b14. advances in plant biochemistry lectures l27.2 l27.1 ... · b14. advances in plant biochemistry...

2nd Congress of Biochemistry and Cell Biology, September 5th–9th, 2011, Kraków, Poland

B14. Advances in plant biochemistry

Lectures

L27.1

Polyisoprenoids — secondary metabolites or physiologically important superlipids?Liliana Surmacz, Ewa Swiezewska

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Polande-mail: Ewa Swiezewska <[email protected]>

The polyisoprenoid alcohols (dolichols and polyprenols) are found in all living organism, from bacteria to mammals. In animal and yeast cells polyisoprenoids are derived from the cytoplasmic mevalonate (MVA) pathway while in plants two biosynthetic pathways, the MVA and the plastidial methylerythritol phosphate (MEP) pathway provide pre-cursors for polyisoprenoid biosynthesis. The key enzymes of polyisoprenoid synthesis are cis-prenyltransferases (CTPs), responsible for construction of the long hydro-carbon skeleton. CPTs elongate a short all-trans precursor, oligoprenyl diphosphate, by sequential addition of the de-sired number of isopentenyl diphosphate molecules which results in formation of a stretch of cis units. Several genes encoding CPT have been cloned from bacteria, plants and mammals. Interestingly, in Arabidopsis, the tissue-specific expression of ten putative cis-prenyltransferases was ob-served. In contrast to polyisoprenoid phosphates serving as cofactors in the biosynthesis of glycoproteins, glucosyl phosphatidyl inositol (GPI) anchor or bacterial peptidog-lycan, the biological importance of polyprenols and doli-chols still remains a question of debate besides their func-tion of reservoir of substrates for kinase. These extremely hydrophobic superlipids are postulated to be involved in intracellular traffic of proteins and in cellular defense against adverse environmental conditions. Recent publica-tions show a direct link between the dolichol biosynthetic pathway and congenital disorders of glycosylation (CDG). These discoveries highlighting the cellular significance of polyisoprenoids simultaneously establish the background for future pharmacological interventions. Our mini-review summarizes the results of recent studies on polyisopre-noids.

L27.2

Mechanisms of chlorophyllase regulationM. Bojko, M. Michalik, J. Fiedor, A. K. Banaś, J. Łabuz, O. Sztatelman, H. Gabryś, L. Fiedor

Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Polande-mail: L. Fiedor <[email protected]>

A key step in chlorophyll biodegradation, one of the most spectacular biological processes on Earth, is mediated through chlorophyllase, catalyzing the removal of the C20 phytyl moiety. A question remains open as how the activity of chlorophyllase is regulated in vivo, especially in species which constitutively posses the enzyme. Our study on the detergent-reconstituted enzyme shows that chlorophyllide a and phytol, the products of chlorophyll a hydrolysis, af-fect the activity, but their effects are antagonistic: chloro-phyllide a activates chlorophyllase while phytol acts as the inhibitor. This indicates the existence of effector (chloro-phyllide) binding site on the enzyme, different from the catalytic site. The inhibitory activity of phytol is likely to be due to a negative feedback. Such regulation seems neces-sary to keep the rate of chlorophyllide generation in pace with the successive catabolic reactions leading to the deg-radation of this highly phototoxic product of chlorophyll biodegradation.In the in vivo study, we found that the levels of mRNA of two chlorophyllases involved in chlorophyll catabolism in Arabidopsis thaliana, products of the AtCLH1 and AtCLH2 genes, dramatically increase upon illumination with white light. Their expression is not related to photosynthesis but rather mediated by photoreceptors. In order to identify the photoreceptors involved, we used various light treatments and photoreceptor mutants (cry1, cry2, cry1cry2, phot1, phot2, phot1phot2, phyA phyB, phyAphyB). Our results demonstrate, for the first time, that the expression of enzymes involved in chlorophyll catabolism is light-controlled.

260 B14. Advances in plant biochemistry


L27.3

The role of COP (constitutive photomorphogenesis) proteins in the regulation of chlorophyll biosynthesis in ArabidopsisPrzemysław Malec, Paweł Jedynak, Elżbieta Turek, Beata Myśliwa-Kurdziel, Andrzej Waloszek

Department of Plant Physiology and Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Kraków, Polande-mail: Przemysław Malec <[email protected]>

In Arabidopsis, genes belonging to COP/DET/FUS pleio-tropic gene class, encode proteins constituting components of signalling pathways mediating the overall gene expres-sion response to light. In particular, the eight-subunit pro-tein complex (COP9 signalosome, CSN) and COP1 pro-tein have been found to be central regulators in seedling deetiolation.In angiosperms, during deetiolation, the light-triggered photoreduction of protochlorophyllide (Pchlide) to chlo-rophyllide (Chlide) has a key role in the regulation of chlorophyll (Chl) biosynthesis. In Arabidopsis, this pho-toreduction is catalyzed by 3 isoforms of light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (LPOR’s): the LPOR A — expressed in etiolated seedlings and active transiently at the beginning of illumination; the LPOR B — expressed and active both in etiolated seed-lings and green plants and the LPOR C — expressed spe-cifically upon illumination.The accumulation of Pchlide forms was studied in mutants of constitutive photomorphogenic phenotype (5-days-old etiolated seedlings), deficient in COP1 (cop1-4) and some of CSN subunits: CSN8 (cop9), CSN7 (fusca5) and CSN1 (cop11), by both low-temperature fluorescence spectrosco-py (at 77K) and HPLC. The remarkable overaccumulation of the total Pchlide has been found in cop1-4. In all analyzed mutants the dominant Pchlide form was Pchlide633. The lack of Chlide formation after flash illumination revealed that functional Pchlide:LPOR: NADPH complexes were absent in etiolated seedlings of both cop1-4 and csn mutants. Upon illumination with continuous light, a subsequent deg-radation of Pchlide633 was observed in csn mutants, where-as cop1-4 was able to accumulate Chlide. The formation of both Chl a and Chl b was observed in cop1-4 mutant. In contrast, csn mutants overaccumulated Chl a only in a dim light (5 µmol m-2 s-1). These observations correlated with the altered expression of LPOR isoforms in mutants. Our results indicate that both COP1 and CSN subunits are proteins essential for greening of Arabidopsis seedlings, playing partially overlapping role in the control of chloro-phyll biosynthesis pathway. In particular COP1 is a negative regulator of Pchlide precursor synthesis in the dark and, together with CSN complex, a positive regulator of LPOR activity in the light.

