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

Summary: UDP-glucose pyrophosphorylase is a common plant enzyme and plays a key role in many processes connected with carbohydrates metabolism in plant tissues. This enzyme catalyse the reversible reaction of synthesis/degradation of UDP-glucose and inorganic pyrophosphate from glucose-1-phosphate and UTP. UDP-glucose is the precursor of many carbohydrates like sucrose, starch, pectin, cellulose. The second product of UGPase activity is PPi, the alternative to ATP energy donor which supply Pi. The highest activity of UGPase was observed in cytosol. In mature leaves UGPase takes part in synthesis of sucrose and other carbohydrates (in cooperation with sucrose phosphate synthase). In sink tissues UGPase cooperates with sucrose hydrolytic enzyme – sucrose synthase in sucrose degradation. It seems that it also plays an important role in endosperm development and sulfolipids biosynthesis in chloroplasts. UGPase plays role in cold sweetening of potato tubers, which were stored in cold temperature, and that process is unfavourable in food industry. There are many external and internal factors, which have an influence on UGPase genes expression and activity. Oligomeryzation is an internal factor. UGPase could occur in few forms but only monomer is an active form in plant tissues. External factors include environmental condition such as light, temperature, nutrient (phosphorus, iron) and other factors like availability of sucrose, mannose or okadaic acid. Stress conditions significantly change expression of UGPase genes, enzyme activity and regulate sucrose and the other carbohydrates synthesis/degradation. It was observed that reduction of UGPase activity had an visibly influence on sugars content. Such modification in carbohydrates metabolism are essential to plant acclimatisation in changing environmental conditions. That is the reason, why UGPase which was not perceived as an important enzyme in carbohydrates metabolism, takes the value. In last few years the knowledge about UGPase has increased – especially about control of genes expression, protein structure and enzyme activity regulation. This review presents short description of role UDP-glucose pyrophosphorylase in metabolic processes in plant tissues.

Key words: UDP-glucose pyrophosphorylase, gene expression, UDP-glucose, sucrose metabolism, stress conditions.

Ph1 Locus, a Supressor of Homoeological Pairing of Chromosomes in Hexaploid Wheat (Triticum Aestivum L.)

Summary: This paper presents literature data regarding molecular structure and function of Ph1 locus. In this review, we focus on the current understanding of the Ph1 influence on subtelomeric and centromeric chromatin remodeling, required for meiotic homologous chromosome pairing in wheat. Wheat is an allopolyploid having two or three different sets of chromosomes (allotetraploid wheat – 28 chromosomes, AABB genome and allohexaploid wheat – 42 chromosomes, AABBDD genome). During the course of meiosis homologous chromosomes have to somehow recognize each other and do not pair with homoeologous chromosomes. Homologous and homoeologous chromosomes contain the same genes but the differences between them concern repeated sequences of DNA. Although the genomes of the tetraploid and hexaploid wheat are extremely complex 1A chromosome pairs with 1A, 1B with 1B and 1D with 1D, this rule refers to all seven pairs of chromosomes of each genome, indicating that the plant behaves like diploid and the inheritance is disomic. The process of diploidization of alloploid wheat, beside other factors, is controlled by Ph1 locus which is localized on a long arm of 5B chromosome. Detailed studies by means of comparative genomics and deletions mapping was performed. The results showed that Ph1 locus is a cluster of seven Cdk-like genes (CDKL2) and inside of this cluster a part of subtelomeric heterochromatin is inserted. This dominant gene arose as a consequence of a tandem duplication during the process of polyploidization. Locus on 5B chromosome has a suppressing effect on equivalent loci on 5A and 5D. Cdk-like genes revealed their close homology to mammalian Cdk2 and budding yeast Ime2. Cdk2 has an influence on DNA replication and also affects chromatin remodeling and recombination. At the onset of meiosis, before pairing of homologous chromosomes occurs, in the presence of Ph1 locus subtelomeric and centromeric heterochromatin under-goes remodeling, changing the binding properties of HP1 protein, and as a result heterochromatin elongate. Remodeling is synchronous and the pairing is complete in homologous chromosomes with the same size of heterochromatin regions. If slight differences in subtelomeric heterochromatin amount occur, remodeling is not synchronous. In this case, instead of complete pairing, „pegging” and „zipping” takes place and chromosomes pair in „chain type” manner. However, if large portion of subtelomeric heterochromatin shows differences, synchronous remodeling would not occur and most of the meiocytes would contain unpaired chromosomes. Elucidation of Ph1 locus molecular structure is of great importance in wheat breeding. In perspective the high homology of Ph1 locus to mammalian Cdk2 gene together with the fact that chemical factors turning off Cdk2 activity are known make possible switching off the Ph1 locus in crosses between wheat and its wild relatives. This allows for homoeological pairing and homoeological recombination. This could enable introducing new, beneficial genes from wild species to bread wheat.

