High-scale analysis of pathogenicity determinants of Potato mop-top virus

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus characterized by a tripartite, single-stranded, positive-sense RNA genome. However, PMTV differs from the other pomoviruses by having an open reading frame (ORF) for a 8-kDa cysteine-rich protein (CRP) on RNA 3, downstream the triple gene block. Site-directed mutagenesis of infectious cDNA clones of PMTV showed that the CRP ORF is not needed for the systemic infection in Nicotiana benthamiana and several other hosts, but it has an effect on the symptom development of PMTV. CRP of PMTV was not capable of RNA silencing suppression in agroinfiltration tests. However, the expression of CRP using vectors based on Potato virus X (PVX) or Tobacco mosaic virus (TMV) resulted in necrotic symptoms on N. benthamiana and tobacco plants, respectively. Sequencing of PMTV isolates obtained from the field revealed significant sequence variability of the CRP. These data suggest that the functions of CRP may be expressed only in certain hosts or at certain phases of the PMTV infection cycle.

intermediates of glycolic pathway decreased. In Lathyrus leaves under investigation we revealed intensive label incorporation into intermediates of the Calvin cycle and its decrease in the metabolites of the glycolic pathway. We found minor quantities of 14 C contents in sugars and PEP-products. It is supposed that the UV-rays influence the activity of RUBISCO and other enzymes of the Calvin cycle. The mechanisms of UV-rays influence on carboxylation system and the possibility of their regulatory role in high mountainous plants are under discussion. Nuclear DNA is arranged into loop domains by periodical attachment of chromatin fibers to nuclear matrix at highest level of chromatin compactisation. Chromatin loops represent the basic structural components of higher order chromatin folding, which are maintained during cell cycle and in differentiated cells. An increasing number of evidences suggests strong relationship between chromatin structure compactisation and functional activity of cell nucleus. We showed previously that regular DNA cleavage into high molecular weight (HMW) DNA fragments can be induced in intact nuclei, resulting in formation of~300 kb and 50-100 kb fragments. The patterns of induced cleavage were essentially similar both in intact and high-salt-extracted nuclei suggesting that HMW-DNA fragments represent DNA loop domains (or their associates). Biochemical properties of induced HMW-DNA cleavage suggest involvement of matrixassociated topoisomerase II in excision of DNA loop domains. We demonstrated further that patterns of HMW-DNA cleavage are distinct in plant tissues differed on proliferative activity and differentiation status. The least fragmentation was found in the embryos of dry quiescent seeds, whereas the induction of growth and development was accompanied by increase in HMW-DNA fragmentation. The study of cell nuclei during natural and accelerated ageing of rye seeds demonstrated that loss of germination capacity was associated with decreased excision of chromatin loop domains.

BioMed Central
Taken together, our data demonstrate that changes in physiological status of plant tissues are accompanied with the changes in patterns of HMW-DNA fragmentation. The HMW-DNA fragments are generated by the scaffold-associated topo II-like endonuclease activity that cleaves nuclear DNA probably at the positions of its attachment to nuclear scaffold. We hypothesize that HMW-DNA cleavage activity is related to some DNA metabolic processes in cell nuclei and is necessary for maintaining active physiological status of plant tissues. Background: At their natural environment plants are exposed to high fluxes of photosynthetically active and UV radiation. The increased synthesis of UV-absorbing compounds, such as flavonoids and other polyphenols, are accepted as a plant response to increased UV radiation. The increase of leaf flavonoid content can be a protection against the harmful effect of UV-B radiation as they function as antioxidant and UV filters in plants. Differences between sun and shade leaves (adapted to high light and low light respectively) of some tree species are analysed, taking in consideration differences on the UV-A absorption via fluorescence imaging of leaves. Materials and methods: Leaves of beech (Fagus sylvatica L.), maple (Platanus hybrida L.) and poplar (Populus nigra L.) were analysed. The Karlsruhe flash-lamp fluorescence imaging system (FL-FIS) was used to measure images at the fluorescence bands blue, green, red and far-red. The decline of chlorophyll fluorescence from Fp to the steady-state Fs was imaged too [1]. Results: Shade leaves exhibited a much higher chlorophyll fluorescence yield than sun leaves. Also differences in the distribution of the fluorescence over the whole leaf were detected which are due to the lower chlorophyll content of shade leaves and a lower reabsorption of the emitted F690 band. Shade leaves are characterized by lower values of the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) than sun leaves. Both ratios are early indicators of stress to the photosynthetic apparatus. The fluorescence ratio red/far-red expressed lower values in sun leaves and is an indicator of their higher chlorophyll content (curvilinear inverse correlation). The R Fd -images (R Fd = (Fp-Fs)/Fs) as indicators of photosynthetic activity allow to evaluate differences in photosynthetic quantum conversion. Conclusion: The observed differences in the fluorescence images and chlorophyll fluorescence images allow to characterize the different properties of sun and shade leaves related to their adaptation to high light and high UV-radiation or low light and low UV-radiation.   BMC Plant Biology 2005, 5(Suppl 1) http://www.biomedcentral.com/bmcplantbiol/5/S1/ important parameters are solar zenith angle, total ozone content, amount and type of aerosols, altitude above sea level and albedo of the ground [1]. In addition, attenuation and in special cases amplification by clouds has to be considered. Based on detailed measurements under a large number of different conditions, the effects of the individual parameters on global UV irradiance are quantified. Most of these effects depend strongly on wavelength and many of the effects are most pronounced in the erythemally weighted UV-range. The effects of the most important parameters are: 1% decrease in ozone results in an increase of erythemally weighted UV irradiance of about 1.1% [2]; aerosols can attenuate erythemally weighted UV irradiance by up to 30%, but this depends strongly on amount and type of aerosols [3]; the increase of erythemally weighted UV irradiance with an increase in altitude of 1000 m amounts between 15% and 25%, for UVA-radiation this increase is about half that much [4]; an increase of ground albedo by 0.5 will increase erythemally weighted irradiance by about 17%, UVA irradiance by about 10% [5]; a complete cloud cover attenuates UV irradiance about 40% less than total irradiance [6]. Long-term changes of solar UV radiation are a complex combination of various effects, where stratospheric ozone depletion, increase of tropospheric ozone levels and effects of global climate change work together. Therefore predictions for future levels of UV radiation are limited to the assumptions of specific scenarios. Future measurements will be necessary to monitor any changes on a local and on a global scale.
programs but also continuously kill many of their own cells through activation of programmed cell death (PCD). The earliest functions of PCD in plant life are fulfilled during embryogenesis. Here PCD governs two major developmental processes. First is the elimination of a transient embryonic structure -suspensor, which functions at early stages of embryo development as a conduit of nutrients and growth factors, but is not required at later stages [1]. Another function of embryonic PCD applies to embryo abortion, which is not only a response to stress or mutagens, but also a normal feature of those plant species, which produce polyembryonic seeds. In the latter case competition among multiple embryos for survival often induces PCD resulting in the elimination of all but one embryo in a seed [2]. At the demise, both suspensor and entire embryos display a gradient of successive stages of PCD along apical-to-basal axis [2,3]. This PCD implicates active role of autophagy in complete removal of cell protoplast. Autophagosomes are formed via Golgi and proplastids [1]. Autophagocytosis depends on the dynamic reorganization of the cytoskeleton. Microtubule network is disrupted early in PCD pathway. F-actin is gradually reorganised into thick longitudinal cables and is present till the vacuole collapse and fragmentation of nuclear DNA [1,3]. Type II metacapase is critically involved in the regulation of the cell death pathway, which is essential for normal embryo development. Metacaspase gene silencing results in the suppression of cell death and failure of embryonic pattern formation [4]. Solar UV interacts with and alters a number of cellular components. Among these, DNA is perhaps the most critical and irreplaceable target. The majority of UV-induced damage takes the form of pyrimidine dimers. These lesions act both as blocks to the progression of both replicative DNA polymerases and the RNA polymerase holoenzyme. Expression of ''dimer bypass'' DNA polymerases may also lead to permanent changes (mutations) in DNA sequence, by inserting potentially incorrect bases opposite the lesion. Thus the excision or reversal of UV-induced damage is important both in the soma, as it is required for the maintenance of gene expression, and in the germline, where error-free repair pathways maintain genomic integrity. The basic mechanisms of photoreactivation and excision repair of UV-induced dimers are well understood in plants, although we would like to learn more about the tissuespecificity and environmental regulation of these important UV-protective mechanisms. Field experiments suggest that repair of UV-induced dimers is not essential to the survival of Arabidopsis plants, nor is the expression of the sinapic acid esters that act as natural sunscreens. However, plants that are defective in both repair and sunscreen production die within hours of exposure to natural light. Progression through the cell cycle in the presence of unrepaired DNA damage products leads to a progressive deterioration of the genome. During S phase, persisting lesions are either miscopied or produce daughter strand gaps opposite dimers, which are difficult to repair in an error-free manner. For this reason, cells respond to persisting DNA damage by arresting the cell cycle in order to provide time prior to the initiation of S phase (G1/S arrest), the continuation of S phase (intra-S arrest) or progression into M (G2/M arrest). These DNA damage-induced cell cycle ''checkpoints'' are now being characterized in plants. Given the homologies between plant and mammalian damage checkpoint genes, it is likely that many aspects of cell cycle regulation by UV-induced damage are shared between plants and animals. One UV-induced mammalian response to persisting DNA damage is the induction of programmed cell death. This actively induced apoptosis and necrosis of damaged cells leads to the inflammation observed in sunburn. The induction of cell death has two beneficial effects: the resulting bursts of radicals are thought to further stimulate the repair response of neighboring cells, and the suicide of damaged cells precludes their possible progression into cancer. Plant cells can also be killed by very high doses of UV light, in what might be a programmed response (as the genomic ''laddering'' characteristic of programmed cell death occurs), but it is not clear that such a response occurs under natural light. Given plants natural resistance to the lethal effects of cancer, it is possible that plants differ from mammals in this aspect of UV-response, and lack a sensitive apoptotic response to DNA damage. reaction center complex [1,2]. In intact cells the inhibited PSII activity can be restored via de novo synthesis of the D1 subunit [3]. The FtsH protease has been shown to play an important role in the turnover of the D1 protein when induced by visible light both in higher plants and in cyanobacteria [4,5]. However, the role of FtsH in the damage and repair of PSII under UV-B irradiation has not been clarified. Here we studied this question by using a mutant strain of Synechocystis 6803 cyanobacterium, which lacks the slr0228 gene that encodes one of the FtsH homologues. Materials and methods: Synechocystis sp. PCC 6803 cells were propagated in BG-11 growth medium in a rotary shaker at 30˚C under a 5% CO 2 -enriched atmosphere. The MFtsH mutant was constructed by interrupting the slr0228 gene with a chloramphenicol resistance cassette as described in Ref. [5]. UV-B irradiation was performed in open glass containers in which the cell suspension of 6.5 Mg Chl ml -1 formed a layer of 10 mm height. A Vilbert-Lourmat VL-215M lamp was used as UV-B light source, with maximal emission at 312 nm, in combination with an 0.1 mm cellulose acetate filter (Clarfoil, Courtaluds Chemicals, UK) in order to screen out any UV-C contribution. The UV-B intensity was 4.8 Wm -2 (& 13 MEm -2 s -1 ) at the surface of the cell suspension. UV-induced changes in the transcript level of the FtsH homologue genes were detected by quantitative RT PCR. Light-saturated steady-state rate of oxygen evolution was measured using a Hansatech DW2 O 2 electrode at a light intensity of 1000 MEm -2 s -1 in the presence of 0.5 mM 2,5-dimethyl-pbenzoquinone as electron acceptor.
