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

Epigenetic Control of Plant Developement by Polycomb Group and Tritorax Proteins

Summary: Epigenetic mechanisms control gene expression programs during animal and plant development. This control can be mediated by DNA methylation/demethylation or histone H3 and H4 specific lysine methylation/demethylation. In general, Polycomb group (PcG) proteins are responsible for maintaining genes in a repressed state; on the contrary, tritorax group (trxG) proteins maintain genes in an active state. Histone H3 methylation at lysine 9 or 27 and histone H4 at lysine 20 results in gene silencing. Histone methyltransferases (HMTases) are proteins present in PcG. Histone H3 methylation at lysine 4 and 36, as well as histone H3 acetylation at lysine 9 and 14, lysine 5 and 8 in histone H4 take place in transcriptionally active chromatin; HMTases and acetylases are present in trxG proteins. PcG proteins were first discovered in Drosophila melangaster. In this fruit fly PcG proteins are indispensable for the control od appropriate expression of homeotic genes, while trxG proteins maintain homeotic genes activity necessary in particular body segments during development. PcG and trxG complexes constitute a general mechanism evolutionary conserved from flies to mammals and plants. PcG and trxG proteins act at the same target genes, mainly on homeotic genes both in animal and plant kingdoms. The knowledge of the role of PcG and trxG in plants is based mainly on the studies of Arabidopsis thaliana. PcG in Arabidopsis contains several homologs of PcG proteins of Drosophila and mammals, (see tab. 1) and a homologue animal Heterochromatin Protein1 (HP1), LHP1 (like HP1). In animals, PcG mechanism is based on two multiprotein complexes, PRC1 and PRC2 (Polycomb Repressive Complexes). PRC2 contains HMTase for histone H3K27me3. In plants there are some PRC2-like complexes. It is suggested that in plants LHP1 has an analogous function to PRC1 in animals, i.e. participates in chromatin remodeling. In Arabidopsis, reproductive development is subjected to regulation by the PRC2-like complexes that control femal gametophyte (embryo sac), endosperm and embryo development. It is suggested that two kinds of PRC2-like complexes regulate different target genes, but the floral homeotic genes are the main group of target genes for PcG proteins (see tab. 2). Arabidopsis Tritorax1 (ATX1) is a close homologue of TRX protein from mouse. ATX-SET domain probably shows a low activity of HMTase for histone H3K4me. Activity of HMTase for histone H3K4me3 and histone H3K36me3 is displayed by some other protein from trxG complex. Some floral homeotic genes and FLC gene involved in vernalization process are the target genes for trxG (see tab. 3). DNA methylation and histone modification resulting in gene silencing are responsible for parent-of-origin-dependent expression of imprinted genes. In Arabidopsis thaliana and Zea mays, among eleven genes studied, only two are inactivated by proteins from PcG – one maternal and one parental allele; in five parental alleles inactivation in caused by DNA methylation, and the mechanisms of silencing of three other parental alleles in unknown (see tab. 3). In spite of the fact that during the past decade the studies on PcG and trxG in plants were extremely fruitful, as well, as in animal material, the fundamental question by which molecular mechanisms PcG prevent transcription is not resolved. It is not clear till now if core histone H3 and H4 repressive methylation is the sole target of PcG proteins or whether modifications of basic transcription machinery are responsible for repression.

