Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

Diagnosis of the functional state of the photosynthetic apparatus Taraxacum officinale (L.) Weber ex F. H. Wigg. by the method of chlorophyll fluorescence induction

The informativeness is established of the method of induction of chlorophyll fluorescence for bioindication in the conditions of anthropogenic influence on the example of T. officinale plants, by the parameters: F0, Fm, Fst, Fv, Fv/Fm та k1, k2. The main parameters of the induction curve can be used as test indicators to determine the resistance of plants to adverse factors. Changes in these indicators characterize the processes associated with the influence of the environment on the course of both light and dark phases of photosynthetic processes in chloroplasts. The photosensitivity of T. offic- inale leaves, which indicates the physiological state of plants, decreases with increasing adverse external influences. It is established that the plants of T. officinale have the maximum intensity of photosynthesis, which grow in more favorable ecological conditions control. Therefore, the use of the device «Floratest» to determine the condition of plants at different levels of anthropogenic load is promising and requires further research.

Field Study of the Effects of Two Different Environmental Conditions on Wheat Productivity and Chlorophyll Fluorescence Induction (OJIP) Parameters

Wheat is one of the main cereal crops for ensuring food supply. Thus, increasing grain yield is a major target for plant breeders, where insights into wheat productivity can be gained by studying the activity of the components of photosynthetic apparatus. The objectives of this study were to evaluate the agronomical performance of three winter wheat varieties and test photosynthetic efficiency over two different locations. Chlorophyll fluorescence was used to evaluate the maximum quantum yield of photosystem II (PSII) (TR0/ABS) and performance index on absorption basis (PIabs) of flag leaves and glumes of heads at the flowering stage until the mid-senescence stage. The grain yield of all varieties on average was significantly higher at Osijek compared to Tovarnik. Variety Tika Taka exhibited the highest yield reduction (27.1%) at Tovarnik compared to Osijek, followed by El Nino (20.5%) and Vulkan (18.7%), respectively. A higher amount of precipitation in June at Tovarnik provoked higher Fusarium head blight disease intensity, which could be seen as the bleaching of plant heads at the plots and resulted in an earlier decrease in photosynthetic activity. Therefore, earlier senescence and contracted grain fill duration could occur.

Application of Optimal Combined Microfertilizers of Boron, Molybdenum, and Copper Improves Root Tuber Yield Trait and Photosynthetic Response Characteristics in Pseudostellaria heterophylla

In the actual cultivation process, blind fertilizer application was widespread, resulting in a serious decline in the yield of Pseudostellaria heterophylla. We used the 3414 fertilizer experiment design to study the effects of combined Boron (B), Molybdenum (Mo), and Copper (Cu) on the growth indexes, diurnal changes of photosynthesis, and rapid fluorescence induction dynamics in P. heterophylla. Our results show that the optimal combination of B, Mo, and Cu simultaneously promoted the growth of underground and aboveground parts, and significantly improved the quality of single root tuber and yield per unit area. The best combination was treatment 9 (T9 = B, 1 g/L; Mo, 0.08 g/L; Cu, 0.05 g/L), and resulted in a 35.1% increase in yield per unit area compared with the control group (T1). Although the optimal combined application of microfertilizers did not change the bimodal trend of diurnal variation of photosynthesis, it effectively increased the daily average, peak, and valley values of the photosynthetic rate by alleviating the nonstomatal limitation and the photosynthetic midday depression. Pseudostellaria heterophylla leaves showed greater photochemical activity and less photoinhibition of photosystem II in T9. Major effects were that it helped protect the activity of the oxygen-evolving complex to reduce the oxidative damage of chloroplasts and prevent the dissociation of thylakoid. The microfertilizer application also enhanced the electron receiving ability of the QB and plastoquinone (PQ) electronic pools, thereby increasing the ability of electron transfer from QA to QB. The number of reaction centers per unit area was promoted notably by the fertilization treatment.

Combined chlorophyll fluorescence techniques to study environmental impact on the mountain moss Polytrichum commune

Two chlorophyll fluorescence (ChlF) methods were used to study the effects of high light (photoinhibition) and dehydration, common stressors of the alpine environment, on primary photosynthetic processes in the moss Polytrichum commune from the Czech Republic, the Jeseníky Mountains. Photoinhibition (PI) was studied in fully hydrated thalli of P. commune and during the period of spontaneous desiccation. Time courses of Kautsky kinetics (KK) of ChlF and derived parameters: maximum quantum yield (FV/FM), effective quantum yeld (ΦPSII), and non-photochemical quenching parameters, were measured before and after the samples were treated with high light (1500 µmol m-2 s-1 PAR) for 60 min. Dehydration effects were tested in two sets of experiments with a Pulse-Amplitude-Modulation fluorometry (PAM) and Fast Chlorophyll Fluorescence induction curve (OJIP) techniques. In PAM tests, the desiccating samples were exposed to saturating light pulses every 10 min. in order to obtain ΦPSII and non-photochemical quenching (NPQ). In the second dehydration experiment, OJIP transients of ChlF were repeatedly recorded, OJIP-derived ChlF parameters were plotted against relative water content (RWC) monitored during desiccation. Combined ChF techniques provided insights into the mechanisms activated during P. commune desiccation, such as dissipation of excess absorbed energy through heat dissipation, and conformational changes or destructions of the light harvesting complexes. Combination of stressors resulted in amplified interference with the photosynthetic machinery, even when the added stressor (dehydration) was applied in low dose.

