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

Development and cytological characteristics of symbiotic systems.

Symbiotic properties of Rhizobium sp. strain NGR234 mutants, NGRbxqH and dctA were investigated. BxqH is a transcriptional regulator that controls the expression of at least 6 genes in NGR234 of not well known function. Nodules of Psophocarpus tetragonolobus induced by NGR234 wild type and the NGRbxqH have the similar anatomy. Also, the ultrastructure of bacteroidal tissue in both types of nodules was similar, except of the uncommon PHB accumulation in mutant bacteroids. These observations suggest that the metabolism of bacteroids is perturbed when BxqH is missing. In rhizobia, dctA gene codes for a transporter of dicarboxylic acids (Dct). Dicarboxylates such as malate and/or succinate were shown to play a crucial role in fueling the energy requirements of the nitrogenase during symbiotic nitrogen fixation. Comparative analysis of mutant and wild type nodules in both tested plants Vigna unguiculata and Leucena leucocephala did not reveal differences at the ultrastructural level, though Leucena plants inoculated with NGR234dctA are Fix+ while in those of Vigna nitrogen fixation is reduced.

Studies focused on the different aspects of nitrogen fixation effectiveness were also conducted. Correlation between two strains of Rhizobium (effective Sinorhizobium medicae 419 and partially effective Sinorhizobium meliloti 1021) and structure and ultrastructure of Medicago truncatula Gaertn. root nodules was investigated. The influence of high level of nitrogen on root nodule structure was also examined. It was approached by morphometric analyses related to bacteroids and cell organelles content within particular parts of root nodules. The volume of bacteroids was also measured using fluorescent dye binding to nucleic acids. The other part of these investigations deals with elucidation of the role of the water and photoassimilate transport in effective nitrogen fixation. Fluorescent tracers were used to follow the flux of carbon compounds and water from leaves to root nodules and water circulation within root and root nodules. Phloem unloading and post-phloem transport of sugars can proceed symplastically via plasmodesmata or apoplastically via plasma membrane sugar transporters. It is unclear which route – symplastic or apoplastic in the cell of indeterminate root nodule is in favour. Our results indicate that flow of assimilates between uninfected and infected cells outside the vascular bundles in nodules of Medicago truncatula is apoplastic. Uninfected cells may mediate as a pathway to the infected cells.

Research on changes in the structure of Medicago truncatula root nodules in response to stress caused by copper or mercury was also continued and finished. The aim was to recognize the cytological changes that occur in root nodules of plants growing in soils with high copper or mercury concentrations and to indicate changes in their antioxidant system. As a result, it was concluded that glutathione provides antioxidant cover for the meristematic cells of the nodule and for infected cells of a mature bacteroid tissue. Phenolic compounds, which are also important antioxidants, were accumulated by the meristematic cells of the nodule and non infected cells of the bacteroid tissue. On the other hand, in plants treated with mercury ascorbic acid was strongly and evenly accumulated in root nodules, while after being treated with copper ions its level was high in the meristem and mature bacteroid tissue. In all combinations the value of total antioxidant activity was lower than in control plants. After the native electrophoresis only one catalase activity band was present on gels. Four peroxidase activity bands were observed in zymograms. The highest increase in activity was observed in plants growing for 4 weeks in soil with mercury (6 mg/l HgCl₂) and it was 58%higher than in control plants. Three manganese dismutase isoforms (Mn-SOD), two copper-zinc dismutase isoforms (Cu,Zn-SOD), and one iron dismutase (Fe-SOD) were found.

Investigations on comparison of the activity of genes involved in root and nodule meristem maintenance in certain fabaceous species were continued. In order to create a background for the main study of the project, which is the possible involvement of PLETHORA, CLAVATA, SHORT ROOT gene-products in the meristem maintenance in root and nodule meristem of Medicago truncatula, Genista tinctoria and Lupinus angustifolius, the study of root apical meristem (RAM) organisation and ultrastructure was carried out in these species. The microscopic observations of RAM confirmed that in the species investigated, the RAM is of the open type, as in other fabaceans. In the central part of the meristem, the histogens of root cortex, rhizodermis and lateral root cap are not clearly delimited, and stem cells of these parts are difficult to identify merely on the basis of their location within the meristem. The quiescent center cells and stem cells are ultrastructurally similar in the investigated species. A single, large nucleolus was present in the nuclei. The cytoplasm was rich in ribosomes, active Golgi structures with small dictyosomes associated with numerous vesicles, non-differentiated small plastids, mitochondria with an electron-transparent matrix and sparse, electron-dense microbodies. Endoplasmic reticulum was rather non-proliferated and it was of rough type. In the columella stem cells, small starch grains were already formed in plastids. Also, their vacuoles and cytoplasm contained more phenolic deposits. During the differentiation of columella cells, the most characteristic change was formation of amyloplasts with compound starch grains and proliferation of the endoplasmic reticulum within the distal part of the cells. In every differentiated cell, mitochondria became electron-dense, and a clearly discernible, compact dictyosome was formed in the Golgi bodies. At the same time, the amount of vesicles decreased.

