Advances in Cell biology, vol. 32, 2005, issue 1

Summary: The most successful beneficial interaction between genetically distant organisms in natural ecosystems are associations of higher plants with microorganisms. The best examples of such associations are ubiquitous symbiosis of flowering plants with Glomeromycota fungi forming arbuscular mycorrhiza and more specialized endosymbiosis of legumes with Rhizobium species. Fossil records and phylogenetic estimations suggest that mycorrhiza might be as old as the earliest land plants. It was therefore postulated that plant colonization of the lands was dependent on fungal symbiosis. A crucial role in the molecular dialogue between higher plants and microorganisms play chemical signals which induce recognition mechanism with participation of a large number of regulatory proteins. It has recently been documen-ted that plant hormones cytokinins are actively involved in development of symbiotic associations as well as in the morphogenesis of plant symbiotic organ root nodule.

Key words: Fabaceae, Glomeromycota, Rhizobium, arbuscular mycorrhiza, symbiosis, sygnaling

Cucumber's Secrets Disclosed: the Investigation in to Cucumber Genome Sequence Completed

Summary: Complete genomic sequences are valuable source of new knowledge and opens up new scientific horizons for molecular genetics and functional genomics. In the last decade, these new scientific fields grow up expansively due to development of new generations of WGS sequenators. These machines are able to read longer and longer fragments of a DNA sequence with simultaneous reduction of costs of bp. As a result of this technical progress a new initiative, such as Arabidopsis 1001 genomes [61] appeared. In our approach for cucumber genome sequence determination we used BAC library of 32 640 clones. Sequencing of ends of these BAC clones generated 44 276 885 nt of STC, representing ca.12.06% of the whole cucumber genome with coding sequence contents of ca. 23.76%. Subsequently, we sequenced the whole genome 8-times using of GS FLX Titanium sequenator, that result of 8 millions fragments of average length of 375 nt. This method was also used for sequencing of so call „paired ends” that give additional 4-times cover of the whole genome sequence. After all we did genome annotation. We will present some selected and characteristic features of cucumber genome sequence and various research activities due to sequence disclosure.

H1 His to nes in Plants – Unexpected Functions of Universal Elements of the Epigenetic System

Summary: Histone H1 proteins of angiosperm plants have all the features of evolutionarily-conserved linker histones and, as in animals, they occur in chromatin as different non-allelic variants. Due to much simpler organization of plants compared to animals, plants and in particular Arabidopsis thaliana, are uniquely suitable for studying universal mechanisms underlying the biological function of H1 in complex multicellular eukaryotes. The results of recent studies indicate that H1 in plants, in addition to role in modulation of transcriptional activity and chromosomal architecture, could act as coordinator of chromatin and microtubular processes. The recent analyses of global distribution patterns of nucleosomes along DNA also indicate that the lack in plant H1 of a pantapetide GXGAX that is strongly conserved in animals, could be one of the causes of increased developmental plasticity of plant cells.

Key words: TILLING, FOX-hunting, functional analysis of genes, mutagenesis

Plant Micro RNA Gene Structures and Possible Ways of Their Expression Regulation

Summary: Micro RNAs are short (18-24 nt) sequence-specific regulatory molecules of eukaryotic organisms. Micro RNAs control gene expression at the posttranscriptional level by targeting the cleavage of complementary mRNAs or by inhibiting their translation. At least six proteins are involved in the processing of primary miRNA precursors in A. thaliana: DCL1, HYL1, SE, DDL, HEN1 and HASTY. Very little is known about plant pri-miRNAs and MIR gene structures. Until now only 31 Arabidopsis thaliana MIR genes from among 190 known MIR loci are precisely characterized. Plant MIR genes are usually transcripted from their own loci. The analysis of Arabidopsis thaliana MIR genes revealed that most of them are unexpectedly long (the longest, MIR156c – 3108 bp) and contain introns. Plant MIR genes can be also organized in polycistrons and there are examples of micro RNA-coding sequences overlapping with other genes or localized within introns of other, protein-coding plant genes. Pri-miRNAs are heterogenous in length and structures. Micro RNA precursors undergo splicing and alternative splicing, posses alternative polyadenylation sites and their transcription can start at alternative transcription start sites. The cellular level of mature micro RNA molecules can be regulated during transcription and also at posttranscriptional level during processing of pri-miRNAs.

