B14. Advances in plant biochemistry. Lectures L27.1 L27.2. Mechanisms of chlorophyllase regulation

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1 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, Poland Ewa Swiezewska 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 precursors for polyisoprenoid biosynthesis. The key enzymes of polyisoprenoid synthesis are cis-prenyltransferases (CTPs), responsible for construction of the long hydrocarbon skeleton. CPTs elongate a short all-trans precursor, oligoprenyl diphosphate, by sequential addition of the desired 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 observed. In contrast to polyisoprenoid phosphates serving as cofactors in the biosynthesis of glycoproteins, glucosyl phosphatidyl inositol (GPI) anchor or bacterial peptidoglycan, the biological importance of polyprenols and dolichols still remains a question of debate besides their function 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 publications 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 polyisoprenoids. L27.2 Mechanisms of chlorophyllase regulation M. 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, Poland L. Fiedor A key step in chlorophyll biodegradation, one of the most spectacular biological processes on Earth, is mediated through chlorophyllase, catalyzing the removal of the C 20 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, affect the activity, but their effects are antagonistic: chlorophyllide a activates chlorophyllase while phytol acts as the inhibitor. This indicates the existence of effector (chlorophyllide) 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 necessary to keep the rate of chlorophyllide generation in pace with the successive catabolic reactions leading to the degradation 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.

2 260 B14. Advances in plant biochemistry L27.3 The role of COP (constitutive photomorphogenesis) proteins in the regulation of chlorophyll biosynthesis in Arabidopsis Przemysław 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, Poland Przemysław Malec In Arabidopsis, genes belonging to COP/DET/FUS pleiotropic gene class, encode proteins constituting components of signalling pathways mediating the overall gene expression response to light. In particular, the eight-subunit protein complex (COP9 signalosome, CSN) and COP1 protein have been found to be central regulators in seedling deetiolation. In angiosperms, during deetiolation, the light-triggered photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) has a key role in the regulation of chlorophyll (Chl) biosynthesis. In Arabidopsis, this photoreduction 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 seedlings and green plants and the LPOR C expressed specifically 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 spectroscopy (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 Pchlide 633. 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 degradation of Pchlide 633 was observed in csn mutants, whereas 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 chlorophyll 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 conditions Maria Rapala-Kozik Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Poland Rapala-Kozik Plants are the primary source of vitamins for human nutrition. 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 megaloblastic 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 phosphate 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 involves the increased thiamine or TDP production observed in Arabidopsis thaliana and Zea mays plants under drought, salinity, osmotic or oxidative stress conditions. The expression levels of two most important thiamine biosynthesis genes (thi1 and thic) engaged in the independent formation of pyrimidine and thiazole parts of thiamine molecule increase 2 5 fold in stressed plants. A similar up-regulation 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 enzymatic detoxification systems. A similar pattern of vitamin B1 overproduction is observed in plants treated with abscisic acid that suggests an involvement of this hormone in the regulation of thiamine biosynthesis during the perception of environmental changes. The increased level of thiamine in stressed plants is documented by the activation of the pentose phosphate pathway/the Calvin-Benson cycle 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 protectant 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 adaptation processes.