Results: The cyanobacterium Synechocystis 6803 has four homologues of the membrane bound ATP-dependent FtsH protease encoded by the slr0228, slr1390, slr1604 and sll1463 genes [6]. We have checked the effect of UV-B irradiation on the relative transcript level of these genes by quantitative RT PCR. As shown by the data in Figure 1 all four genes were induced more than 2-fold after 90 min UV-B exposure with the most significant effect seen in the slr0228 and slr1604 transcripts.
In search for the role of the FtsH protease we studied the UV-B induced damage in the oxygen evolving activity of PSII in the MFtsH mutant from which the slr0228 gene had deleted. When compared to the WT, the MFtsH strain showed accelerated loss of oxygen evolution under UV-B exposure and almost complete lack of recovery under visible light (Fig. 2.). The loss of oxygen evolution in the MFtsH strain has practically the same kinetics as seen in the WT in the presence of the protein synthesis inhibitor lincomycin (Fig. 2.). Conclusion: Our data demonstrate that the FtsH protease is required for the efficient repair of UV-damaged PSII centers in Synechocystis 6803 cells. Considering that restoration of PSII activity requires de novo synthesis of the D1 reaction center protein [3], we conclude that in the MFtsH strain the degradation and synthesis of the D1 protein is blocked. l l Z, Nagy F and Vass I: Repair of UV-B induced damage of Photosystem II via de novo synthesis of the D1 and D2 reaction centre subunits   Background: RAPD (random amplified polymorphic DNA) analysis is wide used for the genetic mapping, taxonomic and phylogenetic studies of many organisms. It can be also applicable for the detection of DNA alterations after influence of many genotoxical agents as well [1]. UV-radiation can produce several major types of DNA lesions such as cyclobutane-type pirimidine dimers and the 6-4 photoproducts [2]. Another important types of DNA damages such as protein cross-links, DNA strand breaks, deletion or insertion of base pairs can also be induced by UV-irradiation. Whereas, irradiation by g-rays leads to the increasing level of DNA break formation. These different types of DNA damages must be detected by changes in RAPD profiles. The aim of present study was to investigate the possibility mutation changes in plant DNA after the influence of UV-B and g-ray with using RAPD method. The 10-mer primers with single nucleotide substitutions were used in order to estimate these structural alterations in DNA following after genotoxical agents' treatment. Materials and methods: Approximately 0.20 g of plant tissue from alfalfa seedlings, which was irradiated by high doses of UV-B and g-ray (LD 50 ) was frozen in liquid nitrogen, ground with mortar and pestle and incubated in 1.5 ml of extraction buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 50 mM EDTA pH 8, 2% SDS and 0.1 mg/ml proteinase K) during 1.5 h at 37˚C. DNA was extracted twice with chloroform:isoamyl alcohol (24:1) mixture. DNA was precipitated from the aqueous phase with 2 vol. of cold isopropanol at -20˚C during 48 h. DNA pellet was harvested by centrifugation, washed several times in 70˚ethanol, air-dried and dissolved in deionized water. RNAse treatment was performed accordingly to [3]. DNA was analyzed using agarose gel electrophoresis. RAPD amplification involved initial denaturation of DNA template (20 ng per 25 ml reaction mix) at 94˚C for 3 min, followed by 33 cycles of 1 min at 94˚C, 1 min at 36˚C and 2 min at 72˚C in thermocycler ''Tertsik'' (Russia). Amplification was finished with incubation at 72˚C for 7 min. The sequences of 10-mer primers (MedBioservice) with single base substitutions are given in Table 1.
PCR amplification products were analyzed in 2.5% (w/v) agarose gel in 0.5xTBE buffer. PCR products (25 ml per sample) were mixed with 3-5 ml Gel Loading Dye Solution (Fermentas, Lithvuania) and loaded onto the agarose gel, containing ethidium bromide (0.5 mkg/ml). Gene Ruler 100 bp DNA ladder was used for each agarose gel. Electrophoresis was carried out at 80 V for 3.5 h, then the results were visualized under UV light and documented using Canon digital camera.
Results: It was shown, that single nucleotide substitutions in 10-mer primers indeed can be reflected on the amplicons' profiles.
The main changes observed in the RAPD profiles have been resulted both in an appearance (see Figure 1) or disappearance of different bands (see Figure 2) with variation of their intensity as well. Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. In animals, NO is implicated in a number of diverse physiological processes such  [2]. Both NO and ROS are necessary to trigger host cell death (see Figure 1); they are also components of a highly amplified and integrated defence system that involves activation of ion fluxes, changes in protein phosphorylation patterns, extracellular pH, membrane potential, oxidative cross-linking of plant cell wall proteins, and perturbations in the level of cytosolic Ca 2 + that triggers the local expression of resistance genes [3]. NO also functions independently of ROS in the induction of various defence genes including pathogenesis-related proteins and enzymes of phenylpropanoid metabolism involved in the production of lignin, antibiotics and the secondary signal salicylic acid (see Figure 2). The mobile nature of NO and its chemical reactivity with various cellular targets means that downstream effects of NO may be directly induced by interaction with various cellular components, like ion channels or proteins that modulates gene expression, or indirectly following interaction with signalling proteins such as protein kinases. NO signalling functions depend on its reactivity and ROS are key modulators of NO in triggering cell death, although through mechanisms different from those commonly observed in animals [4]. In summary, although several hypotheses still await experimental demonstration, it is now clear that NO is an important component of plant defence systems. Much evidence supports the view that NO plays a key role in disease resistance responses. Moreover, the recent identification of a plant NOS will lead to the characterization and manipulation of mechanisms modulating NO signalling. Thus, the understanding of NO signalling functions at the biochemical, cellular and molecular levels will soon make it possible to discern several important physiological and pathological processes in plants, as has already been demonstrated in mammals.   Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. The objectives of this study were: to examine whether NO is produced in transgenic maize suspension cell cultures and transgenic alfalfa roots exposed to hypoxic growth conditions, to determine whether there is a relationship between a class 1 hemoglobin and the amount of NO detected under hypoxic conditions, to estimate the route of formation and breakdown of NO in the tissue and to determine whether there is a relationship between NO production and aerenchyma formation. Maize suspension cell cultures and alfalfa roots, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class 1 hemoglobin. Up to 500 nmol NO/g fresh weight were detected in maize cells exposed to low oxygen tensions for 24 h. The steady state levels of NO in the different cell lines under hypoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Moreover EPR spectra showed evidence of nitrosylated heme complexes in denatured samples of hypoxically-exposed maize cells from both a wild type cell culture and a culture transformed to overexpress hemoglobin. No EPR signal characteristic of nitrosylated heme complexes was evident under aerobic conditions or in treated maize cells transformed to reduce hemoglobin expression. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO 3 -. Nitrate was shown to be a precursor of NO in hypoxic maize suspension cell cultures by using 15 NO 3 and EPR spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. There was an inverse relationship between the expression of hemoglobin and the formation of aerenchyma under hypoxic conditions. The Hbline displayed strong evidence of aerenchyma formation under hypoxia, whereas an Hb + line showed only slight evidence of cell breakdown characteristic of aerenchyma formation. The levels of NO expressed distal to the root tip were, at least, five fold greater than those found in the tip. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class 1 hemoglobins have a significant function in regulating NO levels. Background: With NASA support this research aimed at evaluating early opportunities in Microgravity Sciences to commercialize space and to develop the biotechnology facility for the International Space Station [1]. The main task was to evaluate the production of taxol (generic name: paclitaxel) with cell suspensions in bioreactors designed for the Space Shuttle. Unexpectedly, this work led to the early demonstration of L-arginine-dependent nitric oxide (NO) bursts in mechanically and gravitationally stressed plant cells, to NO-induced programmed cell death (apoptosis) (reviewed in [2]), and to a model describing how these factors contribute to increased taxol and taxane recovery from conifers. Earlier, when intermediates of the Krebs-Henseleit or urea cycle (See Figure 1) were fed to conifers, several substituted guanidino compounds were derived from uniformly labeled 14 C-L-arginine, and less so from 14 C-L-citrulline [3,4]. At that time, the substituted guanidines were considered mainly as respiratory inhibitors [5,6]. Today, they are natural inhibitors of plant, animal and human nitric oxide synthases (NOSs). NOS substrates are L-arginine and oxygen. NOS products are L-citrulline and NO. Since nitrate and nitrite reductases were also known sources of NO, we obtained an Arabidopsis nitrate reductase double mutant with the aim of finding out if cells could produce NO in the absence of nitrate, nitrite, and their reductases [7]. With this mutant we reaffirmed that the source of NO was putative NOS activity. The production of NO from L-arginine was blocked by D-arginine, and by the NOS inhibitor, N G -monomethyl-L-arginine (L-NMMA) ensuring that NO was produced in the absence of any residual nitrate reductase activity. NOS-dependent NO production in cells was inhibited by other guanidino compounds but not by D-arginine. Subsequently, the discovery by others of two plant NOS genes provided evidence that plant genomes code for NOS. In our work with Taxus cell suspensions, the substituted guanidines offered protection against mechanically induced stress and cell-death or apoptosis. Taxol is an effective anti-cancer agent that was first isolated from the bark of Taxus brevifolia [8]. Taxol binds to microtubules thereby offering a novel mechanism of blocking cell proliferation. It became the best-selling anticancer drug in history. By 2000, commercial sales of taxol were well over $1.5 billion. New models for taxol and taxane biosynthesis emerged [9] so that taxol biosynthesis could be followed at the subcellular level by immunocytochemistry, and by laser confocal and scanning electron microscopy [10,11,12,13,20]. The use of NO donors, NOS substrates, products, inhibitors (substituted guanidines), and NO traps provided new opportunities to control the citrulline-NO cycle (See Figure 1), apoptosis, and taxol production in unit gravity, simulated microgravity, and in hypergravity.
