A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ

<p>Abstract</p> <p>Background</p> <p>Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating) or preparation-intensive (eg. fluorescent staining). In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation.</p> <p>Results</p> <p>The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism <it>Synechocystis </it>sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis.</p> <p>Conclusions</p> <p>The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.</p

Carbohydrate, phenolic and antioxidant level in relation to chlorophyll a content in oilseed winter rape (Brassica napus L.) inoculated with Leptosphaeria maculans

Syftet med föreliggande studien var att undersöka om sjuksköterskors egna rökvanor påverkar attityden till tobakspreventivt arbete på sjukhuset, både till tobakspreventivt arbete med patienterna och attityden till rökfritt sjukhus. Studien är empirisk och utfördes genom kvalitativa intervjuer med sex sjuksköterskor på en vårdavdelning på ett sjukhus i södra Sverige. Data från intervjuerna analyserades och resulterade i sju olika teman: Preventiva rollen, Kunskap om prevention, Vem skall leda det preventiva arbetet, Rökkontroll, Utbildningsnivå och rökning, Sjuksköterskan, en förebild?, Vem ska hjälpa patienten vid rökstopp på sjukhuset?, Är det någon skillnad mellan icke rökande och rökande vad avser rökpreventionen?. Den preventiva rollen hamnade i fokus och skillnader fanns mellan rökande och icke rökande sjuksköterskor både vad gäller preventivt omvårdnasarbete och kontrollThe aim of the present study is to investigate whether nurses smoking habits influence their attitude to tobacco prevention in hospitals, both in their work with patients and regarding their attitude to hospital smoking bans. The following question was posed: is there a difference between smoking and non-smoking nurses in patient-care activities regarding smoking prevention and control? The study is qualitative, based on qualitative interviews with six nurses at a ward of a hospital in Sweden. Interview data were analyzed and eight themes emerged: the role in prevention work knowledge of prevention who is to lead prevention work smoking control smoking and education levels the nurse as a role model who is to help the patient give up smoking possible differences between nonsmoking and smoking nurses regarding smoking prevention. The role in prevention work turned out to be central. Smoking nurses had greater difficulties in connection with preventive work and control, due to their personal experience of how hard it can be to give up smoking habits

Effect of the down-regulation of the high Grain Protein Content (GPC) genes on the wheat transcriptome during monocarpic senescence

<p>Abstract</p> <p>Background</p> <p>Increasing the nutrient concentration of wheat grains is important to ameliorate nutritional deficiencies in many parts of the world. Proteins and nutrients in the wheat grain are largely derived from the remobilization of degraded leaf molecules during monocarpic senescence. The down-regulation of the NAC transcription factor <it>Grain Protein Content </it>(<it>GPC</it>) in transgenic wheat plants delays senescence (>3 weeks) and reduces the concentration of protein, Zn and Fe in the grain (>30%), linking senescence and nutrient remobilization.</p> <p>Based on the early and rapid up-regulation of <it>GPC </it>in wheat flag leaves after anthesis, we hypothesized that this transcription factor is an early regulator of monocarpic senescence. To test this hypothesis, we used high-throughput mRNA-seq technologies to characterize the effect of the <it>GPC </it>down-regulation on the wheat flag-leaf transcriptome 12 days after anthesis. At this early stage of senescence <it>GPC </it>transcript levels are significantly lower in transgenic GPC-RNAi plants than in the wild type, but there are still no visible phenotypic differences between genotypes.</p> <p>Results</p> <p>We generated 1.4 million 454 reads from early senescing flag leaves (average ~350 nt) and assembled 1.2 million into 30,497 contigs that were used as a reference to map 145 million Illumina reads from three wild type and four GPC-RNAi plants. Following normalization and statistical testing, we identified a set of 691 genes differentially regulated by <it>GPC </it>(431 ≥ 2-fold change). Transcript level ratios between transgenic and wild type plants showed a high correlation (<it>R </it>= 0.83) between qRT-PCR and Illumina results, providing independent validation of the mRNA-seq approach. A set of differentially expressed genes were analyzed across an early senescence time-course.</p> <p>Conclusions</p> <p>Monocarpic senescence is an active process characterized by large-scale changes in gene expression which begins considerably before the appearance of visual symptoms of senescence. The mRNA-seq approach used here was able to detect small differences in transcript levels during the early stages of senescence. This resulted in an extensive list of <it>GPC</it>-regulated genes, which includes transporters, hormone regulated genes, and transcription factors. These <it>GPC</it>-regulated genes, particularly those up-regulated during senescence, provide valuable entry points to dissect the early stages of monocarpic senescence and nutrient remobilization in wheat.</p

