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Development of methods for breeding high-lipidcontent algal strain Chlamydomonas reinhardtii using fluorescence-activated cell sorting
Fedorko, Jan ; Búzová, Diana ; Červený, Jan
Green microalgae are among the most widely distributed microorganisms in the biosphere. They are significant contributors to global photosynthetic productivity and are interesting for biotechnology due to their large variety of high-value compound accumulation and range of applications. To achieve profitable microalgae cultures for biotechnology, one wants to combine antagonistic properties: rapid growth and high accumulation of specific compounds. Here, we focus on development of advanced cultivation strategies and breeding methods applied to the model algae Chlamydomonas reinhardtii for optimized production of lipids. For identification, isolation, and subsequent selection of an optimal subpopulation with high lipid content, we used high-throughput fluorescenceactivated cell sorting in combination with imaging flow cytometry on cells stained with lipid-specific fluorescent dye. We observed that post-sort cell viability was not negatively influenced by external parameters used during the sorting procedure (pressure, light quality and quantity, influence of the sorting electromagnetic field, toxic effects of both fluorescent marker and microfluidic system medium composition).
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CN-PAGE as a tool for separating pigment–protein complexes and studying their thermal stability in spruce and barley thylakoid membranes
Kurasová, Irena ; Svrčinová, K. ; Karlický, Václav ; Špunda, Vladimír
The central aim of our study was to develop a method for solubilization and native electrophoretic (colourless native polyacrylamide gel electrophoresis; CN-PAGE) separation of pigment–protein complexes (PPCs) embedded in thylakoid membranes (tBMs) isolated from spruce. Subsequently, we focused on studying the effect of temperature on the composition and PPC stability of two different species: barley and spruce. We found that the mild detergent n-dodecyl β-D-maltoside (β-DM) is suitable for PPC solubilization of spruce tBMs, but longer solubilization and a higher ratio of detergent to total chlorophyll are needed for spruce than are needed for barley. We also unified CN-PAGE protocols to optimize the separation of spruce and barley PPCs that resulted in the separation of photosystem I (PSI) and photosystem II (PSII) supercomplexes (SCs), PSI and PSII core dimers, PSII core monomers, trimeric and monomeric light-harvesting complexes of PSII, and bands with free pigments. Studying the effect of elevated temperature on PPCs using CN-PAGE revealed different thermal stability of PPCs in spruce and barley tBMs. Pronounced PPCs changes were observed at temperatures at or above 40°C. We observed partial disappearance of PSII SCs bands at 44°C in barley and at 52°C in spruce. In addition, spruce PSI SCs exhibited slightly higher thermal stability than did barley PSI SCs. The increased thermal stability of spruce tBMS in comparison to that of barley tBM was also confirmed by the circular dichroism spectra of isolated tBMs at different temperatures (Karlický et al. 2015).
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The thermostability of photosystem II photochemistry is related to maintenance of thylakoid membranes organization
Karlický, Václav ; Kurasová, I. ; Špunda, Vladimír
For higher plant photosynthetic reactions, responses to the temperature changes are important, particularly if we consider global warming and the increasing frequency of extreme temperature fluctuations. High temperature stress decreases photosynthetic assimilation through the inactivation of photosystem II (PSII), the most heat-sensitive component of the oxygen-evolving complex. We have recently found higher thermostability of spruce PSII photochemistry compared to such control plants as Arabidopsis species and barley. In this work, we have therefore attempted to describe the causes of this effect on the level of the organization of pigment–protein complexes (PPCs) in spruce thylakoid membranes using circular dichroism (CD) spectroscopy. We have confirmed higher maximum efficiency of PSII photochemistry (FV/FM) for spruce needles in comparison to barley leaves. Temperature-dependent CD spectra have also demonstrated higher (by about 6°C) PSII thermostability of chiral macro-organization of PPCs in spruce thylakoid membranes compared to those in barley. However, thermal disruption of PPCs did not reveal significant differences. Our results demonstrate that the stability of PSII macro-organization in different plant species correlates with the thermostability of PSII photochemistry in intact needles/leaves and so effective PSII photochemistry is related to the maintenance of PSII macro-organization under high temperature stress.