3 B14. Advances in plant biochemistry 261 Oral presentations O27.1 The C-terminal flexible segment of Rubisco large subunit from Thermosynechococcus elongatus as a hotspot for specificity-changing mutations Beata Gubernator, Andrzej Szczepaniak Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland Beata Gubernator Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the addition of CO 2 to ribulose-1,5-bisphosphate (RuBP) or the binding of O 2 to RuBP. In cyanobacteria, green algae and plants, Rubisco is composed of eight large (L) and eight small (S) subunits forming L 8 S 8 complex. The dimmer L 2 serves as the catalytic unit. The preference of the enzyme for CO 2 versus O 2 is represented 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 CO 2 increases with the rate by which the binding 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 conformation 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 algorithm 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. Substitution A471T should stabilize the closed conformation of active site, resulting in decreasing of enzyme specificity. In this study the wild type of thermophilic Rubisco, L E470P and L A471T mutants were expressed, purified and SF values were measured. The SF values for the wild type Rubisco, L E470P and L A471T mutants were found to be: 83.9±1.9; 95.8±2.5; 56.9±1.2, respectively. The obtained results confirm the t-w hypothesis which explains the molecular basis 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 conjugation Maciej Ostrowski, Anna Jakubowska Institute of General and Molecular Biology, Department of Biochemistry, Nicolaus Copernicus University, Torun, Poland Maciej Ostrowski Indole-3-ylacetyl-aspartate synthetase (IAA-Asp synthetase) was isolated via a biochemical approach from pea seeds during our previously studies. Biochemical and immunochemical properties of the purified enzyme strongly suggested that it is a member of an acyl-adenylate/ thioester-forming GH3 family. Thus, these proteins are ATP-dependent amidosynthetases catalyzing the formation of amide-linked conjugates that are involved in hormone 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. Interestingly, 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 family 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 subjected to the trypsin digestion and the resulting peptides mixture 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-dichlorophenoxyacetic acid (2,4-D) 3,6-dichloro-2-methoxybenzoic acid (Dicamba), and 4-amino-3,5,6-trichloro-2-pyridenocarboxylic acid (Picloram). All tested phytohormones enhanced the accumulation of the PsGH3-5 transcripts in etiolated pea tissues. Moreover, pea seedlings exposed to red, farred, 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 environmental conditions.

4 262 B14. Advances in plant biochemistry O27.3 Identifying of small regulatory RNAs that could be involved in plant response to herbicide stress at the level of translation Aleksandra Szopa 1, Tomasz Twardowski 1,2 1 Institute of Technical Biochemistry, Technical University of Łódź, Poland; 2 Institute of Bioorganic Chemistry, Polish Academy of Science, Poznań, Poland Aleksandra Szopa Plants exposed to different conditions of abiotic and biotic stress, developed several mechanisms allowing them to adapt to adverse environmental conditions based on a regulation of gene expression at transcription and translation levels. Recently it was shown that small non coding RNAs were also involved in plant response to stress conditions. 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 molecular mechanism of plant response to stress causing factors. Testes herbicides are Roundup (glyphosate) and Titus (rimsulfuron). 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 affinity and specificity to ribosomes isolated from Zea mays. The RNA molecules are being selected against ribosomes isolated from three different maize strains treated with herbicides 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 selection. 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 buds Magdalena Pawelkowicz, Katarzyna Chądzyńska, Katarzyna Kurek, Cezary Kowalczuk, Zbigniew Przybecki Warsaw University of Life Sciences, Department of Plant Genetics, Breeding and Biotechnology, Warsaw, Poland Magdalena Pawelkowicz Sex determination is one of the key developmental processes 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 species, 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 following 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 differentional libraries coming from 1 2 mm cucumber s floral 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 cucumber 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 cucumber, monoecious B10 and female 2gg, and female Gy3 and hermaphrodite Hgy3. Some transcripts show the differences in localization and intensity of the signals among buds within a pair of analyzed lines, as well as between developmental 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 arresting 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 morphogenesis and sex determination in cucumber s flowers. Acknowledgements The study was supported by a grant N N