L27.4

Involvement of vitamin B1 (thiamine) in adaptation of plants to stress conditionsMaria Rapala-Kozik

Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Polande-mail: Rapala-Kozik <[email protected]>

Plants are the primary source of vitamins for human nutri-tion. The proper doses of vitamin B1 are critical for the functioning of human nervous system, heart and muscle. Its deficiency is associated with beri-beri disease, Wernicke-Korsakoff syndrome and thiamine responsive megaloblas-tic anemia. The main vitamin B1 compound used by all living organisms is thiamine diphosphate (TDP) that plays the cofactor function for enzymes involved in the key metabolic pathways which in plants include the acetyl-CoA synthesis, the tricarboxylic acid cycle, the pentose phos-phate pathway, the Calvin-Benson cycle and the isoprenoid biosynthesis pathway. As the plants are unable to escape environmental changes they evolved sensitive mechanisms of perception and adaptation that allow them to maintain the homeostasis for survival. One of these mechanisms in-volves the increased thiamine or TDP production observed in Arabidopsis thaliana and Zea mays plants under drought, salinity, osmotic or oxidative stress conditions. The expres-sion levels of two most important thiamine biosynthesis genes (thi1 and thiC) engaged in the independent forma-tion of pyrimidine and thiazole parts of thiamine molecule increase 2–5 fold in stressed plants. A similar up-regula-tion of the gene encoding thiamine pyrophosphokinase, the sole thiamine-activating enzyme, occurs in the plants under the stress conditions. These changes are correlated with the activation of other defense systems such as the production of antioxidants or the up-regulation of enzy-matic detoxification systems. A similar pattern of vitamin B1 overproduction is observed in plants treated with ab-scisic acid that suggests an involvement of this hormone in the regulation of thiamine biosynthesis during the per-ception of environmental changes. The increased level of thiamine in stressed plants is documented by the activation of the pentose phosphate pathway/the Calvin-Benson cy-cle as well as the tricarboxylic acid cycle. Additionally, the supplementation of plants by exogenous thiamine confers the oxidative protection to plants. Although the mechanism of this protection is not well understood, it corresponds to the findings that thiamine compounds overproduced in bacteria can play a role of stress alarmone or stress pro-tectant to enable the survival in unfavorable environments. In plants under stress conditions, vitamin B1 is active on the early signaling events as well as takes part in the adapta-tion processes.

B14. Advances in plant biochemistry 261


Oral presentations

O27.1

The C-terminal flexible segment of Rubisco large subunit from Thermosynechococcus elongatus as a hot-spot for specificity-changing mutations Beata Gubernator, Andrzej Szczepaniak

Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, Wrocław, Polande-mail: Beata Gubernator <[email protected]>

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Ru-bisco) catalyzes the addition of CO2 to ribulose-1,5-bi-sphosphate (RuBP) or the binding of O2 to RuBP. In cy-anobacteria, green algae and plants, Rubisco is composed of eight large (L) and eight small (S) subunits forming L8S8 complex. The dimmer L2 serves as the catalytic unit. The preference of the enzyme for CO2 versus O2 is represent-ed by specificity factor (SF). A high SF value means more efficient photosynthesis. Recently the molecular basis of Rubisco specificity was investigated in terms of structure and kinetics of the enzyme and the “time-window” (“t-w”) hypothesis was proposed. This model predicts that SF of Rubisco for CO2 increases with the rate by which the bind-ing niche, in the active site, opens. The flexible outer part of the binding niche consists of two elements (located at the L subunit): loop 6 and the C-terminal tail. According to the “t-w” hypothesis, destabilization of closed conforma-tion should lead to increase in SF value. Previously we have found that the Rubisco from thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher SF (SF~84) compared with the mesophilic Rubisco (SF~46). The analysis revealed that in Rubisco from T. el. internal salt bridge from residue 474 to 470 is weaker compared with mesophilic Rubisco due to the K474T substitution. Likewise, the substitution R131K seem to weaken the salt bridge to E470. Apparently, the thermophilic enzyme has C-terminal strand which is less well anchored on the protein surface what explains an enhanced specificity. SeqCat algo-rithm based on the “t-w” hypothesis pointed aminoacide substitutions in the sequence of L subunit of thermophilic Rubisco which could change the enzyme specificity. E470P mutation should weaken the stabilization of the mobile segment during catalysis causing acceleration of closing/opening cycle, simultaneously increasing enzyme SF. Sub-stitution A471T should stabilize the closed conformation of active site, resulting in decreasing of enzyme specificity. In this study the wild type of thermophilic Rubisco, LE470P and LA471T mutants were expressed, purified and SF values were measured. The SF values for the wild type Rubisco, LE470P and LA471T mutants were found to be: 83.9±1.9; 95.8±2.5; 56.9±1.2, respectively. The obtained results con-firm the “t-w” hypothesis which explains the molecular ba-sis of Rubisco specificity in term of the rate by which the binding niche, of catalytic center, opens.

O27.2

Indole-3-ylacetyl-aspartate synthetase from pea seeds — a new GH3 protein involved in IAA conjugationMaciej Ostrowski, Anna Jakubowska

Institute of General and Molecular Biology, Department of Biochemistry, Nicolaus Copernicus University, Torun, Polande-mail: Maciej Ostrowski <[email protected]>

Indole-3-ylacetyl-aspartate synthetase (IAA-Asp syn-thetase) was isolated via a biochemical approach from pea seeds during our previously studies. Biochemical and im-munochemical properties of the purified enzyme strong-ly suggested that it is a member of an acyl-adenylate/thioester-forming GH3 family. Thus, these proteins are ATP-dependent amidosynthetases catalyzing the forma-tion of amide-linked conjugates that are involved in hor-mone homeostasis. Recent genetic studies indicated the involvement of some GH3 genes in light responses via phytochrome A and B, and cryptochrome pathways or through cross-talk between auxin and light signaling. In-terestingly, there is some evidence that synthetic derivatives of IAA, so-called auxinic herbicides may also induced the GH3 expression.In this study we provide evidence that IAA-Asp synthetase from seeds of pea is a new member of GH3 protein fam-ily and report the effect of some phytohormones, auxinic herbicides and various light conditions on gene expression in vegetative tissues of pea.The purified IAA-Asp synthetase preparation was subject-ed to the trypsin digestion and the resulting peptides mix-ture was analyzed using LC/MS/MS. The comparison of the received results with NCBI protein sequence database confirmed that IAA-Asp synthetase belongs to the GH3 protein family. We have found 453-bp fragment of Pisum sativum genomic GH3-5 sequence tagged that is predicted to encode a GH3 polypeptide fragment containing 154 amino acids. BLAST searches of GenBank and Swiss-Prot showed that PsGH3-5 amino acid sequence fragment was similar to other plant IAA-amido synthetases.The influence of auxin (1-NAA), other phytohormones (ABA, BR, GA, JA, SA, kinetin), auxinic herbicides (2,4-D, Picloram, Dicamba) and different light conditions on expression of PsGH3-5 in pea seedlings was shown using RT-PCR after treatment each of effectors for 30 minutes. The obtained results indicated that the PsGH3-5 expression increased after treating with 5 µM of 2,4-dichlorophenoxy-acetic acid (2,4-D) 3,6-dichloro-2-methoxybenzoic acid (Dicamba), and 4-amino-3,5,6-trichloro-2-pyridenocarbox-ylic acid (Picloram). All tested phytohormones enhanced the accumulation of the PsGH3-5 transcripts in etiolated pea tissues. Moreover, pea seedlings exposed to red, far-red, blue and white light conditions exhibited an increase in PsGH3-5 expression. Based on these results, it has been suppose that the GH3 proteins may control the growth and development of plants to adapt themselves to environmen-tal conditions.