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Elevated temperature stimulates light-induced processes that contribute to protecting photosystem II against oxidative stress
Materová, Z. ; Štroch, Michal ; Holubová, I. ; Sestřenková, J. ; Oravec, Michal ; Večeřová, Kristýna ; Špunda, Vladimír
We focused on elucidating the impact of elevated temperature on rapid induction of zeaxanthin (Z)- dependent photoprotection in two different plant species. The dynamics of violaxanthin (V) de-epoxidation under different illumination regimes was studied together with chlorophyll a fluorescence transients in Picea abies seedlings and Arabidopsis thaliana leaves pre-acclimated to temperatures ranging from 20 to 40°C. Whereas for spruce seedlings the rapid phase of V de-epoxidation (induced by either 10 s illumination or 10 light pulses 1 s in duration at 1 min intervals) was gradually stimulated upon increasing temperatures, for A. thaliana leaves considerable acceleration of V de-epoxidation occurred only at 40°C. Moreover, only for spruce seedlings was a considerable amount of Z accumulated after 10 × 1 s illumination. Elevated temperatures stimulated rapid formation of Z-dependent non-radiative dissipation of excitation energy within photosystem II (NRD) induced by 1 s light pulses only for spruce seedlings. The possible role of a specific fatty acid composition in spruce thylakoid membrane lipids in facilitated V de-epoxidation and NRD induction at elevated temperatures is discussed.
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High night temperature-induced accelerated maturation of rice panicles can be detected by chlorophyll fluorescence
Šebela, David ; Quiňones, C. ; Olejníčková, Julie ; Jagadish, K. S. V.
Rice panicle maturation is considered to be highly sensitive to environmental conditions. Since one of the factors accompanying global climate change is increases in minimum night temperatures more pronounced than those in maximum day temperatures, the effect of high night temperature (HNT) on rice panicle maturation was investigated. Two rice genotypes with contrasting HNT responses, N22 (highly tolerant) and Gharib (susceptible), were exposed to control temperatures (ca 23°C) and HNTs (ca 29°C) from flowering until maturity. Loss of photosynthetic activity and/or pigments during rice panicle maturation were evaluated temporally by measuring (i) effective quantum yield of photosystem II efficiency (ΦII), and (ii) steady-state chlorophyll fluorescence level (FS). To prove the accuracy of the new approach presented in this study, several vegetative indices were calculated from reflectance measurements and correlated with fluorescence parameters. It has been observed that ΦII tracks the accelerated maturation of rice panicles exposed to HNT better than does FS. Employing a newly identified chlorophyll fluorescence-based parameter could potentially enable larger genetic diversity scans and identification of novel genotypes with longer panicle maturation periods so as to increase rice yields directly under field conditions.
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Effects of vegetation season and needles’ position in spruce canopy on emissions of volatile organic compounds
Večeřová, Kristýna ; Holišová, Petra ; Pallozi, E. ; Guidolotti, G. ; Calfapietra, Carlo ; Urban, Otmar
The main objective of this study was to investigate seasonal changes and vertical distribution in emissions of biogenic volatile organic compounds (BVOCs) within a Norway spruce canopy profile. Emissions were measured on current-year needles from the upper and lower canopy in early July and late August. Our results show that total BVOC emissions under standardized conditions (light intensity 1,000 µmol m–2 s–1, temperature 30°C) are higher in July than they are in August. BVOC emissions from upper canopy needles were approximately 3 times higher than were those from lower canopy needles. This difference was observed in July but not in August. The monoterpenes α-pinene, camphene, and terpinolene showed the most significant differences between emissions from upper and lower canopy needles.
