Microalgal photosynthesis induces alkalization of aquatic environment as a result of H+ uptake independently from CO2 concentration - New perspectives for environmental applications.

The photosynthetic process in microalgae and the extracellular proton environment interact with each other. The photosynthetic process in microalgae induces a pH increase in the aquatic environment as a result of cellular protons uptake rather than as an effect of CO2 consumption. The photosynthetic water photolysis and the reduction/oxidation cycle of the plastoquinone pool provide lumen with protons. Weak bases act as "permeant buffers" in lumen during the photosynthetic procedure, converting the ΔpH to Δψ. This is possibly the main reason for continuous light-driven proton uptake from the aquatic environment through cytosol and stroma, into the lumen. The proton uptake rate and, therefore, the microalgal growth is proportional to the light intensity, cell concentration, and extracellular proton concentration. The low pH in microalgae cultures, without limitation factors related to light and nutrients, strongly induces photosynthesis (and proton uptake) and, consequently, growth. In contrast, the mitochondrial respiratory process, in the absence of photosynthetic activity, does not substantially alter the culture pH. Only after intensification of the respiratory process, using exogenous glucose supply leads to significantly reduced pH values in the culture medium, almost exclusively through proton output. Enhanced dissolution of atmospheric CO2 in water causes the phenomenon of ocean acidification, which prevents the process of calcification, a significant process for numerous phytoplankton and zooplankton organisms, as well for corals. The proposed interaction between microalgal photosynthetic activity and proton concentration in the aquatic environment, independently from the CO2 concentration, paves the way for new innovative management strategies for reversing the ocean acidification.

Bioenergetic reprogramming plasticity under nitrogen depletion by the unicellular green alga Scenedesmus obliquus.

MAIN CONCLUSION: Simultaneous nitrogen depletion and 3,4-dichlorophenol addition induce a bioenergetic microalgal reprogramming, through strong Cyt b 6 f synthesis, that quench excess electrons from dichlorophenol's biodegradation to an overactivated photosynthetic electron flow and H 2 -productivity. Cellular energy management includes "rational" planning and operation of energy production and energy consumption units. Microalgae seem to have the ability to calculate their energy reserves and select the most profitable bioenergetic pathways. Under oxygenic mixotrophic conditions, microalgae invest the exogenously supplied carbon source (glucose) to biomass increase. If 3,4-dichlorophenol is added in the culture medium, then glucose is invested more to biodegradation rather than to growth. The biodegradation yield is enhanced in nitrogen-depleted conditions, because of an increase in the starch accumulation and a delay in the establishment of oxygen-depleted conditions in a closed system. In nitrogen-depleted conditions, starch cannot be invested in PSII-dependent and PSII-independent pathways for H2-production, mainly because of a strong decrease of the cytochrome b 6 f complex of the photosynthetic electron flow. For this reason, it seems more profitable for the microalga under these conditions to direct the metabolism to the synthesis of lipids as cellular energy reserves. Nitrogen-depleted conditions with exogenously supplied 3,4-dichlorophenol induce reprogramming of the microalgal bioenergetic strategy. Cytochrome b 6 f is strongly synthesized (mainly through catabolism of polyamines) to manage the electron bypass from the dichlorophenol biodegradation procedure to the photosynthetic electron flow (at the level of PQ pool) and consequently through cytochrome b 6 f and PSI to hydrogenase and H2-production. All the above showed that the selection of the appropriate cultivation conditions is the key for the manipulation of microalgal bioenergetic strategy that leads to different metabolic products and paves the way for a future microalgal "smart" biotechnology.

Bioenergetic strategy of microalgae for the biodegradation of tyrosol and hydroxytyrosol.

Olive mill wastewater has significant polluting properties due to its high phenolic content [mainly tyrosol (trs) and hydroxytyrosol (htrs)]. Growth kinetics and a series of fluorescence induction measurements for Scenedesmus obliquus cultures showed that microalgae can be tolerant of these phenolic compounds. Changes in the cellular energy reserves and concentration of the phenolic compounds adjust the "toxicity" of these compounds to the microalgae and are, therefore, the main parameters that affect biodegradation. Autotrophic growth conditions of microalgae and high concentrations of trs or htrs induce higher biodegradation compared with mixotrophic conditions and lower phenolic concentrations. When microalgae face trs and htrs simultaneously, biodegradation begins from htrs, the more energetically demanding compound. All these lead to the conviction that microalgae have a "rational" management of cellular energy balance. Low toxicity levels lead to higher growth and lower biodegradation, whereas higher toxicity levels lead to lower growth and higher biodegradation. The selection of appropriate conditions (compatible to the bioenergetic strategies of microalgae) seems to be the key for a successful biodegradation of a series of toxic compounds, thus paving the way for future biotechnological applications for solving complicated pollution problems, like the detoxification of olive mill wastewater.