O27.3

Identifying of small regulatory RNAs that could be involved in plant response to herbicide stress at the level of translationAleksandra Szopa1, Tomasz Twardowski1,2

1Institute of Technical Biochemistry, Technical University of Łódź, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Science, Poznań, Polande-mail: Aleksandra Szopa <[email protected]>

Plants exposed to different conditions of abiotic and bi-otic stress, developed several mechanisms allowing them to adapt to adverse environmental conditions based on a regulation of gene expression at transcription and transla-tion levels. Recently it was shown that small non coding RNAs were also involved in plant response to stress condi-tions. So far many of RNAs were identified to take part in protecting the plant from stress causing factors.Various strains of Zea mays displayed different reactions to stress caused by using the herbicides in plant culturing. It is an important issue for agriculture to understand molecu-lar mechanism of plant response to stress causing factors. Testes herbicides are Roundup (glyphosate) and Titus (rim-sulfuron).We aim to identify small RNAs that can be involved in maize response to herbicide stress through the protein biosynthesis regulation at the level of translation. We use in vitro selection to obtain RNAs that bind with high af-finity and specificity to ribosomes isolated from Zea mays. The RNA molecules are being selected against ribosomes isolated from three different maize strains treated with her-bicides Roundup and Titus, displaying various reactions to stress conditions.We isolated ribosomes and enzymatic fractions from maize strains and the combinatorial library was designed. The bioinformatic analysis of library was carried out to avoid hybridization primers as well flanking sequences between eachother. So far we have carried out seven cycles of selec-tion. After seventh cycle we cloned and subjected obtained RNA molecules to sequencing.

O27.4

Localization of expression of cDNA sequences related to flower morphogenesis in cucumber’s (Cucumis sativus) floral budsMagdalena Pawelkowicz, Katarzyna Chądzyńska, Katarzyna Kurek, Cezary Kowalczuk, Zbigniew Przybecki

Warsaw University of Life Sciences, Department of Plant Genetics, Breeding and Biotechnology, Warsaw, Polande-mail: Magdalena Pawelkowicz <[email protected]>

Sex determination is one of the key developmental process-es in plant sexual reproduction. Elucidation of mechanism of plant sex determination has great practical significance in breeding, horticulture and agriculture. The mechanism by which this process is controlled is still poorly understood. Cucumber is an economically important monoecious spe-cies, and also a good model for study of sex determination. Several genes are involved in this process, but to date the molecular mechanism of sex determination in cucumber is unknown At the early stages of development, the cucumber floral primordia are initially bisexual, containing initials of both anthers and pistils, and sex determination occurs fol-lowing the selective arrest of the development of either the staminate or the pistillate primordial. The aim of the study was to localise the expression pattern of chosen genes in whorls of cucumber floral buds by in vitro and in situ RT-PCR methods. This transcript were selected from the dif-ferentional libraries coming from 1–2 mm cucumber’s flo-ral buds. Afters comparison to data base it was occurred that eight of the chosen transcripts were similar to EST sequences from 1–2 mm cucumber’s floral buds and ten clones were homologous to EST sequences from cucum-ber’s fruits. The signals of the transcripts were observed in two developmental stages of cucumber flower buds 1–2 mm and 3–5 mm of two pairs of isogenic lines of cucum-ber, monoecious B10 and female 2gg, and female Gy3 and hermaphrodite Hgy3. Some transcripts show the differenc-es in localization and intensity of the signals among buds within a pair of analyzed lines, as well as between develop-mental stages of a single line. Transcript CsB10dhB2_268 was expressed only in staminate primordial of B10 male floral bud, which may suggest that it is connected with ar-resting female generative primordial in male flowers. The expression pattern of transcript CsGy3dhGH_420 in 1–2 mm floral bud of Hgy3 line shows that it may be connected with anther development. Transcript Cs2gggd2B_12_Bam, which expression was observed in the staminate and the pistillate primordial of B10 male flower buds, may act in two ways, arresting development of stamen and promoting development of anthers. In conclusion the sixteen out of eighteen transcripts probably are involved in morphogen-esis and sex determination in cucumber’s flowers.AcknowledgementsThe study was supported by a grant N N302 3633 33.



Posters

P27.1

In vitro study on the spontaneous mechanism for the insertion of cytochrome b6 protein into the thylakoid membraneMalgorzata Piskozub, Jaroslaw Kroliczewski

University of Wroclaw, Faculty of Biotechnology, Wroclaw, Polande-mail: Jarosław Króliczewski <[email protected]>

The thylakoid membrane contains the major photosyn-thetic complexes: PS I, PS II, the cytochrome b6f and the ATP synthase. Each of them consists of several subunits. Formation of these functional complexes requires coordi-nated transport towards the membrane, insertion into or translocation across the membrane, and subsequent assem-bly into high-molecular-weight structures. Thylakoid mem-brane proteins are inserted by different mechanisms: the cpSRP, cpSec or the apparently spontaneous pathway. The SRP pathway appears to be a specialized mechanism for the post-translational insertion of many members of the light-harvesting complex super-family and requires binding of SRP to the substrate, GTP hydrolysis, and the assist-ance of proteins FtsY and Alb3. By contrast, the spontane-ous insertion pathway don’t require the presence of other proteins including Alb3 and in the absence of energy. In vitro assays for the transport and the spontaneous insertion of the chloroplast encoded cytochrome b6 by isolated pea thylakoids have been study. We used native or denatured cytochrom b6: isolated from Synechocistis sp. PCC 6803 or overexpressed in E. coli and synthetic cytochrome b6 with signal sequence from OE33. As a control we used PsbW a single-span protein inserted into the thylakoid membrane in an unassisted spontaneous manner. We have been unable to demonstrate posttranslational import of cytochrome b6 into isolated thylakoids membrane with or without stro-mal extract. In contrast, efficient import of preOE33-cy-tochrom b6 has been observed on the presence of stromal extract and also insertion of PsbW protein. Strong pos-sibility is that the import of cytochrom b6 involves the operation of at least one stromal element of the transloca-tion machinery. We also don’t observe incorporation of cytochrome b6 during in vitro transcription and translation experiments in the presence of thylakoid membranes. Fur-ther studies are required to analyze in detail other aspects of the mechanisms by which these proteins are translocat-ed across the thylakoid membrane.