Haploid egg cells from female trees of T. brevifolia were selected as the experimental material [12,19]. These cells are easily screened without the effects of dominance, recessive, and epistatic interactions characteristic of diploid cells. Lethal genes are directly expressed, and removed by apoptosis making cell populations genetically more uniform. Diploid cell suspensions were also established from T. cuspidata [13] needles on 3-year-old stock obtained in 1995 from Zelenka Nursery (Grand Haven MI), and from seeds of T. chinensis. This provided better comparisons with all other published work with using diploid cells. Cell suspensions were maintained in darkness at 25 ± 2˚C on semisolid media, in 125 ml Erlenmeyer flasks (60 rpm), 1 L nippled flasks on a clinostat (1 rpm simulating 2 6 10 -4 6 g, but with significant convective mixing of the gaseous and liquid environment), 100 ml high-aspect rotating vessels (HARV 12.5 cm dia.), and rotating cylindrical culture vessels (RCCV 7.5 cm dia.) both at ca.10 -2 6g (Synthecon, Houston TX). The HARV and RCCV were used by NASA in early space shuttle experiments, and in a minipayload integration center, designed as an in-flight laboratory. In all cell assays, apoptotic cells were distinguished morphologically and by the TUNEL reaction [14,15]. Free taxol, taxanes, and baccatin III in cells and the culture medium, or bound taxol and taxanes, released after xylanase activity, were determined with competitive inhibition enzyme-linked immunoassays kits from Hawaii Biotechnology. Cells were examined by laser confocal microscopy (Zeiss LSM 410 Invert Scan Microscope) using single or double-labeling immunocytochemical fluorescence (FITC, Cy3) and colloidal gold to reaffirm the subcellular locations of taxol, the taxane ring (baccatin III), the C-13 side chain of taxol, and taxanes in general. For samples larger than 10 g fresh biomass, these compounds were determined by HPLC using authentic standards and taxil columns (MetaChem Technologies Inc.) [12]. Results: With T. cuspidata suspension in RCCV bioreactors (10 -2 6 g), and in 1 L nippled flasks (3 wks in darkness at 24˚C), free taxol comprised 42 and 21 percent of the total taxanes (1.1 and 2.4 mg taxol/kg air-dry biomass weight), respectively [13]. Centrifugation of cells at 3 and 24 6 g greatly increased taxane content but reduced taxol recovery. T. cuspidata and T. brevifolia responded similarly to mechanical stresses, simulated microgravity, and hypergravity by producing NO bursts, apoptosis, and by the overproduction of taxol and taxanes. T. brevifolia heat-killed cells did not produce NO. Generally 19% of the live cells (unit gravity in shaker flasks for 3 h) were always positive for NO as assayed with DAF-2DA. With the addition of 10 -4 mM SNP, a NO donor, or after centrifugation at 150 6 g for 3 h, nearly all cells produced NO. However, if 0.5 mM L-NMMA (NOS inhibitor) was added, only 26% of the cells produced NO. When 0.5 mM D-NMMA, a non inhibitor of NOS, was added, 92% of the cells produced NO [14]. The greater the NO produced and released by cells, the more cells became apoptotic. Additions of SNP (10 -6 to 10 -2 mM) increased apoptosis from 18 to 75%. Centrifugation at 150 g yielded 35% apoptosis. This was reduced to less than 10% by the addition of the NOS inhibitor, L-NMMA. Substituting D-NMMA for L-NMMA resulted in apoptosis (32% cells), but this was not significantly different from the cells exposed to 150 g [14]. The NOS requirement for oxygen connected NO production to the stress-induced and damaging increases in free radicals, viz., reactive oxygen species and reactive nitrogen species [2]. The lesson learned offered guanidino compounds as countermeasures to NO-mediated stress and apoptosis. The reactions of the Krebs-Henseleit (urea) cycle, and their relations to NOS activity. Enzymes: 1. ornithine carbamoyl transferase, 2. argininosuccinate synthetase, 3. argininosuccinate lyase, 4. arginase, 5. nitric oxide synthase, 6. arginine deiminase, 7. arginine decarboxylase, 8. numerous enzymes contributing to the formation of substituted guanidino compounds. Reactions 1 to 4 comprise the urea cycle. Reactions 2, 3, 5, may account for NOS activity in plants (citrulline-NO cycle). Arginine deiminase was reported in chloroplasts but is mostly found in microorganisms. Reactions 7 and 8 comprise decarboxylation, oxidation, methylation, transamidination, phosphorylation, keeping the guanidino group intact or modifying it by methylation, phosphorylation, etc. They remove L-arginine as a substrate from the urea cycle, and from reactions 5 and 6. L-arginine represents an important branch point that links nitrate and ammonium nutrition to protein synthesis and turnover (not shown), to the urea cycle, to a postulated citrulline-NO cycle, and to the formation of guanidino compounds (substituted and non substituted). Through oxygen requirements, the stress-induced NOS activity links respiration to NO, ROS and RNS production, their signaling pathways and damaging reactions, e.g., the nitration of phenols and tyrosines residues in cell regulatory proteins, and to apoptosis.

BMC Plant Biology 2005, 5(Suppl 1)
http://www.biomedcentral.com/bmcplantbiol/5/S1/ At unit gravity and after 10 d, egg cells exposed to SNP (10 -4 mM) produced 18 mg taxol/Kg fresh weight. This taxol yield was now 64% higher than the controls without SNP. The number of cells producing NO with SNP was 86.7% higher than the controls without SNP. By contrast, the addition of L-NMMA (0.5 mM) decreased NO production by 87.3%. Taxol yield was reduced by 75% (4.1 mg/Kg fresh weight) (unpublished results). In a separate study at unit gravity with cells supported on filter paper, the effects of SNP (10 -4 mM), sodium tungstate, (a nitrate reductase inhibitor at 1 mM), and L-NMMA (0.5 mM) were used to evaluate taxol yields after 5 d. SNP added alone, or tungstate with SNP together, yielded taxol at 8.0 and 8.1 mg/Kg fresh weight, respectively, i.e., the differences were not significantly different. This reaffirmed that nitrate reductase activity was not a contributor to NO-dependent taxol production. L-NMMA alone, or together with added tungstate, reduced taxol recovery to 2.1 and 2.0 mg/kg fresh weight, respectively. Here again, NOS was responsible for the NO that increased taxol production without a significant contribution from nitrate reductase. Removing and starving the control egg cells only slightly increased taxol recovery. The tetracyclic diterpenoid ring of taxol is synthesized by cyclization of geranylgeranyl diphosphate to give taxa-4(5),11 (12(-diene) in plastids and in light and darkness [9,11]. This basic ring structure gives rise to over 300 different taxanes. One of these, baccatin III, can provide the ring structure for the biosynthesis of taxol. Although the mechanisms are not yet clear, taxol formation also requires the addition of a C-13 side-chain originating from phenylalanine. Additional and finishing structural changes for taxol involve reaction in the endoplasmic reticulum and Golgi stacks. Using specific antibodies, the biosynthesis of the taxol ring in plastids, and the cytoplasmic assembly of bound taxol and related taxanes were visualized by laser confocal and scanning electron microscopy. Gold labeling was especially useful to identify taxol and taxanes in transport vesicles, the plasmalemma, cell walls, and in the culture medium. Colloidal 40 nm-gold-immunolabeled antibodies to taxanes, taxol, and baccatin III were visualized with 7 to 21 nM sensitivity, with and without gold backscatter (laser confocal and scanning electron microscopy). In egg cells, baccatin III (taxane ring) was detected in gravisensing amyloplasts and vesicles associated with plastid membranes. Baccatin III, taxol, and other taxanes were transported through the cytoplasm by these vesicles, which then released their contents to the plasma membrane. Plastids, that moved and docked at the plasma membrane, also released bound taxol and taxanes into the plasma membrane. The bound taxol and taxanes at the cell-surface were deposited in newly formed cell walls or released into the culture medium. The drug-productive cells and materials in the culture medium containing bound taxol, baccatin III, and taxanes were collected with antibody-labeled paramagnetic beads [16]. Immunogold-labeled taxol, taxanes and baccatin III were detected in transport vesicles, plasma membrane fragments, and on materials recovered from the culture medium. Bound taxol and taxanes were recovered from xylanase (commercial and purified) hydrolysates of cell biomass, debris in the culture medium, and from the wood of trees. Taxol and other taxanes were characterized and identified by HPLC using authentic standards. Results indicated that recovered taxol was bound to xyloglucan oligosaccharides. Probes of cells with an antibody to xyloglucan endotransglycosylases implied that 'touch' genes normally expressed under mechanical forces [18], could have provided additional sites for the attachment of taxol and other taxanes. In hypergravity (3 to 150 6 g), the taxol and taxanes released from cells by syneresis, were recovered on hydrophobic PVDF (polyvinylidene fluoride) filters. Cyclodextrins added to the culture medium enhanced biomass yield and altered the solubility of taxanes to improve the recovery of taxol and taxanes. The addition of cyclodextrins to the culture medium improved cell biomass and taxol recovery [17]. Further details are available in US Patents [16,17,18,20].
11. Durzan DJ: Gravisensing, apoptosis, and drug recovery in Taxus  In diploid parthenogenesis, 'proembryos' developed from binucleate egg-equivalents containing an egg nucleus and an apoptotic ventral canal nucleus. As the axial tier of 'early embryos' formed, the cell regulatory proteins in proembryonal cells were ubiquitinated and turned over as suspensors differentiated. Axial tier formation was blocked at high levels of chlorsulfuron, an inhibitor of acetolactate synthase. This enzyme is required for the biosynthesis of branched-chain amino acids, which are especially abundant in ubiquitin. The block of acetolactate synthase led to the accumulation of free 8-aminon-butyrate. The overall behavior of branched-chain amino acids revealed rigid and linear relations over all chlorsulfuron levels. The proliferating cell nuclear antigen (PCNA), required for DNA synthesis, was detected in rapidly cycling proembryonal cells. PCNA appeared to serve as a factor maintaining the cell replication typical of rapidly growing early embryos. Less than 0.01% of nuclei reacted with epitopes to anti-p53 and anti-p21 that are commonly associated with cell cycle arrest and DNA damage. The cleavage sites of early embryos involved apoptosis and contributed to their multiplication. Chlorsulfuron contributed to aborted axial tier development due to disrupted patterns for the ubiquitination of cell regulatory proteins as revealed by changes in the soluble amino acids.

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New approach to radiation amplification factor Don J Durzan 1  One important factor for the estimation of climate change is the Radiation Amplification Factor (RAF) defined as the percentage increase in UVbio that would result from 1% decrease in the column amount of atmospheric ozone [1,2]. RAF can generally be used only to estimate effects of small ozone changes, e.g. of a few percent, because the relationship between ozone and UVbio becomes non-linear for larger ozone changes. In other words RAF is ratio of relative changes of UVbio and the ozone column. We can say also that RAF is differential slope of UVbio to the ozone column relationship in log-log scale.