Summary: TILLING (Targeting Induced Local Lesions IN Genomes) and FOX-hunting (Full-length cDNA Over-eXpressing gene hunting system) are new research methods which enable quite fast functional analysis of gens on a mass scale. Accelerating of functional analysis of gens is required in biological sciences. It is crucial for systematization of data gathered from programs of genomes, genes, and EST (Expressed Sequence Tag ) sequencing as well as from studies of gene expression profiles, based on DNA microarray analysis. Classical methods of functional studies have been fulfilled using Top-Down (Forward) approach. Firstly genetic markers cosegregating or at least strongly conjugated with the trait of interest are searched for, using quantitative or qualitative mapping methods. Subsequently those markers are mapped physically on contigs of genomic library clones spanning the region between cosegregating markers in order to select the clone carrying the gene to be tested by complementation. Positive result of that test confirms biological functions of the gen, however lack of changes in transformant phenotypes do not negate the assumptions due to frequent gene silencing. Newer breakthrough tactic in gene functional analysis is the bottom-up approach. This procedure assumes generation of mutants followed by mutant population screening and analysis. The TILLING technique is a combination of traditional chemical mutagenesis with modern and sensitive method of point mutations – SNP (Single Nucleotide Polymorphism) identification. The TILLING bases on PCR amplification of template DNA which is a mixture of equal amounts of mutants DNA, followed by digestion with endonuclease Cel1, recognizing unpaired bases of the double-helix DNA. Crucial and difficult preliminary step of this method is generation of appropriate population of mutants, which depends not only on chemical mutagen but also on genome to be modified. The second crucial step is preparation of a template DNA, which is collected in 96-well microplates. For single PCR reaction DNAs are combined from each row of wells into 8 test-tubes and then DNA from the columns into 12 tubes so that the material from one plate can be analyzed in 20 (12+8) not in 96 reactions. DNA analysis from several plates can be additionally improved by collecting the DNAs from the same wells of different plates. Finally, in order to identify mutants, each of PCR reaction products is denatured and then renatured which induces heteroduplex formation with unpaired bases in the point of mutation. Digestion of this DNA by Cel1 endonuclease, specific to unpaired bases, enables their identification after the electrophoresis on sequencing gel. In the TILLING method the identification of SNP mutants is faster than in the classical approach because: i/ the standard electrophoresis on sequencing gels of Cel1 digested, renatured PCR products is run instead of costly and time-consuming sequencing reaction; ii/ the PCR reaction is carried out on a mass scale due to the DNA template which is a mixture of few/several different DNAs. The FOX-hunting is a novel gain-of-function system, which enables the plant genes overexpression as well as genes isolation and sequencing parallelly to functional analysis. This method is a modification of the protocol of A. thaliana transformation by floral dip in a suspension of A. tumefaciens carrying the full-length cDNA library in binary vectors. The binary vector used in the FOX-hunting system contains between the border sequences: i/ two tandem repeats of sequence strengthening the transcription (5'-upstream sequence of CaMV 35S promoter –419 to –90 bp); ii/ P35S, CaMV 35S promoter –90 to –1 bp; iii/ W – 5'-upstream sequence of TMV, which increases translation effectiveness of inserted gen; iv/ a gene cassette flanked by sequences of PCR starters to identify the gene in the mutant and restriction sites for SfiI endonuclease; v/ polyadenylation signal of nopaline synthase gene from Ti plasmid; vi/ hygromycin resistance gene. To construct the library in the binary vector, single copies of each gen gathered in cDNA libraries (in Lambda ZAP and Lambda FLC-1-B vectors) are used. The advantages of FOX-hunting system: i/ low percentage of gene cosuppression due to use of full length cDNA clones and normalized libraries in binary vectors; ii/ reduction of the expression of housekeeping genes in normalized libraries; iii/ simplification in phenotype analysis due to A. thaliana's short life cycle; iv/ quite easy genes isolation and sequencing. The disadvantage of FOX-hunting system is limitation of functional analysis only to the genes selected to construction of the library in A. tumefaciens. Both methods, TILLING and FOX-hunting enable more rapid functional analysis of genes. Projects using one of that protocol can be used as an additional source of diversified material for plant breeding. The TILLING system, developed to study of chemically induced mutants, enables immediate and direct implementation of gathered results to breeding programs. FOX-hunting system which generates valuable mutants with gene overexpression gives the new perspectives to functional analysis.

Summary: The development of molecular biology methods opened a new area in genetic diagnosis and thus, allowed to reveal small deletions and duplications of the genomic DNA fragments (CNV – Copy Number Variants) which constitute about 5,5% of all pathogenic alterations. MLPA – Multiplex Ligation-dependent Probe Amplification which was first described in 2002, is one of the most powerful methods for detection of microaberrations (deletions and/or duplications). This method is also useful in studies on aneuploidy and also copy number variation analyses and methylation profiling in cancers. MLPA is a molecular technique based on ligation and PCR that enables analysis of 40 –45 different genomic sequences in one reaction. Specific probes are used to hybridize to target DNA sequences. Each probe consists of two oligonucleotides which, after hybridization to a complementary sequence in patient's DNA, are ligated and subsequently used as a template for the polymerase in PCR. After PCR with fluorescently labeled primers products are separated by capillary electrophoresis and relative quantification of the fluorescence intensity of amplified products according to control probes is prepared. Thanks to low costs and short time for analysis as well as possibility of simultaneous analysis of many probes in one reaction, MLPA has become one of most widely applied methods in genetic studies.

