Head of Department: Dr Sławomir Orzechowski

Phone: 0 48 22 59 32 560

e-mail:kbioch@sggw.pl

• Dr Bartoszewicz Katarzyna Senior Lecturer
• Dr Bielawski Wiesław Full Professor
• Dr Drzymała Adam Assistant
• Dr Grabowska Agnieszka Assistant Professor
• Dr Grudkowska Małgorzata Assistant Professor
• Dr Gutman Wojciech Assistant Professor
• Dr Jankiewicz Urszula Assistant Professor
• Dr Kwinta Joanna Assistant Professor
• Dr Miazek Anna Assistant
• Dr Orzechowski Sławomir Assistant Professor
• Dr Paszkowski Andrzej Associate Professor
• Dr Piszczek Ewa Assistant Professor
• Dr Prabucka Beata Assistant Professor
• M.Sc. Sitnicka Dorota Assistant
• M.Sc. Szawłowska Urszula Assistant
• Dr Zagdańska Barbara Associate Professor
• Dr Zdunek-Zastocka Edyta Assistant Professor

Postgraduate Students

• M.Sc. Głos Joanna
• M.Sc. Kendziorek Maria
• MSc. Maciąga Marcin
• M.Sc. Malinowska Magdalena
• MSc. Szkop Michał
• MSc. Szwost Dagmara

The research conducted in the Department of Biochemistry in respect to the biochemical aspects of the productivity of cultivated plants includes the analysis of biochemical mechanisms conditioning the productivity of cultivated plants in control conditions and in conditions unfavourable for their growth. With regard to that, there are studies carried out in the in the Department of Biochemistry concerning the molecular and biochemical characterization of the key enzymes responsible for assimilation and metabolic transformation of nitrogen and carbon in the case of selected cultivated plants, with particular emphasis put on the degradation of the plants’ energy storage. The research embraces also selected chemical substances that, by regulating adaptive mechanisms, significantly affect crop quality not only in control conditions, but primarily in stress conditions. This issue includes the following research subtasks:

• ·Cloning and mRNA level investigations of Triticum aestivum type II metacaspase in abiotic stress conditions
• ·Degradation of storage proteins during the development and germination of seeds of cereals.
• ·Glyoxylate aminotransferases in C3 i C4 plants.
• ·Proteolytic activity of seedlings of spring wheat (Triticum aestivum L.)
• ·Siderophores of bacteria Pseudomonas
• ·The role of nitrogen metabolism enzymes in cereals in response to abiotic stress.

Abstracts of major scientific subject

Bielawski W., Prabucka B., Drzymała A. Szawłowska U. Degradation of storage proteins during the development and germination of seeds of cereals

Hydrolysis of storage proteins involves the participation of various groups of peptidases, among others, cysteine endopeptidases and serine carboxypeptidases. The cooperation of these enzymes affects the initial growth of seedlings. Our previous research demonstrated that at least a few enzymes of each of these groups are crucial in the prolamins breakdown in triticale grains. One of such enzymes is carboxypeptidase III (previously partially purified and characterized) which is synthesized and secreted by the aleurone layer to starchy endosperm during germination with the influence of gibberellins. A series of experiments has been conducted in order to determine the kinetic parameters of the enzyme (kcat, kcat/Km) in reactions of peptides hydrolysis; the impact of pH on the Km, kcat and kcat/Km changes has also been studied. Moreover, studies aiming at determining the changes of the enzyme’s mRNA expression in germinating triticale seedlings have been commenced and the research attempting of obtain full length nucleotide sequences of carboxypeptidases I and III by means of the RACE method has been continued. The method contributed also to the works aiming to determine the full sequence encoding endopeptidase EP8, the enzyme participating in total hydrolysis of storage proteins during triticale grain germination. Besides, we have carried on with experimental works on the purification of other endopeptidases of germinating triticale grains in order to establish the parts of amino acid sequence of these enzymes.