5 B14. Advances in plant biochemistry 263 Posters P27.1 In vitro study on the spontaneous mechanism for the insertion of cytochrome b 6 protein into the thylakoid membrane Malgorzata Piskozub, Jaroslaw Kroliczewski University of Wroclaw, Faculty of Biotechnology, Wroclaw, Poland Jarosław Króliczewski The thylakoid membrane contains the major photosynthetic complexes: PS I, PS II, the cytochrome b 6 f and the ATP synthase. Each of them consists of several subunits. Formation of these functional complexes requires coordinated transport towards the membrane, insertion into or translocation across the membrane, and subsequent assembly into high-molecular-weight structures. Thylakoid membrane 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 assistance of proteins FtsY and Alb3. By contrast, the spontaneous 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 b 6 by isolated pea thylakoids have been study. We used native or denatured cytochrom b 6 : isolated from Synechocistis sp. PCC 6803 or overexpressed in E. coli and synthetic cytochrome b 6 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 b 6 into isolated thylakoids membrane with or without stromal extract. In contrast, efficient import of preoe33-cytochrom b 6 has been observed on the presence of stromal extract and also insertion of PsbW protein. Strong possibility is that the import of cytochrom b 6 involves the operation of at least one stromal element of the translocation machinery. We also don t observe incorporation of cytochrome b 6 during in vitro transcription and translation experiments in the presence of thylakoid membranes. Further studies are required to analyze in detail other aspects of the mechanisms by which these proteins are translocated across the thylakoid membrane. P27.2 A cpsecy is not involved in chloroplastencoded cytochrome b 6 protein biogenesis Małgorzata Piskozub, Andrzej Szczepaniak, Jarosław Króliczewski University of Wroclaw, Faculty of Biotechnology, Wrocław, Poland Malgorzata Piskozub In chloroplast the major complexes responsible for electron 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 encoded 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 independent 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 minimally of SecA, SecE and SecY is also involved in the cotranslational 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 b 6 is a multispanning membrane core subunit of the cytochrome b 6 f complex encoded by a chloroplast gen petb, like many other thylakoid integral proteins, operated with uncleaved targeting signals for thylakoid integration. Because current results are not explaining insertion of cytochrome b 6 into thylakoid membrane in this study, we analyzed the interplay between the cpsecy and the chloroplast encoded cytochrome b 6 protein by isolation of RNC (ribosome nascent protein chain) complexes from chloroplasts and using crosslinking factors and antibodies together with mass spectroscopy (MS), SDS/PAGE and Western Blot analyses. From the whole pool of RNC using immunoprecipitation method and appropriate antibody we will isolate the RNC complex containing nascent cytochrome b 6. Isolated complexes were then separated by SDS-PAGE and identified by Western blotting. Furthermore, cytochrome b 6 or cpsecy protein found in stained band, was excised from a polyacrylamide gel and analyzed by MS. cpsecy were remarkably absent from the crosslinked complex contained cytochrome b 6 suggesting that cpsecy is either not cross-link to other components and is not involved in cytochrom b 6 integration.

6 264 B14. Advances in plant biochemistry 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, Poland Renata Bączek-Kwinta 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 activity. It was established that the more advanced germination, the more intensive superoxide radical synthesis and the higher non-specific peroxidase (POX) activity (a several-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 H 2 O 2 scavenging during germination of lupine seeds. P27.4 Diadenosine polyphosphates (Ap 3 A and Ap 4 A), putative alarmones, trigger synthesis of enzymes of the phenylpropanoid pathway in Arabidopsis thaliana Małgorzata Pietrowska-Borek 1, Katarzyna Nuc 2, Małgorzata Zielezińska 1, Andrzej Guranowski 2 1 Department of Plant Physiology and 2 Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poznań, Poland Andrzej Guranowski It is known that the cells being under stress accumulate different 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 observation links these two phenomena and provides an example of metabolic addressee of the alarm raised by diadenosine triphosphate or diadenosine tetraphosphate, Ap 3 A or Ap 4 A. Seedlings of Arabidopsis thaliana incubated with 5 µm Ap 3 A or Ap 4 A increased the synthesis of such enzymes of the phenylpropanoid pathway as phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase and chalcone 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 Ap 3 A or Ap 4 A. Acknowledgements This study was supported by the Ministry of Science and Higher Education (grant no. N N ).