It is necessary to note that the main peculiarity of this approach is its dimensionless properties. Reactive oxygen species (ROS) can arise from normal metabolic activity such as organelle-based electron transport or be intermediates in signal transduction pathways activated by plant respiratory burst oxidase homologue (Rboh). UV-B exposure induces a pathogenesis-like response in leaves that can be abrogated by ROS scavengers [1]. A local signal is propagated by hydrogen peroxide and is sensitive to the application of catalase [2]. Similarly, local ROS production initiated by elicitors or pathogens can arise from stimulation of superoxide producing Rboh activity [3,4]. In this case as well, propagation of a ROS signal to adjoining cells is sensitive to catalase. Antisense of Rboh homologues in tomato lead to reduced ROS production [5]. Organogenic nodules have been studied in several plant species as an additional morphogenic pathway for regeneration strategies, automated micropropagation, and genetic transformation for desirable characteristics. This process has been described in hop [1]. Lipoxygenases (LOX) have several functions in plants such as response to wounding stress and pathogen attack, and growth and development [2]. Western blotting analysis showed a de novo synthesis of LOXs isoenzymes in response to wounding and during development of nodules. Immunogold labelling detected LOX in peroxisomes of some nodular cells, probably related to programmed degradation of these organelles [3]. Nodule separation into ''daughter nodules'' was initiated by the formation of necrotic layers at the future region of nodule separation which accumulated reactive oxygen species (ROS) as revealed by NBT staining. Peroxisomes may have a function in plant cells as a source of signal molecules like nitric oxide and superoxide radicals [4]. Low levels of nitric oxide and ROS are involved in many physiological processes, including growth and development [4]. Preliminary studies showed that internodes cultured with 0.3 mM of N-acetylcysteine, an inhibitor of ROS, did not undergo nodule formation. LOX presence in peroxisomes may act as a source of ROS. MAPK homologues belonging to the Extracellular-Regulated Kinases cascade were active either in the nucleus or cytoplasm depending on cellular type and developmental stage of nodule formation. ERK2 was activated upon wounding of internodes, apparently due to the formation of reactive oxygen species. ERK1 expression increased during nodule formation and plantlet regeneration [5]. During prenodule formation, ERKs were detected in the cytoplasm and nucleus of dividing cells, co-localizing with activated MAPKs. The presence of ERKs in the nucleus of prenodular cells may be related to the initiation of cell differentiation into nodules. Prenodules showed elevated levels of ROS. In vacuolated cells of nodules, ERK1/2 appeared in the cytoplasm and nucleus whereas in meristematic nodular cells they appeared only in the cytoplasm. These different cellular localization patterns may be related to proliferation and/or differentiation processes occurring during organogenic nodule formation. In our research we found out that ultraviolet B irradiation increases the activity of the pure hydrolase (b-amylase, E.C.3.2.1.1) and Merck peroxidase (E.C.1.11.1.7) by free radicals generated from synthetic polymers walls of the experimental tubes. The activation was higher after UVB irradiation than after UVA. UVB and UVA increase the intensity of cell structure destruction by free radical-activated hydrolase determined on nude alga Tetraselmis suecica, cultivated bacteria Escherichia coli O 157 (exponential phase), Acinetobacter calcoaceticus and total germs after the short time of exposure in the thermo-stated conditions. The nude alga T. suecica was more resistant and stores starch and lipids. This alga was able to convert the energy of irradiation into chemical energy of synthetic products. Some individuals of T. suecica become encysted through creating of thick inhomogeneous subsilique, under which a new silique appears. Other cells increase their glucide (intra-plastid starch granules) and lipid reserves of provisions (oleosoma appear in the central part of the cell and affect the tillakoid lamellar structure; plasto-globules appear as well). T. suecica, which lives in the surface region of the sea's waters, has 10 times higher growth resistance to natural solar irradiation. After 10 min. of exposure, it succeeds to convert energy of the absorbed radiation into the energy of the provision products stored as starch and lipid granules. Cultivated bacteria on poor specific media developed very slowly after irradiation (transmittance 300-800 nm). If the bacteria were cultivated on reach media, which absorb UV (Martin medium) they are developed by n 3 rule, instead n 2 in the first stage, after irradiations of bacteria culture (transmittance 235-800 nm).  [1,2]. Results: The human caspase-1 inhibitor Ac-YVAD-CMK and the broad range caspase inhibitor Z-Asp-CH 2 -DCB, abolished the cell death of Cd-treated and CPT-treated cells (See Table 1). This strongly suggests that the cell death pathway that is induced by cadmium employs caspase-like proteases and gives a reason to assume that in tomato suspension cells Cd-triggered cell death most probably resembles features of programmed cell death. The amount of hydrogen peroxide increased in response to Cd and CPT. Efficient inhibition of cell death occurred at the application of antioxidants and calcium channel blocker (See Table 1). The inhibition of NADPH oxidase by imidazole, quercetin and kaempferol significantly reduced the percentage of dead cells. Treatments with ethylene further decreased both Cd-and CPTreduced cell viability. Comparative experiments with Cd-or CPT-treated cells revealed an analogy in cell response to the ethylene inhibitor AVG (See Table 2). AVG greatly reduced the cell death that was enhanced in response to Cd or CPT. An increase of endogenous ethylene production (measured by laser photoacoustics) occurred in cadmium-treated cells. The data are a clear demonstration of ethylene involvement in Cd-and CPT-triggered cell death. Administration of IP 3 cycle inhibitors showed a strong inhibition to Cd-induced cell death. Conclusion: Evidence is accumulating that caspase-like cysteine proteases showing functional similarity to animal caspases, participate in the programmed cell death in plants. In addition to discoveries that caspase-like proteases are involved in cell death in response to pathogen invasion, abiotic stresses and chemical elicitation, our data show that cell death induced by cadmium is also a form of programmed cell death mediated by caspase-like proteases. We have established a key role of hydrogen peroxide and calcium in cadmium-induced apoptotic cell death and have demonstrated that oxidative stress is associated with both cadmium and camptothecin-triggered cell death. We have also shown that polyamine spermine can effectively preserve the cell viability at conditions of chemical stress.

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Ethylene was found to be an important mediator of plant cell death.
The finding that ethylene greatly stimulated cadmium-induced cell death and that cadmium treatment enhanced endogenous ethylene production indicated that ethylene participates in cadmium-induced cell death in tomato suspension cells. The application of specific inhibitors of phospholipase C, phospholipase D, inositolphosphate monophosphatase, inositol-3-phosphate kinase and phosphatidic acid caused considerable decrease of Cd-stimulated cell death and are the first more detailed evidence that Cd-triggered cell death in plants involves the phospholipid pathway. Collectively, the cell response to cadmium elicitation and the inhibitors indicate that Cd-triggered cell death is analogous to cell death in response to CPT treatment [1,2,3,4] and involves caspase-like proteases, oxidative stress and ethylene. Cdinduced cell death in plant cells exhibits similarities to HR [5] and cell death induced by known apoptosis inducing chemicals and to its effect in animal systems.    Figure 1). The single spike of chemiluminescence was observed after a lagphase of 20-40 sec. After [Ca 2+ ] cyt was increased from 0.1 mM to 1.3 mM and in 1.5-2 min was returned to a basal level. Our data are in accordance with data obtained by Price et al [7]. Results: Pre-treatment of seedlings with NOR-1 led to a single Ca 2+ -spike (see Figure 2) similar to that observed during oxidative stress. However, the level of [Ca 2+ ] cyt was increased only to 0.7 MM.
In this way, the increase in [Ca 2+ ] cyt was by 50% lower as compared with control ones. These data testify that NOR-1 interferes with [Ca 2+ ] cyt elevations caused by the oxidative stress in seedlings and that a potential communication point for cross-talk between signal transduction pathways (NO signal transduction and signal transduction during oxidative stress) is the [Ca 2+ ] cyt . Conclusion: These data suggest that NO plays an important role in the activation of plant defense responses after oxidative stress. It may partly explain that NO is able to inactivate directly the reactive oxygen species (ROS) [8]. The presence of an unpaired electrons within the NO molecule gives it its reactive species properties and is also the origin of its duality. At physiological concentrations NO may play a protective role acting as a chain inhibitor to limit the damage.   Background: Herbicide application for weed control is an integral part of the method for chemical plant protection in agriculture. Herbicides are known to be used partially only for weed destruction (as to various assessments from 5 to 40% of applied preparation amount) and the rest of their amount remains in an environment and due to high stability is not able to undergo quick physicochemical and biological degradation. Being a factor of man's impact on the environment, herbicides can play a role of toxicants under certain conditions, with manifestation of their effect on different organisation levels of living organisms. Methods: A comparative estimation of mutagenity for different commercial herbicide preparations was performed. Gesagard, granstar, zenkor, semerone, simazine and treflan were used for application on few plant test models such as Allium cepa L., Hordeum vulgare L. and Pisum sativum L. Crop seeds were germinated in water solutions of respective herbicide preparations. Seeds germinated in distilled water were used as the control. Concentration of the substances under test were chosen experimentally with due account of recommended doses, their solubility, as well as the influence on seed germination. Results: It was established that simazine caused an increase in the number of chromosome aberrations. Gesagard, zenkor, semeron and treflan affected to some or other extent cytoskeleton structures and chromosome disjunction during mitosis. Cells with apoptotic characters were detected in plants seedlings treated with herbicides (see Figure 1). Herbicides observed by us under pesticide effect indicate that cells undergo a state of oxidative stress. Active oxygen forms (AOFs) emerging under oxidative stress are known to affect the cytoskeleton structure [1]. In their turn, disorders in the cytoskeleton result in a disorganisation of intracellular transport and oxygen consumption resulting in intracellular hyperoxia and the higher AOFs [2].
Disorders in the stability of cytoskeleton proteins under oxidative stress cause inhibition of mitotic events and resulted in cell death. The glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzyme of the formaldehyde detoxification system in eukaryotes. In Arabidopsis FALDH is coded by a single gene, which is constitutively expressed [1]. By immunolocalization experiments on Arabidopsis root and leaf sections, we demonstrate that the pattern of expression of the enzyme is cell specific. By using tobacco BY-2 cell cultures we show that FALDH co-localizes with tubulin on the cortical microtubules and the microtubules figures (preprophase band, mitotic spindle and phragmoplast), which suggests a role for FALDH in some plant-specific function during cell division. Overexpression of FALDH in Arabidopsis plants results in a 25% increase in the efficiency of elimination of exogenous formaldehyde, whereas plants with reduced levels of FALDH, bearing antisense constructs, show a reduced ability and slower rate in formaldehyde The importance of FALDH has been greatly increased by the discovery of its potent activity toward S-nitrosoglutathione, the condensation product of glutathione and nitric oxide (NO) [3,4,5]. NO and NO-related metabolites, such as S-nitrosothiols (SNOs) play a central role in signal transduction and host defense [6]. We have investigated the gene response to mechanical wounding and plant hormones involved in the signal transduction pathway, showing that the gene is down-regulated by wounding in a JA-dependent pathway, and that it is transcriptionally activated by salicylic acid [7]. This is the first time that regulation of FALDH in response to signals associated with plant defense has been demonstrated.   BMC Plant Biology 2005, 5(Suppl 1) http://www.biomedcentral.com/bmcplantbiol/5/S1/ oxidation caused by UV irradiation, chemical oxidants or pathogen attack or other kinds of stress. Involved are phenolic compounds from different classes such as numerous phenol carboxylic acids, hydroxylated flavonoids such as flavones, flavonols, leucocyanidins, anthocyanins and procyanidins as well as isoflavonoids. Many of these substances have been isolated from plant species possessing valuable and intensively studied medicinal properties. For assessment of the free radical scavenging and antioxidant capacity of phenolic complexes in plants the chemical in vitro (cell free) tests can be used for their relative simplicity and sometimes reasonable cost. Here, we describe the application of several antioxidant and anti-free radical spectrophotometric assays for testing the antioxidant abilities of some rarely studied plant species containing different classes of polyphenols. In addition, the antimutagenic bacterial assays were used to examine the in vivo genoprotective activity of these compounds against chemical mutagens. Among the investigated compounds there are lipophilic flavones and their glucuronides from Scutellaria baicalensis and Iridaceae-type isoflavonoids from Belamcanda chinensis. Phenolic acids, procyanidins and flavonols containing Lamiaceae species such as Leonurus sp, Lamium sp., Stachys officinalis, Marrubium vulgare, Galeopsis speciosa have been also studied to comprise wider spectrum of different types of polyphenolics. The assays used address the different aspects of antioxidant properties such as: free radical scavenging in aqueous and nonaqueous environment (ABTS and DPPH colorimetric tests), scavenging of enzymatically generated superoxide anion radical, transition metal reduction ability by phosphomolybdenum complex formation [2], protection against hydroxyl radical induced polyunsaturated lipid peroxidation in the Fenton reaction system. It is important to employ several antioxidant assays for each object as there are usually different mechanisms of the antioxidation involved that results in varying outcome depending on the test used. For example the polyphenolic mixture from Stachys officinalis showed the weakest potential in the DPPH discoloration test whereas was the strongest one in molybdate reduction assay what clearly indicates the complexity of the involved mechanisms. The antimutagenic activity of the extracted phenol complexes and isolated compounds correlates with free radical scavenging.
