Master Regulatory Transcription Factors in ß-Aminobutyric Acid-Induced Resistance (BABA-IR): A Perspective on Phytohormone Biosynthesis and Signaling in Arabidopsis thaliana and Hordeum vulgare.

The endogenous stress metabolite ß-aminobutyric acid (BABA) primes plants for enhanced resistance against abiotic and biotic stress by activating a complex phytohormone signaling network that includes abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). In this study, through stringent filtering, we identify 14 master regulatory transcription factors (TFs) from the DOF, AHL, and ERF families that potentially regulate the biosynthesis and signaling of these phytohormones. Transcriptional analysis of BABA-treated Arabidopsis thaliana and Hordeum vulgare suggests that DOF family TFs play a crucial role in stress response regulation in both species. BABA treatment in A. thaliana upregulates the TFs MNB1A and PBF and enhances the expression of the genes ICS1, EDS5, and WIN3 in the SA biosynthesis pathway, potentially boosting NPR1 and PR1 in the SA signaling pathway. Conversely, in H. vulgare, the BABA-induced upregulation of TF DOF5.8 may negatively regulate SA biosynthesis by downregulating ICS1, EDS5, and PR1. Additionally, in A. thaliana, BABA triggers the expression of TF PBF, which may result in the decreased expression of MYC2, a key gene in JA signaling. In contrast, H. vulgare exhibits increased expression of ERF2 TF, which could positively regulate the JA biosynthesis genes LOX and Tify9, along with the COI1 and JAZ genes involved in the JA signaling pathway. These findings offer new perspectives on the transcriptional regulation of phytohormones during plant priming.

Treatments with Diquat Reveal the Relationship between Protein Phosphatases (PP2A) and Oxidative Stress during Mitosis in Arabidopsis thaliana Root Meristems.

Reversible protein phosphorylation regulates various cellular mechanisms in eukaryotes by altering the conformation, activity, localization, and stability of substrate proteins. In Arabidopsis thaliana root meristems, histone post-translational modifications are crucial for proper cell division, and they are also involved in oxidative stress signaling. To investigate the link between reactive oxygen species (ROS) and mitosis, we treated various Arabidopsis genotypes, including wild-types and mutants showing dysfunctional PP2A, with the ROS-inducing herbicide diquat (DQ). Studying the c3c4 double catalytic subunit mutant and fass regulatory subunit mutants of PP2A provided insights into phosphorylation-dependent mitotic processes. DQ treatment reduced mitotic activity in all genotypes and caused early mitotic arrest in PP2A mutants, likely due to oxidative stress-induced damage to essential mitotic processes. DQ had a minimal effect on reversible histone H3 phosphorylation in wild-type plants but significantly decreased phospho-histone H3 levels in PP2A mutants. Following drug treatment, the phosphatase activity decreased only in the stronger phenotype mutant plants (fass-5 and c3c4). Our findings demonstrate that (i) the studied PP2A loss-of-function mutants are more sensitive to increased intracellular ROS and (ii) DQ has indirect altering effects of mitotic activities and histone H3 phosphorylation. All these findings underscore the importance of PP2A in stress responses.

Production of Polyphenolic Natural Products by Bract-Derived Tissue Cultures of Three Medicinal Tilia spp.: A Comparative Untargeted Metabolomics Study.