Key words: MIR gene, micro RNA, pre-miRNA, pri-miRNA, alternative splicing, polyadenylation, polycistron, overlapping genes

Summary: Plant Pathogenesis-Related proteins of class 10 (PR-10) are structurally well characterized but their physiological role remains obscure. The discovery that Cytokinin-Specific Binding Proteins (CSBP) have PR-10 fold suggests that hydrophobic ligand binding could be the general biological role of all PR-10 proteins. This conjecture is corroborated by the crystal structures of cytokinin complexes of the yellow lupine LlPR-10.2B protein. Surprisingly, the proteins are capable of binding in their huge internal cavity (1100–4500 ³) of multiple copies of the ligand molecule, despite the fact that kinetic measurements indicate 1:1 stoichiometry. It appears that the ability to bind multiple ligands in diverse modes is controlled by the C-terminal helix a3 of the PR-10 fold, which shows a high degree of structural plasticity and a low level of sequence conservation, and can in consequence adapt to the requirements of the binding partner. The discovery of the structure of PR-10 proteins and of their complexes with small-molecule ligands redefines our understanding of such phenomena as molecular recognition or binding specificity.

Key words: plant pathogenesis-related proteins, cytokinins, zeatin, protein-ligand recognition, allergens, yellow lupine

Summary: Lipoxygenases are enzymes widely spread in the animal and plant kingdoms. These enzymes catalyze dioxygenation of the long chain of fatty acids, which contain a cis, cis-1,4-pentadiene structure. In this article the structure and functions of plant lipoxygenases are discussed. There are also described biochemical and molecular properties of lipoxygenases including reactions, which proceeded with participation of the enzyme in plant cells. Into account were taken main and side pathways of metabolism of lipid hydroperoxy fatty acid. Beyond the discussion of the physiological function of lipoxygenases in plants, the role of plant lipoxygenases in food technology and industry is described.

Key words: lipoxygenases, plant cells, structure of lipoxygenases, function of lipoxygenases

Summary: Bacterial cellulose is a nanobiomaterial with many interesting characteristics, deciding of its numerous applications in medicine and other areas. This material is biotechnologically obtained and shows several important features: is extremely hygroscopic, chemically pure, uniquely structured, biocompatible, biofunctional and non-toxic. However, its biosynthesis by Gluconacetobacter xylinus has not been completely elucidated at the molecular level. The knowledge on sequences and functions of known genes and proteins that are directly involved in the metabolic pathway producing the b-1,4-glucan is insufficient to understand the overall network of the interplay and regulation influencing the intensity of bacterial cellulose synthesis and its properties. It is possible that this mechanism involves some global gene expression regulators which are known from described earlier mechanisms controlling formation of biofilms by model organisms. Due to its applicable potential both in native and modified form, exceeding its external and internal uses in medicine, bacterial cellulose is a material of future. Expected discoveries in a field of molecular control systems of BC biosynthesis can give rise to its much larger production in industrial scale.

Summary: Life span of the adult cells is restricted and senescent/undergoing apoptosis cells are replaced by new ones that originate from population of stem cells. An example is the hemopoietic system in the bone marrow. Stem cells in the adult organism may be identified by markers that are expressed by both embryonic stem cells and tissue-specific stem cells. Oct3/4, CXCR4, Nanog, CD133 and CD34 are examples of such markers. The stem cells share several common properties such as they may: 1) replicate and give rise to new stem cells, 2) differentiate into heterogenous tissue and organ specific cells, and finally 3) stem cells are self-protected against various toxic agents and radiation. Stem cells reside in the adult tissues in specialized sites (niches) that are identified e.g., in the bone marrow, skin, digestive tract epithelium, and skeletal muscles. Stem cells that reside in stem cell niches are anchored by chemokines and adhesion molecules. An important role in retention of stem cells in niche plays -chemokine SDF-1 (Stroma Derived Factor-1) and stem-cell expressed receptor CXCR4. Several stress factors may attenuate SDF-1-CXCR4 axis in the bone marrow, what leads to release of stem cells from their niches into circulation. Another important stem cell niche-anchoring mechanism is interaction of Wnt (ligand with Frizzled LRP (receptor). Recent evidence accumulated, that malignancy arises from maturation arrest of stem cells and their mutation rather than the dedifferentiation of somatic cells. Cancer stem cells are responsible for tumor growth, its re-growth and metastasis. The first direct evidence for the existence of cancer stem cells came from observations of the acute myeloblastic leukemia. Currently cell population enriched for cancer stem cells were isolated from several tumors. The cancer stem cells represent less than 1% of tumor cells in the mouse models.