In the Ames bacterial assays [1] the direct mutagenesis by chemical mutagens can be distinguished from the mutagenesis induced by activation of pro-mutagen with cytochrome P-450 enzymatic fractions. The aglycones were clearly more efficient than glycosides in inhibition of mutagenesis, the lipophilic flavone from Scutellaria baicalensis-baicalein being the most efficient. Other flavonoids were effective in inhibition of indirect mutagenesis that can be attributed to the inhibitory action against the pro-mutagen activating enzymes [3]. Free radical scavenging by the low molecular weight compounds can play an important role as the last line of defense against oxidative damage of the cells for they are more stable than enzymatic antioxidant apparatus and can be easily accumulated in stress conditions (e.g deposited in the cell wall or the vacuole). Superoxide scavenging can protect the cells against the production of deleterious peroxynitrite upon reaction of the relatively harmless superoxide with an important signaling molecule-nitric oxide.
The activity of complex extracts is sometimes stronger than individual compounds, which can be interpreted as the necessity for preserving the native composition, more effective when acting in oxidation/reduction cascades and thereby able to reduce the formation of harmful oxidation end-products. The in planta function of the antioxidant and antigenotoxic compounds should be further explored in order to obtain the complete insight into their role in protecting the plant cell.  3-Nitrotyrosine, the product of interaction between the free tyrosine with nitric oxide, is characterized by its posttranslational incorporation into carboxyl terminus of a-tubulin molecule via tubulin-tyrosine ligase, similar to unmodified tyrosine residue. However, a-tubulin C-terminal nitrotyrosination is irreversible unlike an enzymatically regulated tyrosination/detyrosination cycle. The nitrotyrosination of a-tubulin results in an appearance of some changes in animal cell morphology and their microtubule organization, a loss of some cellular functions, and in intracellular redistribution of cytoplasmic dynein. Since, nitrotyrosination in plant cell has not been investigated, the aim of our study was to reconstruct the spatial structure of nitrotyrosinated plant a-tubulin with following modeling of possible mechanisms of this posttranslational modification influence on cellular processes. http://www.biomedcentral.com/bmcplantbiol/5/S1/ The last 10-12 amino acid residues of a-tubulin C-terminal region, unlike the main part of the molecule, form a free oscillating tail that can be considered as individual structural domain. The results of reconstructing the spatial structure of detyrosinated, tyrosinated and nitrotyrosinated goosegrass (Eleusine indica (L.) Gaerth.) a-tubulin forms and studies of their behavior testify that these modifications change substantially the level of mobility of flexible a-tubulin C-terminal domain. We found that tyrosination increases substantially the mobility of C-terminus (see Figure 1). The root mean square deviation (RMSD) in coordinates of amino acid residues composing this region grows from 0.48 A for detyrosinated form, to 0.55 A for tyrosinated a-tubulin as was calculated on the basis of molecular fluctuations trajectory in 500 ps interval. These results correlate with data about predomination of detyrosinated a-tubulin in long-time living microtubules and vice versa. The breakdown of tyrosination/ detyrosination cycle due to incorporation the nitrotyrosine into C-terminus should resulted in predomination of short-time living microtubules over long-time living microtubules. It is necessary to note that mobility of nitrotyrosinated terminus is lower than the tyrosinated one, but it is higher than the detyrosinated terminus. In the last case RMSD is 0.52 À . It can lead to the appearance of microtubules with a certain ''intermediate'' lifetime and, as a consequence, modify the microtubules organization in cells.
It is known that the tubulin C-terminal region is responsible for interactions with structural and motor MAPs. In combination with the above mentioned data about the redistribution of cytoplasmic dynein after nitrotyrosination of a-tubulin we can speculate that nitrotyrosination can influence processes of tubulin-MAP recognition and interaction. This assumption is confirmed by our results on modeling the interaction of a-tubulin with the MT-binding domain of dynein heavy chains. The high content of dicarboxylic amino acid residues, especially glutamate residues, is the hallmark of the flexible C-terminal region of both tubulin subunits. It has to be correlated with the necessity of presence, a surface enriched with diamidate amino acid residues in the dynein MT-binding domain structure. The surface of dynein MT-binding domain formed by residues Lys3148, Lys3151, Lys3154, Lys3155, Arg3162, Lys3287, Lys3300, Arg3327, Lys3328 is correspondent to this factor. The surface is flanked by residues Asp3144, Asp3146 and Asp3320, Asp3321 on both sides. Included in nitrotyrosine nitro-group possesses an additional negative charge, that is not typical for dynein-tubulin interaction, and locates close to flanking residues. This location results in a strong decrease in the affinity between the dynein contact surface and a-tubulin C-terminal region. Nitric oxide is known to act as a biological messenger in divers signal transduction pathways in animal organisms. Initial investigations suggest that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. Especially, the siganiling role of NO during plant defense reactions is well established. However, mounting evidences support the hypothesis that NO is a more general effector of plant growth and development. In our laboratory, alfalfa cell cultures were used to investigate the possible involvement of NO in the regulation of cell division and differentiation in plant cells. The homogenous population of leaf protoplasts were cultured in the presence of a NO donor, sodium nitroprusside (SNP) and/or an inhibitor, NG-monomethyl-L-arginine (L-NMMA). BrdU incorporation frequency into the nuclei of the protoplast-derived cells indicated that the entry into the S-phase of the cell cycle is enhanced by SNP and inhibited by L-NMMA treatments, respectively (see Figure 1). Experiments have also been carried out with continuously dividing cell suspension cultures. The obtained data indicated that these type of cells are insensitive to similar treatments shown to affect protoplast-derived cell division (see Figure 2). In addition to cell cycle progression, the effect of the above drugs on the auxin-induced formation of embryogenic competent cells from leaf protoplasts (for more details see [1,2]) have been followed. The promotive effect of SNP and the inhibitory effect of L-NMMA have been observed on the process, especially at low exogenous auxin (0.02 mM 2,4-dichlorophenoxyacetic acid) supplementation. Conclusion: The obtained experimental data indicated that NO is required for and can stimulate auxin-mediated activation of cell division as well as embryogenic cell formation from differentiated leaf cells (protoplasts), but is not involved in the regulation of cell cycle progression of continuously dividing cells.

Figure 1 (abstract S27)
BrdU incorporation frequency into the nuclei of leaf protplast-derived alfalfa cells during the third day of culture. The cells were treated by the indicated drugs affecting endogenous NO formation. For cell culture details see [2]. Different stress conditions, of biotic and abiotic nature, enhance the cellular production of reactive oxygen species (ROS) [1]. Due to their reactive nature, ROS are potentially harmful to all cellular components. Apart their destructive nature, ROS behave as metabolic regulators, being considered as secondary messengers. Indeed, ROS can trigger pathways aimed at saving cells from demise; however, under certain conditions, they can also impair the cellular red/ox balance as to activate a programmed cell death (PCD) process [2,3]. These differences in the ROS-dependent responses seem to be due to different localization, timing and level of ROS production under different stimuli [4,5]. Moreover, the possibility that the co-production of other reactive species is a critical point for the activation of different defence responses has been recently underlined. Increasing attention has been paid to nitric oxide (NO) as a signal molecule synergically acting with ROS in the activation of PCD [6,7]. The level of ROS and the cellular redox homeostasis are regulated by different antioxidant systems; among these, ascorbate (ASC) plays a pivotal role, it being both a direct scavenger of ROS and the electron donor of ascorbate peroxidase (APX), a key enzyme for scavenging hydrogen peroxide in plant cells [8,9].
Here we report data showing that in tobacco cultured cells a moderate oxidative stress did not lead to apoptotic or necrotic events whereas, when H 2 O 2 production was increased over a certain range of concentration, an induction of cell death with the features of necrosis was achieved. On the other hand, when the tobacco cells were simultaneous treated with NO and H 2 O 2 generators a PCD program was triggered. The scavengers ASC and APX change differently under the various stress conditions. During the moderate oxidative stress a transient increase in APX activity occurred whereas, in the induction of cell necrosis, the activity of APX decreased proportionally to cell death. Under such conditions, no alteration in the APX gene expression was evident and 24 hours after the generation of the oxidative stress, APX activity was significantly increased in the surviving cells, in order to overcome the oxidative stress and to avoid further cell death. When the PCD program was triggered in these cells by the contemporary increase of NO and H 2 O 2 the suppression of APX occurred both at the translation or post-translation level. The decrease in APX seems to be one of the first alteration in the redox regulating systems induced in the plant cells in route to PCD.  Background: Nitric oxide (NO) is a unique ubiquitous molecule in animals and plants [1]. It has been demonstrated that NO mediates ABA-induced stomatal closure and interferes with ethylene during the maturation and senescence of plant tissues. Recently published data [2,3] suggested that NO is also involved in cytokinin signaling. However the above-mentioned data did not provide defenitive proof of a role of NO in cytokinin signaling. In order to obtain more conclusive evidence we have performed a study using Parr5::GUS transgenic Arabidopsis plants which express the GUS reporter gene under the transcriptional control of the cytokinin-responsive ARR5 promoter. Additional experiments were also performed using Amaranthus seedlings. Materials and methods: The Arabidopsis and Amaranthus assay systems were described in detail earlier [4,5]. The cytokinin effects were measured quantitatively in both systems. For RNA blot analysis, 3-day-old Arabidopsis seedlings were incubated with BA (with oor without tested compounds) for 35 min. Total RNA was isolated and separated in a 1.5% agaroseformaldehyde gel, transferred to nylon membrane (Amersham) and hybrydized with radioactive-labeled DNA probe. For radioautography blots were exposed with BioMax MS films (Kodak) for 1-3 days. Quantification of signal was done on an integrative densitometer CD-50 (Desaga). As a control for loading, the blot was rehybridized with an actin 2 gene probe. Results: In our experiments, a strong competitive inhibitor of all three isoforms of animal NO synthase (NOS), L-NMMA, inhibited the accumulation of GUS activity in transgenic Arabidopsis harboring the GUS gene driven by a cytokinin-sensitive ARR5 promoter. In accordance with earlier publication [ ], this inhibitor also suppressed cytokinin-dependent betacyanin accumulation in Amaranthus. In the Arabidopsis assay system, the effectiveness of L-NMMA was higher than in Amaranthus one: 1-2 mM L-NMMA inhibited cytokinininduced effect in Arabidopsis at about 80-90%. In Amaranthus, similar inhibition was observed with 5 mM L-NMMA. In addition to L-NMMA we have used its D-isomer (D-NMMA) which does not affect animal NOS. Experiments with L-and D-isomers have shown that both isomers were able to inhibit cytokinin action with similar effectiveness. Thus no functional difference between the active (in animals) L-form and its inactive D-analog was revealed in the Arabidopsis assay system. To obtain more direct evidence, we treated Arabidopsis seedlings with NO. Arabidopsis seedlings were incubated in the presence of the NO donors NOR3 or SNAP. In our experiments neither SNAP nor NOR3, both tested in a wide concentration range, caused a cytokinin-like effect, namely the enhancement of GUS activity. This result argues against a role for NO as a direct messenger of the cytokinin signal. However, it does not exclude the possibility that NO has a role in a some parallel transduction pathway, which could be indispensable to effective cytokinin signaling. This possibility was tested experimentally by adding NO donors to Arabidopsis plants which were treated with BA and L-NMMA. However experiments showed that NO did not alleviate the L-NMMA inhibition of cytokinin-induced GUS activity. This result also does not support the participation of NO in cytokinin signal transduction to primary response genes, at least in Arabidopsis.
