• Dr Borucki Wojciech Assistant Professor
• Dr Brzezicka-Szymczyk Krystyna Senior Lecturer
• Dr Chachulski Łukasz Assistant Professor
• Dr Fudali Sylwia Assistant
• Dr Garbaczewska Grażyna Associate Professor
• Dr Golinowski Władysław Full Professor
• Dr Górecka Mirosława Assistant
• Dr Grymaszewska Grażyna Senior Lecturer
• Dr Janakowski Sławomir Assistant Professor
• Dr Kopcińska Joanna Senior Lecturer
• Dr Kurek Wojciech Senior Lecturer
• Dr Łotocka Barbara Assistant Professor
• Dr Otulak Katarzyna Assistant
• Dr Rudzińska-Langwald Anna Senior Lecturer
• Dr Sobczak Mirosław Assistant Professor
• Dr Sujkowska Marzena Assistant

• M.Sc. Górska-Czekaj Magdalena
• M.Sc. Kolenda Daniel
• M.Sc. Pisarzewska Anna
• M.Sc. Różańska Elżbieta
• M.Sc. Wiśniewska Magdalena

Main fields of research

• Anatomical and ultrastructural aspects of plant/pathogen and plant/symbiont interactions.
• The structure and biodiversity of grassland phytocenoses.

Golinowski W., Borucki W., Górska-Czekaj M., Kopcińska J., Łotocka B., Sujkowska M.: Development and cytological characteristics of symbiotic system rhizobia/Fabaceae

Investigations on phenotypic effects of mutations in pss genes of Rhizobium leguminosarum bv. trifolii were continued in cooperation with the Institute of Microbiology, University of Lublin (UMCS). Bacterial pss genes are responsible for synthesis and translocation of exopolysaccharides (EPS), which play a significant role in establishment of effective symbiosis between rhizobia and legumes. R. leguminosarum bv. trifolii pssP mutant did not produce any detectable amount of EPS and induces non-nitrogen-fixing nodules in clover. Compared to the parental RtTA1 strain, pssP mutant nodulated clover roots with 14-day delay. In the nodules induced by mutant, except for the meristematic zone, none of the developmental zones typical for cylindrical nodules was observed. The enlarged infection threads have been found mainly in the root-hairs and sporadically in the meristematic cells of the nodule. The infection thread walls in pssP mutant nodules had numerous outgrowths (protuberances) and were not uniformly fine-fibrillar, as occurs in the wild type nodules. Inside their cell walls discontinuous homogenous regions were surrounded by electron dense fibrillar layers. The release of bacteria from infection threads was rarely observed. Localization of hydrogen peroxide (H2O2) was carried out cytochemically during the first stage of wild type RtTA1 and pssP mutant infection. Electron-dense deposits of cerium perhydroxides were found in matrix, infection thread walls and in cell walls of the nodule primordia.

Concerning the study of genistean plants nodulation (Sarothamnus scoparius, Genista tinctoria, Lembotropis nigricans, Ulex europaeus and Chamaecytisus ratisbonensis) it was shown that the structure of root nodules was similar in the investigated species. The most important feature of the nodules was a meristem of indeterminated type, which contained cells infected with rhizobia. Genistean root nodules exhibit a mixture of traits characteristic of two different types of indeterminated nodules-cylindrical and collar ones. At the same time, the genistean nodules had numerous features unique for the tribe, i.e.: formation of apoplast enclaves, which allow the rod-shaped rhizobia that are never transformed into bacteroids to survive within the nodule till the saprophytic zone differentiates; presence of two forms of rhizobia in infected bacteroid tissue–typical bacteroids within symbiosomes and small bacteria embedded in membrane-bound matrix packets. The bacteroids inside symbiosomes were curled within peribacteroid membrane, while the cells in packets were rod-shaped.