Cytological characteristics of plant reaction to infection with viruses.

Investigations were focused on ultrastructural and anatomical analysis of Tobacco rattle virus strain PSG infection in situ introduced into potato and tobacco tissues by Trichodorus primitivus. TRV genus Tobravirus is able to infect a broad range of plant species, possesses worldwide distribution and naturally infects a very large number of cultivated as well as ornamental plants. Members of the plant ectoparasitic nematode genera Trichodorus and Paratrichodorus transmit TRV in semi-persistent way in non-replicative process, where virus particles are transferred to host-plant through vector’s feeding on root epidermal and root hair cells. Our anatomical observations indicate necrotic changes of rhizodermis, cell wall deformations with hypertrophy of cells in primary cortex parenchyma and external phloem layer as a consequence of the interaction Trichodorus-TRV-host plants. Ultrastructural analyses revealed TRV particles in rhizodermis, cortex and vascular tissues in potato and tobacco roots. These results indicate that TRV PSG is transferred into the root and also transported from cell to cell in all root tissues. Complete TRV PSG particles of two lengths were present in companion cells, phloem fiber and parenchyma cells, as well as in immature and mature xylem tracheary elements and xylem parenchyma cells. These findings suggest that TRV was systemically transported from the place of direct transfer by vector to above-ground plant organs (especially leaves). The presence of TRV PSG particles in mesophyll and vascular tissues of leaves confirmed our hypothesis. Moreover, not only phloem was responsible for TRV systemic movement, because virus particles were often present in xylem parenchyma cells and xylem tracheary elements.

 Development and characteristics of the pathogenic system: nematodes/crop plants.

Analyses of expression of genes coding for wall associated kinases (WAKs), WAK-like (WAKL) and arabinogalactan proteins (AGPs) were conducted on mRNA isolated from root segments containing syncytia induced in Arabidopsis thaliana roots by the beet cyst nematode (Heterodera schachtii). With the exception of WAK2 and WAKL22 genes, the expression levels of examined WAK and WAKL genes in syncytia were relatively low and comparable to their levels of expression in uninfected roots. The expression of WAK2 was up-regulated only in 10 day old syncytia. It was apparently a response to degradation of short-living syncytia associated with males as it is well-known that WAK and WAKL proteins are involved in plant responses to stress factors. Expression level of WAKL22 gene was elevated in root segments containing syncytia collected 3 and 7 days after infection. In the case of AGPs genes coding for proteins involved in cell wall synthesis and modifications and thus apparently important for syncytium development, RT-PCR analyses indicated up-regulation of AGP1, -9, -10 and -20 gene expression in root segments containing syncytia. AGP1 and -10 genes were up-regulated during entire time of syncytium development. Expression of AGP20 reached its maximum in syncytia collected at 7 and 15 days after infection. Expression of AGP9 gene was also up-regulated in root segments containing syncytia but the level of its expression was apparently lower than these of AGP1, -10 and -20.

Basing on results of gene expression obtained by in vitro RT-PCR method, WAKL22 and AGP1 genes were selected for in situ analyses of gene expression. In situ RT-PCR confirmed that transcripts of WAKL22 gene were present abundantly in syncytial elements, especially these located next to the vessels. In later stages of syncytium development the transcripts of WAKL22 were detected also in parenchymatous cells of the vascular cylinder adjacent to the syncytia. In situ RT-PCR localisation of AGP1 expression indicated elevated amounts of AGP1 transcripts only in syncytial elements during entire syncytium development.

Reactive oxygen radicals (ROS) were localised during induction and early developmental stages of syncytia induced in roots of A. thaliana by H. schachtii as well as in syncytia induced by Globodera rostochiensis in roots of resistant and susceptible tomato lines. The localisation method employed confocal laser scanning microscopy and hydrogen peroxide-specific fluorescent H2DCF-DA probe. Two and five days after syncytium induction in Arabidopsis root, a specific signal characteristic for the hydrogen peroxide was observed around the nematode head and developing syncytium. Similar staining pattern indicating production of hydrogen peroxide was observed also around syncytia induced in roots of resistant and susceptible tomato lines. However, during syncytium induction and early stages of its development the signal intensity around syncytia induced in roots of resistant tomato plants was much higher than in susceptible one. Later, the signal intensity was comparable in both lines.

Phytosociological characteristics of pasture communities.

The succession of plant communities due to changes of water regime was investigated on lowlands Baniocha where the small retention system was established in 2009. 74 releve were made on experimental plots established in 2009. The data collected in 2009 and 2011 were compared. The analysis of species structure of community Deschampsia caespitosa and the association Filipendulo-Geranietum showed that the share of plants characteristic for All. Calthion and Filipendulion drastically decreased during last three years. Most of experimental plots was covered with the association Caricetum gracilis. On the plots with higher level of water, original rushes of Caricetum gracilis were replaced with rush communities dominated by sedges with wide leafs like Carex acutiformis or Carex riparia. The above changes were followed by changes in agricultural exploitation of Baniocha meadows.