Medicinal plant tissue cultures are potential sources of bioactive compounds. In this study, we report the chemical characterization of the callus cultures of three medicinal Tilia spp. (Tilia cordata, Tilia vulgaris and Tilia tomentosa), along with the comparison to bracts and flowers of the same species. Our aim was to show that calli of Tilia spp. are good alternatives to the calli of T. americana for the production of polyphenols and are better sources of a subset of polyphenolic metabolites, compared to the original organs. Calli were initiated from young bracts and grown on woody plant medium containing 1 mg L-1 2,4-D and 0.1 mg L-1 BAP. For chemical characterization, a quality-controlled untargeted metabolomics approach and the quantification of several bioactive compounds was performed with the use of LC-ESI-MS/MS. While bracts and flowers contained flavonoid glycosides (astragalin, isoquercitrin) as major polyphenols, calli of all species contained catechins, coumarins (fraxin, esculin and scopoletin) and flavane aglyca. T. tomentosa calli contained 5397 µg g DW-1 catechin, 201 µg g DW-1 esculin, 218 µg g DW-1 taxifolin and 273 µg g DW-1 eriodictyol, while calli from other species contained lower amounts. T. cordata and T. tomentosa flowers were rich in isoquercitrin, containing 8134 and 6385 µg g DW-1, respectively. The currently tested species contained many of the bioactive metabolites described from T. americana. The production of catechin was shown to be comparable to the most efficient tissue cultures reported. Flowers and bracts contained flavonoid glycosides, including tiliroside, resembling bioactive fractions of T. americana. In addition, untargeted metabolomics has shown fingerprint-like differences among species, highlighting possible chemotaxonomic and quality control applications, especially for bracts.

Microcystin-LR, a cyanotoxin, modulates division of higher plant chloroplasts through protein phosphatase inhibition and affects cyanobacterial division.

Microcystin-LR (MC-LR) is a harmful cyanotoxin that inhibits 1 and 2A serine-threonine protein phosphatases. This study examines the influence of MC-LR on chloroplast division and the underlying mechanisms and consequences in Arabidopsis. MC-LR increased the frequency of dividing chloroplasts in hypocotyls in a time range of 1-96 h. At short-term exposures to MC-LR, small-sized chloroplasts (longitudinal diameters ≤6 µm) were more sensitive to these stimulatory effects, while both small and large chloroplasts showed stimulations at long-term exposure. After 48 h, the cyanotoxin increased the frequency of small-sized chloroplasts, indicating the stimulation of division. MC-LR inhibited protein phosphatases in whole hypocotyls and isolated chloroplasts, while it did not induce oxidative stress. We show for the first time that total cellular phosphatases play important roles in chloroplast division and that particular chloroplast phosphatases may be involved in these processes. Interestingly, MC-LR has a protective effect on cyanobacterial division during methyl-viologen (MV) treatments in Synechococcus PCC6301. MC-LR production has harmful effects on ecosystems and it may have an ancient cell division regulatory role in stressed cyanobacterial cells, the evolutionary ancestors of chloroplasts. We propose that cytoplasmic (eukaryotic) factors also contribute to the relevant effects of MC-LR in plants.

"B" Regulatory Subunits of PP2A: Their Roles in Plant Development and Stress Reactions.

Protein phosphatase PP2A is an enzyme complex consisting of C (catalytic), A (scaffold) and B (regulatory) subunits. B subunits are a large family of proteins that regulate activity, substrate specificity and subcellular localization of the holoenzyme. Knowledge on the molecular functions of PP2A in plants is less than for protein kinases, but it is rapidly increasing. B subunits are responsible for the large diversity of PP2A functioning. This paper intends to give a survey on their multiple regulatory mechanisms. Firstly, we give a short description on our current knowledge in terms of "B"-mediated regulation of metabolic pathways. Next, we present their subcellular localizations, which extend from the nucleus to the cytosol and membrane compartments. The next sections show how B subunits regulate cellular processes from mitotic division to signal transduction pathways, including hormone signaling, and then the emerging evidence for their regulatory (mostly modulatory) roles in both abiotic and biotic stress responses in plants. Knowledge on these issues should be increased in the near future, since it contributes to a better understanding of how plant cells work, it may have agricultural applications, and it may have new insights into how vascular plants including crops face diverse environmental challenges.

Microcystin-LR, a Cyanobacterial Toxin, Induces Changes in the Organization of Membrane Compartments in Arabidopsis.