7 B14. Advances in plant biochemistry 265 P27.5 Changes in the activity of hydrolytic enzymes in Nicotiana tabacum BY-2 cells deprived of sucrose, nitrogen or phosphorus Aneta Domańska, Agnieszka Kobylińska, Mirosław Godlewski University of Łódź, Department of Plant Cytology and Cytochemistry, Łódź, Poland Aneta Domańska Many plant developmental, physiological and metabolic processes are regulated by nutrient availability while limitation 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 carbohydrates 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 respiration and significantly disrupted cellular metabolism (Yu SM, 1999, Plant Physiol 121: ). Similarly limitation of nitrogen or phosphorus severely reduces the basic metabolism of plant cells and restricts their growth and development. 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 Mizushima N, 2007, Genes Dev 21: ). 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 parameters of cell culture growth (cell number, cell viability, fresh weight, morphological changes) and activity of hydrolytic 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 cyanobacteria Angelika Kluk, Marcin Łoś, Grzegorz Węgrzyn Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland Angelika 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, anticancer or cytotoxic activities. This makes cyanobacteria attractive objects of genetic and biotechnological studies, 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 problematic 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 procedure, allowed us to obtain a high yield ( µg/g of fresh weight pallet, depending on the cyanobacterial strain) of pure genomic DNA (A 260 /A 280 ratio of about 1.8 and A 260 /A 230 ratio of about 2.0). We suggest that this method can be successfully used in procedures leading to cyanobacterial gene cloning, construction of genomic libraries and any experiments in which highly purified cyanobacterial DNA is required.

8 266 B14. Advances in plant biochemistry P27.7 Nutrient starvation inhibits cell death of Nicotiana tabacum BY-2 cells Aneta Domańska 1, Agnieszka Kobylińska 1, Ewa Nowakowska-Świrta 2, Mirosław Godlewski 1 1 Department of Plant Cytology and Cytochemistry, University of Łódź, Łódź, Poland; 2 Nofer Institute of Occupational Medicine, Centre of Occupational Allergy and Environmental Health, Allergological Diagnostics Laboratory, Łódź, Poland Aneta Domańska 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): apoptosis and autophagy (van Doorn WG & Woltering EJ, 2005, Trends Plant Sci 10: ). Apoptosis occurs in response to irreversible damage and various stimuli including 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: ). 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 dysfunction 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 complete 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. Studies were performed for 10 days: the number of cells, their fresh weight, cell vitality and morphology as well as proteolytic activity in the cell homogenates were analyzed. Additionally, Western blot technique was used to assay proapoptotic 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 proteolytic 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 conditions expression of Bax protein drastically dropped. P27.8 Solvent-dependent opposite effects of quercetin on gamma irradiationinduced oxidative modifications in human red blood cells Adam Antosik, Alicja Cichoń, Michał Bijak, Paweł Nowak, Halina M. Żbikowska University of Lodz, Department of General Biochemistry, Lodz, Poland Adam Antosik Quercetin, a natural plant polyphenol, exhibits a strong antioxidant activity and it is reported to be a radioprotective agent mostly due to its direct hydroxyl radical scavenging 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 quercetin 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 4 o C. 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 activity 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-dependent 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 radiopreventive activity when dissolved in ethanol. It may be concluded that the solvent or other experimental conditions might significantly affect beneficial radioprotective properties of quercetin.

9 B14. Advances in plant biochemistry 267 P27.9 UDPG: indole-3-ylacetyl β-dglucosyltransferase (IAGlc synthase) from immature pea seeds purification and kinetic characterization Maciej Ostrowski, Anna Hetmann, Anna Jakubowska, Stanisław Kowalczyk Institute of General and Molecular Biology, Department of Biochemistry, Nicolaus Copernicus University, Torun, Poland Anna Hetmann UDPG: indole-3-ylacetyl β-d-glucosyltransferase (IAGlc synthase) catalyzes the reaction: IAA+UDPG 1-O-IAglucose+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 monocotyledonous 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 preparative polyacrylamide gel electrophoresis in non-denaturing conditions. 1-O-IA-glucose synthase activity was indicated in the fraction at R f =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 IAGlc synthase preparation exhibited 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 conjugating activity was observed on other natural and synthetic 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, (+) abscisic acid, (±) jasmonic acid, gibberelic acid, salicylic acid, brassinolide, and zeatin). The kinetic parameters of the enzyme on IAA were: K m =0.52 mm, V max =2.97 U mg -1 of protein, k cat =2.27 s -1, k cat /K m =4.36 mm s -1. A competitive 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 inhibition of the enzyme from 5.0 up to 7.0 mm concentration. The inactive isomer of synthetic auxin, 2-naphthalene acetic acid (2-NAA) was a competitive inhibitor of IAGlc synthase (K i =0.038 mm, K m app =6.0 mm). The biological significance of the UDPG-dependent IAGlc synthase in pea tissues remains unknown. To date, no enzyme activity 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 coronatus Marta Ligęza, Mieczysława I. Boguś Institute of Parasitology PAS, Warszawa, Poland Marta Ligeza Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological 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 occurring 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, centrifuged (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 ( µg fungal proteins/ ml). Into control cell-culture wells IPS was added. Observation of cell morphology was held on inverted contrast phase microscope. We also did injections from the homogenate (7.5 µg of fungal protein) to last instar G. mellonella 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 addition 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 appearance, 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 suggest that natural toxins can act very selectivly and do not threaten the biodiversity