Investigations on reactions of plant/symbiont interaction on short- and long-term aluminium stress were continued. Localization of aluminium in pea root nodules was performed using haematoxylin and fluorochrome Morin staining. Both methods indicated that aluminium was mainly accumulated in the cell walls of nodule cortex. The activity of antioxidant enzymes (SOD, CAT, and POD) in the nodules was evaluated using spectrophotomeric method applied after native gel electrophoresis. Strong increase of POD and SOD activities in nodules during Al stress occurred, while the CAT activity decreased.

An influence of abiotic stress conditions caused by copper, manganium or mercury on symbiotic interaction between Medicago truncatula cv. “Jemalong” and Sinorhizobium meliloti 1021 was studied. Distribution of copper and mercury within alfalfa nodules was analysed using scanning electron microscopy. An accumulation of both copper and mercury on nodule surface was proved. The results indicated an existence of a barrier protecting nodule interior from heavy metals. An increase in the level of autofluorescence in meristematic zone and nodule cortex of copper or mercury treated nodules was observed which indicated increased amount of phenolics. It seems that phenolics may play a role in protection of nodules against heavy metals.

In co-operation with research group of Prof. Ryszard Russa (Department of General Microbiology of Maria Curie-Sklodowska University in Lublin), an influence of O-polysaccharides upon induction, development and effectiveness of Lotus corniculatus root nodules was investigated. Immunogold assay revealed a reduced number of lipopolysaccharide (LPS) epitopes on the mutant bacteroids comparing with wild type strain. It can result from decreased number of O-antigenic determinants in mutant LPS bacteria or from structural modification of epitopes caused by alterations in acetylation or methylation of sugar residues.

Garbaczewska G., Rudzińska-Langwald A., Otulak K., Pisarzewska A.: Cytological characteristics of plant reaction to infection with viruses and phytoplasma

Investigations on immunocytological localisation of PVY capsid protein (CP) using specific antibodies against CP-PVY were continued on potato and tobacco plants expressing different levels of resistance to necrotic strains of PVY. In potato mesophyll cells electron microscopy observations revealed the antigen of capsid protein inside mitochondria, cell nuclei and nucleoli. Highly abundant labelling was found also in phloem parenchyma cells near the small PVY particles. Small virus particles were not labelled, but a lot of colloidal gold grains was located directly in their neighbourhood. Three days after infection with PVY, during hypersensitive response, single gold particles attached to small PVY virions, especially when virions were bound to ER membranes. The colloidal gold grains attached to ER cisternae, mitochondria and inner membrane of chloroplasts confirmed the presence of CP-PVY antigen during compatible as well as incompatible interactions. The PVY capsid protein was also detected inside phloem sieve elements, companion cells and xylem tracheary elements.

We investigated immunocytological localisation of plants calreticulin (CRT) in potato and tobacco infected with PVY using antibodies specific against C-domain of CRT. The C-domain of CRT was detected inside mitochondria and cell nuclei in PVY infected and non-infected potato and tobacco tissues. The colloidal gold grains were present near the plasmalemma and in the space between cell walls and plasma membranes in potato mesophyll cells. Abundant labelling occurred next to plasmodesmata in mesophyll cells, along the ER cisternae and on surface of cytoplasmic inclusions of PVY (laminated and pinwheels inclusions, especially in their core region). C-domain of CRT was also detected in sieve elements near the sieve plates, close to phloem P-protein. In general, intensity of labelling with antibodies directed against C-domain of CRT suggests that CRT is more abundant in plants tissues infected with necrotic strains of PVY than in non-infected plants.

Ultrastructural examinations of plants from Solanaceae family infected with CDV were continued. CDV particles were observed inside nuclei and mitochondria of infected cells. Filamentous viral particles were found associated with outer membranes of mitochondria, cisternae of endoplasmic reticulum and nuclear pores. Viral particles were also often found near and in aperture of plasmodesmata. Additionally, virus particles and cylindrical inclusions were found within all phloem and xylem elements.