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Comparison of emissions of biogenic volatile organic compounds from leaves of three tree species
Holišová, Petra ; Večeřová, Kristýna ; Pallozi, E. ; Guidolotti, G. ; Esposito, R. ; Calfapietra, Carlo ; Urban, Otmar
Biogenic volatile organic compounds (BVOCs) play many roles in plants’ ecophysiology and have the potential to affect atmospheric quality due to their chemical reactivity. Rates of BVOC emissions are highly variable depending on plant species and growing condition. Our study evaluated the amounts and spectra of BVOCs emitted from three tree species. We investigated BVOC emissions from the leaves of mature Norway spruce and sessile oak saplings grown in the field and from 1-year-old cuttings of hybrid poplar grown under laboratory conditions. Emitted BVOCs were sampled on desorption Tenax tubes in parallel with gas-exchange measurements. After subsequent thermal desorption of Tenax tubes, BVOC profiles were estimated by gas chromatography coupled with mass spectrometry. The tree species showed substantial differences in BVOC emission rates per unit leaf area ranging between 2.33 and 25.67 nmol m–2 s–1. Spruce trees had the lowest BVOC emissions and oak had slightly higher BVOC emissions on average than did poplar. Isoprene composed more than 97% of total BVOC emissions from oak and poplar, while no isoprene emissions from spruce needles were detected. Spruce BVOC emissions were mainly composed of such monoterpenes as α-pinene, β-pinene, and limonene.
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Diurnal changes of monoterpene fluxes in Norway spruce forest
Juráň, Stanislav ; Fares, S. ; Křůmal, Kamil ; Večeřa, Zbyněk ; Urban, Otmar
Biogenic volatile organic compounds (BVOCs) are important components of biosphere–atmosphere exchange. Their emissions depend on various meteorological parameters and stresses. Diurnal fluxes of different monoterpenes were studied within a Norway spruce (Picea abies) mountain forest to investigate their dependence on temperature and global radiation. Fluxes of monoterpenes, the most abundant BVOCs in spruce, were modelled using an inverse Lagrangian transport model, and representative diurnal variation trigged by both temperature and light was observed. This research enables future parametrization and quantification of various factors driving bidirectional fluxes.
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Surface water temperature modelling to estimate Czech fishery productivity under climate change
Svobodová, Eva ; Trnka, Miroslav ; Kopp, R. ; Mareš, J. ; Spurný, P. ; Pechar, L. ; Beděrková, I. ; Dubrovský, M. ; Žalud, Zdeněk
Freshwater fish production is significantly correlated with water temperature, which is expected to increase under climate change and affect fish growth, productivity, and survival. This study deals with estimating the change in water temperature in productive ponds and its impact on fishery in the Czech Republic. The target fish species were common carp (Cyprinus carpio), maraena whitefish (Coregonus maraena), northern whitefish (Coregonus peled), and rainbow trout (Oncorhynchus mykiss). It was hypothesized that there would be an increasing risk of high water temperature stress for fish. Water temperature calculations based on 3-day means of air temperature were tested in several ponds in three major fish production areas. The verified model was applied to the climate change conditions determined by standardized scenarios derived from the five global circulation models MPEH5, CSMK3, IPCM4, GFCM21, and HADGEM. The results for changed climate indicated limitations for Czech fish farming in terms of prolonged periods with fish temperature stress as well as the increased number of stress periods and increased number of days within these periods. It is very likely that Czech fishery will have to change the fish species farmed in particular productive areas. In particular, higher altitudes are likely to become less suitable for the Salmonidae.
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The influence of reduced precipitation supply on spring barley yields and the ability of crop growth models to simulate drought stress
Pohanková, Eva ; Orság, Matěj ; Hlavinka, Petr
This paper evaluates the first year (2014) of results from a field experiment with spring barley (cultivar Bojos) under reduced precipitation supply. The field experiment was carried out at an experimental station in the Czech Republic and consisted of small plots in two variants and three repetitions. The first variant was uncovered, and the second was partly covered to exclude rain throughout the entire vegetation season. For plots’ partial covering, a material was used to divert rainwater away from 70% of the plots. The main aim was to determine whether there are any differences in soil water content or in grain yield size between uncovered and partly covered plots and to compare the results obtained. Data measured in this field experiment were used to compare simulations of this field experiment in the DAISY crop growth model. Subsequently, the crop growth model’s ability to simulate grain yield, which was affected by drought stress, was explored. In reality, differences in phenological development and grain yield size were not evident. Reducing precipitation supply in DAISY by about 70% led to simulations of covered plots with reduced grain yield in accordance with the initial hypothesis. Agreement between observed and simulated grain yield was evaluated using selected statistical indicators: root mean square error (RMSE) as a parameter of average magnitude of error and mean bias error (MBE) as an indicator of systematic error. RMSE of grain yield was 2.6 t ha−1. MBE revealed grain yield undervalued by 2.6 t ha−1.
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