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga Ostreococcus tauri

© 2017, The Author(s).Introduction: The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism of this alga. Objectives: In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness. Methods: Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling. Results: Of 111 metabolites quantified over light–dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 μM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri. Conclusions: This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified

Nitric oxide triggers a transient metabolic reprogramming in Arabidopsis

[EN] Nitric oxide (NO) regulates plant growth and development as well as responses to stress that enhanced its endogenous production. Arabidopsis plants exposed to a pulse of exogenous NO gas were used for untargeted global metabolomic analyses thus allowing the identification of metabolic processes affected by NO. At early time points after treatment, NO scavenged superoxide anion and induced the nitration and the S-nitrosylation of proteins. These events preceded an extensive though transient metabolic reprogramming at 6&#8201;h after NO treatment, which included enhanced levels of polyamines, lipid catabolism and accumulation of phospholipids, chlorophyll breakdown, protein and nucleic acid turnover and increased content of sugars. Accordingly, lipid-related structures such as root cell membranes and leaf cuticle altered their permeability upon NO treatment. Besides, NO-treated plants displayed degradation of starch granules, which is consistent with the increased sugar content observed in the metabolomic survey. The metabolic profile was restored to baseline levels at 24&#8201;h post-treatment, thus pointing up the plasticity of plant metabolism in response to nitroxidative stress conditions.This work was supported by grants BIO2011-27526 and BIO2014-56067-P from the Spanish Ministry of Economy and Competitiveness and FEDER funds. We thank support and comments from Danny Alexander (Metabolon Inc., USA) on metabolomic analyses.Leon Ramos, J.; Costa-Broseta, Á.; Castillo López Del Toro, MC. (2016). Nitric oxide triggers a transient metabolic reprogramming in Arabidopsis. Scientific Reports. 6:1-14. doi:10.1038/srep37945S1146Arc, E., Galland, M., Godin, B., Cueff, G. & Rajjou, L. Nitric oxide implication in the control of seed dormancy and germination. Front. Plant Sci. 4, 346 (2013).Beligni, M. V. & Lamattina, L. Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 210, 215–221 (2000).Lozano-Juste, J. & León, J. Nitric oxide regulates DELLA content and PIF expression to promote photomorphogenesis in Arabidopsis. Plant Physiol. 156, 1410–1123 (2011).He, Y. et al. Nitric oxide represses the Arabidopsis floral transition. Science 305, 1968–1971 (2004).Tsai, Y. C., Delk, N. A., Chowdhury, N. I. & Braam, J. Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering. Plant Signal. Behav. 2, 446–454 (2007).Manjunatha, G., Lokesh, V. & Neelwarne, B. Nitric oxide in fruit ripening: trends and opportunities. Biotechnol. Adv. 28, 489–499 (2010).Liu, F. & Guo, F. Q. Nitric oxide deficiency accelerates chlorophyll breakdown and stability loss of thylakoid membranes during dark-induced leaf senescence in Arabidopsis. PLoS One 8(2), e56345 (2013).Du, J. et al. Nitric oxide induces cotyledon senescence involving co-operation of the NES1/MAD1 and EIN2-associated ORE1 signalling pathways in Arabidopsis. J. Exp. Bot. 65, 4051–4063 (2014).Siddiqui, M. H., Al-Whaibi, M. H. & Basalah, M. O. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248, 447–455 (2011).Arasimowicz-Jelonek, M. & Floryszak-Wieczorek, J. Nitric oxide: an effective weapon of the plant or the pathogen? Mol. Plant Pathol. 15, 406–416 (2014).Thomas, D. D. Breathing new life into nitric oxide signaling: A brief overview of the interplay between oxygen and nitric oxide. Redox Biol. 5, 225–33 (2015).Groβ, F., Durner, J. & Gaupels, F. Nitric oxide, antioxidants and prooxidants in plant defence responses. Front. Plant Sci. 4, 419 (2013).Astier, J. & Lindermayr, C. Nitric oxide-dependent posttranslational modification in plants: an update. Int. J. Mol. Sci. 13, 15193–15208 (2012).Hess, D. T. & Stamler, J. S. Regulation by S-nitrosylation of protein post-translational modification. J. Biol. Chem. 287, 4411–4418 (2012).Guerra, D. D. & Callis, J. Ubiquitin on the move: the ubiquitin modification system plays diverse roles in the regulation of endoplasmic reticulum- and plasma membrane-localized proteins. Plant Physiol. 160, 56–64 (2012).Skalska, K., Miller, J. S. & Ledakowicz, S. Trends in NO(x) abatement: a review. Sci. Total Environ. 408, 3976–3989 (2010).Pilegaard, K. Processes regulating nitric oxide emissions from soils. Phil. Transac. Royal Soc. London. Ser. B, Biol. Sci. 368, 20130126 (2013).Jaegle, L., Steinberger, L., Martin, R. V. & Chance, K. Global partitioning of NOx sources using satellite observations: Relative roles of fossil fuel combustion, biomass burning and soil emissions. Faraday Discus. 130, 407–423 (2005).Gupta, K. J., Fernie, A. R., Kaiser, W. M. & van Dongen, J. T. On the origins of nitric oxide. Trends Plant Sci. 16, 160–168 (2011).Mur, L. A. et al. Nitric oxide in plants: an assessment of the current state of knowledge. AoB Plants 5, pls052 (2013).Correa-Aragunde, N., Foresi, N. & Lamattina, L. Nitric oxide is a ubiquitous signal for maintaining redox balance in plant cells: regulation of ascorbate peroxidase as a case study. J. Exp. Bot. 66, 2913–2921 (2015).Noctor, G., Lelarge-Trouverie, C. & Mhamdi, A. The metabolomics of oxidative stress. Phytochemistry 112, 33–53 (2015).Allan, W. L., Simpson, J. P., Clark, S. M. & Shelp, B. J. Gamma-hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms. J. Exp. Bot. 59, 2555–2564 (2008).Romero, L. C., Aroca, M. Á., Laureano-Marín, A. M., Moreno, I., García, I. & Gotor, C. Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana. Mol. Plant 7, 264–276 (2014).Noctor, G. et al. Glutathione in plants: an integrated overview. Plant Cell Environ. 35, 454–484 (2012).Feussner, I. & Wasternack, C. The lipoxygenase pathway. Ann. Rev. Plant Biol. 53, 275–297 (2002).Green, M. A. & Fry, S. C. Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 433, 83–87 (2005).Szarka, A., Tomasskovics, B. & Bánhegyi, G. The ascorbate-glutathione-α-tocopherol triad in abiotic stress response. Int. J. Mol. Sci. 13, 4458–4483 (2012).Hurlock, A. K., Roston, R. L., Wang, K. & Benning, C. Lipid trafficking in plant cells. Traffic 15, 915–932 (2014).Blokhina, O., Virolainen, E. & Fagerstedt, K. V. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 91, 179–194 (2003).Yeats, T. H. & Rose, J. K. The formation and function of plant cuticles. Plant Physiol. 163, 5–20 (2013).Lozano-Juste, J. & León, J. Enhanced abscisic acid-mediated responses in nia1nia2noa1-2 triple mutant impaired in NIA/NR- and AtNOA1-dependent nitric oxide biosynthesis in Arabidopsis. Plant Physiol. 152, 891–903 (2010).Hörtensteiner, S. Update on the biochemistry of chlorophyll breakdown. Plant Mol Biol. 82, 505–17 (2013).Pruzinská, A. et al. Chlorophyll breakdown in senescent Arabidopsis leaves: characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol. 139, 52–63 (2005).Hirashima, M., Tanaka, R. & Tanaka, A. Light-independent cell death induced by accumulation of pheophorbide a in Arabidopsis thaliana. Plant Cell Physiol. 50, 719–29 (2009).Zottini, M., Costa, A., De Michele, R., Ruzzene, M., Carimi, F. & Lo Schiavo, F. Salicylic acid activates nitric oxide synthesis in Arabidopsis. J Exp Bot. 58, 1397–1405 (2007).Mainz, E. R. et al. Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection. Analytical Methods 4, 414–420 (2012).Vandelle, E. & Delledonne, M. Peroxynitrite formation and function in plants. Plant Sci. 181, 534–539 (2011).Minocha, R., Majumdar, R. & Minocha, S. C. Polyamines and abiotic stress in plants: a complex relationship. Front. Plant Sci. 5, 175 (2014).Parsons H. T., Yasmin, T. & Fry, S. C. Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism. Biochem. J. 440, 375–383 (2011).Hou, Q., Ufer, G. & Bartels, D. Lipid signalling in plant responses to abiotic stress. Plant Cell Environ. 39, 1029–4108 (2016).Zhou, X. R., Callahan, D. L., Shrestha, P., Liu, Q., Petrie, J. R. & Singh, S. P. Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA. Front. Plant Sci. 5, 41 (2014).Pohl, C. H. & Kock, J. L. Oxidized fatty acids as inter-kingdom signaling molecules. Molecules 19, 1273–1285 (2014).Araújo, W. L., Tohge, T., Ishizaki, K., Leaver, C. J. & Fernie, A. R. Protein degradation-an alternative respiratory substrate for stressed plants. Trends Plant Sci. 16, 489–498 (2011).Sakamoto, W. & Takami, T. Nucleases in higher plants and their possible involvement in DNA degradation during leaf senescence. J. Exp. Bot. 65, 3835–3843 (2014).Del Duca, S., Serafini-Fracassini, D. & Cai, G. Senescence and programmed cell death in plants: polyamine action mediated by transglutaminase. Front. Plant Sci. 5, 120 (2014).Franco, M. C. & Estévez, A. G. Tyrosine nitration as mediator of cell death. Cell. Mol. Life Sci. 71, 3939–3950 (2014).Palumbo, A., Fiore, G., Di Cristo, C., Di Cosmo, A. & d’Ischia, M. NMDA receptor stimulation induces temporary alpha-tubulin degradation signalled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis. Biochem. Biophys. Res. Commun. 293, 1536–1543 (2002).Wang, Y. Y., Lin, S. Y., Chuang, Y. H., Mao, C. H., Tung, K. C. & Sheu, W. H. Protein nitration is associated with increased proteolysis in skeletal muscle of bile duct ligation-induced cirrhotic rats. Metabolism 59, 468–472 (2010).Castillo, M. C., Lozano-Juste, J., González-Guzmán, M., Rodriguez, L., Rodriguez, P. L. & León, J. Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants. Sci. Signal. 8(392), ra89 (2015).Blaise, G. A., Gauvin, D., Gangal, M. & Authier, S. Nitric oxide, cell signaling and cell death. Toxicology 208, 177–192 (2005).Brüne, B. Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ. 10, 864–869 (2003).Wang, Y., Chen, C., Loake, G. J. & Chu, C. Nitric oxide: promoter or suppressor of programmed cell death? Prot. Cell 1, 133–142 (2010).Serrano, I., Romero-Puertas, M. C., Sandalio, L. M. & Olmedilla, A. The role of reactive oxygen species and nitric oxide in programmed cell death associated with self-incompatibility. J. Exp. Bot. 66, 2869–2876 (2015).Huang, S., Hill, R. D. & Stasolla, C. Plant hemoglobin participation in cell fate determination. Plant Signal. Behavior 9, e29485 (2014).Maes, M. B., Scharpé, S. & De Meester, I. Dipeptidyl peptidase II (DPPII), a review. Clin. Chim. Acta 380, 31–49 (2007).Gibbs, D. J. et al. Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors. Mol. Cell 53, 369–379 (2014).Kitamura, K. Inhibition of the Arg/N-end rule pathway-mediated proteolysis by dipeptide-mimetic molecules. Amino Acids 48, 235–243 (2016).Duek, P. D., Elmer, M. V., van Oosten, V. R. & Fankhauser C. The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1. Curr Biol. 14, 2296–2301 (2004)