Investigations on distribution of phytoplasma from Aster Yellows group in infected Catharanthus plants were continued. They were focused on companion cell structure during phytoplasma pathogenesis. In phloem of Catharanthus roseus plants infected by grafting with AY-phytoplasma obtained from naturally infected Acer negundo plant, phytoplasma cells were present only in sieve tubes but structural changes of companion cells were also observed. Influence of phytoplasma infection on companion cells structure was observed in both transport and collection phloem. The relationship between severe and mild infection and companion cells ultrastructure was observed. Similar reactions occurred also in Tagetes patula and Gladiolus x hybridus plants infected by other phytoplasmas from Aster Yellows group. These observations indicate crucial role of companion cells in this pathogenesis.

Golinowski W., Fudali S., Górecka M., Grymaszewska G., Janakowski S., Kolenda D., Kurek W., Różańska E., Sobczak M.: Development and characteristics of the pathogenic system: nematodes/crop plants

The analysis of anatomy and ultrastructure of syncytia induced in transgenic tomato lines carrying constructs silencing LeEXPA5 gene under control of CaMV35S or AtSUC2 promoters was continued in collaboration with the Institute of Plant Protection (University of Natural Resources and Applied Life Sciences, Vienna, Austria). In addition, in collaboration with Laboratory of Nematology (Wageningen University) the experiments on the responses of transgenic potato lines possessing constructs silencing cellulase 7 or 9C1 genes were also continued. Previous studies revealed that activation of all silenced genes (LeEXPA5, cel7 and cel9.1) in parasitized root plays an important role in the development of syncytia during compatible plant-nematode interaction. Microscopic observations have shown that silencing of any of the studied gene did not affect the process of root infection by invasive juveniles of the nematode. In the transgenic lines, similarly to the situation observed during compatible interaction, syncytia were induced in parenchyma or endodermal cells and then they spread towards vascular cylinder via incorporation of pericyclic and cambial cells. Cambial cells neighbouring xylem vessels or sieve tubes fused with developing syncytia. However, syncytia induced in transgenic lines were smaller and were composed of fewer and less hypertrophied cells than those induced during compatible interaction. In the cell walls between syncytial elements the lower number of cell wall openings was observed. Moreover, the cell wall of syncytium was significantly and irregularly thickened.

Investigations on localisation of calcium ions in roots of Brassicaceous plants infected with Heterodera schachtii were continued. The investigation was conducted on model plants Arabidopsis thaliana applying the method based on potassium antimonite. Calcium antimonite precipitates appeared in late developmental stages of syncytium. The early developmental stages of syncytium require more detailed investigations.

Analysis of changes in dynamics of actin and tubulin cytoskeletons in syncytia induced in A. thaliana roots upon infection with H. schachtii were continued. Immunolocalization of actin and tubulin on macrosections of syncytia at different developmental stages was carried out using specific commercially available antibodies. Obtained results indicated that cytoskeleton participates in plant response to infection with sedentary nematodes. Crucial time point when rearrangement of actin and tubulin cytoskeleton occurred was 1 to 3 days after inoculation. This data were supported by in situ hybridisation expression analysis of actin ACT2, ACT7 and ACT8 genes. The most important time point appeared to be the 3rd day after inoculation, when the level of expression of investigated genes was the highest.

The studies on the involvement of plant hormones, gibberellins, in the interaction between potato cyst nematode and tomato were commenced. To verify their potential role during induction and differentiation of syncytium, tomato mutants (gib-1 and gib-3) being unable to synthesise gibberelic acid were used. Lack of the endogenous gibberellins did not notably affect the process of root penetration by invasive juveniles. In the roots growing on medium with or without GA3 the similar number of infection was counted. However, on the roots of mutant plants more juveniles developed into females and formed cysts than on the same plants grown under control conditions, where the wild phenotype was restored by addition of exogenous GA3. The experiments revealed that lack of endogenous gibberellins in tomato roots have a positive effect on the development of the potato cyst nematode.

