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.

Deep Sequencing of Porcine Reproductive and Respiratory Syndrome Virus ORF7: A Promising Tool for Diagnostics and Epidemiologic Surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major concern worldwide. Control of PRRSV is a challenging task due to various factors, including the viral diversity and variability. In this study, we evaluated an amplicon library preparation protocol targeting the ORF7 region of both PRRSV species, Betaarterivirus suid 1 and Betaarterivirus suid 2. We designed tailed primers for a two-step PCR procedure that generates ORF7-specific amplicon libraries suitable for use on Illumina sequencers. We tested the method with serum samples containing common laboratory strains and with pooled serum samples (n = 15) collected from different pig farms during 2019-2021 in Hungary. Testing spiked serum samples showed that the newly designed method is highly sensitive and detects the viral RNA even at low copy numbers (corresponding to approx. Ct 35). The ORF7 sequences were easily assembled even from clinical samples. Two different sequence variants were identified in five samples, and the Porcilis MLV vaccine strain was identified as the minor variant in four samples. An in-depth analysis of the deep sequencing results revealed numerous polymorphic sites along the ORF7 gene in a total of eight samples, and some sites (positions 12, 165, 219, 225, 315, 345, and 351) were found to be common in several clinical specimens. We conclude that amplicon deep sequencing of a highly conserved region of the PRRSV genome could support both laboratory diagnosis and epidemiologic surveillance of the disease.

Supplementation of the Plant Conditioner ELICE Vakcina® Product with ß-Aminobutyric Acid and Salicylic Acid May Lead to Trans-Priming Signaling in Barley (Hordeum vulgare).

Plant immunological memory, priming, is a defense mechanism that can be triggered by external stimuli, leading to the activation of biochemical pathways and preparing plants for disease resistance. Plant conditioners improve yield and crop quality through nutrient efficiency and abiotic stress tolerance, which is enhanced by the addition of resistance- and priming-induced compounds. Based on this hypothesis, this study aimed to investigate plant responses to priming actives of different natures, including salicylic acid and beta-aminobutyric acid, in combination with the plant conditioning agent ELICE Vakcina®. Phytotron experiments and RNA-Seq analyses of differentially expressed genes using the combinations of these three investigated compounds were performed in a barley culture to investigate possible synergistic relationships in the genetic regulatory network. The results indicated a strong regulation of defense responses, which was enhanced by supplemental treatments; however, both synergistic and antagonistic effects were enhanced with one or two components, depending on the supplementation. The overexpressed transcripts were functionally annotated to assess their involvement in jasmonic acid and salicylic acid signaling; however, their determinant genes were highly dependent on the supplemental treatments. Although the effects overlapped, the potential effects of trans-priming the two supplements tested could be largely separated.

Cryptogamic communities on flatroofs in the city of Debrecen (East Hungary).

Cryptogams of ten urban flatroofs, contrasting in their age and size, were studied between 2016 and 2018. Siliceous (bituminous felt, gravel, brick) and calcareous (concrete) substrata occurred at each site. Microclimate (T, RH) at two sites of contrasting shading was monitored from September 2016 to January 2017. Biomass of two differently aged, exposed flatroofs was sampled in October 2018. Taxa of Cladonia and Xanthoparmelia have been identified by spot tests and HPTLC. A total of 61 taxa (25 bryophytes, 36 lichens), mostly widespread synanthropic species, have been detected with an explicit difference of species composition between shaded and exposed sites. Floristically interesting species included acidophilous bryophytes (Hedwigia ciliata, Racomitrium canescens) and lichens (Xanthoparmelia conspersa, Stereocaulon tomentosum) of montane character. The most widespread lichen is Cladonia rei which accounted for a significant part of the biomass at selected sites. Species-area curves for bryophytes at exposed sites have become saturated at 100-150 m2. In contrast, saturation of lichen diversity has not been reached even at the largest sites. Flatroofs with traditional roofing techniques can harbour relatively diverse microhabitats and species-rich synanthropic vegetation. It is urgent to study these sites before renovation with modern roofing techniques eliminates them. Diversification of urban surroundings is possible in the future via application of various substrats in renovated and newly constructed roofs.

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" 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.

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 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.

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.

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.

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.

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.

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.