Next we explored whether L-NMMA inhibits cytokinin signaling at an early stage (before gene activation) or acts posttranscriptionally. To this end we analysed the steady state levels of GUS transcripts 35 min after cytokinin treatment of Arabidopsis seedlings. Results demonstrated that with no cytokinin treatment GUS gene expression was very low, the radioactive signal being hardly detectable. 35 min after cytokinin treatment a more than 30-fold increase of the GUS transcript was detected. 5 mM L-NMMA, which strongly inhibits the cytokinin-induced accumulation of GUS activity, had no influence on the cytokinininduced accumulation of GUS transcripts. This result shows that L-NMMA acts posttranscriptionally.
Conclusion: Together the obtained results suggest that NOS inhibitor acted after the cytokinin signal transduction stage and NO has no direct role in eliciting the primary cytokinin response in plants, at least on cytokinin primary response genes in Arabidopsis.
ozone measurements) collected at Belsk (52N,21E), Poland, in the period 1992-2003. The transfer function from the erythemal weighted UV radiation to the UV radiation convolved with the plant action spectrum is constructed using total ozone observations (from the Brewer spectrophotometer) and UV spectral and UV erythemal data (from the Brewer spectrophotometer and the broadband UV biometer SL 501 for the erythemal effects) taken during the intercomparison campaign of the spectrophotometers in Warsaw, May 2004. The long-term variations in the reconstructed data are discussed concerning the impact of total ozone and cloudiness fluctuations on the UV radiation for the period 1992-2003. Conclusion: Reasonable reconstruction of the UV irradiance weighted by the higher plants action spectrum is possible if the erythemal UV irradiance and total ozone data are available. The erythemal irradiance is measured for many European sites by various broadband instruments. Thus, for many stations having also total ozone data (ground-based or from satellite observations) we can calculate the UV irradiance weighted by various biological effects. Discovery of characteristic proteins expressed by a given cell type and how these proteins change during development or pathology, has only been feasible very recently. Powerful separation techniques coupled with the sensitivity and selectivity of mass spectrometry, are used to identify these proteins.
Computer-based, bioinformatics techniques are used to match unique peptides to the genome.
We recently received a Major Research Instrumentation award from the National Science Foundation (USA) to obtain new state-of-the-art protein analysis equipment. Specifically, we have acquired a research grade hybrid quadrapole ESI-MS/MS mass spectrometer interfaced with a capillary liquid chromatograph (cap-LC). This system (Micromass V-QTM) is a sensitive (femtomole), high resolution, time of flight mass spectrometer (Tof) with electrospray ionization (ESI) sample introduction interface for the capLC. Tandem quadrapole mass spectrometric capabilities and high resolution permits the determination of amino acid sequence of peptides. Software associated with the spectrometer is used to unsort multiple charged versions of the same peptides and proteins, drive the LC separations, and handle other, expected complications in data analysis. As part of this proposal, we received funds to develop an education and outreach module for educators. The proposed module involves proteomics-based experiments conducted for the study of cell death in plants. The daylily flower (Hemerocallis spp.) provides an ideal model system for the study of non-stress related programmed cell death in plants. The entire genetic program/cascade occurs over a compact 24-h period and can be studied in a series of specific sequential steps. We are currently analyzing changes in the daylily petal proteome during the various stages of cell death begining at 12 h prior to flower opening and ending at the final stages of cell death 12 h after opening. A shotgun proteomics approach is currently being utilized. Data derived from this study will not only provide valuable insight in programmed cell death in plants, but also be used to teach proteomics and the study of biological processes to educators involved in biological instruction.   The main targets for UV irradiation in biological cells are DNA molecules [1]. Photochemical reactions in DNA caused by UV irradiation are the general processes that we use, in cell biology, to study the DNA damaged area. We analyzed: • known mechanisms of UV induced DNA damage in cells: excited by UV light, nucleotide bases begin to take part in photochemical reactions of cyclobutane-pyrimidine dimmer and (4-6) adducts formation.
• absorption DNA spectrum behaviour in the range 200 -300 nm changes of absorption by bases under UV irradiation.
• photoluminescence charge transport behaviour of ds-DNA molecules in wet, dry adsorbed layers with bases changes under UV irradiation. Then the experimental investigations were carried out on 2 mM plant ds-DNA polymerized molecules (Eris, France [11]) that were dissolved in 3 mM NaOH buffer and 2 mM ss-DNA with 15 oligonucleotides bases: 3'-CCA CCG CTG CTG AGG-5', length 5,4 nm, wide 1 nm (Jena BioScience, Germany) were dissolved in 100 mM carbonate/bicarbonate buffer [2]. We prepared ds-DNA polymerized molecules water solutions dissolving 0,1 ml of DNA in buffer solution in 1, 40, 50 ml of water showed a pH of 7,4 (the volume ratios are 1:1, 1:40, 1:50, correspondingly) and ss-DNA water solution dissolving 1 ml of DNA in buffer solution in 5 ml of water showed pH of 7,4. The solutions in quartz cuvettes were irradiated by UV (# = 200-400 nm) during 5-90 min with the light power 10 20 photon/(cm 2 s). From absorption spectra of different concentration of ds(ss)-DNA molecules in water solution the influence of UV irradiation on ds(ss)-DNA molecule absorption were revealed. Photochemical reactions in the nucleotide base pairs with formation of dimmers and/or (4-6) adducts, that lead to the change of electronic levels structure of ds-DNA molecule and were revealed in absorption spectra (this spectrum is similar to presented in Figure 1): absorption maximum at 265 nm (4.7 eV) shifts to 269 nm (4.6 eV) -this caused by additional absorption on formed dimmers; maximum at 285 nm (4.3 eV) is absentabsorption on cytosine base becomes minimal: we assume that cytosine was used in dimmer formation. The dependence of ds-DNA absorption intensity (for wavelengths 252 nm) on UV radiation time is represented in Figure 2b. Almost all changes in absorption take place during first 30 min of UV radiation, in the comparison with ds-DNA it could be caused by smaller number of adjacent T and C in ss-DNA bases sequence. The band in short wavelengths (l < 235 nm) with maximum at 220 nm is not sensitive to UV irradiation as and for ds-DNA. These results prove that the UV effect in DNA becomes apparent in the excitation of nucleoside bases (absorption maxima is 235-280 nm) and photochemical reactions in DNA chains. Photoluminescence spectra of wet dsDNA layer with networks have maxima at 432, 440 and 454, 463 nm before and after UV irradiation with 337 nm and 365 nm during one hour, respectively. The presence of photochemical reactions appear in the decreasing of photoluminescence intensity in this layer. Then it is possible intensive emission with participation of electronic states corresponds to molecular orbital systems in the nucleotide base pairs and sides of the ladder having a periodic structure with alternating sugar and phosphate groups. A part of the electron levels that determine photoluminescence spectrum can correspond the formation of dsDNA molecular networks.
The presence of the photochemical reactions appear in the decreasing of dry absorbed ds-DNA molecular layer with networks conductivity under periodical switched UV irradiation: as a result the conductivity decreases after first radiation reflecting the reducing of pyrimidine bases (that formed dimmers) contributing to ds-DNA conductivity. The increase of the conductivity value after UV  BMC Plant Biology 2005, 5(Suppl 1) http://www.biomedcentral.com/bmcplantbiol/5/S1/ irradiation could be caused by the particular reparation of ds-DNA under applied voltage of 1 V. Conclusion: UV induced ds(ss)-DNA damage in buffer solution and in wet, dry absorbed layer, using optical and photoluminescence spectroscopy, as well as conductivity was revealed. Models of UV induced ds(ss)-DNA damage were accounted in the photochemical reactions of cyclobutanepyrimidine dimmer and (4-6) adduct formation were used to recognise ds(ss)-DNA damage in their absorption spectra and wet ds-DNA damage in photoluminescence spectra and in the behaviour of conductivity under periodical switched of UV irradiation. But it's important to develop offered methods for its further ability to direct evident possible photochemical reaction that took place in ds (ss)-DNA. Cultivated sunflower (Helianthus annuus L.), together with soybean, oil seed rape and peanut is one of the four most important crops for oil production and has a cultivated area that range from Russia, Eastern and Central Europe to Central America. Wild sunflower species served as genetic base for the modern sunflower. Cultivated sunflower was selected from a very narrow genetic base. Enlarging the genetic base will allow it to be better adapted to environmental conditions. Many wild species are used to introduce useful traits, such as disease resistance (Phomopsis stem cancer, Sclerotinia wilt), cytoplasmic male sterility and increased oil content to the cultivated crop by introgression. Sexual hybridisation is limited in most cases to cultivars within a species or at best to a few wild species closely related to a cultivated crop. Species barrier thereby limits the genetic improvement by classic breeding. Somatic hybridisation, or somatic cell fusion technique, leading to the formation of viable cell hybrids is one of the methods used to overcome sexual incompatibility. In order to eliminate the unfavourable wild species traits in the somatic hybrids, a number of back crossings to the cultivated sunflower would be necessary. A method to reduce the backcrossings by reducing the amount of DNA from wild species in the hybrids, is the asymmetric fusion of the protoplasts. This technique is used to introduce fragments of the nuclear genome from the wild species into the complete genome of cultivated ones. A fragmentation of the donor genome can be induced by UV irradiation of the donor protoplasts prior to protoplast fusion. The effect of UV-light treatment was determined on the basis of Helianthus mollis protoplast viability and division capacity. The isolation and purificartion of protoplasts was according to Krasnyanski and Menczel [1] protocol. The suspension of purified protoplasts was placed under a UV lamp during 60 min (wavelength of UV irradiation was 254 nm). Irradiation intensity was 2 Mmol m -2 s -1 . Protoplast viability was determined every 5 min, on 100 protoplasts, using FDA, under fluorescent microscope. The protoplasts were cultured in agarose droplets in VKM media. Plating efficiency (the number of cells dividing of total number of cells) was determined at 4 and 8 days in culture. We observed the UV irradiation affects both, viability and division capacity of H. mollis protoplasts. Efficiency of UV-light treatment depends on its intensity and duration, a treatment in our conditions of more than 10 min decreases significantly the viability of the tested cells. Introduction: Over the past two decades, it has been recognized that nitric oxide (NO) plays an important role in diverse mammalian physiological processes. NO regulates physiological processes by modulating the activity of proteins principally by nitrosylation, a process referring to the binding of NO to a transition metal centre or cysteine residues [1]. An important class of proteins that constitutes key targets of NO is that of the Ca 2+ channels including plasma membranes as well intracellular Ca 2+ channels. NO modulates these channels directly by nitrosylation, but also indirectly via the second messenger cyclic GMP (cGMP) and/or cyclic ADP ribose (cADPR). Therefore, NO emerges as a key messenger governing the overall control of Ca 2+ homeostasis [2]. In the late 1990s, NO also became an increasingly popular target for investigation in plants. As in mammals, NO fulfils a broad spectrum of signaling functions in (patho)physiological processes in plants [3]. Here, we summarise studies published in recent years that provide novel insights into the signaling functions of NO produced by plant cells exposed to abiotic stresses and biotic stress (pathogen-derived elicitors). It focuses particularly on the cross-talk operating between NO and Ca 2+ .