Key words: wheat, Ph1 locus, Cdk-like genes, subtelomeric and centromeric heterochromatin remodeling.

Genetic Polymorphism of the Estrogen Biosynthesis Pathway Key Enzymes in Women

Summary: Endogenous estrogens play an important role in women's organism: they control menstrual cycle through the influence on foliculogenesis, ovary's steroidogenesis and growth, and endometrium transformations. But their role is not only confined to ovaries and testis action. Number of researches report about systemic role of these hormones in women. They are involved in lipids and carbohydrates metabolism, bone mineralization, vascular functions. Estrogens are synthesized not only in gonads, but also in adipose cells, bones, brain, vasculature and adrenal cortex. Many enzymes are involved in estrogen biosynthesis pathway. It seems that two of them are one of the most important – aromatase cytochrome P450, complex of the 17a-hydroxylase and 17,20-lyase. They are products of the CYP19 and CYP17 genes respectively. The CYP19 gene encodes the aromatase cytochrome P450 enzyme, which is responsible for final steps in biosynthesis of estrogens. The CYP17 gene encodes complex of the 17a-hydroxylase and 17,20-lyase enzymes, which catalyze transformation from pregnenolone and progesterone to DHEA and androstendione respectively, which are major precursors of the estrogens. A number of publications report about influence of the genetic variation across these genes on reproductive system functions such as estrogen concentration, age at the natural menopause, breast and endometrial cancer risk in women.

RNA Editing in Chloroplast Genome. What is Known About the Regulation of This Process

Summary: The RNA editing is one of the post-transcriptional modifications which prepare RNA for fulfilling its function. It is well known that editing is a common process for most of eukaryotic organisms (from protozoan to human) and for some groups of viruses, however its mechanism is specific for species, genera and kingdoms. Up to now RNA editing is well characterized for few species only. It is suggested that substitutional RNA editing is conducted by deaminase so as it does not lead to the sugar-phosphate backbone brakeage. The sequence, usually 20 to 30 nucleotides long, placed directly upstream of the editing site is believed to be a cis-acting element and to play the crucial role in indicating the site undergoing editing. Trans-acting factors, preferably the proteins from the large family of PPR (Pentatrico Peptide-Repeat) proteins, should be able to recognize and attach to cis-acting elements initiating the editing process. In the plant kingdom keeping so complicated and energy consuming process seems to be superfluous because all the changes introduced by RNA editing could be placed directly in DNA without any harmful effects. For that reason several hypothesis concerning editing evolution, significance and mechanisms were developed. Currently the studies concerning the RNA editing in plants focus on determining any similarities and/or differences between sequences adjacent to the same editing sites in different species and these placed in the different parts of the genome. At the same time some effort is taken to develop efficient in vitro systems to study the RNA editing and to prepare some tools able to detect directly the editing defects, systematically control the RNA editing reaction state and analyse this process in vitro with use of fluorescent dyes. In this article we attempted to put together known facts referring to the RNA editing in plants with an emphasis on the chloroplast genome (particularly on regulation of the mechanism of RNA editing).

Key words: chloroplasts, RNA editing, RNA maturation, post-transcriptional processing.

Key words: replication, endoreduplication, cell cycle, pre-replication complex, Geminin, Cdk, APC, SCF.