10 268 B14. Advances in plant biochemistry P27.11 Correlation of changes in global DNA methylation level and maize resistance of herbicide stress Anna M. Kietrys, Adrian Czaban, Tomasz Twardowski Institute of Bioorganic Chemistry Polish Academy of Sciences, Protein Biosynthesis Team, Poznan, Poland Anna Kietrys In recent years, it has become clear that dynamic changes in chromatin properties like DNA methylation and histone modification also contribute to transcriptional and posttranscriptional regulation of gene expression important for stress responses. The different strains of the maize displayed the natural resistance 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 m 5 C level in plants that reveal differences in stress resistance, we applied two-dimensional TLC separation method of labelled nucleotides from enzymatic hydrolysate of DNA. We quantified signals of spots of m 5 C, C and T and measured the amount of m 5 C in relation to pyrimidines C and T and evaluated with the Graph Pad Prism Software. In our conclusions we imply that there is statistically significant 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 drought Apolonia Sieprawska 1, Maria Filek 2, Barbara Kreczmer 1, Jolanta Biesaga-Kościelniak 1, Anna Barbasz 1, Elżbieta Rudolphi-Skórska 1 1 Institute of Biology, Pedagogical University, Kraków, Poland; 2 Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland Apolonia Sieprawska 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 complex 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-protectants and non-enzymatic antioxidants. Water stress was produced in hydroponic cultures by PEG (ψ= 1.5 MPa) addition 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 osmoptotectants), of tocopherols and carotenes (as non-enzymatic antioxidants) and of polyamines - putrescine, spermidine and spermine (as hormonal substances) were analyzed. Differences 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, tocopherols 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 characteristic for wheat varieties less sensitive to drought stress. Acknowledgements This work was partly supported by the COST project No. 556/COST- N/2009/0.

11 B14. Advances in plant biochemistry 269 P27.13 Anticoagulant effect of plants preparation isolated from Asteraceae family M. Bijak 1, J. Saluk 1, I. Pawlaczyk 2, A. Antosik 1, H. Żbikowska 1, P. Nowak 1 1 Department of General Biochemistry, University of Łódź, Pomorska Łódź, Poland; 2 Division of Medicinal Chemistry and Microbiology, Wrocław University of Technology, Wrocław, Poland M. Bijak 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 transformation. Dysfunctions of mechanisms controlling the generation and activity of thrombin may contribute to the formation and development of many blood circulation system diseases. A lot of studies indicate that polyphenols may protect against cardiovascular diseases. The healing properties 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 preparations 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 sodium 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 preparations (0.5; 5; 50 µg/ml) at 25 o C. After 10 min of preincubation the thrombin-induced fibrin polymerization, thrombin time (TT), and amidolytic activity of thrombin were measured. The in vitro experiments showed that both extracts prolonged thrombin time in the dose-dependent manner. The Arnica extract (50 µg/ml: 33.90±1.26, 5 µg/ml: ±1.13 vs. control 27.40±0.80) Echinacea extract (50 µg/ml: 31.40±0.99, 5 µg/ml: ±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 activity of thrombin was observed (Arnica IC 50 ~ 6.0 µg/ml, Echinacea IC 50 ~ 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 preparations on coagulation system may help in prevention against the pathological states where hemostasis disorders are observed. 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, Poland Magdalena Pawelkowicz Sex determination in plants involves a variety of mechanisms. Cucumber is used as a model plant to study sex expression 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 several EST sequences has been pointed that differ cucumber 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 positive result of hybridization were as a probe the cdna from floral buds and shoot apex were used. The pattern of transcript 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 meristem and floral buds at 1 2 and 3 5 mm stage, confirmed that clones were up regulated only in line B10. To precisely 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 difference. 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 expression 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. Acknowledgements The study was supported by a grant N N