Methyl Jasmonate Protects the PS II System by Maintaining the Stability of Chloroplast D1 Protein and Accelerating Enzymatic Antioxidants in Heat-Stressed Wheat Plants

The application of 10 µM methyl jasmonate (MeJA) for the protection of wheat (Triticum aestivum L.) photosystem II (PS II) against heat stress (HS) was studied. Heat stress was induced at 42 °C to established plants, which were then recovered at 25 °C and monitored during their growth for the study duration. Application of MeJA resulted in increased enzymatic antioxidant activity that reduced the content of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) and enhanced the photosynthetic efficiency. Exogenous MeJA had a beneficial effect on chlorophyll fluorescence under HS and enhanced the pigment system (PS) II system, as observed in a JIP-test, a new tool for chlorophyll fluorescence induction curve. Exogenous MeJA improved the quantum yield of electron transport (ETo/CS) as well as electron transport flux for each reaction center (ET0/RC). However, the specific energy fluxes per reaction center (RC), i.e., TR0/RC (trapping) and DI0/RC (dissipation), were reduced by MeJA. These results indicate that MeJA affects the efficiency of PS II by stabilizing the D1 protein, increasing its abundance, and enhancing the expression of the psbA and psbB genes under HS, which encode proteins of the PS II core RC complex. Thus, MeJA is a potential tool to protect PS II and D1 protein in wheat plants under HS and to accelerate the recovery of the photosynthetic capacity.

Respiration Interacts With Photosynthesis Through the Acceptor Side of Photosystem I, Reflected in the Dark-to-Light Induction Kinetics of Chlorophyll Fluorescence in the Cyanobacterium Synechocystis sp. PCC 6803

In cyanobacteria, the photosynthetic prokaryotes, direct interaction between photosynthesis and respiration exists at plastoquinone (PQ) pool, which is shared by the two electron transport chains. Another possible point of intersection of the two electron transport chains is NADPH, which is the major electron donor to the respiratory chain as well as the final product of the photosynthetic chain. Here, we showed that the redox state of NADPH in the dark affected chlorophyll fluorescence induction in the cyanobacterium Synechocystis sp. PCC 6803 in a quantitative manner. Accumulation of the reduced NADPH in the dark due to the defect in type 1 NAD(P)H dehydrogenase complex in the respiratory chain resulted in the faster rise to the peak in the dark-to-light induction of chlorophyll fluorescence, while depletion of NADPH due to the defect in pentose phosphate pathway resulted in the delayed appearance of the initial peak in the induction kinetics. There was a strong correlation between the dark level of NADPH determined by its fluorescence and the peak position of the induction kinetics of chlorophyll fluorescence. These results indicate that photosynthesis interacts with respiration through NADPH, which enable us to monitor the redox condition of the acceptor side of photosystem I by simple measurements of chlorophyll fluorescence induction in cyanobacteria.

Phytoplankton Photophysiology Utilities: A Python Toolbox for the Standardization of Processing Active Chlorophyll-a Fluorescence Data

The uptake and application of single turnover chlorophyll fluorometers to the study of phytoplankton ecosystem status and microbial functions has grown considerably in the last two decades. However, standardization of measurement protocols, processing of fluorescence transients and quality control of derived photosynthetic parameters is still lacking and makes community goals of large global databases of high-quality data unrealistic. We introduce the Python package Phytoplankton Photophysiology Utilities (PPU), an adaptable and open-source interface between Fast Repetition Rate and Fluorescence Induction and Relaxation instruments and python. The PPU package includes a variety of functions for the loading, processing and quality control of single turnover fluorescence transients from many commercially available instruments. PPU provides the user with greater flexibility in the application of the Kolber-Prasil-Falkowski model; tools for plotting, quality control, correcting instrument biases and high-throughput processing with ease; and a greater appreciation for the uncertainties in derived photosynthetic parameters. Using data from three research cruises across different biogeochemical regimes, we provide example applications of PPU to fit raw active chlorophyll-a fluorescence data from three commercial instruments and demonstrate tools which help to reduce uncertainties in the final fitted parameters.

OJIP chlorophyll fluorescence induction profiles and plastoquinone binding affinity of the Photosystem II assembly intermediate PSII-I from Thermosynechococcus elongatus

Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.

The Microalga Chlorella vulgaris as a Natural Bioenergetic System for Effective CO2 Mitigation—New Perspectives against Global Warming

In the present contribution, the differentiation in the molecular structure and function of the photosynthetic apparatus of the unicellular green alga Chlorella vulgaris was studied at several light intensities (0–400 μmol m−2 s−1) and various CO2 concentrations (0.04–60% CO2), in completely autotrophic conditions. Asymmetries that occur by different light intensities and CO2 concentrations induce metabolic and functional changes. Using chlorophyll fluorescence induction techniques (OJIP test), we showed that Chlorella vulgaris tolerates extremely high CO2 levels and converts them photosynthetically into valuable products, including O2 and biomass rich in carbohydrates and lipids. Interestingly, the microalga Chlorella vulgaris under extremely high CO2 concentrations induces a new metabolic state intensifying its photosynthetic activity. This leads to a new functional symmetry. The results highlight a potent CO2 bio-fixation mechanism of Chlorella vulgaris that captures up to 288 L CO2 L PCV−1 day−1 under optimal conditions, therefore, this microalga can be used for direct biological CO2-reducing strategies and other green biotechnological applications. All of the above suggest that Chlorella vulgaris is one of the most prominent competitors for a closed algae-powered bioreactor that is able to consume huge amounts of CO2. Thus, it is a sustainable and natural bioenergetic system with perspectives in dealing with major environmental issues such as global warming. In addition, Chlorella vulgaris cultures could also be used as bioregeneration systems in extraterrestrial missions for continuous atmospheric recycling of the human settlements, paving the way for astrobiological applications.