Summary: P53 is a multifunctional protein of 53 kDa, activated in response to various molecular stressors. It is involved in cell cycle arrest in G1 and induction of apoptosis, regulation of gene transcription, cell differentiation, angiogenesis, but its primary role is DNA repair. Necessary during molecular stress, when genome integrity is threatened, it was titled „the guardian of the genome”. In virus infected cell cellular approach is to prevent virus from gene expression and replication, activating apoptotic pathways, because cell death may defend a whole organism from infection. The aim of a virus is to replicate its genome quickly and delay apoptosis as far as possible to achieve it, as well as to activate cell cycle. Various viruses in course of coevolution with host organisms developed many complex mechanisms of biochemical cellular death pathways modification, in which the main aim of viral attack is p53. This article presents general characteristics of p53 particularly concentrating on viral strategies during infection, especially connected with „the guardian of the genome”.

Key words: p53, apoptosis, virus, infection

Summary: Acid phosphatases (EC 3.1.3.2) are enzymes catalyzing the hydrolysis of different orthophosphate esters. They are abundant among both plant and animal tissues. Acid phosphatases can be divided into few groups of enzymes with differential substrate specifity and cellular localization. High substrate specific acid phosphatases are relatively well described and their role in metabolic processes is well known. On the other hand, role of nonspecific acid phosphatases is still not clear. Acid phosphatases can be divided in terms of localization on extracellular (take part in Pi scavenging from soil) and intracellular (play role in Pi mobilization from internal organic phosphorus sources). Nonspecific acid phosphatases take part in plant reaction not only on phosphorus deficiency but also water stress and pathogen defense. Purple acid phosphatases are the largest group of nonspecific phosphatases containing metal ions in their active site. Plant purple acid phosphatases can be distinguished into small (about 35 kDa) and large (about 55 kDa). They play a significant role not only in Pi releasing from unavailable, organic Pi sources but probably also in reactive oxygen species scavenging during senescence or in stressed tissues. However their role in plant metabolism is currently under investigation. In contrast, phytases are highly specific phosphatases which hydrolyze phytic acid (and phytates). These enzymes hydrolyze storage forms of phosphorus in seeds but they can also be secreted into the rhizosphere by microorganisms and some plants. This review summarizes our current knowledge about diverse roles of acid phosphatases, especially their role in plant phosphorus nutrition.