Could structural similarity of specific domains between animal globins and plant antenna proteins provide hints important for the photoprotection mechanism?

Non photochemical quenching is a fundamental mechanism in photosynthesis, which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. In the last decades hundreds of papers have appeared that describe the role of antenna regulation in protection or the so called qE response. However, the exact quenching site is still obscure. Previously overlooked features of the antenna may provide hints towards the elucidation of its functionality and of the quenching mechanism. Recently it was demonstrated that the catalytic domain of human myoglobin that binds the pigment (i.e. heme) is similar in structure to the domain of the light harvesting complex II of pea that binds Chl a 614 (former known as b3). In addition, it is well accepted that conformational changes of the chlorophyll macrocycle result in reversible changes of fluorescence (the lowest fluorescence corresponds to non planar macrocycle). Here we put forward a hypothesis regarding the molecular mechanism that leads to the formation of a quenching center inside the antenna proteins. Our main suggestion is that a conformational change of helix H5 (known also as helix D) forces conformational changes in the macrocycle of Chl a 614 is implicated in the ΔA535 absorbance change and quenching during photoprotective qE. The specific features (some of them similar to those of heme domain of globins) of the b3 domain account for these traits. The model predicts that antenna proteins having b3 pigments (i.e. LHCII, CP29, CP26) can act as potential quenchers.

Polyamines in cell walls of chlorococcalean microalgae.

Biotechnology of microalgae represents a very attractive alternative as a source of energy and substances of high value when compared with plant cultivation. Cell walls of green microalgae have an extraordinary chemical and mechanical resistance and may impede some steps in the biotechnological/industrial exploitation of algae. The aim of the present contribution was to check the presence of polyamines in the cell walls of chlorococcalean green microalgae. Polyamines are nitrogenous compounds synthesized normally in cells and may affect the properties of the cell wall. Our work included strains either forming or not forming the polymer algaenan, allowing us to conclude that algaenan is not a prerequisite for the presence of polyamines in the cell walls. Polyamines were detected in isolated cell walls of Scenedesmus obliquus, Chlorella fusca, Chlorella saccharophila, and Chlorella vulgaris. Their concentration in isolated cell walls ranged between 0.4 and 8.4 nmol/mg dry weight.

Acclimation mechanism of microalgal photosynthetic apparatus under low atmospheric pressures - new astrobiological perspectives in a Mars-like atmosphere.

This study reveals a new acclimation mechanism of the eukaryotic unicellular green alga Chlorella vulgaris in terms of the effect of varying atmospheric pressures on the structure and function of its photosynthetic apparatus using fluorescence induction measurements (JIP-test). The results indicate that low (400mbar) and extreme low (2 atmosphere (simulating the Mars atmosphere), reveals that the impact of extremely low atmospheric pressure on PQ mobility within the photosynthetic membrane, coupled with the low density of an almost 100% CO2 Mars-like atmosphere, results to a similar photosynthetic efficiency to that on Earth. These findings pave the way for the identification of novel functional acclimation mechanisms of microalgae to extreme environments that are vastly distinct from those found on Earth.

Stress-related transcriptomic changes associated with GFP transgene expression and active transgene silencing in plants.

Plants respond to biotic and abiotic stress by activating and interacting with multiple defense pathways, allowing for an efficient global defense response. RNA silencing is a conserved mechanism of regulation of gene expression directed by small RNAs important in acquired plant immunity and especially virus and transgene repression. Several RNA silencing pathways in plants are crucial to control developmental processes and provide protection against abiotic and biotic stresses as well as invasive nucleic acids such as viruses and transposable elements. Various notable studies have shed light on the genes, small RNAs, and mechanisms involved in plant RNA silencing. However, published research on the potential interactions between RNA silencing and other plant stress responses is limited. In the present study, we tested the hypothesis that spreading and maintenance of systemic post-transcriptional gene silencing (PTGS) of a GFP transgene are associated with transcriptional changes that pertain to non-RNA silencing-based stress responses. To this end, we analyzed the structure and function of the photosynthetic apparatus and conducted whole transcriptome analysis in a transgenic line of Nicotiana benthamiana that spontaneously initiates transgene silencing, at different stages of systemic GFP-PTGS. In vivo analysis of chlorophyll a fluorescence yield and expression levels of key photosynthetic genes indicates that photosynthetic activity remains unaffected by systemic GFP-PTGS. However, transcriptomic analysis reveals that spreading and maintenance of GFP-PTGS are associated with transcriptional reprogramming of genes that are involved in abiotic stress responses and pattern- or effector-triggered immunity-based stress responses. These findings suggest that systemic PTGS may affect non-RNA-silencing-based defense pathways in N. benthamiana, providing new insights into the complex interplay between different plant stress responses.