Summary: Brassinosteroids (BR) are hormones displaying high activity in stimulation of plant growth and development. They are present at low concentrations in pollen grains, anthers, seeds, leaves, stems, roots and vegetative tissues undergoing early developmental stages in a broad range of species representing various evolutionary groups. The richest sources of brassinosteroids are pollen grains and immature seeds, whereas leaves and stems contain much lower concentration of these hormones. The first identified representative of this class of hormones was a compound characterized as a steroidal lactone – brassinolide, extracted in 1979 from pollen grains of Brassica napus. Glucosylation, sulphonization and acetylation of brassinosteroids was noted and these chemical modifications enable their transportation, storing and inactivation. Brassinosteroids are polyhydroxylated derivatives of sterols. Intact metabolism of sterols is crucial for proper embryogenesis, xylogenesis and development of shoot and root apical meristems. Biosynthesis of sterols, which precursor is mevalonian, was elucidated to the greatest extent in Arabidopsis thaliana and divided into three stages: as a result of a series of reactions constituting phase A of this pathway an intermediate is produced, which may serve as a substrate of two further alternative synthesis routes: the first one, known as phase B, leads to production of sitosterol and stigmasterol being an important components of phospholipid membranes, influencing their properties. The last stage of sterol biosynthesis pathway, defined as phase C, comprises series of reactions leading to production of brassinosteroids. As far as genetic regulation of sterol biosynthesis is concerned, late stages of this process leading to brassinosteroid production are much better elucidated by identification of genes encoding enzymes catalyzing reactions constituting phase C, together with characterization of mutant phenotypes, displaying defects in this process. Early stages of sterol biosynthesis pathway are relatively much less understood – only some of the genes responsible for these reactions were identified and phenotypes of mutants with abnormalities in this process include impaired embryogenesis. Biochemical and genetic analysis clarified the brassinosteroid signal transduction pathway in A. thaliana, which commences with perception of this hormone on the surface of plasma membrane leading to alteration in genes' expression. Brassinosteroids are perceived by transmembrane polypeptide BRI1 (Brassinosteroid-Insensitive 1) functioning as a serine-threonine kinase, belonging to a vast family of proteins, containing LRR (Leucine-Rich Repeat) domains. As a result of research, several genes were identified, encoding enzymes catalyzing different types of chemical reactions, some of them function as negative regulators of BR signal transduction. Perception of brassinosteroids triggers deactivation of signaling inhibitors and, on the other hand, accumulation and induction of transcription factors, which lead to expression of genes constituting molecular response to perception of this hormone. Brassinosteroid metabolism is maintained in the state of dynamic homeostasis on the basis of feedback mechanism between their synthesis and signaling. Brassinosteroid regulate broad range of physiological processes, including: seed development and germination, cell division and elongation, anther development, microspore germination and pollen-tube growth, differentiation of tracheary elements, proton pumping and membranes polarization, leaf senescence, induction of photosynthesis caused by increase in carbon dioxide assimilation and Rubisco activity. Brassinosteroids modulate plant metabolic response to a broad range of environmental stresses. Positive effect of brassinosteroids on aquaporin activity, responsible for transmembrane water transportation was also reported, as well as stimulation of somatic and microspore embryogenesis described in species from genus Brassica. Brassinosteroids stimulate expression of genes encoding alpha- and beta-tubulin proteins and re-orientation of cortical microtubules, which is essential for proper deposition of cellulose microfibrils, influencing structural properties of a cell wall. Brassinosteroids induce nodulation in Pisum sativum. These hormones when present at high concentration inhibit root growth and lateral root formation. Some of these physiological processes are regulated on the basis of synergistic interactions with auxins. BR regulate photo- and skotomorphogenesis through molecular interactions of brassinosteroid synthesis and signal transduction with photoreceptor-initiated transduction pathway. Alterations in metabolism of brassinosteroids cause abnormalities in morphology and architecture of plants. One of these traits is dwarfism of cereals, which gained an economical importance taking into account that it enables an increase in fertilizers dosage and as a consequence greater yield because of elevated lodging resistance of short stature plants. This paper presents a comprehensive review on the genetic and molecular basis of brassinosteroid metabolism and physiological effects of these hormones on a broad range of morphogenetic processes.