To evaluate the effects of the cyanobacterial toxin microcystin-LR (MCY-LR, a protein phosphatase inhibitor) and diquat (DQ, an oxidative stress inducer) on the organization of tonoplast, the effect of MCY-LR on plastid stromule formation and on mitochondria was investigated in wild-type Arabidopsis. Tonoplast was also studied in PP2A catalytic (c3c4) and regulatory subunit mutants (fass-5 and fass-15). These novel studies were performed by CLSM microscopy. MCY-LR is produced during cyanobacterial blooms. The organization of tonoplast of PP2A mutants of Arabidopsis is much more sensitive to MCY-LR and DQ treatments than that of wild type. In c3c4, fass-5 and fass-15, control and treated plants showed increased vacuole fragmentation that was the strongest when the fass-5 mutant was treated with MCY-LR. It is assumed that both PP2A/C and B" subunits play an important role in normal formation and function of the tonoplast. In wild-type plants, MCY-LR affects mitochondria. Under the influence of MCY-LR, small, round-shaped mitochondria appeared, while long/fused mitochondria were typical in control plants. Presumably, MCY-LR either inhibits the fusion of mitochondria or induces fission. Consequently, PP2A also plays an important role in the fusion of mitochondria. MCY-LR also increased the frequency of stromules appearing on chloroplasts after 1 h treatments. Along the stromules, signals can be transported between plastids and endoplasmic reticulum. It is probable that they promote a faster response to stress.

B" and C subunits of PP2A regulate the levels of reactive oxygen species and superoxide dismutase activities in Arabidopsis.

The serine-threonine protein phosphatases PP2A regulate many cellular processes, however their role in oxidative stress responses and defence is less known. We show the involvement of its C (catalytic) and B" (a regulatory) subunits. The c3c4 (C subunit) and fass (B") subunit mutants and Col wt of Arabidopsis were used. Controls and treatments with the PP2A inhibitor microcystin-LR (MCY-LR) and reactive oxygen species (ROS) inducer diquat (DQ) were employed. ROS levels of primary roots were largely genotype dependent and both C and B" subunit mutants had increased sensitivity to MCY-LR and DQ indicating the involvement of these subunits in oxidative stress induction. Superoxide dismutases (SOD), mainly the Cu/Zn-SOD isoform, as key enzymes involved in ROS scavenging are also showing altered (mostly increased) activities in both c3c4 and fass mutants and have opposite relations to ROS induction. This indicates that the two types of subunits involved have partially different regulatory roles. In relation to this, control and MCY-LR/DQ treated B" subunit mutants were proven to have altered levels of phosphorylation of histone H2AX. γH2AX, the phosphorylated form indicates double stranded DNA damage during oxidative stress. Overall we point out the probable pivotal role of several PP2A subunits in the regulation of oxidative stress responses in plants and pave the way for future research to reveal the signaling pathways involved.

Microcystin-LR, a Cyanobacterial Toxin, Induces DNA Strand Breaks Correlated with Changes in Specific Nuclease and Protease Activities in White Mustard (Sinapis alba) Seedlings.

There is increasing evidence for the induction of programmed cell death (PCD) in vascular plants by the cyanobacterial toxin microcystin-LR (MC-LR). Our aim was to detect the occurrence of PCD-related DNA strand breaks and their possible connections to specific nuclease and protease activities. DNA breaks were studied by the deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method in the photoperiodically grown dicot model of white mustard (Sinapis alba). In-gel nuclease and protease activity assays showed changes in the activities of specific isoenzymes during treatments with MC-LR. Strand breaks occurred both in the developing root epidermis and cortex. Several isoenzyme activities were related to these breaks, for example: an increase in the activity of neutral 80-75 kDa, acidic high MW (100-120 kDa) and, most importantly, an increase in the activity of neutral 26-20 kDa nucleases, all of them having single-stranded DNA cleaving (SSP nuclease) activities. Increases in the activities of alkaline proteases in the 61-41 kDa range were also detected and proved to be in relation with MC-LR-induced PCD. This is one of the first pieces of evidence on the correlation of PCD-related DNA strand breaks with specific hydrolase activities in a model dicot treated with a cyanobacterial toxin known to have environmental importance.