Polyamines induce aggregation of LHC II and quenching of fluorescence in vitro.

Dissipation of excess excitation energy within the light-harvesting complex of Photosystem II (LHC II) is a main process in plants, which is measured as the non-photochemical quenching of chlorophyll fluorescence or qE. We showed in previous works that polyamines stimulate qE in higher plants in vivo and in eukaryotic algae in vitro. In the present contribution we have tested whether polyamines can stimulate quenching in trimeric LHC II and monomeric light-harvesting complex b proteins from higher plants. The tetramine spermine was the most potent quencher and induced aggregation of LHC II trimers, due to its highly cationic character. Two transients are evident at 100 µM and 350 µM for the fluorescence and absorbance signals of LHC II respectively. On the basis of observations within this work, some links between polyamines and the activation of qE in vivo is discussed.

Modeling the dynamic modulation of light energy in photosynthetic algae.

An integrated cell-based dynamic mathematical model that take into account the role of the photon absorbing process, the partition of excitation energy, and the photoinactivation and repair of photosynthetic units, under variable light and dissolved inorganic carbon (DIC) availability is proposed. The modeling of the photon energy absorption and the energy dissipation is based on the photoadaptive changes of the underlying mechanisms. The partition of the excitation energy is based on the relative availability of light and DIC to the cell. The modeling of the photoinactivation process is based on the common aspect that it occurs under any light intensity and the modeling of the repair process is based on the evidence that it is controlled by chloroplast and nuclear-encoded enzymes. The present model links the absorption of photons and the partitioning of excitation energy to the linear electron flow and other quenchers with chlorophyll fluorescence emission parameters, and the number of the functional photosynthetic units with the photosynthetic oxygen production rate. The energy allocation to the LEF increases as DIC availability increases and/or light intensity decreases. The rate of rejected energy increases with light intensity and with DIC availability. The resulting rate coefficient of photoinactivation increases as light intensity and/or as DIC concentration increases. We test the model against chlorophyll fluorescence induction and photosynthetic oxygen production rate measurements, obtained from cultures of the unicellular green alga Scenedesmus obliquus, and find a very close quantitative and qualitative correspondence between predictions and data.

Polyamines: Α bioenergetic smart switch for plant protection and development.

The present review highlights the bioenergetic role of polyamines in plant protection and development and proposes a universal model for describing polyamine-mediated stress responses. Any stress condition induces an excitation pressure on photosystem II by reforming the photosynthetic apparatus. To control this phenomenon, polyamines act directly on the molecular structure and function of the photosynthetic apparatus as well as on the components of the chemiosmotic proton-motive force (ΔpH/Δψ), thus regulating photochemical (qP) and non-photochemical quenching (NPQ) of energy. The review presents the mechanistic characteristics that underline the key role of polyamines in the structure, function, and bioenergetics of the photosynthetic apparatus upon light adaptation and/or under stress conditions. By following this mechanism, it is feasible to make stress-sensitive plants to be tolerant by simply altering their polyamine composition (especially the ratio of putrescine to spermine), either chemically or by light regulation.

Polyamines stimulate non-photochemical quenching of chlorophyll a fluorescence in Scenedesmus obliquus.

Polyamines (PAs) are small metabolites that are produced and oxidized in chloroplasts with an obscure mode of action. Recently, we showed that qE is stimulated by PAs in higher plants (Nicotiana tabacum) and in genetically modified plants with elevated thylakoid-associated PAs (Ioannidis and Kotzabasis Biochim Biophys Acta 1767:1371-1382, 2007; Ioannidis et al. Biochim Biophys Acta 1787:1215-1222, 2009). Here, we investigated further their quenching properties both in vivo in green algae and in vitro is isolated LHCII. In vivo spermine up-regulates NPQ in Scenedesums obliquus about 30%. In vitro putrescine--the obligatory metabolic precursor of PAs--has a marginal quenching effect, while spermidine and spermine exhibit strong quenching abilities in isolated LHCII up to 40%. Based on available 3D models of LHCII we report a special cavity of about 600 Å(3) and a near-by larger pocket in the trimeric LHCII that could be of importance for the stimulation of qE by amines.

