1,57% glucose and
1,57% fructose and one week subcultivation interval, 3) medium with 3% sucrose and
subcultivation every second, fourth and seventh day in a week, which lead to limitation
of sucrose hydrolysis in medium, 4) medium with 3% sucrose first two days, 2,25%
sucrose next two days and 1,5% sucrose next three days in week.
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Substitution of sucrose in maturation medium by glucose and fructose decrease yield of
mature somatic embryos. Limitation of sucrose hydrolysis in medium lead to partial
decrease of yield of mature somatic embryos. However, the significant decrease in
embryo yield was also observed in cultures grown on a series of media with declining
sucrose/hexoses ratio and three transfers to a new medium per week. Content and
spectrum of endogenous carbohydrates in somatic embryos during maturation were
similar in all variants. Analysis of maturation media confirm, that sucrose presented in
media is completely hydrolyzed to glucose and fructose after one week cultivation.
Dynamics of pH of media during six weeks of maturation of SE was similar in all
variants. There was a decrease of pH from the first week to the lowest value in the third
week of cultivation. From the fourth week, increase of pH of media until the end of the
sixth week was observed. There was not statistic difference in content of storage lipids
and proteins in SE after maturation among variants.
The effect of non-penetrating osmoticum PEG in maturation media on accumulation of
storage proteins and lipid in somatic embryos was studied. It was found, that the content
of storage lipids was higher in somatic embryos treated by PEG during maturation.
Diplomová práce
Autor: Mgr. Nikola DráÏná
Vedoucí diplomové práce:
RNDr. Helena Lipavská, Ph.D
Konzultant Mgr. Martin Kube‰
Místo a datum obhajoby:
Katedra fyziologie rostlin PfiF UK,
Praha, podzim 2006
Effect of Schizosaccharomyces pombe cdc25
expression on morphogenesis in tobacco Nicotiana
tabacum L., cv. Samsun
Lucie Uchytilová
Abstract:
Morphogenetic processes including process of de novo organ
formation is governed by the rate, frequency and orientation
of cell division. Cell cycle is a complex process with
complicated regulation. CDKs (cyclin-dependent kinases) are
taken for the key proteins regulating cell cycle progression.
These proteins are well conserved among eucaryotic
organisms. CDK activity strictly depends on specific subunit –
cyclin. The activity of the CDK-cyclin complex can be further
modified besides others by phosphorylation/dephosphorylation. In yeast (and in most eukaryotic organisms)
activating defosforalation of CDKs is performed by Cdc25
phosphatase. Situation in plants is still not fully elucidated.
A small CDC25-like tyrosine-phosphatase was recently identified in Arabidopsis thaliana
(Arath;CDC25), which could not, however, complement the temperature sensitive
Spcdc25-22 mutant (Landrieu et al., 2004).
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This study is focused on the effect of expression of the foreign gene (S. pombe) coding
for Cdc25 phosphatase on the morphogenesis, especially on the ability of de novo
organ formation, in tobacco. Plant material used for experiments: Nicotiana tabacum
L., cv. Samsun, wild type, two independent lines transformed with Spcdc25 under
a constitutive promotor (designated as lines A and C) (Bell et al., 1993) and inducible
line with Spcdc25 under tetracycline-inducible promotor (designated as Tx) (McKibbin
et al., 1998). Cultivation of internode stem segments proceeded on MS medium with or
without growth regulators.
The transformation led to dramatic change in process of de novo organogenesis. On
medium without growth regulators the significantly higher amounts of de novo formed
buds were found in A and C lines, in Tx line on medium with tetracycline (inductive
conditions) compared to Tx line on medium without tetracycline and wild type on the
both types of media. Application of tetracycline effected the process of organogenesis
and led to increase in the amounts of de novo formed buds on the segments of lines
A and C. To study Cdc25 involvement during particular stages of de novo organogenesis
different arrangements of inducible condition were used – time of exposure to tetracyclin
was limited to 3, 5 or 7 days out of 21 days of cultivation. The results indicate that the
first 5 - 7 days of cultivation under inductive conditions are essential for the process of
de novo shoot formation. As lovastatin had no effect on de novo organ formation on the
stem segments of Spcdc25 transformed plants, it is likely that Cdc25 affects the process
of organogenesis de novo independently of the endogenous level of cytokinins.
The transformation led also to differences between transformed and wild type plant
material as regards shoot apical meristem structures at very early developmental stages
under in vitro conditions. It could be causation of observed flowering of nodal-stemsegment-derived transformed plants under in vitro conditions.
The expression of Spcdc25 affects organisation of cells and accumulation of starch in
internodal-stem-segment-derived cell suspension cultures.
The results confirmed the hypothesis that the transformation with S. pombe cdc25 has
cytokinin-like effect on morphogenetic processes and support the model of upregulation of a plant Cdc25 homolog by cytokinin.
Diplomová práce
Autor: Mgr. Lucie Uchytilová
Vedoucí diplomové práce:
RNDr. Helena Lipavská, Ph.D
Konzultant Mgr. Petra Ma‰ková
Místo a datum obhajoby:
Katedra fyziologie rostlin PfiF UK,
Praha, podzim 2006
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