Subcellular Alterations Induced by Cyanotoxins in Vascular Plants-A Review.

Phytotoxicity of cyanobacterial toxins has been confirmed at the subcellular level with consequences on whole plant physiological parameters and thus growth and productivity. Most of the data are available for two groups of these toxins: microcystins (MCs) and cylindrospermopsins (CYNs). Thus, in this review we present a timely survey of subcellular cyanotoxin effects with the main focus on these two cyanotoxins. We provide comparative insights into how peculiar plant cellular structures are affected. We review structural changes and their physiological consequences induced in the plastid system, peculiar plant cytoskeletal organization and chromatin structure, the plant cell wall, the vacuolar system, and in general, endomembrane structures. The cyanotoxins have characteristic dose-and plant genotype-dependent effects on all these structures. Alterations in chloroplast structure will influence the efficiency of photosynthesis and thus plant productivity. Changing of cell wall composition, disruption of the vacuolar membrane (tonoplast) and cytoskeleton, and alterations of chromatin structure (including DNA strand breaks) can ultimately lead to cell death. Finally, we present an integrated view of subcellular alterations. Knowledge on these changes will certainly contribute to a better understanding of cyanotoxin-plant interactions.

Microcystin-LR, a cyanobacterial toxin affects root development by changing levels of PIN proteins and auxin response in Arabidopsis roots.

Microcystin-LR (MCY-LR) is a heptapeptide toxin produced mainly by freshwater cyanobacteria. It strongly inhibits protein phosphatases PP2A and PP1. Functioning of the PIN family of auxin efflux carriers is crucial for plant ontogenesis and their functions depend on their reversible phosphorylation. We aimed to reveal the adverse effects of MCY-LR on PIN and auxin distribution in Arabidopsis roots and its consequences for root development. Relatively short-term (24 h) MCY-LR treatments decreased the levels of PIN1, PIN2 and PIN7, but not of PIN3 in tips of primary roots. In contrast, levels of PIN1 and PIN2 increased in emergent lateral roots and their levels depended on the type of PIN in lateral root primordia. DR5:GFP reporter activity showed that the cyanotoxin-induced decrease of auxin levels/responses in tips of main roots in parallel to PIN levels. Those alterations did not affect gravitropic response of roots. However, MCY-LR complemented the altered gravitropic response of crk5-1 mutants, defective in a protein kinase with essential role in the correct membrane localization of PIN2. For MCY-LR treated Col-0 plants, the number of lateral root primordia but not of emergent laterals increased and lateral root primordia showed early development. In conclusion, inhibition of protein phosphatase activities changed PIN and auxin levels, thus altered root development. Previous data on aquatic plants naturally co-occurring with the cyanotoxin showed similar alterations of root development. Thus, our results on the model plant Arabidopsis give a mechanistic explanation of MCY-LR phytotoxicity in aquatic ecosystems.

Impacts of Microcystins on Morphological and Physiological Parameters of Agricultural Plants: A Review.

Cyanobacteria are a group of photosynthetic prokaryotes that pose a great concern in the aquatic environments related to contamination and poisoning of wild life and humans. Some species of cyanobacteria produce potent toxins such as microcystins (MCs), which are extremely aggressive to several organisms, including animals and humans. In order to protect human health and prevent human exposure to this type of organisms and toxins, regulatory limits for MCs in drinking water have been established in most countries. In this regard, the World Health Organization (WHO) proposed 1 µg MCs/L as the highest acceptable concentration in drinking water. However, regulatory limits were not defined in waters used in other applications/activities, constituting a potential threat to the environment and to human health. Indeed, water contaminated with MCs or other cyanotoxins is recurrently used in agriculture and for crop and food production. Several deleterious effects of MCs including a decrease in growth, tissue necrosis, inhibition of photosynthesis and metabolic changes have been reported in plants leading to the impairment of crop productivity and economic loss. Studies have also revealed significant accumulation of MCs in edible tissues and plant organs, which raise concerns related to food safety. This work aims to systematize and analyze the information generated by previous scientific studies, namely on the phytotoxicity and the impact of MCs especially on growth, photosynthesis and productivity of agricultural plants. Morphological and physiological parameters of agronomic interest are overviewed in detail in this work, with the aim to evaluate the putative impact of MCs under field conditions. Finally, concentration-dependent effects are highlighted, as these can assist in future guidelines for irrigation waters and establish regulatory limits for MCs.