Biotechnology under extreme conditions: Lichens after extreme UVB radiation and extreme temperatures produce large amounts of hydrogen.

The present study demonstrates biotechnological applications of the lichen Pleurosticta acetabulum, specifically the production of large amounts of hydrogen even after the lichen exposure to extreme conditions such as a) extreme UVB radiation (1.7 mW/cm2 = 1000 J m-2 min-1) over different time periods (4, 20 & 70 h) and b) combined exposure of the lichen to high intensity UVB radiation and extreme low (-196 °C) or extreme high temperatures (+70 °C). The results highlight that the extremophilic and polyextremophilic behavior of lichens both in dehydrated and in regenerated form, under extreme conditions not necessarily recorded on earth, is compatible with their biotechnological uses. The lichen viability was measured using fluorescence induction techniques (OJIP-test), which record changes in the molecular structure and function of the photosynthetic mechanism, while its ability to produce molecular hydrogen was measured through thermal conductivity gas chromatography (GC-TCD) analysis. Hydrogen is a promising fuel for the future. The exciting result of a lichen micro-ecosystem is its ability to expel its moisture and remain in an inactive state, protecting itself from extreme conditions and maintaining its ability to high yield hydrogen production in a closed system, with the sole addition of water and without the need for additional energy. Our results expand the potential use of lichens for future biotechnological applications in extreme Earth environments, but also in environments on other planets, such as Mars, thus paving the way for astrobiotechnological applications.

Hydrogen gas as a central on-off functional switch of reversible metabolic arrest - New perspectives for biotechnological applications.

Metabolism is the sum of all chemical reactions that sustain life. There is an ongoing effort to control metabolic rate, which correlates with the maximum lifespan potential and constitutes one of the oldest scientific questions. Herein, we report on the complete reversible arrest of cellular metabolism and cell growth in a series of organisms, from microalgae to yeast upon exposure to a 100 % hydrogen atmosphere. We also report a tolerance of the microalgae under these conditions against extreme stress conditions, like high salt concentrations. The addition of oxygen or air almost completely restores the metabolic rate and cell growth. Molecular dynamics simulations are employed to decipher this phenomenon at atomic scale. Various proteins, including photosynthetic and respiratory complexes (LHCII, cytochrome c5) are probed in the interaction with hydrogen. Exposure to hydrogen, as opposed to oxygen, decreases the fluctuations of protein residues indicating thermostability. According to the above mechanism, an absolute hydrogen atmosphere can preserve biological products (e.g. fruits) for a long time without consuming any energy. By combining biological, chemical and computational methods, in this research we provide the basis for future innovative studies and advances in the field of biotechnology.

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase.

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing epsilon-(gamma-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIalpha) at the expense of centers located mainly in stroma thylakoids (PSIIbeta). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.

Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants.

BACKGROUND: Expression of exogenous sequences in plants is often suppressed through one of the earliest described RNA silencing pathways, sense post-transcriptional gene silencing (S-PTGS). This type of suppression has made significant contributions to our knowledge of the biology of RNA silencing pathways and has important consequences in plant transgenesis applications. Although significant progress has been made in recent years, factors affecting the stability of transgene expression are still not well understood. It has been shown before that the efficiency of RNA silencing in plants is influenced by various environmental factors. RESULTS: Here we report that a major environmental factor, light intensity, significantly affects the induction and systemic spread of S-PTGS. Moreover, we show that photoadaptation to high or low light intensity conditions differentially affects mRNA levels of major components of the RNA silencing machinery. CONCLUSIONS: Light intensity is one of the previously unknown factors that affect transgene stability at the post-transcriptional level. Our findings demonstrate an example of how environmental conditions could affect RNA silencing.

Combinational system for biodegradation of olive oil mill wastewater phenolics and high yield of bio-hydrogen production.