P27.2

A cpSecY is not involved in chloroplast-encoded cytochrome b6 protein biogenesisMałgorzata Piskozub, Andrzej Szczepaniak, Jarosław Króliczewski

University of Wroclaw, Faculty of Biotechnology, Wrocław, Polande-mail: Malgorzata Piskozub <[email protected]>

In chloroplast the major complexes responsible for elec-tron transport contain subunits that are encoded by both the nuclear and chloroplast genomes. Numerous regulatory factors are required during formation for the coordinated transport and assembly of the subunits. Nuclear encod-ed thylakoid precursor proteins are imported across the chloroplast envelope into the chloroplast stroma by a Toc/Tic complex. To date import into or across the thylakoid membrane are thought to occur through four independ-ent precursor specific thylakoid transport pathways (cpTat, cpSec, cpSRP and the spontaneous integration pathway). The cpSec pathway evolved from the general secretory pathway involved in export of Sec-dependent proteins to the periplasm in bacteria which in E. coli consists mini-mally of SecA, SecE and SecY is also involved in the co-translational insertion of SRP-dependent proteins into the plasma membrane. Our knowledge of the cpSec pathway in chloroplasts is limited, with current models being mainly based on homology to the bacterial Sec system. Little is known about the role of the chloroplast Sec translocase in the insertion of proteins into the thylakoid membrane. Cytochrome b6 is a multispanning membrane core subunit of the cytochrome b6f complex encoded by a chloroplast gen petB, like many other thylakoid integral proteins, oper-ated with uncleaved targeting signals for thylakoid integra-tion. Because current results are not explaining insertion of cytochrome b6 into thylakoid membrane in this study, we analyzed the interplay between the cpSecY and the chloroplast encoded cytochrome b6 protein by isolation of RNC (ribosome nascent protein chain) complexes from chloroplasts and using crosslinking factors and antibod-ies together with mass spectroscopy (MS), SDS/PAGE and Western Blot analyses. From the whole pool of RNC using immunoprecipitation method and appropriate anti-body we will isolate the RNC complex containing nascent cytochrome b6. Isolated complexes were then separated by SDS-PAGE and identified by Western blotting. Further-more, cytochrome b6 or cpSecY protein found in stained band, was excised from a polyacrylamide gel and analyzed by MS. cpSecY were remarkably absent from the cross-linked complex contained cytochrome b6 suggesting that cpSecY is either not cross-link to other components and is not involved in cytochrom b6 integration.



P27.3

Oxidoreductive processes during germination of white lupine (Lupinus albus L.) and narrow-leafed lupine (Lupinus angustifolius L.)Renata Bączek-Kwinta, Marta Dziedzic, Małgorzata Borek

University of Agriculture in Kraków, Faculty of Agriculture and Economics, Plant Physiology Department, Kraków, Polande-mail: Renata Bączek-Kwinta <[email protected]>

The pattern of changes in oxidoreductive processes during germination of white and narrow-leafed lupine seeds was studied. Physiological bases for these assays were soluble sugar and soluble protein contents, and acid proteases ac-tivity. It was established that the more advanced germina-tion, the more intensive superoxide radical synthesis and the higher non-specific peroxidase (POX) activity (a sever-al-fold POX increase in 3 days). However, catalase (CAT) and ascorbate peroxidase (APX) activities dropped by 25-50% during germination, and neither superoxide dismutase (SOD) nor phenolics were altered. Hence, non-specific peroxidases play an important role in H2O2 scavenging during germination of lupine seeds.

P27.4

Diadenosine polyphosphates (Ap3A and Ap4A), putative alarmones, trigger synthesis of enzymes of the phenylpropanoid pathway in Arabidopsis thalianaMałgorzata Pietrowska-Borek1, Katarzyna Nuc2, Małgorzata Zielezińska1, Andrzej Guranowski2

1Department of Plant Physiology and 2Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poznań, Poland e-mail: Andrzej Guranowski <[email protected]>

It is known that the cells being under stress accumulate dif-ferent dinucleoside polyphosphates; the compounds that had been suggested to function as alarmones. In plants, the phenylpropanoid pathways yield metabolites protecting these organisms against various type of stress. Our obser-vation links these two phenomena and provides an example of metabolic “addressee” of the “alarm” raised by diad-enosine triphosphate or diadenosine tetraphosphate, Ap3A or Ap4A. Seedlings of Arabidopsis thaliana incubated with 5 µM Ap3A or Ap4A increased the synthesis of such en-zymes of the phenylpropanoid pathway as phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase and chal-cone synthase. The adenine mononucleotides (AMP, ADP or ATP) or adenosine, used at the same concentration, did not evoke such effects. The investigated enzymes assayed in vitro were not affected by Ap3A or Ap4A. AcknowledgementsThis study was supported by the Ministry of Science and Higher Educa-tion (grant no. N N303 068634).



P27.5

Changes in the activity of hydrolytic enzymes in Nicotiana tabacum BY-2 cells deprived of sucrose, nitrogen or phosphorusAneta Domańska, Agnieszka Kobylińska, Mirosław Godlewski

University of Łódź, Department of Plant Cytology and Cytochemistry, Łódź, Polande-mail: Aneta Domańska <[email protected]>

Many plant developmental, physiological and metabolic processes are regulated by nutrient availability while limita-tion or lack of nutrients can ultimately lead to cell death. In particular, alterations in the availability of soluble sugars, inorganic nitrogen and phosphorus help regulate a diverse array of processes. It was shown that no access to carbohy-drates led to an immediate inhibition of proliferation and cell growth, caused a quick intake of cellular carbohydrates, degradation of proteins and lipids, fall in glycolytic enzyme activity and consequently reduced the intensity of respi-ration and significantly disrupted cellular metabolism (Yu SM, 1999, Plant Physiol 121: 687-693). Similarly limitation of nitrogen or phosphorus severely reduces the basic metabo-lism of plant cells and restricts their growth and develop-ment. In plant cells as in yeast and animal ones starvation (primarily sugar starvation) triggers proteolysis and in the “starved” plant cells autophagy is induced (for review Mi-zushima N, 2007, Genes Dev 21: 2861-2873).In our study we investigated culture growth and activity of hydrolytic enzymes of tobacco BY-2 cells in complete or nutrient-deprived media.Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cell cultures were maintained by weekly passaging of the cell suspension at the stationary growth phase to a fresh Linsmaier and Skoog (LS) medium. Nutrient starvation was attained by the passage of 7 day-old cell suspension into the complete LS medium (control) or into that depleted of sucrose (-suc), nitrogen (-N) or phosphate (-P). The pa-rameters of cell culture growth (cell number, cell viability, fresh weight, morphological changes) and activity of hy-drolytic enzymes in the cell homogenates (proteases and alpha-amylases activity) were analyzed.Our results have revealed that: i) lack of particular nutrients in the medium arrested or significantly reduced the BY-2 cell proliferation; ii) the absence of sucrose, nitrogen or phosphorus in the medium inhibited the ability of cells to proliferate but did not cause their death; iii) in comparison with the control accumulation of numerous starch grains was found in the cells grown on the -N and -P media; iv) the activities of proteolytic enzymes in the starved cells were several times higher than in the control, however v) the activity of alpha-amylase was increased only in the sucrose-starved cells but it was significantly decreased in those growing on the -N or -P media.

P27.6

An improved method for efficient isolation and purification of genomic DNA from filamentous cyanobacteriaAngelika Kluk, Marcin Łoś, Grzegorz Węgrzyn

Department of Molecular Biology, University of Gdańsk, Gdańsk, Polande-mail: Angelika <[email protected]>

Cyanobacteria, a group of photosynthetic Gram-negative prokaryotes, are considered as a natural source of various biologically active compounds. Some of their secondary metabolites have antifungal, antibacterial, antivirus, an-ticancer or cytotoxic activities. This makes cyanobacteria attractive objects of genetic and biotechnological stud-ies, including construction of genomic libraries in order to search for genes encoding enzymes responsible for synthesis of the compounds mentioned above. However, there are serious problems with obtaining pure DNA from filamentous cyanobacteria due to the presence of large amounts of polyphenols and polysaccharides in their cell envelopes. These compounds interfere with standard DNA purification procedures, resulting in contamination of DNA samples and subsequent problems with gene cloning. Several methods have been developed to obtain high yield of good quality genomic DNA. Unfortunately current methods ensuring efficient removal of polyphenols and polysaccharides cause DNA damage, which is prob-lematic in procedures of gene cloning. In this report we present development of an improved method for isolation and purification of DNA from filamentous cyanobacteria. This method, which employs a modified phenol-based pro-cedure, allowed us to obtain a high yield (64–612 µg/g of fresh weight pallet, depending on the cyanobacterial strain) of pure genomic DNA (A260/A280 ratio of about 1.8 and A260/A230 ratio of about 2.0). We suggest that this method can be successfully used in procedures leading to cyano-bacterial gene cloning, construction of genomic libraries and any experiments in which highly purified cyanobacte-rial DNA is required.