Key words: purple acid phosphatases, phosphate deficiency, phytase, secretion

Summary: It is well known that both, macro- and microelements are required for normal growth and development of all organisms. However, elevated concentrations of both, essential and non-essential metals are very toxic for cell metabolism. Plants as sessile organisms are frequently subjected to different biotic and abiotic stresses, including heavy metals stress, so they have evolved various mechanisms that protect their cells from the toxicity resulting from metal excess. They involve the regulation of the uptake, distribution and sequestration of metal ions within the cells and tissues. In the last few years various proteins specific for metals have been identified and initially characterized in plants, including active transport systems comprising pumps and secondary transporters that transfer ions across cellular membranes. Among them, Ca²⁺/cation antiporter (CaCA) family constitute integral membrane proteins that transport Ca²⁺or other cations using the H⁺ or Na⁺ gradient. They have been divided into following five major families according to their similarity and function: the Na⁺/Ca²⁺ antiporters (NCX), the K⁺-dependent Na⁺/Ca²⁺antiporters (NCKX and CCX), the YRBG transporters found in Procaryotes, and the cation exchangers, but the CAXs (CAation eXchangers) family is the best characterized. Genes of CAX family have been found in plants, fungi and bacteria but their homologs are absent in animal organisms, including human. Previous studies indicated that there are at least 11 CAX genes (AtCAX1–11) in Arabidopsis genome. Further phylogenetic analysis revealed that the subfamily forms rather two distinct groups: typical (CAX1-6) and untypical CAX (CAX7-11) proteins with the latter showing strong similarity to the potassium dependent Na⁺/Ca²⁺ antiporter family. Typical CAX proteins have been further classified into 2 distinct types: IA and IB in Arabidopsis thaliana and Oryza sativa. The open reading frames of CAX genes encode for the proteins consisting of approximately 400 amino acids and spanning the membrane 7–12 times. Within the sequences various specific motifs has been identified which are characteristic for the CaCA family, as well as for the CAX type. It is assumed that these domains play critical role in the regulation of proteins activity and substrate specificity. Two crucial domains: c-1 and c-2 have been proposed to act as a filter for cation selection. Other specific CAXs motifs involve: autoinhibitory domain, which participates in the control of CAX activity, the CaD domain, the Mn²⁺ domain, the D domain and the acidic motif, which probably determine substrate specificities of these transporters. Heterological expression of plant CAXs in yeast and phenotypic analyses of available plant cax mutants have been the most common tools for functional characterization of CAX transporters. They revealed that CAXs made up the essential component of calcium arrangement in plant and yeast cells. Moreover, it was well proved that these transporters show affinity to more than one different metals. The transformation of cax yeast mutants with different CAX genes from plants restored Cd²⁺, Ca²⁺, Zn²⁺ and Mn²⁺ resistance of yeasts to elevated concentration of these metals. It seems that CAX family comprises proteins with multiple functions determining the normal growth and development of plants. They probably contribute in the maintenance of metal and other ions homeostasis (Mn, Ni and Cd). The single cax1 mutants accumulated less Zn²⁺ and Mn²⁺ than wild type, however it was observed that the level of PO₄^3–, Mn²⁺ i Zn²⁺ in cax1/cax3 double mutants was even higher. It has been shown that CAX transporters are involved in the acclimation to cold as well as in the response to salt stress: the AtCAX1 transcript level increased significantly after cold treatment (4°C), and the AtCAX1-4 gene expression was considerably enhanced in the presence of salt in the environment. It was also suggested, that CAX transporters may be functionally associated with each other as well as with distinct membrane proteins. Tissue expression analyses of the individual genes of Arabidopsis thaliana shown different or similar expression pattern of all the CAX members during different stages of plant ontogenesis. In the view of their functional multiplicity, CAX genes seem to be suitable targets for engineering of cultivated plants, which can enrich humans diet in the indispensable elements. Engineered tomato and carrot plants expressing a high number of CAX proteins accumulated more calcium than a wild type. The over-expression of CAX could thus improve calcium content of potato, tomato and carrot and result in the sequestration of this element in central vacuole. On the other hand, CAX proteins can be also used to improve the phytoremediation process: cleaning up polluted soils from toxic heavy metals.

Key words: secondary transporters, CAX proteins, detoxication, phytoremediation, heavy metals, heterological expression

Summary: By binding to its receptor, insulin plays a very important role in maintaining the whole organism's homeostasis. Insulin receptors are present in all cells of vertebrates, reflecting the diversity of regulatory processes in which this hormone is involved. There are many different factors which may influence the level of insulin receptor expression, including the sole insulin or stage of development and mutations in the receptor leading to the development of insulin resistance that differ in the level of severity and is associated with such disorders as diabetes mellitus, hypertension, cardiovascular disorders, heart failure, metabolic syndrome and even infertility in women. More than 50 mutations in insulin receptor gene have already been known. These mutations are associated with rare forms of insulin resistance like leprechaunism, insulin resistance type A or Rabson-Mendenhall syndrome. Molecular analysis of insulin receptor gene may lead to better under-standing of molecular mechanism underlying various types of insulin resistance and help to develop much more efficient treatment modalities in patients.

Key words: insulin receptor, INSR gene, alternative splicing, insulin receptor isoforms, hybrid receptors, insulin resistance