Chachulski Ł.: Phytosociological characteristics of pasture communities

The investigations of plant communities in subalpine zone of Svydovets (Pokucko-Marmaroskie Mt.) were conducted. As a result 270 relevé of meadows with Festuca amethistina and shrub communities of Juniperus nana at different stages of degradation caused by natural factors and pasture utilization were collected. It was found, that there are two types of shrub communities dominated by Juniperus nana on the investigated area. The first one is a primary association described by other authors as a Juniperetum nanae. It occurs at the habitats similar to those occupied by association Pinetum mugi carpaticum in neighbouring massifs. The second community belongs to the dynamic circle of spruce forest. Their characteristic traits are: small coverage of shrub layer, high number of species characteristic for alliance Nardion and class Betulo-Adenostyletea. Collected material is a basis for further analysis of secondary succession pathways of plant communities unique for this region.

Łotocka B.: Biology of flowering and fructification of Asian pear

Within a framework of a grant obtained by Department of Pomology and Basic Natural Sciences in Horticulture, the study of Asian pear flowering and fruit set were carried out. As a part of the project, some embryological aspects were investigated by B. Łotocka in Department of Botany. In 2008, the photographic documentation of stigmas (ultra)structure was completed, as well as the SEM observations of stigma surface during anthesis.

Wiśniewska M., Golinowski W.: Anatomical and cytological characteristics of arbuscular mycorrhiza in tobacco

The investigations were aimed at immunolocalization of NtEXPA5 protein in roots of Nicotiana tabacum L. mycorrhized by Glomus mossae (BEG12) at different stages of symbiotic interaction, in order to assess the role of expansins in development of arbuscular mycorrhiza. Localisation of NtEXPA5 was confirmed by immunogold electron microscopy and immunofluorescence microscopy. Using specific polyclonal antibody expansin protein was localized in interface zone, predominantly around the active arbuscule branches. The relatively weak signal or no labelling was found in the interface enclosing collapsing arbuscule branches. We hypothesize that expansins are implicated in expansion of cell walls during mycorrhization process, a key determinant of interface development.

Łotocka B.: The taxonomy and molecular phylogeny of Robinia pseudoacacia (L.) microsymbionts

The investigation of root nodule (ultra)structure was initiated. The preliminary microscopic observations indicate that the nodules were of indeterminate type. However, morphology of their cortex was typical for determinate nodules. It together with spherical shape of young nodules could result in erroneous classification of Robinia nodules reported in earlier papers.

Golinowski W., Sobczak M., Janakowski S., Fudali S., Kurek W., Różańska E., Kolenda D.: Exploiting genomics to understand plant-nemotode interactions – EU grant within COST 872 Action

The aim of this project is to coordinate research conducted in over a dozen of European laboratories on plants/parasitic nematodes interaction. The investigations covering among others such issues like reprogramming of plant gene expression during the pathogen invasion, comparison of genomes of different plant-parasitic and non-parasitic nematodes, identification of new nematode genes involved in the interaction creates the basis for invention of new strategies to engineer new crop lines with durable resistance to nematode pests. An important mission of this project is also training and education of young scientists in this scientific field. It is done via short-term scientific missions, workshops and annual meetings of representatives of all groups involved in the action. In February 2008 D. Kolenda participated in workshop organised by the Institute of Plant Protection of the University of Natural Resources and Applied Life Sciences (Vienna, Austria) that was devoted to practical implementations of micrarrays in research on plant-nematode interactions. In May 2008 a group of 5 scientists from our Department took part in the Second Annual Conference of COST 872 Action that was held in Postoyna (Slovenia).

Golinowski W., Fudali S., Janakowski S., Kolenda D., Kurek W., Różańska E., Sobczak M.: Cytological and molecular analyses of susceptible and resistant responses of selected genotypes of Arabidopsis, potato and tomato to infection with nematodes from the genera Heterodera and Globodera. Research project within COST 872 Action “Exploiting genomics to understand plant-nemotode interactions”

The project covers the costs of research carried out in the frames of COST 872 Action “Exploiting Genomics to Understand Plant-Nematode Interactions”.