P27.7

Nutrient starvation inhibits cell death of Nicotiana tabacum BY-2 cellsAneta Domańska1, Agnieszka Kobylińska1, Ewa Nowakowska-Świrta2, Mirosław Godlewski1

1Department of Plant Cytology and Cytochemistry, University of Łódź, Łódź, Poland; 2Nofer Institute of Occupational Medicine, Centre of Occupational Allergy and Environmental Health, Allergological Diagnostics Laboratory, Łódź, Polande-mail: Aneta Domańska <[email protected]>

Programmed cell death (PCD) is an integral part of plant development and defence. Two main categories of PCD have been recognized in plants (similarly as in animals): ap-optosis and autophagy (van Doorn WG & Woltering EJ, 2005, Trends Plant Sci 10: 117-122). Apoptosis occurs in re-sponse to irreversible damage and various stimuli includ-ing biotic and abiotic stresses. Autophagy is a mechanism for the degradation of cellular contents in order to recycle nutrients or break down damaged or toxic material. It is a conserved physiological process that is thought to promote cell survival however, under certain conditions autophagy has also been shown to promote cell death (Galluzzi L et al., 2008, Biochem Soc Trans 36: 786-790).To sustain growth and survival plant cells need a constant supply of precursors in order to produce macromolecules. Lack or limited access to nutrients leads to metabolic dys-function of cells, limiting their growth and proliferation and consequently may lead to cell death. Under conditions of starvation a lot of intracellular signaling pathways switch on which can lead to cell death or may activate pro-survival mechanisms, including autophagy.The aim of this study was to examine the growth rate and viability of cells in tobacco BY-2 cultures growing on com-plete or nutrient-deprived media.Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cell cultures at stationary growth phase were passaged into the complete Linsmaier and Skoog (LS) medium (control) or into that depleted of sucrose, nitrogen or phosphate. Stud-ies were performed for 10 days: the number of cells, their fresh weight, cell vitality and morphology as well as pro-teolytic activity in the cell homogenates were analyzed. Ad-ditionally, Western blot technique was used to assay proa-poptotic protein Bax expression under nitrogen starvation conditions.The obtained results have revealed that: i) growth rate of tobacco BY-2 cell cultures was almost completely inhibited already after 24 h of starvation; ii) lack of specific nutrients in the culture medium limited the cell ability to proliferate but did not lead to cell death; iii) the activities of proteo-lytic enzymes in the starved cells were several times higher than in the control starting from the first day of culture starvation, which may be connected with the induction of autophagy; iv) in the cells cultured under nitrogen-free con-ditions expression of Bax protein drastically dropped.

P27.8

Solvent-dependent opposite effects of quercetin on gamma irradiation-induced oxidative modifications in human red blood cellsAdam Antosik, Alicja Cichoń, Michał Bijak, Paweł Nowak, Halina M. Żbikowska

University of Lodz, Department of General Biochemistry, Lodz, Polande-mail: Adam Antosik <[email protected]>

Quercetin, a natural plant polyphenol, exhibits a strong antioxidant activity and it is reported to be a radioprotec-tive agent mostly due to its direct hydroxyl radical scaveng-ing potency. However, quercetin has also been found to be genotoxic and to show prooxidant properties. Moreover, a therapeutic use of quercetin has been heavily hampered by its extreme water-insolubility. Gamma irradiation of red blood cells (RBCs) to prevent graft versus host disease has been well-established practice in transfusion medicine.The aim of the study was to investigate the effects of quer-cetin on post-irradiated changes generated in the stored RBCs. The cells were irradiated with a dose of 50 Gy in the presence of quercetin (2–50 µM) or without antioxidant and stored at the 4oC. Quercetin was dissolved in DMSO or in ethanol. At the 1-, 10- and 20-day of storage the level of lipid peroxidation (TBARS), glutathione (GSH) and ac-tivity of extracellular lactate dehydrogenase (LDH) were measured.It was shown that in irradiated and nonirradiated RBCs quercetin dissolved in DMSO resulted in the increase of the extracellular LDH activity, TBARS concentration and in the decrease of GSH concentration in a storage time-de-pendent manner, compared to RBCs non-pretreated with quercetin. Contrary, quercetin dissolved in ethanol resulted in the reduction of the extracellular LDH activity, TBARS concentration and in the elevation of GSH concentration in a time of storage-dependent manner, in irradiated and nonirradiated RBCs. The observed changes were quercetin concentration-dependent. The results suggest that in RBCs quercetin showed cytotoxic and prooxidant properties when dissolved in DMSO and antioxidant and radiopre-ventive activity when dissolved in ethanol.It may be concluded that the solvent or other experimental conditions might significantly affect beneficial radioprotec-tive properties of quercetin.



P27.9

UDPG: indole-3-ylacetyl β-D-glucosyltransferase (IAGlc synthase) from immature pea seeds – purification and kinetic characterizationMaciej Ostrowski, Anna Hetmann, Anna Jakubowska, Stanisław Kowalczyk

Institute of General and Molecular Biology, Department of Biochemistry, Nicolaus Copernicus University, Torun, Polande-mail: Anna Hetmann <[email protected]>

UDPG: indole-3-ylacetyl β-D-glucosyltransferase (IAGlc synthase) catalyzes the reaction: IAA+UDPG↔1-O-IA-glucose+UDP. It is well known that the formation of the acyl alkyl acetal — 1-O-IA-glucose (IAGlc) is the first step during IAA-ester conjugates biosynthesis in monocotyle-donous plants. However, IAGlc synthase activity was also identified in immature seeds of some dicotyledonous plants. In this study the enzyme was partially purified from immature seeds of pea by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and prepara-tive polyacrylamide gel electrophoresis in non-denaturing conditions. 1-O-IA-glucose synthase activity was indicated in the fraction at Rf=0.38 obtained after native PAGE. Analysis of the pH dependence of the 1-O-IA-glucose formation showed that the enzyme was active within the range of pH 6.5–9.0. IAGlc synthase preparation exhib-ited the hightest enzymatic activity on IAA. 1-Naphthalene acetic acid (1-NAA) and indole-3-propionic acid (IPA) were found to be a poor glucose moiety acceptors (24% and 22% of the relative activity, respectively) and no con-jugating activity was observed on other natural and syn-thetic auxins (indole-3-butyric acid (IBA), phenylacetic acid (PAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picloram, dicamba) as well as on the rest phytohormones, (+) ab-scisic acid, (±) jasmonic acid, gibberelic acid, salicylic acid, brassinolide, and zeatin). The kinetic parameters of the enzyme on IAA were: Km=0.52 mM, Vmax=2.97 U mg-1 of protein, kcat=2.27 s-1, kcat/Km=4.36 mM s-1