Summary: Heavy metals these which are essential for life microelements and these which do not fulfil physiological roles, pose a serious threat for health of human and animals. Human activity has entailed that large amounts of these metals are deposited in soils all over the world. Because of the toxicity of heavy metal there is an urgent necessity to develop efficient and inexpensive methods of soil remediation. The conventional methods of remediation are rather ineffective and expensive, and often may destroy natural habitats. As a result some alternative methods have arisen. One of this methodologies is phytoremediation. In this method plants are used as cleaners of soils from heavy metal. Phytoremediation comes into several forms. For metal-contaminated sites may be useful techniques like phytoextraction, phytostabilisation or phytovolatilisation. Phytoextraction refers to the uptake pollutants which are then accumulate within the shoots. In contrast, phytostabilisation do not allow to remove pollutants from soils. They are stabilized and not available for other organisms. Phytovolatilisation allows to biologically convert metals into gaseous form and then release them into the atmosphere. Although there are some plant species able to phytoremediation, their efficiency in remediation of degraded areas is limited. Natural phytoremediators are perfect model for studies of cellular mechanism involved in natural resistance for high concentration of heavy metal ions. Indicating genes involved in heavy metal resistance allows getting in the future transgenic plants which will be able to phytoremediation large desolate sites. At the moment the most popular genes using for creating transgenic phytoremediators are genes encoded metal-binding ligands, transporters of metal ions or enzymes involved in converting mercury into gaseous form. The results remain elusive. However because of the rising need of obtainment effective and inexpensive method of soil remediation works are still intensified. In the nearest future phytoremediation may become inexpensive and effective method of soil remediation.

Key words: phytoremediation, heavy metals, genetic engineering, environmental remediation

Summary: Nitric oxide (NO) is a small gaseous radical molecule previously studied primarily as an air pollutant and metabolic product of certain bacteria. NO's uptake into leaves, as well as its metabolism and phytotoxicity are well documented. It was subsequently demonstrated that plants not only respond to atmospheric NO but also produce substantial amount of nitric oxide. After such discovery our appreciation of NO dramatically changed. Nowadays there is no doubt that nitric oxide has emerged as animportant molecule in plant signal transduction pathways, where NO may directly or indirectly interact with other signaling molecules such as cyclic nucleotides (cAMP, cGMP), H₂O₂, salicylic acid, and cytosolic Ca²⁺. It is likely that concentration of biologically active molecule such as nitric oxide must be precisely regulated by its synthesis and removal. There are many possible sources of nitric oxide. It can be generated by nitric oxide synthase from L-arginine or from nitrite via nitrate reductase. Moreover, NO can be generated non-enzymaticly as a byproduct of denitrification-, nitrogen fixation and respiration. Simple chemical reactions and some compounds such as superoxide anions, glutathione, transition metals and non-symbiotic haemoglobins are responsible for quick NO removal from the solution. Various experimental data indicate that in plants nitric oxide plays important signaling role in diverse (patho)physiological processes from reduction of seed dormancy and promotion of seed germination, regulation of plant senescence, suppression of floral transition, stomatal movement as an intermediate downstream of abscisic acid signaling to programmed cell death and xylogenesis. Moreover, nitric oxide mediates a multiple plant responses toward a variety of biotic (pathogen infection) and abiotic (drought, salt, heat, UV light irradiation, heavy metals, herbivores, mechanical wounding) stresses. All stresses mostly induce NO production in plants. NO alleviates the harmfulness of reactive oxygen species and reacts with other target molecules such as salicylic acid, calcium ions and cyclic GMP. It also regulates the expression of stress responsive genes. In the present review, we introduce how NO is produced and removed in plants and highlight the recent progress that provides novel insights into the functions of NO under abiotic stresses. Moreover, interactions of NO signaling with other signaling molecules in regulation of stomatal closure in responses to dehydratation were also discussed.

Key words: abiotic stress, nitric oxide, reactive oxygen species, signal transduction

Summary: Genomic instability is an effect of damage of DNA repair systems and is an important step in neoplastic transformation. The BRCA1 and BRCA2 proteins which mutations have been identified in breast and ovarian cancer are involved in many cellular processes essential to maintain genome integrity. Fundamental process is the repair of double strand breaks (DBSs) which are arguably the most important class of DNA damage. Failure to repair DNA double strand breaks may lead to either loss of genetic material or cell death. The efficiency of DNA double strand break repair affects the genomic stability. The BRCA1 and BRCA2 proteins interact indirectly or directly with RAD51 protein, a homolog of the RecA of Escherichia coli and Rad51 of Saccharomyces cerevisiae. These three proteins play a key role in DNA double strand breaks repair by homologous recombination (HR), by the mechanism that retrieves genetic information from undamaged, homologous DNA molecule. At the site of DNA damage nuclear foci containing BRCA1, BRCA2 and RAD51 proteins, together with other proteins engaged in homologous recombination process, are forming. The initiation of this multistep process of DNA damage repair has involved MRN comlex of MRE11, RAD50, NBS1 proteins which function is to generate single stranded DNA. The BRCA1 protein transmits signal of DNA damage to the other proteins involved in DNA repair, interacts with MRN complex and modulates RAD51 protein activity. The BRCA2 protein enable interaction of RAD51 protein with single stranded DNA at the site of damage and promotes DNA double strand breaks repair. RAD51 protein forms nucleoprotein filament which searches the undamaged DNA for homologous repair template and invades undamaged, homologous DNA duplex, in a process referred to as DNA strand exchange and heteroduplex assembly.