The Protein Phosphatase PP2A Plays Multiple Roles in Plant Development by Regulation of Vesicle Traffic-Facts and Questions.

The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP-GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.

Metal element alteration in the lung by cisplatin and CV247 administration.

Despite significant nephrotoxicity, cisplatin is still used in the therapy of various tumors. We were interested in how metal ion composition is altered by cisplatin and whether platinum accumulates in the non-tumorous lung. We also aimed to study metal ion changes after treatment with a veterinary medicament CV247 with antioxidant property (containing Cu and Mn gluconate, ascorbic acid, Na salicylate), and whether CV247 alters pulmonary platinum accumulation in the healthy lung. Male Wistar rats were randomly selected into 4 groups (n = 10/group): control group, cisplatin-treated group, CV247-treated group, cisplatin + CV247-treated group. Inductively coupled plasma optical emission spectrometry and mass spectrometry were used for measuring Al, As, B, Ba, Ca, Cd, Co, Cu, Cr, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Pt, S, Sb, Se, Sn, Sr, and Zn in the lung and the redox state was measured in the plasma. Cisplatin influenced the element homeostasis in the lung. Pt, Mn, Se accumulation and Ca, Mg excretion were observed after treatment with cisplatin. The antioxidant CV247 supplementation modified the Mn concentration; however, the concentration of Cu did not change despite the Cu content of the product, and CV247 did not affect other metal concentrations in the lung of the cisplatin-treated group. In conclusion, cisplatin has a systemic impact on the metal element metabolism, and this effect was demonstrated in the healthy lung, too. The results indicate the importance of supplementing some essential elements, such as Ca and Mg during cisplatin cancer therapy.

The Role of Serine-Threonine Protein Phosphatase PP2A in Plant Oxidative Stress Signaling-Facts and Hypotheses.

Abiotic and biotic factors induce oxidative stress involving the production and scavenging of reactive oxygen species (ROS). This review is a survey of well-known and possible roles of serine-threonine protein phosphatases in plant oxidative stress signaling, with special emphasis on PP2A. ROS mediated signaling involves three interrelated pathways: (i) perception of extracellular ROS triggers signal transduction pathways, leading to DNA damage and/or the production of antioxidants; (ii) external signals induce intracellular ROS generation that triggers the relevant signaling pathways and (iii) external signals mediate protein phosphorylation dependent signaling pathway(s), leading to the expression of ROS producing enzymes like NADPH oxidases. All pathways involve inactivation of serine-threonine protein phosphatases. The metal dependent phosphatase PP2C has a negative regulatory function during ABA mediated ROS signaling. PP2A is the most abundant protein phosphatase in eukaryotic cells. Inhibitors of PP2A exert a ROS inducing activity as well and we suggest that there is a direct relationship between these two effects of drugs. We present current findings and hypotheses regarding PP2A-ROS signaling connections related to all three ROS signaling pathways and anticipate future research directions for this field. These mechanisms have implications in the understanding of stress tolerance of vascular plants, having applications regarding crop improvement.

Attack of Microcystis aeruginosa bloom on a Ceratophyllum submersum field: Ecotoxicological measurements in real environment with real microcystin exposure.