12 270 B14. Advances in plant biochemistry 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, Poland Kouhei Yagi Fluorescence in situ hybridization (FISH) is powerful tool for making the physical mapping for genes and for chromosome identification in plants and animals. Signals derived 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 repetitive 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 Science, USA) by standard nick translation reactions. Chromosomes 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 using 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 repetitive sequences that will cause to detect signals on centromere/ telomere region in wheat (Zhang et al., 2004). Especially, cucumber has the highest density of SSRs and was the richest in AT dinucleotides among other species (Cavagnaro 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. Acknowledgements The study was supported by a grant N N P27.16 Isolation of Chlamydomonas reinhardtii CW15 cell strains resistant to copper excess Bartosz Pluciński, Andrzej Waloszek, Katarzyna Łucka, Dagmara Fronczek, Kazimierz Strzałka Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Bartosz Pluciński 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 Cu 2+ (20 times higher than in standard). Three homogenous cell strains CuET (Cu-Excess Tolerant) were isolated from this population using classical microbiological method of maximum dilution of synchronous cultures. In parallel, three control strains, cultured in the same conditions excepting Cu 2+ excess were obtained. Multiplication rates, photosynthetic pigment content and ratios, efficiency of light phase of photosynthesis, gas exchange parameters and response to strong light were analysed in CuET and control strains. *Cu-tolerant strains were able to multiplicate in Cu 2+ concentration as high as 1.28 mm, whereas in lower concentrations they multiplied faster than control strains. *In 640 μm Cu 2+ 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 significantly increased in 640 μm of Cu 2+, however photosynthetic 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. Acknowledgements This work was partially financed from the budget of the Polish Ministry of Science for the years under project No: 2P04C

13 B14. Advances in plant biochemistry 271 P27.17 Expression violaxanthin de-epoxidase from Escherichia coli and two steps purification of active enzyme Monika Olchawa-Pajor 1, Dariusz Latowski 1, Paulina Kuczyńska 1,2, Monika Bojko 1, Kazimierz Strzałka 1 1 Department of Plant Physiology and Biochemistry, Jagiellonian University, Kraków, Poland; 2 Department of Plant Physiology, Institute of Biology, Pedagogical University, Kraków, Poland Olchawa-Pajor Violaxanthin de-epoxidase (VDE) is one of two enzymes of the xanthophyll cycle, an important and widely distributed 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 thaliana 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 digestion. 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 Latowski 1, Reimund Goss 2, Kazimierz Strzałka 1 1 Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2 Institute of Biology I, University of Leipzig, Leipzig, Germany Latowski Dariusz Violaxanthin de-epoxidation and diadinoxanthin deepoxidation 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 several groups of algae, involves interconversion between: violaxanthin (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 diadinoxanthin (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 dependence 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 dependent 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.

14 272 B14. Advances in plant biochemistry P27.19 Biochemical changes induced by karrikinolide (KAR 1 ) in dormant Avena fatua L. caryopses Danuta Cembrowska-Lech 1, Jan Kępczyński 1, Johannes Van Staden 2 1 University of Szczecin, Chair of Plant Physiology and Biotechnology, Szczecin, Poland; 2 Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu- Natal Pietermaritzburg, South Africa Danuta Cembrowska-Lech The smoke derived from burning plant, smoke saturatedwater and their active components such as karrikinins are known to stimulate the release seed dormancy and germination of a number of plant species from fire- and nonfire-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 (butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, KAR 1 ) in dormancy releasing and seed germination of several weeds including Avena fatua. Karrikinolide applied at M effectively stimulated germination of wild oat dormant caryopses in darkness at temperatures from 5 25 C. KAR 1 increased DNA synthesis prior to coleorhiza protrusion after imbibition at 20 C. KAR 1 -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 H 2 O 2 content were increased by karrikinolide.


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