An enigma in the genetic responses of plants to salt stresses

Soil salinity is one of the main factors restricting crop production throughout the world. Various salt tolerance traits and the genes controlling these traits are responsible for coping with salinity stress in plants. These coping mechanisms include osmotic tolerance, ion exclusion, and tissue tolerance. Plants exposed to salinity stress sense the stress conditions, convey specific stimuli signals, and initiate responses against stress through the activation of tolerance mechanisms that include multiple genes and pathways. Advances in our understanding of the genetic responses of plants to salinity and their connections with yield improvement are essential for attaining sustainable agriculture. Although a wide range of studies have been conducted that demonstrate genetic variations in response to salinity stress, numerous questions need to be answered to fully understand plant tolerance to salt stress. This chapter provides an overview of previous studies on the genetic control of salinity stress in plants, including signaling, tolerance mechanisms, and the genes, pathways, and epigenetic regulators necessary for plant salinity tolerance

Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence

Stay-green mutants are delayed in leaf senescence and have been identified from different plant species, including many crops. Functional stay-greens have the potential to increase plant productivity. In cosmetic stay-greens, however, retention of chlorophyll during senescence is uncoupled from a decline of photosynthetic capacity in these mutants. For many cosmetic stay-green mutants, including Gregor Mendel's famous green cotyledon pea variety, molecular defects were recently identified in orthologous stay-green genes. Stay-green genes encode members of a new family of chloroplast-located proteins, which are likely to function in dismantling of photosynthetic chlorophyll-apoprotein complexes. Their activity is considered as a prerequisite for both chlorophyll and apoprotein degradation during senescence