Results: Over the past few years, we have studied the functions of NO in plant cells challenged with elicitors of defense responses. One elicitor has been used primarily: cryptogein, a 10 kDa elicitor produced by the oomycete Phytophthora cryptogea [4].
Using the NO sensitive fluorophore 4,5-diaminofluorescein diacetate (DAF-2DA), we reported the real-time imaging of NO production in epidermal tobacco cells treated with cryptogein [5]. After elicitation with the elicitor, the earliest burst of NO was in the chloroplasts, where NO production occurred within 3 minutes. The level of fluorescence increased with time, and after 6 minutes NO was also found along the plasma membrane, in the nucleus and most probably in peroxisomes. To investigate the signaling events that mediate NO production, and to analyse NO signaling activities in the cryptogein transduction pathway, a spectrofluorometric assay using DAF-2DA was developed to follow NO production in tobacco cultured cells. As observed in tobacco epidermal tissue, cryptogein induced a fast and transient NO production in tobacco cell suspensions [6]. This production was completely suppressed in the presence of the NO scavenger cPTIO, and was reduced by 55 to 85% by mammalian nitric oxide synthase inhibitors. By contrast, inhibitors of nitrate reductase inhibitors, a plant NO source, had no effect on cryptogein-induced NO production. Calcium signals are thought to play an important role in the tobacco cells response to cryptogein [7]. ] cyt in aequorin-transformed tobacco cells [6]. As predicted from a pharmacological study, the channels responsible for NO-induced [Ca 2+ ] cyt elevation include voltage-dependent Ca 2+ channels of the plasma membrane and intracellular Ca 2+ channels sensitive to RYR and IP 3 R inhibitors. This observation paralleled the situation encountered in animal cells in which almost all the molecules involved in the control of Ca 2+ homeostasis seem to be modulated by NO [2]. Recent evidence from our laboratory suggest that NO mediates [Ca 2+ ] cyt through multiple mechanisms including phosphorylation-dependent processes, cADPR and plasma membrane depolarisation.
Our data, along with those from other studies, highlight the crucial role of NO in protecting plants against pathogens by promoting Ca 2+ mobilization but also defense-and stressrelated gene expression and HR [6]. Besides pathogen attack, abiotic stressors, such as drought, salinity and extreme temperature are serious threats to agriculture. In the recent years, a significant amount of work has gone into investigating NO synthesis and functions in plants exposed to abiotic stressors. For example, it was shown both in tobacco leaf peels and tobacco suspension cells that high temperature, osmotic stress, or salinity, generate a rapid and significant surge in NO levels [8]. In contrast, light stress and mechanical injury had no apparent effect on NO production in tobacco and/or tomato. Background: Microtubules are necessary for a wide spectrum of cellular functions, which include cell division, intracellular transport, organelle positioning and generating of cell polarity. The major component of microtubules is tubulin heterodimeric protein which is consist of two subunits: aand b-tubulin. Both tubulin subunits can be extensively altered by post-translational modifications, including detyrosination/tyrosination, acetylation/deacetylation, polyglutamylation, polyglycylation, and phosphorylation. As for the different tubulin isotypes, the functionality of the post-translational modifications is still a matter of debate. Although, it is known that some of them are associated with stable/dynamic populations of microtubules, while others seem to influence the binding of motor proteins [1]. One of post-translational modifications, tubulin phosphorylation, is not a widely observed and its precise function is unknown both in animal and plant cells.
It was shown recently that animal tubulin can be phosphorylated by different systems of cyclic nucleotide-dependent (cAMP-and cGMP-dependent) protein kinases, Ca 2+ -dependent protein kinases (including Ca 2+ -calmodulin-dependent and Ca 2+ -dependent, phospholipid-stimulated types of enzymes), casein kinases and tyrosine kinases, too [2,3]. The combined data demonstrate that plant tubulin can also undergo extensive phosphorylation by different types of protein kinases and that the phosphorylation on serine\threonine as well as at tyrosine residues can participate in the generation of high level of polymorphism of plant tubulin [4]. It is interesting to establish a functional role of this tubulin modification as phosphorylation is a universal posttranslational modification which is typical for most of the proteins. The effects of different activators and inhibitors of protein kinases on microtubule dynamics and cell cycle progression in plan cells are present in this report. Materials and methods: Two plant lines, Arabidopsis thaliana [5] and tobacco BY-2 cell culture [6] (kindly handed over by Prof. J.-P. Verbelen, University of Antwerp, Belgium) both expressing GFP-tubulin as well as A. thaliana and Nicotiana tabacum wild types were used in this research. GFP-labeled microtubules in A. thaliana and BY-2 cells were analyzed by confocal laser scanning microscopy. The root tips of 3-day old Allium cepa seedlings were also used in this study. The primary mouse monoclonal antibodies TU-01 (against a-tubulin) and TU-06 (against b-tubulin) (kindly provided by Drs. V. Viklicky and P. Draber, Institute of Molecular Genetics, Prague, Czech Republic) were used for visualisation of microtubules in onion meristematic root tip cells by immunofluorescence microscopy. FITC-conjugated anti -mouse antibody (Sigma, USA) was used as a secondary one. The fixation and staining of microtubules by antibodies were performed as described by us early [7]. As regulators of protein kinases, dibutyryl-cAMP (Serva, Germany) in combination with ATP, polymyxin B (Serva, Germany), trifluoperazine (Serva, Germany) and okadaic acid (Sigma, USA) were used. Results: For more detailed analysis of the functional role of tubulin phosphorylation in plant cells several specific inhibitors and activators of different types of protein kinases were used in our research. Dibutyryl-cAMP (10 mM in combination with 100 mM ATP) as an activator of cAMP-dependent phosphorylation, polymyxin B (5 mM) as an inhibitors of the protein kinase C, trifluoperazine (5 mM), as an inhibitor of the Ca 2+ -calmodulindependent protein kinase, and okadaic acid (inhibitor of protein phosphatase type 2A, PP2A), in concentration 1-30 nM, were investigated with regard to their ability to affect microtubule dynamics and to induce structural changes of microtubules. The root tips of seedlings were treated with each of these compounds. The effects of these regulators of protein kinases on the structural reorganisation of interphase and mitotic microtubules were studied after exposure of plant material in the presence of activator or inhibitor during 6, 12 and 24 h. Immunofluorescence analysis of microtubules showed that treatment by cAMP causes the disruption of both interphase and mitotic microtubules and accumulation of depolymerised tubulin around the nuclei in the cells. The treatment of onion cells by trifluoperazine caused the reorganization of microtubules and change of their spatial organization from a transverse to a longitudinal orientation and formation of thick longitudinal arrays. The treatment of A. cepa cells with polymyxin B caused the same effects on microtubular organisation as trifluoperazine.
Confocal laser scanning and light microscopy of A. thaliana and N. tabacum cells revealed that okadaic acid arrested cell growth, alter cell morphology, and affected the organization of microtubules. Conclusion: It was reviewed by us that plant tubulin can undergo extensive phosphorylation by different types of protein kinases, and that tubulin phosphorylation participates in regulation of the plant cell cycle [4]. Many studies shown that different protein phosphatase inhibitors effect microtubules in animal and plant cells. For instance, it was shown that the treatment of Tradescantia stamen hair cells with okadaic acid and other protein phosphatase inhibitors caused changes of the metaphase transit times and the pattern of sister chromatid separation [8]. The treatment of Arabidopsis shoots with inhibitors of serine/threonine protein phosphatases (okadaic acid or calyculin A) provoked the destruction of root morphology, that can be explained by the influence of these compounds on cortical microtubules function [9]. The same authors later proved that phosphatase inhibitors as well as protein kinase inhibitors destroy not only root morphology but that cortical microtubules also become disorganized after exposure to some types of inhibitors [10]. In particular, these effects were characteristic of protein phosphatases such as calyculin A and cantaridin. The protein kinase inhibitor staurosporine also had similar effect in plant cells [11,12,13]. The disruption of microtubules was found recently after calyculin A and okadaic acid treatment in Lilium [14]. Thus, literature indicates that phosphorylation and dephosphorylation represent a part of the molecular mechanism responsible for both the organization of the cortical microtubular networks and of mitotic function.