. A competi-tive inhibitor of β-glucosidase activity — D-gluconic acid lactone caused activation of the 1-O-IA-glucose activity from 0.05 up to 0.3 mM concentration, but caused inhi-bition of the enzyme from 5.0 up to 7.0 mM concentra-tion. The inactive isomer of synthetic auxin, 2-naphthalene acetic acid (2-NAA) was a competitive inhibitor of IAGlc synthase (Ki=0.038 mM, Km

app=6.0 mM). The biological significance of the UDPG-dependent–IAGlc synthase in pea tissues remains unknown. To date, no enzyme activ-ity transferring indole-3-ylacetyl moiety from IAGlc has been found in dicotyledonous plants. Future studies will be needed to investigate what kind of reaction is coupled with formation of IAGlc which pushes this energetically unfavourable synthesis.

P27.10

Differential reaction of insect cells cultured in vitro on homogenate from entomopathogenic fungus Conidiobolus coronatusMarta Ligęza, Mieczysława I. Boguś

Institute of Parasitology PAS, Warszawa, Polande-mail: Marta Ligeza <[email protected]>

Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biologi-cal control agents of insect pests due to production of toxic metabolites. We have tested toxicity of spores and mycelial homogenates of Conidiobolus coronatus on hemocytes from Galleria mellonella (primary cell cultures) and ovarian cells from Spodoptera frugiperda (cell line Sf9).These insects are considered as pests: G. mellonella (bee hives), S. frugiperda (grain crops). C. coronatus is naturally oc-curring parasitic fungus with high insecticidal potential.C. coronatus was grown for 5 days on Sabouard agar plate, afterwards spores and mycelium were collected, suspended in chilled IPS (insect physiological saline), sonicated, cen-trifuged (1500 g, 10°, 4°C), filtered (0.2 µm) and stored in –21°C until use.Fungal homogenate was added to insect cells cultured in vitro at various concentrations (50–200 µg fungal proteins/ml). Into control cell-culture wells IPS was added. Obser-vation of cell morphology was held on inverted contrast phase microscope. We also did injections from the ho-mogenate (7.5 µg of fungal protein) to last instar G. mel-lonella larvae to check how toxic is used homogenate in vivo. Our results were surprising. Immediately after adding fungal homogenate cells from S. frugiperda bagan to change their shape, the cell membrane started to form bubbles and create tabs. But during 30 minutes everything was back to normal. Cells were rounded and divide properly suggesting toxin neutralization. In G. mellonella hemocytes cultures ad-dition of fungal homogenate resulted in rapid detachment of plasmatocytes from the plate surface and finally all cells seemed to be dead. Meanwhile in these cultures bacteria started to appear and multiply quickly — probably from disintegrating plasmatocytes — which do not managed to kill them by phagocytosis. Cells in control looked normal.Injected larvae after 4h had visible changes in their appear-ance, black spots on cuticule and swollen body witch led to the death (100% mortality).Our study shows how cells can react differently on the same toxins from pathogenic fungus. Cell line Sf9 is much more resistant than hemocytes from G. mellonella. This may be caused by the fact that cell line is carefully selected and has a great potential for proliferation. These results sug-gest that natural toxins can act very selectivly and do not threaten the biodiversity



P27.11

Correlation of changes in global DNA methylation level and maize resistance of herbicide stressAnna M. Kietrys, Adrian Czaban, Tomasz Twardowski

Institute of Bioorganic Chemistry Polish Academy of Sciences, Protein Biosynthesis Team, Poznan, Polande-mail: Anna Kietrys <[email protected]>

In recent years, it has become clear that dynamic changes in chromatin properties like DNA methylation and histone modification also contribute to transcriptional and post-transcriptional regulation of gene expression important for stress responses.The different strains of the maize displayed the natural re-sistance to herbicides. We examinined the changes in gene expression level which determine this feature. We focus on epigenetic mechanism of this process.To investigate the effect of stress conditions on m5C level in plants that reveal differences in stress resistance, we ap-plied two-dimensional TLC separation method of labelled nucleotides from enzymatic hydrolysate of DNA. We quantified signals of spots of m5C, C and T and measured the amount of m5C in relation to pyrimidines C and T and evaluated with the Graph Pad Prism Software.In our conclusions we imply that there is statistically signifi-cant difference in global DNA methylation level in plants sensitive and resistant to herbicide stress.

P27.12

The role of antioxidants in seedlings of spring wheat exposed to osmotic stress induced by short-term droughtApolonia Sieprawska1, Maria Filek2, Barbara Kreczmer1, Jolanta Biesaga-Kościelniak1, Anna Barbasz1, Elżbieta Rudolphi-Skórska1

1Institute of Biology, Pedagogical University, Kraków, Poland; 2Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Polande-mail: Apolonia Sieprawska <[email protected]>

Drought is one of the major factor of yield reduction of spring wheat varieties. Water imbalance in plant cells results in the osmotic changes and leads to induction of oxidative stress. Acclimation of plants to water deficit requires com-plex and rapid adjustments of their physiological status. Understanding of the basis of drought tolerance of crop plants is a prerequisite for developing superior genotypes through conventional breeding. Short-time treatment by stress factor can be helpful in selection of plant genotypes more tolerant to this kind of stress. The aim of experiments was to study the mobilization of wheat plants exposed to drought stress by an increase of the level of osmo-protect-ants and non-enzymatic antioxidants. Water stress was pro-duced in hydroponic cultures by PEG (ψ=–1.5 MPa) ad-dition to Hoagland (1:1 with water) nutrient during 2 days on 3-leaves seedlings of 10 varieties of spring wheat. The changes in levels of proline and sucrose (as osmoptotect-ants), of tocopherols and carotenes (as non-enzymatic anti-oxidants) and of polyamines - putrescine, spermidine and spermine (as hormonal substances) were analyzed. Differ-ences in fresh weight and water content in stressed plants in comparison to control plants (non-treated by PEG) were chosen as indicator of wheat seedlings tolerance to drought. It was found that PEG-induced osmotic stress caused an increase of levels of proline, sucrose, tocophe-rols and each of polyamines, oppositely to carotenes which content diminished in all investigated plants. In wheat genotypes which were classified as more tolerant, smaller changes of proline, sucrose and carotenes concentrations and higher levels of tocopherol were observed. Among polyamines, larger increase of contents of putrescine and spermine and smaller one of spermidine were characteris-tic for wheat varieties less sensitive to drought stress.AcknowledgementsThis work was partly supported by the COST project No. 556/COST-N/2009/0.