Jankiewicz U., Stroiwąs L., Siderophores of bacteria Pseudomonas

The aim of these studies was to examine how the siderophor-pyoverdine synthesized by soil bacteria Pseudomonas inhibited the growth of pathogenic fungi and induced systemic resistance – ISR in cereal plants. The results of some investigations point to the significant role of pyoverdine in biocontrol by bacteria Pseudomonas. In order to determine whether the presence of bacteria synthesizing pyoverdine triggers ISR, wheat seedlings were cultivated for three weeks in controlled conditions of light and humidity. Wheat seeds were sterilized and subsequently inoculated with Pseudomonas bacteria. After 7 days, the plants were sprinkled with Pyrenophora tritici repentis or Septoria tritici. In plants treated with pathogens, whose seeds were inoculated with Pseudomonas, considerably fewer infection symptoms could be observed than in the case of plants treated only with fungi. No higher activity of chitinase and 1,3-glucanase, used as ISR protein markers, was detected in leaves and roots.

Kwinta J., Grabowska A., Sitnicka D., Batory E. The role of nitrogen metabolism enzymes in cereals in response to abiotic stress

The impact of exogenous sucrose (50mM) on the absorption and assimilation of inorganic nitrogen by triticale seedlings was investigated. Subject to research was also the dynamics of changes in the activity of nitrate reductase (NR), glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in plant roots and shoots after the 6-, ,12-, 24- and 48-hour growth on sucrose-enriched medium. After 6 and 12 hours, an increase in NR, GS and GDH activities in shoots was observed, as well as an increase in NR and GDH activities in roots. Throughout the whole experiment, GS activity in roots decreased. Accumulation of sucrose in roots and shoots of plants grown on sucrose-enriched medium was reported. Accumulation of sucrose in seedlings was accompanied by a decrease in nitrates contents. It appears that the presence of sucrose in the medium and its accumulation in plants altered the uptake and assimilation of inorganic nitrogen.

Paszkowski A., Kendziorek M., Glyoxylate aminotransferases in C3 i C4 plants

The Km values for L-glutamate as amino donor and pyruvate as amino acceptor for all four, purified, AlaAT isoenzymes were determined and the values of Vmax as well as of kcat and kcat/Km were calculated. The activity of two isoenzymes exhibiting glutamate: glyoxylate aminotransferase activity was tested with different pairs of aminoacid: 2-oxoacid substrates. The reaction rate with L-glutamate and glyoxylate as substrates was distinctly higher than that with L-alanine and glyoxylate for both GGAT1 and GGAT2. The rate of reverse reactions between glycine and 2-oxoglutarate or pyruvate was very low. As a preparation to study the mRNA level, four fragments of cDNA encoding four AlaAT isoenzymes were cloned by means of the PCR method, they were inserted in the pCR 4-TOPO vector and after they multiplied in the E. coli bacteria they were sequenced. The sequences obtained will serve to study the changes in four glyoxylate aminotransferase isoenzymes expression under abiotic stress.

Piszczek E., Zdunek-Zastocka E., Nowak G., Cloning and mRNA level investigations of Triticum aestivum type II metacaspase in abiotic stress conditions

The research was conducted on wheat (T. aestivum) seedlings. The total RNA was isolated from this material (Chomczyński method). On its template, cDNA wheat has been obtained by the reaction of reverse transcription. Using a known sequence of the gene encoding type II metacaspase from rice Oryza sativa, GenBank database has been searched in order to find ESTs of wheat metacaspases. On the basis of the artificial sequence obtained by merging several T. aestivum EST (expressed sequence tag) sequences, using the cDNA template in PCR reactions with specific oligo (dT) primers, a fragment of about 800 bp has been amplified. The amplification product has been subcloned to plasmid vector of pGEM®-T Easy and sequenced. The level of type II metacaspase transcript has been examined during abiotic stress conditions: wounding and heat shock treatment by semiquantitative PCR. The aim of research in the nearest future is to obtain the full-length cDNA of type II metacaspase from wheat (T. aestivum) by means of the RACE PCR method and to obtain its expression in Escherichia coli.

Zagdańska B., Miazek A., Grudkowska M., Podres-Kolbuszewska W., Proteolytic activity of seedlings of spring wheat (Triticum aestivum L.)