Keywords: brassinosteroids, synthesis, signaling, metabolism, physiological effects.

Ubiquitin Ligases Involved in Auxin, Jasmonate and Gibberellin Signal Transduction Pathways

Summary: Plant growth and development, which determinate plant form, require the integration of a variety of environmental signals with the intrinsic genetic program. Fundamental to this process are several growth regulators called the plant hormones or phytohormones. In accordance with definition, the plant hormones are signal molecules produced within the plant and occur in extremely low concentrations. Also, like in animals, they are often synthesized in one part of the plant and are transported to another location through the plant vasculature system or, at least in the case of auxin, through a complex cell-to-cell transport system. They interact with specific target tissues to cause physiological responses and unlikely to animals responses are often the result of two or more hormones acting together. Central to comprehending hormonal control of plant growth and development is the understanding of how the hormones are perceived and the signal is transduced. Despite decades of study, only recently receptors for several of these substances have been identified, providing plant physiologists with a much clearer picture of hormonal control. Moreover, studies have revealed a quite novel model of signal transduction in which ubiquitin ligases function as hormone receptors. In eukaryotes, ubiquitin ligases operate in the ubiquitin-proteasome system (UPS) participating in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their polyubiquitylation and later degradation by the 26S proteasome. In Arabidopsis thaliana more than 1400 genes encode components of the UPS. About 90% of these genes encode subunits of the E3 ubiquitin ligases comprise large family of protein or protein complexes containing a RING-finger, U-box domain or a HECT domain. Analysis of nucleotide sequences have revealed also that the UPS is strongly conserved in plants kingdom. The most thoroughly studied in plants is the SCF class of E3 ubiquitin ligases. The name of this class is derived from three of its four subunits: SKP1, cullin and the F-box protein (FBP). FBP represent the largest superfamily in Arabidopsis, comprising 2,7% of this plant genome. The recent study revealed that plant proteins: AtTIR1 (A. thaliana Transport Inhibitor Response 1), OsGID1 (O. Sativa Giberylin Insensitive Dwarf 1) and AtCOI1 (A. thaliana CORonative Insensitive 1), which play important role respectively in auxin, jasmonate and gibberellin signal transduction, are F-box proteins (AtTIR1, AtCOI1) or closely related (OsGID1), whereas the role of the SCF is to degrade repressors of hormone response. In the case of auxin, auxin-responsive genes are repressed by AUX/IAA proteins heterodimerizing with ARF transcription factors. Upon an auxin stimulus, the TIR1 binds auxin, enabling the recruitment of AUX/IAA proteins to the SCF complex for ubiquitination. Subsequently, AUX/IAA degradation by the 26S proteasome derepresses the ARF transcription factors. In the case of jasmonate, JAZ proteins negatively regulate jasmonate response by repressing MYC2 transcriptional activity. After binding of jasmonate, the SCF^COI1 ubiquitin-ligase targets JAZ proteins for ubiquitin-mediated proteolysis, derepressing MYC2. In addition to auxin and jasmonate, gibberellin is yet another plant hormone whose perception involves an SCF ubiquitin ligase complex. DELLA proteins repress GA response by negatively regulating GAMyb, PIF3, PIF4, and presumably other transcription factors that control the expression of GA-inducible genes. DELLA proteins also promote the expression of several GA-repressible genes, some of which encode GA biosynthetic enzymes and components of the response pathway including the GID1 receptors. In the consequence of binding GA, the GID1 receptor interacts with DELLA and the new created complex (GA-GID1-DELLA) is recognized by the SCF^GID2/SLY1 ubiquitin-ligase, which targets DELLA for ubiquitin-mediated degradation. It is seen, that GAs is not directly sensed by an F-box protein but instead by an extension molecule of the SCF substrate receptor subunit. Given the importance of SCFs to signal transduction, it is not surprising that SCF assembly and function are highly regulated. So far, tree proteins or protein complexes have been implicated in SCF regulation: RUB1 (Related to UBiquitin 1), CAND1 (Cullin Associated Neddylation Dissociated 1) and the COP9 signalosome (CSN). The mechanism of hormonal signal transduction, presented in this paper has been described only in plants, however, due to extensive occurrence of ubiquitin ligases in eukaryotic cells, it can be supposed, that this novel hormone-signaling mechanism may also exist in other organisms.