P27.13

Anticoagulant effect of plants preparation isolated from Asteraceae familyM. Bijak1, J. Saluk1, I. Pawlaczyk2, A. Antosik1, H. Żbikowska1, P. Nowak1

1Department of General Biochemistry, University of Łódź, Pomorska Łódź, Poland; 2Division of Medicinal Chemistry and Microbiology, Wrocław University of Technology, Wrocław, Polande-mail: M. Bijak <[email protected]>

Blood coagulation is a physiological process to prevent loss of blood caused by vascular injury. The essence of this process is the conversion of dissolved in the plasma fibrinogen into insoluble fibrin clot. The main enzyme of hemostasis — thrombin is responsible for this transforma-tion. Dysfunctions of mechanisms controlling the genera-tion and activity of thrombin may contribute to the forma-tion and development of many blood circulation system diseases. A lot of studies indicate that polyphenols may protect against cardiovascular diseases. The healing prop-erties of plants of the Asteraceae family have been used for years in a Polish folk medicine. These plants contain many compounds of polyphenol structure.The aim of our study was to examine the effects of prepa-rations from 2 plants of Asteraceae family — Arnica montana L. and Echinacea purpurea L., on plasma coagulation and amidolytic activities of thrombin.Human blood from healthy donors was collected into so-dium citrate and immediately centrifuged to obtain plasma. Fresh human plasma or thrombin (0.75 U/mL in a 50 mM TBS buffer, pH 7.4) were incubated with plant prepara-tions (0.5; 5; 50 µg/ml) at 25oC. After 10 min of pre-incubation the thrombin-induced fibrin polymerization, thrombin time (TT), and amidolytic activity of thrombin were measured.The in vitro experiments showed that both extracts pro-longed thrombin time in the dose-dependent manner. The Arnica extract (50 µg/mL: 33.90±1.26, 5 µg/mL: 30.00 ±1.13 vs. control 27.40±0.80) Echinacea extract (50 µg/mL: 31.40±0.99, 5 µg/mL: 29.40 ±1.06 vs. control 27.40±0.80). Both preparations also decreased the maximal velocity of thrombin-induced fibrin polymerization (at the highest concentration: 75% of control rate). In the presence of the plant preparations inhibitory effect on amidolytic activ-ity of thrombin was observed (Arnica IC50~ 6.0 µg/mL, Echinacea IC50~ 6.5 µg/mL at 0.75 U/mL concentration of enzyme).Our results showed a health prevention role for plants of the Asteraceae family. Inhibitory effect of the plant prepara-tions on coagulation system may help in prevention against the pathological states where hemostasis disorders are ob-served. These results also may help in promoting Polish herbal medicine.

P27.14

The expression of putative lipid binding protein is influenced by the sex gene Gy/gy in cucumber (Cucumis sativus L.)Cezary Kowalczuk, Magdalena Pawelkowicz, Justyna Witkowicz, Katarzyna Chądzyńska, Agnieszka Paziewska, Jerzy Ostrowski, Zbigniew Przybecki

Warsaw University of Life Sciences, Department of Plant Genetics, Breeding and Biotechnology, Warsaw, Polande-mail: Magdalena Pawelkowicz <[email protected]>

Sex determination in plants involves a variety of mecha-nisms. Cucumber is used as a model plant to study sex ex-pression in plants. Its type of gender is controlled by four genetic loci F, M, Gy and A. It was found that two loci — F and M — are key enzymes in ethylene biosynthesis — ACC synthase. In this studies we present a genes, with a strong correlation to Gy gene. In our previous research the sev-eral EST sequences has been pointed that differ cucum-ber near isogenic lines. Nine of them, based on similarity, were identified as a putative lipid binding protein. In our studies we used a pair of cucumbers near isogenic lines — monoecious B10 (ffMMGyGyAA) line versus gynoecious 2gg (ffMMgygyAA) and various techniques to trace clone expression.In physical mapping on cucumber genome it was shown that all of isolated EST sequenced are mapping on the same contig and they are coming from one gene.In macroarray analysis only three EST fragments give posi-tive result of hybridization were as a probe the cDNA from floral buds and shoot apex were used. The pattern of tran-script occurrence was very specific due to its presence only in B10 (Gy) line. No signal was observed in 2gg (gy) line. The localization of transcript checked by in-situ RT-PCR method, shows that clear signal was observed in floral buds 1–2 and 3–5 mm long in second, third and forth floral whorls only in B10 line.The real time RT-PCR analysis performed on apical mer-istem and floral buds at 1–2 and 3–5 mm stage, confirmed that clones were up regulated only in line B10. To precise-ly the quantity of RNA in generative primordia we applied the LCM (Lasser Capture Microdessection) analysis. The primordia of carpels and stamens from bud were dissected from floral buds. The isolated RNA was used in qRT-PCR analysis. Comparison of expression between carpels of line B10 and 2gg show that expression in 1–2 mm bud is higher in B10 line, while at 3–5 mm there was no significant dif-ference. In stamen it was similar but in B10 stamen in line 3–5 mm expression was up regulated. Comparison among B10 line shows that in the bud 1–2 and 3–5 mm the expres-sion in stamen was higher then in carpel in both stages. This research shows that genes coded lipid binding protein strongly correlate with cucumbers dominant Gy gene and could stimulate development of male flower organs.AcknowledgementsThe study was supported by a grant N N302 3633 33.



P27.15

Cytogenetic mapping using chromosome-specific BACs probes in Cucumis sativus L. ‘B10’Kouhei Yagi, Rafał Wóycicki, Magdalena Pawełkowicz, Zbigniew Przybecki, Stefan Malepszy, Wojciech Pląder

Warsaw University of Life Sciences; Department of Plant Genetics, Breeding and Biotechnology, Warsaw, Polande-mail: Kouhei Yagi <[email protected]>

Fluorescence in situ hybridization (FISH) is powerful tool for making the physical mapping for genes and for chro-mosome identification in plants and animals. Signals de-rived from bacterial artificial chromosomes (BACs) were used widely as chromosome-specific cytogenetic DNA markers for chromosome identification in plants (Jiang et al., 1995; Gomez et al., 1997; Fransz et al., 2000; Jackson et al., 2000). In cucumber, construction of BACs and fosmid libraries and BAC or fosmid FISH has been reported (Nam et al., 2005; Gutman et al., 2008; Meyer et al., 2008; Huang et al., 2009). In present study, we performed BAC FISH using probe based on BAC library for Cucumis sativus L. ‘B10’.First, we selected BAC clones which had less tandemly re-petitive sequences according to whole cucumber genome sequence data. BAC DNA was extracted by PhasePrep IM BAC DNA Kit (SIGMA). BAC probes were direct-labeled with tetramethyl-rhodamine-5-dUTP (Roche Applied Sci-ence, USA) by standard nick translation reactions. Chromo-somes preparation method for FISH was modified based on Tagashira et al. (2009). Sequential BAC FISH method was conducted following Tagashira and Kondo (2001), and the hybridization mixture was prepared according to Jenkins and Hasterok (2007) with Cot 1 sequences. BAC FISH signal was detected as red colour and counterstained DAPI was detected as blue colour under UV filter. Imaging pictures were overlayed and analysis were performed us-ing Adobe Photoshop. Chromosome numbering followed Hoshi et al. (1998; 1999).BAC clone probes were detected as chromosome specific and centromere specific signals. For example, the BAC clone 024-D19 was detected on interstitial regions of short arm on chromosome 1. BAC clone 007-M21 and 022-G02 showed hybridization at the interstitial regions of long arm on chromosome 2. These two clones which were located to similar region belonged to the same contig No. c2720. Some BAC clones were hybridized on centromeric region on all chromosomes. BAC DNA contains dispersed repeti-tive sequences that will cause to detect signals on centro-mere/ telomere region in wheat (Zhang et al., 2004). Espe-cially, cucumber has the highest density of SSRs and was the richest in AT dinucleotides among other species (Cav-agnaro et al., 2010). In addition, cucumber centromere had been known to consist of AT rich sequence such as tandem repeat type III (Han et al., 2008). Specific markers will be useful for further analysis.AcknowledgementsThe study was supported by a grant N N302 083 139.