Studies on animal cells clearly demonstrated that okadaic acid and other protein phosphatase inhibitors induce mitotic arrest [15,16], premature chromosome condensation [17,18], microtubule disassembly [18,19], DNA fragmentation [20,21] and apoptosis [16,17,20,21]. Summarizing our data obtained we can conclude that the changes in the spatial organisation of microtubules after treatment by cAMP and the protein kinase inhibitors lead to disturbances of cell cycle progression and it is most likely to launch of the cell death program in plant cells.
mitosis, the process of chromosomal division to form new cells. Microtubule dynamics are highly regulated during the cell cycle by endogenous cellular regulators. In addition, many antimitotic drugs and natural compounds alter the polymerisation dynamics of microtubules, blocking mitosis, and consequently, inducing cell death by apoptosis [1]. The drugs bind to diverse sites on tubulin and at different positions within the microtubule, and they have diverse effects on microtubule dynamics. However, by their common mechanism of suppression microtubule dynamics, they all block mitosis at the metaphase/anaphase transition, and induce cell death. After treatment with different anti-tubulin drugs, it was also demonstrated that apoptosis occurs either in the interphase or in mitosis depending on the cell type and experimental conditions [2,3]. The relationship between cell cycle disturbance due to the use of these agents and apoptosis is poorly understood. Insight into the binding and mode of action of most of antimitotic drugs have been obtained by the identification and mapping of mutation in appropriate resistant lines. Unfortunately, up to now there is no strong evidence about mode of action N-phenyl carbamates, effective anti-mitotic herbicides. Although, recently it was shown that phenylcarbamates and benzimidazoles, can compete for the site(s) of binding on b-tubulin. Benzimidazoles interact very specifically with different eukaryotic b-tubulin and resistance to these anti-microtubule drugs may be caused by point mutations in b-tubulin which replace Glu-198 with either Ala, Asp, Gln, Lys, Val, or Gly, or which replace Phe-200 with Tyr [4]. As it was found amino acid substitutions in position 198 of fungal b-tubulin, lead to negative cross-resistance to phenylcarbamates, but changes Phe from Leu-250, Val from Ala-165, and Ala from Thr-237 are responsible for phenylcarbamate crossressistance [5]. To clarify the sensitive/resistance of isopropyl-N-phenylcarbamate (IPC) resistant N. sylvestris mutants [6] to benzimidazole the influence of different concentration of nocodazole and its diverse effects on microtubule dynamics and cell cycle progression were studied. Two different apoptotic responses and patterns in control and mutant lines were found after nocodazole treatments. Results: In both control and IPC-resistant N. sylvestris lines various nocodazole concentration treatments led not only to increase the mitotic indexes from about 7% to~12-13%, but also resulted in an appearance of mitotic figure disturbances during different phases of mitotic division (see Table 1 and Table  2). No any disturbances were found in prophases of IPC-resistant plants after drug effects, whereas nocodazole in concentration from 10 to 500 MkM revealed them in control plant cells on dose-depended manner (Table 1). It was found that metaphases of both lines were more sensitive to nocodazole treatments, but nevertheless the per cent of BMC Plant Biology 2005, 5(Suppl 1) http://www.biomedcentral.com/bmcplantbiol/5/S1/ disturbances in metaphases, anaphases and telophases was about in two-three times more in control line as compare to mutant one (see Table 1 and 2). It was found also that different nocodazole concentrations rapidly induced apoptotic processes in both control and mutant lines, but cell responses after nocodazole treatments were completely different. Even low concentration of nocodazole with consequent more frequent anomalous situations (for example, multipolar mitoses, and as a result of this, an appearance of star anaphases). No any DNA fragmentation was observed in IPC-resistant lines after any concentration of nocodazole treatment. Conclusion: It is well known that treatment of cells with nocodazole and other microtubule-interfering agents evokes the activation of stress response pathways, cell cycle arrest, and the induction of apoptosis. Early, nocodazole was frequently used to examine the role of microtubules in vesicle transport [7], to induction of polyploidy [8], aneuploidy [9], and certainly to study an apoptosis in different cells [8,9,10,11,12,13]. Very often nocodazole effects result in DNA fragmentation, one hallmark of apoptosis. In our data nocodazole treatments revealed DNA fragmentation in control, sensitive to IPC, N. sylvestris line only. It was not found any DNA fragmentation in mutant plan cells after nocodazole exposure. Nevertheless, cell death was observed in both lines. We established that nocodazole increased the mitotic index in both lines but it also enhanced an apoptotic response. These results are coincided with data obtained early on animal cells [10]. We also found that IPC-resistant N. sylvestris mutant cells possessed less sensitivity to nocodazole treatments that control  and DNA fragmentation. It provokes also cell death process in IPC-resistant cells, but on another distinct way of cell degradation. It is possible to conclude that the same nocodazole treatments could induce, in two different plant lines, cytostatic effects followed by probably two different patterns of plant death. Recently, two different patterns of cell death were also found on two different cell animal lines after same antimicrotubular drug effects [3]. It has already been reported early that nocodazole possesses pleiotropic effects that are independent of microtubules [7], or probably different apoptotic responses depend from cell types as it was indicated by us above. It is possible to presume that the higher apoptotic rate in control plant cells after nocodazole treatments may be also a consequence of absence mutant microtubule protein(s) or mutant microtubule associated protein(s) which confer(s) resistance to IPC in N. sylvestris mutant lines. Experiments on nocodazole effects on cells of model plant lines are continuing now.  Background: Microtubules provide structural support for cells and play a key role in mitosis and meiosis. They also play a key role in plant cell differentiation, new tissue and organ formation through a coordinated program of symmetric or asymmetric cell division. It is well known that microtubule-damaging agents suppress microtubule dynamics, leading to disruption of the mitotic spindle in dividing cells, cell cycle arrest at M-phase, and the late apoptosis [1]. A better understanding of the processes coupling microtubule damage to the onset of apoptosis will help to find out in process of higher plants development. Recently, the mutant Nicotiana lines with high resistance to antimitotic herbicide isopropyl-N-phenyl carbamate (IPC) were produced [2,3]. Detailed analysis of IPC-resistant Nicotiana sylvestris plants revealed some abnormalities in microsporogenesis leading to partial pollen sterility. Here we report that these mutant N. sylvestris lines conferring resistance to IPC demonstrate clearly programmed cell death (PCD) events in male gamethophyte formation and development. Materials and methods: IPC-resistant and control (sensitive) N. sylvestris plants grown in in vitro conditions and greenhouse were used in this study. Sterile plants of both lines were placed from in vitro conditions to soil for adaptation, and then they were grown in greenhouse. For cytological analysis the roots of control and mutant N. sylvestris lines were treated with 30 mkM IPC for 24 h, fixed in an ethanol/acetic acid mixture (3:1) for 12 h, stained with acetoorcein (1% solution in 45% acetic acid) for 24 h and examined. Bud seeds and anthers were fixed accordingly protocols [4]. After washing the samples were kept in 70% ethanol. The samples were underwent an alkali and enzyme treatment before staining with acetoorcein.
Results: The resistance to IPC in mutant N. sylvestris lines were confirmed by us by series of tests and morphocytochemical analyses. For instance, the morphocytochemical analysis of control and mutant lines revealed 6-8 times more resistance to IPC of mutant line as compared with control one. It was found also that the increasing doses of IPC lead to dramatic changes in nucleus and microtubule network in sensitive plants. In control plants tripolar anaphases and telophases were found after treatment with 30 MkM IPC. IPC-treatment resulted also in high number of metaphase+anaphase (from 32.48% in control cells BMC Plant Biology 2005, 5(Suppl 1) http://www.biomedcentral.com/bmcplantbiol/5/S1/ without treatment to 80.07% after treatment) and accumulation of multinuclear cells and lobbed nuclei in treated control cells (see Figure 1d-f). Threshold level of IPC induced an apoptotic process, characterised by cell cycle arrest, hypercondensation of chromatin, an appearance of lobbed nuclei; abnormal, multipolar mitotic spindles, post-mitotic micronuclei formation due to nuclei fragmentation in the root cells of sensitive lines. However, root meristematic cells from resistant plants revealed no disturbances in cell cycle progression after treatment with 30 MkM IPC (see Figure 1a-c). Further investigations shown that control and mutant N. sylvestris plants possessed some morphological differences (see Figure 2). Mutant plants characterised in certain depression resulted in reduction of sizes of vegetative and generative organs, and of plant bodies on the whole. There were slight differences in the appearance of flowers (see Figure 3a) and more visible differences in the length of stamens and pistils between control and mutant lines (see Figures 3b and 3c). More detailed analysis of reproductive organs development of IPC-resistant mutants revealed that microspores in anthers are subjected to degenerative process, leading to pollen sterility. It was found also the chromatin agglutination in nuclei of most part of microsporocytes.
During study the microsporogenesis in IPC-mutant plants it was revealed the formation of microspores of different sizes (gigantic, medium and dwarfic: 15.05 ± 0.06 mkm, 7.50 ± 0.23 mkm and 5.15 ± 0.13 mkm in size, respectively) and in different numbers (dyads, triads and polyads). In the same time normal tetrads produced in control N. sylvestris plants (data not shown). Variation in sizes and numbers indicates on disturbances in chromosome separation in I and II meiotic divisions, loss the second division in one (in the case of triads) and in both dyads, the formation of multipolar spindles (in the case of polyads) in mutants. It was established that pollen grains of control plants possessed the high level of fertility (97.12 ± 0.33%); they were homogenous and synchronous in development. Whereas, genetically non-balanced microscopes of mutant lines after tetrad dissipation were unable to further development. They did not synthesise the fats and new cytoplasm and underwent typical cell death. Only 14.73 ± 1.09% formed microspores got down later to division with vegetative and generative cells formation. As a result, the common number of pollen was reduced in anthers of mutant plants. About 2.88 ± 0.33% of bicellular pollen grains were hydrated and stopped in development. Consequently, the mature pollen of mutant plants was morphologically and functionally non-homogenous. Its fertility in different flowers was from 7.63 to 27.93%. Sterile pollen grains were small, weakly stained or non-stained after incubation with dye (see Figure 5b). They also had reduced contents of cytoplasm as compared with control plants.
The results of female gametophyte study in mutant and control N. sylvestris plants demonstrated that ovary development after pollination was lower in 1.5-2 times in mutant plants as compare with control ones (see Figure 6). The delay in growth and development was connected with the high number of nonfertilized bud seeds (see Figure 7) in mutant plants. We found strong correlation between fertility level of pollen and a number of fertilized bud seeds in these plants.
Analysis of fertilized bud seeds of N. sylvestris mutants shown that bud seeds were different in the sizes that indicates on their non-homogeneous and asynchronous development as compare with control plants. It was found that a speed of embryos development in mutant plants was lower than that in control lines, and sometimes an embryo development stopped on early globular stage. Therefore, the seed production in mutant plants was very low due to both high pollen sterility and decreased viability of bud seeds development.
Conclusion: Programmed cell death has been observed to occur during many stages of plant reproduction [5] as PCD is essential for development and survival of plants. But there are still a lot of open questions about genes and their products which might function as signals to evoke a cell suicide program, in particular, during male gametophyte development. Comprehensive reviews about the mutants known to affect meiosis [6] and about the genes controlled of male gametophyte  BMC Plant Biology 2005, 5(Suppl 1) http://www.biomedcentral.com/bmcplantbiol/5/S1/ development [7] help to understand and clarify certain moments in this issue, but not completely. We suggest also that cytoskeleton proteins, in particular, different isoforms of alpha-, beta-and gamma-tubulins as well as other microtubule organising centre (MTOC) proteins may play a crucial role in many processes of microsporogenesis. Summarising obtained results we can consider that all established abnormalities in male reproductive organ development leading to PCD in pollen production can be arise from a mutation of the microtubule protein(s) in IPC-resistant N. sylvestris lines. We hypothesize also that the mutant protein (s) may represent(s) a key biological trigger in the expression of physiological death at the cellular level in these plants.
The analysis of IPC-resistant N. sylvestris plants is continuing to determine the role of certain mutant cytoskeletal gene(s) which is/are express(es) in male gametophyte and lead to PCD during pollen production for better understanding the male reproductive organ development.    Ovaries of N. sylvestris control (at the top of the picture) and mutant (below on the picture) lines straight away after pollination, and after one, two, three and four weeks of development.