Key words: plant hormones, ubiquitin ligases, signal perception and transduction.

Summary: Breast cancer is one of the most often female cancers. From the last few years in Poland morbidity increased about 4–5%. This cancer is also cause of the major part of deaths caused by malignant tumors. Etiology of most cases of breast cancer is not possible to determine. The most important risk factors are age and breast cancer occurrence in first or second step relatives. Germinal mutations in two major high penetrance genes, BRCA1 and BRCA2 are responsible for high risk of cancer development but they constitute less than 5% of all cases of this cancer. Breast cancer can be a result of genomic instability resulted from presence of DNA double strand breaks. DNA double strand breaks are one of the most dangerous DNA damage. Unrepaired can cause amplification or lost of genetic material, which in turn can cause neoplastic transformation by oncogene activation, inactivation of suppressor genes or loss of heterozygosity. Epithelial cells of mammary gland, in consideration of estrogen exposition are remarkable exposed to induction of different DNA damage, including also double strand breaks. These breaks are usually repaired with high fidelity by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). Disorders of double strand DNA repair increase the breast cancer risk, in familiar as well as sporadic one. Differences in efficacy of DNA repair processes resulting from naturally occurred polymorphisms can also affect of breast cancer risk. Polymorphic genes of DNA repair are in great part included to low penetrance genes, with means that single gene product most often slightly affects the disease occurrence risk, but accumulation of changed alleles can have essential significance for it development. There are about 3 millions of single nucleotide polymorphisms (SNP) in human genome, which consist about 90% of all differences in the sequence. In the article were displayed information of significance of single nucleotide polymorphic variants of genes coding for proteins participating in DNA double strand breaks repair for breast cancer risk. In relatively small numbers of accessible articles, small number of SNPs for selected genes, coding for proteins of both DNA repair pathways, HRR as well as NHEJ were examined. The statistically important increase of the breast cancer occurrence risk was shown in case of persons, in which the occurrence of polymorphic variants was shown: rs1801320, rs2412546, rs4417527, rs861539, rs144848 in genes coding for RAD51, XRCC3 and BRCA2 proteins, taking a part in homologous recombination and in case of variants: rs2267437, rs2075685 in Ku70 and XRCC4 genes, coding for proteins taking a part in DNA repair by non homologous end joining. Breast cancer is the polygenic disease, therefore is particularly interesting to investigate of SNPs in multiple loci from the same or metabolically connected genes. In case of genes coding for proteins of the DNA repair by homologous recombination is particularly worthy to notice the fact, that the same polymorphic variants differentially affect breast cancer risk in carriers of specified mutations in BRCA1 and BRCA2 genes and in persons in which did not discovered such mutations and polymorphisms. These differences can reflect diffe-rent significance of SNP for occurrence and development of sporadic and hereditary breast cancers. The inclusion of SNP variant in disease risk factors is not possible only on the basis on occurring the statistically important difference in polymorphism frequency in groups of sick and healthy persons. From clinical point of view polymorphic variant can be accepted as the real disease development factor if we know physiological mechanism of its influence on the disease origin. At present stage, investigations focus first of all on the selection of all possible polymorphic variants and their combinations, potentially able to contribute in breast cancer development. The great progress in this area surely provide DNA microarrays, that allow in short time period genotype of hundreds polymorphic sites.

Key words: genetic polymorphism, DNA double-strand breaks, DNA repair, breast cancer.