Research carried out in 2008 focused on the characteristics of protein profile in maize (Zea mays L.) seedlings, differing in sensitivity to abiotic stress (soil drought) as well as to biotic stress (subjecting the seedlings to two-spotted spider mite, Tetranychus urticae). The protein content in maize leaves was genotype-dependent. In response to soil drought conditions, the content of soluble protein decreased by about 30%, irregardless of the sensitivity of the maize to dehydration. No differences in the reactions to the two-spotted spider mite were observed. However, the activity of antioxidant enzymes has changed. Higher activity of copper and zinc superoxide dismutase (Cu/Zn-SOD) and guaiacol peroxidase caused by water deficiency prevented the development of oxidative stress in the leaves of the line sensitive to dehydration. Higher activity of guaiacol peroxidase and catalase in leaves feeding two-spotted spider mute indicates the effective removal of hydrogen peroxide, especially in the leaves of the maize line that was sensitive to dehydration.

Bielawski W., Głos J., Inhibitors of cysteine endopeptidases in the forming and germinating triticale grains (x Triticosecale Wittm.).

Project no. N N310 151335

The aim of the project is to learn one of the most natural mechanisms of control of the activity of cysteine endopeptidases, co-responsible for the sprouting of triticale grains. The most direct regulators of the activity of these enzymes are protein inhibitors of the cystatin superfamily, known as phytocystatins. These inhibitors, synthesised also in the initial phases of ripening grains, probably participate in the storage protein protection against premature degradation while sprouting prior to harvest. So far, four different cDNA fragments encoding triticale phytocystatins have been amplified, and afterwards they were cloned to the bacteria vector pGEM-T Easy and sequenced. The obtained cDNA fragments bear resemblance to known phytocystatins of cereal plants (52%-98%). An analysis of the transcript level (RT-PCR method) of two of four identified inhibitor gene fragments was also conducted, at all stages of the grain formation.

Bielawski W., Jankiewicz U., Transferable genetic elements in bacteria – molecular analysis and the use of new biotechnological industry tools for construction

Project no PBZ-MNiSW-04/I/2007, within this project – participation in topic 5 of the task: Overproduction and Purification of the Selected Endoproteases of Phages P1 and A5W

Within the project, research aiming at obtaining overproduction of peptidase of the phage Staphylococcus aureus A5W in the cells of E. coli has been conducted. Enzymatic protein obtained in such a way will be purified and used in further research concerning its detailed biochemical characterization. For this purpose, specific primers were designed for the sequence of the orf65 gene, which encodes potential peptidase of phage A5W. Nucleotides encoding the polyhistidine tail were additionally designed in the primers. The PCR product obtained was cloned in the pGBT32 plasmid and was sequenced in order to check the nucleotide sequence concordance. Currently, research aiming at optimizing the conditions of the gene orf 65 expression in the E.coli cells under transformation is being conducted.

Bielawski W., Kwinta J., Grabowska A., The role of the glutamine synthetase and glutamate dehydrogenase in the yield of winter triticale

Project no N310301134

Within the project, researchis conducted concerning the efficiency of the nitrogen assimilation mechanisms in two varieties of winter triticale: Witon – of high protein content in grain and Kazo – of low protein content in grain. The research is intended to explain the role of isoenzymes of the glutamine synthetase and glutamate dehydrogenase in the crops of winter triticale.

The experiment plants were cultivated in vases with low, optimal and high rate of nitrogen fertilisation, maintaining the vernalization period. Ripe grains were harvested. In the vegetation period, samples for study were collected during the tillering stage (25 according to Zadoks scale), during the shooting stage (52 according to Zadoks scale), during the milk maturity stage (75 according to Zadoks scale) and the ripe grain (92 according to Zadoks scale).