Summary: Clinical proteomics is an analytical strategy functioning at the edge between chemistry, biochemistry, and medicine. This approach utilizes modern and ultrasensitive techniques, such as microextraction and separation of complex biological samples, identification with the aid of mass spectrometry, and advanced bioinformatics. Nowadays proteomics is based on basic research and clinical investigations. Between those disciplines there is a constant exchange of information (translation) and this process is part of the systems biology, i.e. understanding how human organism works. In reality, i.e. in a daily laboratory practice this assumption, however ambitious, meets many methodological ambiguities. The major issue is a lack of the commonly accepted, standard (or optimal) analytical strategy to handle biological material, and to monitor specific diseases. There is also an urgent need to evaluate quality criteria for this multi-task approach. Without such widespread norms, data comparison between various laboratories is out of question. Many ambiguities already appear during material collection phase. This is associated with the overall dynamics of such complex biological system as living organism, influence of age, medications, environment, a way the sample is collected and stored, etc. on the final result of the entire analysis. Additionally, several proteins comprise 96% of the protein content in plasma and the dynamic range of molecules reaches nearly 1010, which obscures less abundant components having important biological impact. Thus, simultaneous analysis of a huge amount of raw data is far beyond capabilities of modern instrumentation and software, and, even more important, cannot be fully interpreted by operators and translated to a common biological sense. One spectacular example may here be cited, where authors honestly reported analysis of a mixture of 6 standard proteins, which resulted in a printout containing „identification” of ca. 800 proteins! How the above contributes to the reliable and reproducible analysis?

Summary: The selected members of Rab subfamilies (Rab1, Rab3, Rab4, Rab5, Rab6, Rab7, Rab9 and Rab11) from different cell types were described in this review. Extensive search of literature enabled to collect the data on expression and localization of many Rab isoforms and Rab isotypes – products of paralogous genes deriving from duplication events. They are involved in endo- and exocytosis, transport, signaling, proliferation and differentiation interacting with the specific effectors and motor proteins. Isoforms and isotypes of some Rab proteins were found to cooperate in the regulation of these processes, whereas others have distinct functions that may be correlated either with the cell type or with the life cycle stage as in the case of parasitic species in which some peculiarities both in the structure and function of their respective Rab GTP-ases were observed by many authors. A great expansion of Rabs is correlated with diversification of trafficking machinery and some evolutionary aspects are discussed. Involvement of Rab7 GTPases in neuropathies and metabolic disorders was emphasized. Interestingly, distinct functions of the particular Rab proteins were found in plants in comparison with other cell types, especially for Rab3, Rab7 and Rab11.

Key words: Rab proteins, isoforms, isotypes, endocytosis, exocytosis, expression, localization, function, neuropathies, metabolic disorders, parasites

Summary: Interaction of anticancer drugs doxorubicin (DOX) and paclitaxel (PTX) with the plasma membrane of MCF-7 human breast carcinoma cells was studied by fluorescence spectroscopy technique. TMA-DPH and DAUDA fluorescent probes were employed to examine fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. Our data showed entirely different effects of DOX and PTX on membrane fluidity of MCF-7 cancer cells. DOX and PTX penetrated differently surficial and hydrophobic parts of the cell membrane. DOX caused similar and concentration-dependent changes in fluidity of both areas of lipid bilayer: low drug concentrations had a fluidizing effect, an increasing rigidization effect was observed with increasing drug concentrations. PTX mainly disturbed the structure of the inner part of the cell membrane and showed rigidization effect that was independent on drug concentration. The same concentrations of DOX and PTX induced different extent of alterations in the lipid bilayer, which could stem from their distinct interactions with the lipid components of the plasma membrane. Of both drugs PTX induced significantly greater changes in plasma membrane fluidity and at much lower concentrations showing at the same time considerably higher cytotoxicity towards MCF-7 cells than DOX. A rapid, over 70% decrease in cell survival with concomitant striking decrease in membrane fluidity were observed with the same concentration range of PTX (0.01–1 µM). 60% decrease in survival of MCF-7 cells treated with 0,05–5 µM concentration range of DOX, was also associated with either fluidization or rigidization of the plasma membrane. These results imply that changes in membrane fluidity occurring in the presence of DOX and PTX noticeably disturb cellular proliferation and suggest that correlation between the cytotoxicity of investigated drugs and the extent of the damage to the cell membrane they cause might exist. Combined effect of DOX and PTX on MCF-7 membrane fluidity was highly dependent on their concentration and molar ratio and was markedly different from the effects the drugs showed alone at the same concentrations. No synergistic or additive effect of DOX and PTX on the plasma membrane properties of MCF-7 human breast carcinoma cells was observed. At some of the investigated concentration ranges and molecular ratios DOX and PTX, however, showed antagonizing effect.