P27.16

Isolation of Chlamydomonas reinhardtii CW15 cell strains resistant to copper excessBartosz Pluciński, Andrzej Waloszek, Katarzyna Łucka, Dagmara Fronczek, Kazimierz Strzałka

Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Polande-mail: Bartosz Pluciński <[email protected]>

The population with increased resistance to copper excess have been obtained as a result of 4-year long culture of cell wall-free Chlamydomonas reinhardtii mutant CW15 in nutrient solution containing 5 μM Cu2+ (20 times higher than in standard).Three homogenous cell strains CuET (Cu-Excess Tolerant) were isolated from this population using classical micro-biological method of maximum dilution of synchronous cultures.In parallel, three control strains, cultured in the same condi-tions excepting Cu2+ excess were obtained.Multiplication rates, photosynthetic pigment content and ratios, efficiency of light phase of photosynthesis, gas ex-change parameters and response to strong light were ana-lysed in CuET and control strains.*Cu-tolerant strains were able to multiplicate in Cu2+ con-centration as high as 1.28 mM, whereas in lower concentra-tions they multiplied faster than control strains.*In 640 μM Cu2+ the content of photosynthetic pigments per one cell was significantly higher in control strains in comparison to CuET cells.*The metabolic activity in cells of control strains was sig-nificantly increased in 640 μM of Cu2+, however photo-synthetic electron transport efficiency was highest in CuET cells in this concentration.*Photoinhibition but not photooxidation was observed during strong light (3.5 mE m-2 s-1) treatment of both CuET and control strains.AcknowledgementsThis work was partially financed from the budget of the Polish Ministry of Science for the years 2004–2008 under project No: 2P04C 020 27.



P27.17

Expression violaxanthin de-epoxidase from Escherichia coli and two steps purification of active enzymeMonika Olchawa-Pajor1, Dariusz Latowski1, Paulina Kuczyńska1,2, Monika Bojko1, Kazimierz Strzałka1

1Department of Plant Physiology and Biochemistry, Jagiellonian University, Kraków, Poland; 2Department of Plant Physiology, Institute of Biology, Pedagogical University, Kraków, Polande-mail: Olchawa-Pajor <[email protected]>

Violaxanthin de-epoxidase (VDE) is one of two enzymes of the xanthophyll cycle, an important and widely distrib-uted photoprotective mechanism in plant. VDE catalyzes de-epoxidation of violaxanthin (Vx) via the intermediate antheraxanthin (Ax). VDE is isolated mainly from different plants. In spite of using a lot of plant materials the amount of isolated VDE and their purity are low. In the presented studies isolation and purification of VDE from transgenic Escherichia coli strain BL 21 have been developed. E. coli was transformed by the gene of VDE from Arabidopsis thal-iana tagged with 6xHis. VDE gen effective expression was evidenced by SDS/PAGE electrophoresis. Purification of VDE had been realized using TALON Superflow Metal Affinity Resin before and after removal by thrombin diges-tion. Presence and purity of VDE in eluted fractions of first chromatography and in washed fraction after removed 6xHis tag was analyzed by SDS/PAGE.

P27.18

Activity of violaxanthin and diadinoxanthin de-epoxidases in different types of inverted micelles Dariusz Latowski1, Reimund Goss2, Kazimierz Strzałka1

1Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2Institute of Biology I, University of Leipzig, Leipzig, Germanye-mail: Latowski Dariusz <[email protected]>

Violaxanthin de-epoxidation and diadinoxanthin de-epoxidation are light dependent steps in one of the most important photoprotecting processes called respectively violaxanthin and diadinoxanthin cycle. Violaxanthin cycle which operates in all higher plants, ferns, mosses and sev-eral groups of algae, involves interconversion between: vi-olaxanthin (Vx), antheraxanthin (Ax) and zeaxanthin (Zx). These reactions are catalyzed by violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase. Diadinoxanthin cycle is present in diatoms in which interconversion between diadi-noxanthin (Ddx) and diatoxanthin (Dtx) occurs. Enzymes catalyzing these reactions are diadinoxanthin de-epoxidase (DDE) and diatoxanthin epoxidase. In the present studies influence of lipids on de-epoxidation of Vx and Ddx was investigated. In particular, the depend-ence between the conversion of Vx into Ax and Zx as well as Ddx to Dtx and the molecular dynamics of hydrophobic fraction of aggregates formed by inverted micelles, which are necessary for de-epoxidation, was studied. Thickness of the hydrophobic fraction of the aggregates, size of the inverted micelles, suggested by mathematical description of these structures and solubility of Vx and Ddx in various kind of lipids were the next tested parameters. Obtained results show that the rate of de-epoxidation is strongly de-pendent on physical/chemical properties of lipids. The key role for VDE and DDE activation play non-bilayer lipids and the parameters of inverted micelles created by them, such as thickness, diameter and molecular dynamics of their hydrophobic core. Mutual orientation of enzyme and substrate molecules and dilution of pigments by lipids are postulated as main mechanisms to explain the results.



P27.19

Biochemical changes induced by karrikinolide (KAR1) in dormant Avena fatua L. caryopsesDanuta Cembrowska-Lech1, Jan Kępczyński1, Johannes Van Staden2

1University of Szczecin, Chair of Plant Physiology and Biotechnology, Szczecin, Poland; 2Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, South Africae-mail: Danuta Cembrowska-Lech <[email protected]>

The smoke derived from burning plant, smoke saturated-water and their active components such as karrikinins are known to stimulate the release seed dormancy and germi-nation of a number of plant species from fire- and non-fire-prone ecosystems.Weeds are a major constraints for agricultural production particularly in the developing world. Seeds of arable-weed species Avena fatua L. are dormant after harvest and may exist in the soil for several years.Little is known about the role of karrikinolide (buteno-lide, 3-methyl-2H-furo[2,3-c]pyran-2-one, KAR1) in dor-mancy releasing and seed germination of several weeds including Avena fatua. Karrikinolide applied at 10–9–10–8 M effectively stimulated germination of wild oat dormant caryopses in darkness at temperatures from 5–25°C. KAR1 increased DNA synthesis prior to coleorhiza protrusion after imbibition at 20°C. KAR1-stimulated germination was also associated with a increased in α-amylase and total dehydrogenases activities. Likewise, and enzymes activities of antioxidant system such as superoxide dismutase (SOD) and catalase (CAT) as well as H2O2 content were increased by karrikinolide.

b14. advances in plant biochemistry lectures l27.2 l27.1 ... · b14. advances in plant biochemistry...

Documents