At the initial stage of the experiment, the total activity of the glutamine synthetase (GS) was determined in roots, stems, leaves, flag leaf, ears and grains coming from different phases of the plants’ development. Very high GS activity was reported in flag leaves, ca. 200% higher than the activity determined in other leaves. Such a tendency occurred in both varieties of triticale, regardless of the fertilizer dose. The higher enzyme activity in flag leaves was observed during the milk maturity phase in comparison to the shooting phase. Moreover, the increase in the total activity of GS in ears in comparison to stems and roots of the researched plants is noteworthy.

In the same samples the protein contents was determined. In the leaves taken in the shooting phase, the amount of protein was ca. twice as big as in the leaves in the tillering phase. It also turned out that very high GS activity in the flag leaf in stage 51 of the Zadoks scale is accompanied by a few times (ca. 2.5) greater protein content in relation to the remaining leaves. Such a tendency occurred in both of the researched varieties of triticale.

In order to establish the full length of the genes encoding glutamate dehydrogenase (GDH) and glutamine synthetase (GS) the method RACE 5’ and 3’ (Rapid Amplification of cDNA Ends) was applied. As a result of the procedure, a full-length gene encoding GDH has been obtained. It has been named tcGDH. The length of the obtained sequence amounts to 1740 bp, including 125 bp of the probable 5’-UTR, open reading frame 1257 bp and 358 bp of 3’-UTR. In the case of GS chloroplast form, a cDNA sequence of the length of 1570 bp was obtained. The achieved sequence embraces 285 bp of the 3’-UTR region. In the case of the GS cytoplasm form (GS1) five different 5’ ends were obtained, which constitutes evidence of the existence of a few encoding genes GS1.

Bielawski W., Szawłowska U., Molecular and biochemical characteristics of proline iminopeptidase from winter triticale (x Triticosecale Wittm.) and its role during abiotic stress

Project no N310 0329 33

Proline iminopeptidase is an enzyme which is present in every organ of triticale plant. Its activity depends on the tissue location and stage development. The highest activity of iminopeptidase was observed in young and developing plant’s parts. High transcript level and activity of proline iminopeptidase was also observed in triticale plants during abiotic streses. The proline iminopeptidase was purified 485 times to the homogeneity by the six-step procedure with recovery ranging up to 6%. Proline iminopeptidase is a tetrameric enzyme with molecular weight 228.3 kDa and 54 kDa per subunit. The enzyme has the highest activity against prolino-β-naphtylamide in pH 7.5 in the temperature of 37ºC. Its activity was strongly inhibited by inhibitors of serine peptidases. Obtained fragments of amino acid sequence and full nucleotide sequence of proline iminopeptidase from triticale exhibit the highest homology to corresponding enzymes from Oryza sativa and Arabidopsis thaliana.

Starch phosphorylating enzymes are necessary for normal mobilization of starch in tubers of Solanum tuberosum. GWD (glucan, water dikinase) is the enzyme that catalyzes the phosphorylation of starch by a reaction in which the β–phosphate of ATP is transferred to the C-6 position of the glucosyl residue of amylopectin. PWD (phosphoglucan, water dikinase) has been so far discovered and described only in Arabidopsis thaliana. This enzyme phosphorylates starch granule previously prephosphorylated by GWD. The enzyme transfers the β– phosphate of ATP to the C-3 position of the glucosyl residue of amylopectin. The degree of phosphorylation of the surfaces of starch granules while they are degrading initiates the transition from the crystalline glucans to the glucan chain accessible for hydrolytic enzymes. The purpose of the current work is to clone the second isoform of glucan, water dikinase from Solanum tuberosum. A full length of GWD2 was cloned. The obtained sequence was used to design specific probes and to define the daily changes in the GWD2 gene expression in Solanum tuberosum leaves by means of the relative RT-PCR method. The tissue localization of the GWD2 gene transcripts in different potato organs was also carried out. It was conducted using the following methods: semi-quantitative RT-PCR, in situ RT-PCR and in situ hybridization. Plant material consisted of S. tuberosum leaves, collected in certain determined parts of the day, as well as of tubers. On the basis of the results achieved it was established that diurnal changes in the GWD2 gene expression in leaves are analogical to the changes in the expression of other genes which participate in starch degradation. In situ experiments confirmed that the expression of the described dikinase is also present in tubers.