Overproduction of toxic cyanobacteria is a type of harmful algal blooms (HABs). The heptapeptide microcystins (MCs) are one of the most common cyanotoxins. There is increasing research concerning the effects of MCs on growth and physiology of vascular plants, however there is a lack of studies on their direct effects on aquatic macrophytes in the real environment. Here we report the occurrence of a MC producing HAB in Lake Bárdos, Hungary in 2012 with harmful effects on cytological, histological and biochemical parameters of Ceratophyllum submersum (soft hornwort) plants naturally growing at the blooming site. Blue-Green Sinapis Test (BGST) showed high toxicity of HAB samples. Cell-free water samples contained a significant amount of MCs (7.31 ±â€¯0.17 µg L-1) while C. submersum plants contained 1.01 ±â€¯0.21 µg g DW-1 MCs. Plants showed significant increases of protein content and decreases of anthocyanin content and carotenoid/chlorophyll ratio, indicating physiological stress- as compared to plants from the control (MC free) sampling site of the same water body. Histological and cytological studies showed (i) radial swelling and the abnormal formation of lateral buds at the shoot tip leading to abnormal development; (ii) the fragmentation of nuclei as well as accumulation of phenolics in the nucleus indicating that the HAB induced cell death and stress reactions at the nuclear level. The most relevant effect was the increase of histone H3 phosphorylation in metaphase chromosomes: since MCs are strong inhibitors of protein phosphatases, this alteration is related to the biochemical targets of these toxins. The HAB decreased peroxidase activity, but increased nuclease and protease activities, showing the decreased capacity of plants to face biotic stress and as the cytological changes, the induction of cell death. This study is one of the first to show the complex harmful changes in aquatic plants that co-exist with HABs.

Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors.

The recently synthesized isocyanonaphtalene derivatives ACAIN and CACAIN are fluorochromes excitable at wavelengths of around 366 nm and bind cysteine-rich proteins with hydrophobic motifs. We show that these compounds preferentially label tonoplasts in living Arabidopsis and tobacco (Nicotiana tabacum SR1) cells. ACAIN-labeled membranes co-localized with the GFP signal in plants expressing GFP-δ-TIP (TIP2;1) (a tonoplast aquaporin) fusion protein. ACAIN preserved the dynamics of vacuolar structures. tip2;1 and triple tip1;1-tip1;2-tip2;1 knockout mutants showed weaker ACAIN signal in tonoplasts. The fluorochrome is also suitable for the labeling and detection of specific (cysteine-rich, hydrophobic) proteins from crude cell protein extracts following SDS-PAGE and TIP mutants show altered labeling patterns; however, it appears that ACAIN labels a large variety of tonoplast proteins. ACAIN/CACAIN could be used for the detection of altered vacuolar organization induced by the heptapeptide natural toxin microcystin-LR (MCY-LR), a potent inhibitor of both type 1 and 2A protein phosphatases and a ROS inducer. As revealed both in plants with GFP-TIP2;1 fusions and in wild-type (Columbia) plants labeled with ACAIN/CACAIN, MCY-LR induces the formation of small vesicles, concomitantly with the absence of the large vegetative vacuoles characteristic for differentiated cells. TEM studies of MCY-LR-treated Arabidopsis cells proved the presence of multimembrane vesicles, with characteristics of lytic vacuoles or autophagosomes. Moreover, MCY-LR is a stronger inducer of small vesicle formation than okadaic acid (which inhibits preferentially PP2A) and tautomycin (which inhibits preferentially PP1). ACAIN and CACAIN emerge as useful novel tools to study plant vacuole biogenesis and programmed cell death.

Allyl-Isothiocyanate and Microcystin-LR Reveal the Protein Phosphatase Mediated Regulation of Metaphase-Anaphase Transition in Vicia faba.