The studies of the ultrastructural changes induced by the invasive juveniles of root-knot nematode (Meloidogyne incognita) or root-knot nematode juveniles’ secretions in the pea root border cells were continued in the collaboration with Rothamsted Research Institute (UK). The cell walls of root border cells after 24-h incubation with juveniles or juveniles’ secretions were significantly thinner and consisted of separated layers of electrontranslucent material. The nuclei of root border cells contained considerable amounts of heterochromatin. In some of the cells, the nuclear envelope was broken down and the nuclear content spread into the cytoplasm. In addition, the root border cells contained numerous mitochondria, lipid bodies and Golgi Apparatuses.

Using RT-PCR method the levels of expression of two expansins genes in roots of nematode susceptible and resistant tomato lines were compared. Examined genes (EXPA4 and EXPA5) are crucial for successful development of syncytia induced in nematode susceptible tomato line. In the case of nematode resistant tomato line upon infection with G. rostochiensis the expression of EXPA4 gene was decreased shortly after syncytium establishment. The expression of EXPA5 was primarily enhanced and then decreased from 5th day after syncytium induction.

The analysis of spatial and temporal expression pattern of promoter of nematode resistance gene was conducted. After infection of GUS expressing transgenic potato plants with virulent and avirulent lines of G. rostochiensis it appeared that expression of the examined promoter was switched off in transgenic plants at about 5th day after infection with virulent line of the nematode.

Golinowski W., Sobczak M., Janakowski S., Różańska E., Kolenda D.: Functional analysis of plant genes involved in plant-nematode interaction – project running within Polish-Austrian agreement on cultural and scientific cooperation

This project is running in cooperation with Institute of Plant Protection, University of Natural Resources and Applied Life Sciences, Vienna. It is aimed at observation of anatomical and ultrastructural changes in syncytia induced by H. schachtii in roots of transgenic Arabidopsis plants carrying constructs silencing different genes encoding cell wall modifying proteins. The structure of syncytia induced in roots of mutant plants devoid of expression of myo-inositol oxygenase genes was analysed. The syncytia induced in mutant plants were smaller than observed in wild type plants. Parenchymatic cells and sieve tubes elements next to syncytia had strongly thickened cell walls and very narrow lumens. The system of cell wall ingrowths at the interface between syncytia and vessels was well developed.

List of publications

1. Łotocka B. 2008. Vascular system within developing root nodules of Lupinus luteus L. 1. Juvenile stage. Acta Biologica Cracoviensia Series Botanica 50: 79-88.
2. Łotocka B. 2008. Vascular system within developing root nodules of Lupinus luteus L. 2. Differentiated nodules. Acta Biologica Cracoviensia Series Botanica 50: 89-103.
3. Borucki W., Sujkowska M. 2008. The effects of sodium chloride-salinity upon growth, nodulation, and root nodule structure of pea (Pisum sativum L.) plants. Acta Physiologiae Plantarum 30: 293-301.
4. Turska-Szewczuk A, Pietras H, Borucki W., Russa R. 2008. Alteration of O-specific polysaccharide structure of symbiotically defective Mesorhizobium loti mutant 2213.1 derived from strain NZP2213. Acta Biochimica Polonica 55: 191-199.
5. Karczmarek A., Fudali S., Lichocka M., Sobczak M., Kurek W., Janakowski S., Roosien J., Golinowski W., Bakker J., Goverse S., Helder J. 2008. Expression of two functionally distinct plant endo-ß-1,4-glucanases is essential for the compatible interaction between potato cyst nematode and its hosts. Molecular Plant-Microbe Interactions 21: 791-798.
6. Szpadzik E., Jadczuk-Tobjasz E., Łotocka B. 2008. Preliminary evaluation of pollen quality, fertility relations and fruit set of selected sour cherry cultivars in Polish conditions. Acta Agrobotanica 61: 71-77.
7. Marciniuk J., Rudzińska-Langwald A. 2008. Morphological diversity of pollen from selected species of the genus Taraxacum, according to their ploidal level. Acta Agrobotanica 61: 65-69.
8. Lichocka M., Golinowski W. 2008. Two tomato endoglucanases have a function during syncytium development. Acta Societatis Botanicorum Poloniae 77: 5-15.