Summary: The plant hormone (phytohormone), auxin plays a crucial role in a wide variety of growth and developmental processes involving cell elongation, division and differentiation. The cellular responses to auxin involve not only electrophysiological changes at the plasma membrane, but also fast alterations of gene expression. Currently, the involvement of auxin in the regulation of gene expression is well-recognized. Using differential screening approaches, a number of auxin-regulated genes have been identified, mainly in elongating tissues and dividing cells. mRNA levels of these genes were altered within minutes after auxin application and were unaffected by treatment with protein synthesis inhibitor, cycloheximide. It means that protein synthesis is not required for their activation, suggesting that the hormonal signal is transmitted to the nucleus via preexisting components. These genes are referred to as early or primary auxin response genes and classified into three major classes known as the Aux/IAA, GH3 and SAUR gene families. Members of the Aux/IAA gene family are involved in light regulation of auxin responses. Several GH3 genes encode acyladenylate-forming enzymes that catalyze conjugation of indole-3-acetic acid, jasmonic acid and salicylic acid to amino acids. The GH3 enzymes regulate auxin homeostasis by conjugating excess hormone to amino acids. Analysis of GH3 mutants indicated the involvement of these genes in photomorphogenesis, root and hypocotyl elongation and both biotic and abiotic stress adaptation responses. GH3 genes are also regulated by light suggesting a role of GH3 proteins in light-auxin interactions. SAUR are small short-lived basic nuclear proteins that physiological functions remain unknown. Some members of SAUR family have been implicated in calcium/calmodulin-mediated auxin responses. The conserved sequences TGTCTC named the auxin response elements (AuxREs) within the promoters of early auxin response genes have been identified and a family of auxin response factors (ARFs) binding to AuxRE has also been characterized. ARF proteins either promote or inhibit target gene expression. Aux/IAA genes encode short lived nuclear proteins that themselves do not directly bind DNA, but bind to ARF proteins resulting in repression of their transcriptional activity. Auxin promotes the interaction between Aux/IAA and TIR1/AFB proteins and increases the degradation rate of Aux/IAA proteins in ubiquitin/proteasom 26S pathway, such that ARF activity is derepressed and numerous auxin-mediated transcriptional changes occur. ARF proteins released from their repressor counterparts regulate the transcription of auxin response genes. This review describes recent advances in studies on early auxin response genes and physiological functions of the proteins encoded.

Key words: auxin, auxin response element, auxin response factor, Aux/IAA, GH3, SAUR.

Metalloproteinase 9 (MMP-9) as a Unique Member of The Matrix Metalloproteinase Family: Role in Influx of Neutrophils and Their Apoptosis During Inflammation

Summary: Matrix metalloproteinase (MMP) family consists of Zn²⁺-dependent endopeptidases. The enzymes degrade structural proteins of basement membranes and extracellular matrix facilitating tissue remodeling and cell mobility thus phenomena accompanying physiological and inflammatory processes as well as cancer diseases. One of the subgroups among MMPs is a subfamily of gelatinases consisting of gelatinase A (MMP-2) and gelatinase B (MMP-9). The two enzymes are characterized by the most complicated structure among MMPs and similar spectrum of substrates, however, they show different expression – constitutive in case of MMP-2 and (usually) inducible of MMP-9. Moreover, one of distinguished features of MMP-9 is the capacity to regulate cytokine and chemokine activity. Bioen-gineering of transgenic mice deficient in gelatinase B (MMP-9^-/-) revealed also physiological functions of MMP-9, for example in reproduction, functioning of nervous system, bone development, remodelling of blood vessels, wound healing and some processes occurring in thymus. Moreover, studies on MMP-9^-/- mice allowed for detailed studies of the role of the enzyme in inflammation. It was shown that apart from its role in tissue infiltration by inflammatory leukocytes (mostly neutrophils), during late stages of inflammation MMP-9 also participates in apoptosis of the cells. Furthermore, it was reported that in certain circumstances MMP-9 might limit neutrophil infiltration by degradation of various chemokines. In addition development of compensatory mechanisms was reported to operate in MMP-9^-/- mice. Thus overall MMP-9 is one of the key enzymes in the development and the course of inflammation.

Key words: matrix metalloproteinase (MMP) family, gelatinase B, transgenic mice, inflammation, neutrophils.