Key words: BRCA1, BRCA2, RAD51, DNA double strand breaks, DNA repair, homologous recombination

Key words: osteoblasts, transcriptional factors, Runx2, Osterix, osteoblastogenesis

Summary: Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that is expressed in the cytoplasm of many cell types, including osteoblasts. FAK is activated when integrin receptors interact with proteins of the extracellular matrix and is then recruited to focal adhesion complexes. The presence of kinase in these structures is strictly associated with its function in cell processes such as adhesion, migration and proliferation. FAK-deficient cells spread more slowly and exhibit reduced migration. Kinase activates various intracellular signaling pathways, including those leading to cell differentiation. When regulating mitogen activating protein kinase (MAPK), FAK influences in vitro cell maturation and differentiation into osteoblasts. The expression of genes characteristic for osteoblast phenotype, such as Runx2 and Osterix, is stimulated by FAK activation, whereas FAK gene deficiency prevents differentiation into osteoblastic cells. This review describes the role that FAK plays in cell biology, with particular attention to osteoblasts.

Key words: focal adhesion kinase (FAK), differentiation, migration, proliferation, apoptosis, osteoblasts

Summary: Polar transport of auxin, mediated by carrier proteins, is a unique mechanism resulting in a controlled distribution of phytohormone that generates higher auxin concentration in specific cells or tissues. Three families of cellular transport proteins, PIN-formed (PIN), P-glycoprotein (PGP/ABCB), and AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) coordinately transport auxin in plants. Among the eight members of the PIN family in Arabidopsis thaliana, five have been experimentally shown to function as auxin efflux carrier. PGP/ABCB proteins, that belong to the ATP-Binding-Cassette transporter superfamily, are the second class of auxin efflux pumps. The best characterized members of A. thaliana PGP/ABCB proteins are the auxin transporters PGP1/ABCB1, PGP19/ABCB19 and PGP4/ABCB4. Multiple lines of evidencesuggest that both PIN and PGP proteins function as independent efflux transporters that can interact in coordinated export of hormone. AUX1 uptake symporter, and three members of the LIKE-AUX1 (LAX) – the functional analogues of AUX1 – participate in auxin influx. The dynamic subcellular trafficking of auxin transport proteins, as an important factor in regulation of auxin polar transport, now focuses much attention. The distribution of transporters in the plasma membrane is crucial for the directional nature of the auxin fluxes and requires a certain degree of flexibility. The constitutive cycling of auxin transporters consists of two repeated steps: internalization of the protein from the plasma membrane into an endosome (endocytosis) and its recycling back to the plasma mebrane (exocytosis). Recent studies directly demonstrated that PINs internalization occurs via clatrin-dependent endocytic mechanism. Transfer of auxin transporters from the distinct endosomal compartments back to the plasma membrane or to vacuole is controlled by small G-proteins, retromers and protein kinases from AGCVIII subfamily. As yet, rapid PINs relocations were observed during embryonic development, lateral root formation, phyllotaxis and during tropic growth. Gravistimulation causes relocalization of PIN3, that exhibits an apolar orientation in root columella cells, but relocates in the direction of auxin movement upon root reorientation into a horizontal position. Similarly, phototropic bending in hypocotyls is thought to results from asymmetric accumulation of auxin in cells distal to the site illumination. It is likely, that PIN3 protein, localized predominantly at the inner lateral sides of endodermal cells of shoot is required for phototropic growth.

Key words: auxin, polar auxin transport, relocalization of auxin transporters