Monographs

3. Łotocka B. 2008. Anatomia rozwojowa i ultrastruktura brodawek korzeniowych o nieograniczonym wzroście i jej specyfika u roślin z plemienia Genisteae. Rozprawy Naukowe i Monografie, Wydawnictwo SGGW, Warszawa. Rozprawa habilitacyjna., pp. 151 + 170 fotografii na stronach nienumerowanych.

Others

1. Sobczak M., Janakowski S., Fudali S., Kerlan M.C., Mugniéry D., Golinowski W. 2008. Histology of defence responses of resistant Solanum sparsipilum and S. spegazzinii lines to infection with Globodera pallida. Proceedings of the second COST 872, “NEMAGENICS”, “Exploiting Genomics to Understand Plant-Nematode Interactions”, Annual Meeting, Postojna, Słowenia, 26.-29.05. 2008, pp 58.
2. Fudali S., Curtis R., Hazelman C., Golinowski W. 2008. The role of root border cells and root tip exudates in the plant-nematode interactions. Proceedings of the second COST 872, “NEMAGENICS”, “Exploiting Genomics to Understand Plant-Nematode Interactions”, Annual Meeting, Postojna, Słowenia, 26.-29.05. 2008, pp 55.
3. Koropacka K., Slootweg E., Roosien J., Sobczak M., Dees R., Sacco M.A., Moffett P., Smant G., Bakker J., Goverse A. 2008. Structure-function analyses of Gpa2-mediated resistance against the potato cyst nematode Globodera pallida. Proceedings of the second COST 872, “NEMAGENICS”, Exploiting Genomics to Understand Plant-Nematode Interactions”, Annual Meeting, Postojna, Słowenia, 26.-29.05. 2008, pp 16.
4. Bogatek R., Rudzińska-Langwald A., Kurek W., Kostrzewa Z., Gniazdowska A. 2008. Inhibition of maize (Zea mays) root growth by m-tyrosine results from cell cycle disruption. 5th World Congress on Allelopathy, Saratoga Springs, USA, 21-25.09. 2008, pp. 68.
5. Sołtys D., Rudzińska-Langwald A., Kurek W., Gniazdowska A., Bogatek R. 2008. Cytotoxic effects of cyanamide on tomato (Lycopersicon esculentum L.) root growth. 5th World Congress on Allelopathy, Saratoga Springs, USA, 21-25.09. 2008, pp. 64.
6. Otulak K., Garbaczewska G. 2008. Ultrastructural calreticulin and calcium ions localization during hypersensitive reaction of potato cultivar Rywal infected PVYNTN. Sympozjum Naukowe “Choroby roślin na tle zmian klimatycznych” oraz XIII Walne Zgromadzenie Członków Polskiego Towarzystwa Fitopatologicznego, Uniwersytet Przyrodniczy Wrocław, 17-19 wrzesień 2008, materiały konferencyjne s. 44.
7. Pisarzewska A., Garbaczewska G. 2008. Cell ultrastructure of Lycopersicon esculentum, Solanum tuberosum and Datura stramonium infected with CDV. Sympozjum Naukowe “Choroby roślin na tle zmian klimatycznych” oraz XIII Walne Zgromadzenie Członków Polskiego Towarzystwa Fitopatologicznego, Uniwersytet Przyrodniczy Wrocław, 17-19 wrzesień 2008, materiały konferencyjne s.47.
8. Wiśniewska M., Golinowski W. 2008. Differential localization of the NtEXPA5 in Nicotiana tabacum L. cell walls in the presence of Glomus mossae. International Plant Microbial Interactions, 2-6 lipiec 2008, Kraków.