Key words: doxorubicin, paclitaxel, plasma membrane fluidity, MCF-7 breast cancer cells

Summary: The discovery of many critical points in signal transduction pathways in the research into plant response to biotic stress was possible owing to the use of mutants of the model plant – Arabidopsis thaliana. Accessibility of knock-out mutants obtained through chemical mutagenesis and lines of insertion mutants make it possible to focus on a specific problem without necessarily using time-consuming production of own mutants. Plant resistance to necrotrophic fungi is mostly dependent on biosynthesis and the signaling pathway of jasmonic acid as well as biosynthesis and accumulation of phytoalexins, low molecular weight secondary metabolites. Phytoalexins effectively suppress fungal growth and colonization of plant tissues. In this paper, we present current knowledge on selected aspects of A. thaliana defense response to necrotrophic fungi dependent on jasmonic acid signaling pathway and conditioned by camalexin production, which were deciphered as a result of using the mutants.

Key words: biotic stress, camalexin, JA-dependent defense response, signal transductio

Summary: An oxygen molecule in its basic form is relatively nonreactive. However, it can be a source of reactive derivatives generally called reactive oxygen species (ROS) which are produced both because of physiological metabolic activity of the cell and after exposing a plant to various stress factors. In a gradual, single-electron reduction of oxygen, partly reduced indirect products are generated. They comprise superoxide anion radical (•O₂–), hydrogen peroxide (H₂O₂) and hydroxyl radical (•OH). An induced oxygen molecule that occurs in triplet state results in the production of singlet oxygen (¹O₂) which can react with alkanes, sulphides and phenols. Free radicals – superoxide anion radical and hydroxyl radical – participate in a range of reactions: they can create covalence bonds in reactions with another free radical, or start a free radical chain reaction with nonradical molecules. Consequently, a serious impairment of macromolecular compounds of cells such as proteins, lipids and nucleic acids can occur. One of the very often investigated ROS is hydrogen peroxide, mainly because it is more stable than other ROS. The process of generating reactive oxygen forms in the host cell, which occurs during the infection of plant with necrotrophic fungi, is basically the result of the modification and/or disorders of the mechanisms sustaining homeostasis in the physiological conditions. These mechanisms include various ROS generation systems in the cell wall, plasmolemma and particularly in organelles, as well as the antioxidative systems removing ROS surplus generated in the physiological conditions or as the side products of other processes, e.g. side products of the metabolism of mitochondria, chloroplasts and peroxisomes. RFT can modulate plant defence response during infection with necrotrophic fungi, the induction of changes in gene expression profile, triggering of phytoalexins synthesis or rearrangement of cell wall structure. The present article contains a review of problems connected with the origin of reactive oxygen species, especially the mechanisms of regulation of their activity and signalling pathways they participate during interactions of plants with necrotrophic fungi.

Key words: necrotrophic fungi, oxidative burst, plant defense response, reactive oxygen species

Summary: For many years, a small flowering plant Arabidopsis thaliana is the most thoroughly studied model organism in plant biology. It has become a major model system for investigating plant molecular, genetic and biochemical processes. A plant transformation method that does not require the use of tissue culture and plant regeneration would greatly reduce the time necessary to produce transgenic plants, and this kind of method was described as in planta transformation almost 20 years ago. Here, we present the development of in planta transformation methodology in recent years.

Key words: Arabidopsis thaliana, in vitro culture, floral dip, vacuum infiltration

Summary: In recent years, simple and efficient procedures for obtaining transformed roots have been developed. Much like Agrobacterium tumefaciens, Agrobacterium rhizogenes, a causative agent of hairy root disease in plants, transfers T-DNA fragment from its root-inducing (Ri) plasmid to plant cells, where it is steadily integrated and expressed. It has to be emphasized, that while it usually takes a relatively short period of time to produce transformed roots following A. rhizogenes-mediated transformation, at least 4–6 months are required to regenerate transgenic plants when A. tumefaciens carrying Ti plasmid is used. Moreover, since each transgenic root represents an independent transformation event, a large number of transformed root lines can be produced and analyzed in only a few weeks. Here, we present the results of selected research on the increase in transformation frequency.

Key words: genetic transformation of plants, Agrobacterium rhizogenes, hairy roots