Horseradish allyl isothiocyanate (AITC, a volatile oil) and cyanobacterial microcystin-LR (MCY-LR, a cyclic heptapeptide) affect eukaryotic cell cycle. MCY-LR inhibits protein phosphatases PP1 and PP2A. We aimed to reveal the mechanisms of their cellular effects in a model eukaryote, Vicia faba. We have shown for the first time that AITC had minor effects on PP1 and PP2A activities in vitro, but it inhibited significantly PP1 in vivo. The combination of 10 µM AITC with 10 µM MCY-LR induced metaphase arrest after short-term (12 h) treatments. 10 µM AITC, 0.2-10 µM MCY-LR and their combinations induced histone H3 hyperphosphorylation, associated with the regulation of metaphase-anaphase transition. This hyperphosphorylation event occurred at any treatment which led to the inhibition of PP1 activity. 10 µM AITC + 10 µM MCY-LR increased the frequency of metaphase spindle anomalies, associated with metaphase arrest. We provide new insights into the mechanisms of metaphase-anaphase transition. Metaphase arrest is induced at the concomitant hyperphosphorylation of histone H3, alteration of metaphase spindle assembly and strong inhibition of PP1 + PP2A activity. Near-complete blocking of metaphase-anaphase transition by rapid protein phosphatase inhibition is shown here for the first time in plants, confirming a crucial role of serine-threonine phosphatases in this checkpoint of cell cycle regulation. Tissue-dependent differences in PP1 and PP2A activities induced by AITC and MCY-LR suggest that mainly regulatory subunits are affected. AITC is a potential tool for the study of protein phosphatase function and regulation. We raise the possibility that one of the biochemical events occurring during AITC release upon wounding is the modulation of protein phosphatase dependent signal transduction pathways during the plant defense response.

Cylindrospermopsin induces biochemical changes leading to programmed cell death in plants.

In the present study we provide cytological and biochemical evidence that the cyanotoxin cylindrospermopsin (CYN) induces programmed cell death (PCD) symptoms in two model vascular plants: the dicot white mustard (Sinapis alba) and the monocot common reed (Phragmites australis). Cytological data include chromatin fragmentation and the increase of the ratio of TUNEL-positive cells in roots, the latter being detected in both model systems studied. The strongest biochemical evidence is the elevation of the activity of several single-stranded DNA preferring nucleases-among them enzymes active at both acidic and alkaline conditions and are probably directly related to DNA breaks occurring at the initial stages of plant PCD: 80 kDa nucleases and a 26 kDa nuclease, both having dual (single- and double-stranded nucleic acid) specificity. Moreover, the total protease activity and in particular, a 53-56 kDa alkaline protease activity increases. This protease could be inhibited by PMSF, thus regarded as serine protease. Serine proteases are detected in all organs of Brassicaceae (Arabidopsis) having importance in differentiation of specialized plant tissue through PCD, in protein degradation/processing during early germination and defense mechanisms induced by a variety of biotic and abiotic stresses. However, knowledge of the physiological roles of these proteases and nucleases in PCD still needs further research. It is concluded that CYN treatment induces chromatin fragmentation and PCD in plant cells by activating specific nucleases and proteases. CYN is proposed to be a suitable molecule to study the mechanism of plant apoptosis.

Cellular Effects of Cylindrospermopsin (Cyanobacterial Alkaloid Toxin) and its Potential Medical Consequences.

Cylindrospermopsin (CYN) is a tricyclic guanidino alkaloid toxin produced by several cyanobacterial genera. It alters cellular functioning in eukaryotes, including animal and plant organisms. Over the past decades, more and more evidence shows its potential hazardous effects on animal and human health. In this review, we give a critical survey and interpretation of data currently available on its biochemical and consequently, cellular effects. CYN is considered to be a cytotoxin. Several reports suggest that it is a potent inhibitor of eukaryotic protein synthesis, though the exact mechanisms are not completely understood. Here we show that the biochemical changes induced by CYN are complex, possibly involving multiple modes of action. Glutathione metabolism and pyrimidine nucleotide synthesis is affected besides the proposed protein synthesis inhibition. Biochemical alterations lead to the following cellular/subcellular alterations both in animals and plants: (i) changes in cell division rates due to perturbations in chromatin and cytoskeleton; (ii) perturbations of structure and functioning of endomembranes including endoplasmic reticulum; (iii) general metabolic alterations leading to genotoxicity and programmed cell death/apoptosis. The underlying mechanisms and possible health consequences are discussed.