Olive oil mill wastewater (OMW) is a significant pollutant in the Mediterranean region. In the present contribution, we showed clearly that microorganisms (microalgae and OMW-microflora) activated the biodegradation of OMW-phenolics and produced a high yield of hydrogen (H2). In a closed incubation system, the appropriate adjustment of OMW-pH leads to the establishment of anoxic conditions through the oxygen consumption of microorganisms during the first incubation day. The biodegradation procedure of OMW-phenolics needs oxygen. Therefore, after the establishment of anoxic conditions, the biodegradation stopped and the activation of hydrogenases started, leading to a continuous high yield of bio-hydrogen production. If the cultivation system re-opened (oxygen enrichment), the OMW-phenolic biodegradation (oxygen dependent process) started again and therefore the detoxified OMW could be used for further biotechnological applications (production of biodiesel, bioalcohols, organic fertilizers, etc.). Apart from the environmental compatibility of the method and the sustainability of such a combinational application (OMW detoxification and high yield of hydrogen production) in the context of a green biotechnology approach, the cost/profit ratio appears to be particularly tempting and guarantees its widespread use in the near future. The present contribution proposes a solution to a major environmental problem by upgrading its solution to a high-value product.

Comparative biodegradation of all chlorinated phenols by the microalga Scenedesmus obliquus - The biodegradation strategy of microalgae.

This work presents the comparative biodegradation of all chlorinated phenolic compounds by the green alga Scenedesmus obliquus and determines the microalgal bioenergetic strategy. The microalga manages its energy reserves rationally by investing them, either on growth or on the biodegradation of the toxic compound. The microalga seems to follow two distinct detoxification strategies. In the first one, when microalgae are surrounded by relatively low toxic phenolic compounds (phenol, monochlorophenols, 2,4-dichlorophenol and 2,6-dichlorophenol), they use all, or at least more of their energy reserves to increase the biomass production and not the biodegradation. In the second one, when surrounded by higher toxic chlorophenols (meta-substituted dichlorophenols, trichlorophenols, tetrachlorophenols and pentachlorophenol) the microalgae invest more, or all of their energy reserves directly in the biodegradation of the toxic compounds, while less or no energy is invested in biomass increase. The microalga biodegraded in five days approximately 9% of the lower toxic phenol and 90% of the higher toxic pentachlorophenol. Considering our ability to interfere with microalgae energy reserves, which define their stress tolerance in the toxic environment, and knowing the microalgal bioenergetic strategy, we could easily use microalgae to biodegrade toxic wastes in the frame of a rational biotechnological approach in the near future.

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro.

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.

A polyamine- and LHCII protease activity-based mechanism regulates the plasticity and adaptation status of the photosynthetic apparatus.

In the present study we aim to dissect the basis of the polyamine mode of action in the structure and function of the photosynthetic apparatus. Although the modulating effects of polyamines in photosynthesis have been reported since long [K. Kotzabasis, A role for chloroplast-associated polyamines? Bot. Acta 109 (1996) 5-7], the underlying mechanisms remained until today largely unknown. The diamine putrescine was employed in this study, by being externally added to Scenedesmus obliquus cultures acclimated to either low or high light conditions. The results revealed the high efficiency by which putrescine can alter the levels of the major photosynthetic complexes in a concerted manner inducing an overall structure and function of the photosynthetic apparatus similar to that under higher light conditions. The revealed mechanism for this phenomenon involves alterations in the level of the polyamines putrescine and spermine which are bound to the photosynthetic complexes, mainly to the LHCII oligomeric and monomeric forms. In vitro studies point out to a direct impact of the polyamines on the autoproteolytic degradation of LHCII. Concomitantly to the reduction of the LHCII size, exogenously supplied putrescine, induces the reaction centers' density and thus the photosynthetic apparatus is adjusted as if it was adapted to higher light conditions. Thus polyamines, through LHCII, play a crucial role in the regulation of the photosynthetic apparatus' photoadaptation. The protective role of polyamines on the photosynthetic apparatus under various environmental stresses is also discussed in correlation to this phenomenon.

Salt stress impact on the molecular structure and function of the photosynthetic apparatus--the protective role of polyamines.

In the present study the green alga Scenedesmus obliquus was used to assess the effects of high salinity (high NaCl-concentration) on the structure and function of the photosynthetic apparatus and the possibility for alleviation by exogenous putrescine (Put). Chlorophyll fluorescence data revealed the range of the changes induced in the photosynthetic apparatus by different NaCl concentrations, which altogether pointed towards an increased excitation pressure. At the same time, changes in the levels of endogenous polyamine concentrations, both in cell and in isolated thylakoid preparations were also evidenced. Certain polyamine changes (Put reduction) were correlated with changes in the structure and function of the photosynthetic apparatus, such as the increase in the functional size of the antenna and the reduction in the density of active photosystem II reaction centers. Thus, exogenously added Put was used to compensate for this stress condition and to adjust the above mentioned changes, so that to confer some kind of tolerance to the photosynthetic apparatus against enhanced NaCl-salinity and permit cell growth even in NaCl concentrations that under natural conditions would be toxic.