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.

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.

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.

Silencing S-Adenosyl-L-Methionine Decarboxylase (SAMDC) in Nicotiana tabacum Points at a Polyamine-Dependent Trade-Off between Growth and Tolerance Responses.

Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.

Putrescine, a fast-acting switch for tolerance against osmotic stress.

During the last decade we showed clearly that abiotic stress changes the cellular composition of polyamines, which in turn regulate the photochemical and non-photochemical quenching of the received light energy in the photosynthetic apparatus and that modulate substantially the level of plant tolerance. In the present contribution, we tried to change the bioenergetics of the leaf discs before the exposure to osmotic stress only by exogenously supplied putrescine, in order to enhance quickly the tolerance against the abiotic stress. Tobacco leaf discs treated with polyethylene-glycol reduced their water content about 24% within 1h. This relatively mild osmotic stress increased endogenous putrescine about 83% and decreased maximum photosystem II photochemical efficiency about 14%. In line with this, here we show that treatment with 1mM exogenous putrescine 1h before polyethylene-glycol addition protects the photochemical capacity and inhibits loss of water, confirming the key role of putrescine in the modulation of plant tolerance against osmotic stress. Furthermore, our recent works indicate that putrescine is accumulated in lumen during light reactions and may act as a permeable buffer and an osmolyte.

Role of plastid transglutaminase in LHCII polyamination and thylakoid electron and proton flow.

Transglutaminases function as biological glues in animal cells, plant cells and microbes. In energy producing organelles such as chloroplasts the presence of transglutaminases was recently confirmed. Furthermore, a plastidial transglutaminase has been cloned from maize and the first plants overexpressing tgz are available (Nicotiana tabacum TGZ OE). Our hypothesis is that the overexpression of plastidal transglutaminase will alter photosynthesis via increased polyamination of the antenna of photosystem II. We have used standard analytical tools to separate the antenna from photosystem II in wild type and modified plants, 6 specific antibodies against LHCbs to confirm their presence and sensitive HPLC method to quantify the polyamination level of these proteins. We report that bound spermidine and spermine were significantly increased (∼80%) in overexpressors. Moreover, we used recent advances in in vivo probing to study simultaneously the proton and electron circuit of thylakoids. Under physiological conditions overexpressors show a 3-fold higher sensitivity of the antenna down regulation loop (qE) to the elicitor (luminal protons) which is estimated as the ΔpH component of thylakoidal proton motive force. In addition, photosystem (hyper-PSIIα) with an exceptionally high antenna (large absorption cross section), accumulate in transglutaminase over expressers doubling the rate constant of light energy utilization (Kα) and promoting thylakoid membrane stacking. Polyamination of antenna proteins is a previously unrecognized mechanism for the modulation of the size (antenna absorption cross section) and sensitivity of photosystem II to down regulation. Future research will reveal which peptides and which residues of the antenna are responsible for such effects.

Evidence that putrescine modulates the higher plant photosynthetic proton circuit.

The light reactions of photosynthesis store energy in the form of an electrochemical gradient of protons, or proton motive force (pmf), comprised of electrical (Δψ) and osmotic (ΔpH) components. Both components can drive the synthesis of ATP at the chloroplast ATP synthase, but the ΔpH component also plays a key role in regulating photosynthesis, down-regulating the efficiency of light capture by photosynthetic antennae via the q(E) mechanism, and governing electron transfer at the cytochrome b(6)f complex. Differential partitioning of pmf into ΔpH and Δψ has been observed under environmental stresses and proposed as a mechanism for fine-tuning photosynthetic regulation, but the mechanism of this tuning is unknown. We show here that putrescine can alter the partitioning of pmf both in vivo (in Arabidopsis mutant lines and in Nicotiana wild type) and in vitro, suggesting that the endogenous titer of weak bases such as putrescine represents an unrecognized mechanism for regulating photosynthetic responses to the environment.

DCL3 and DCL4 are likely involved in the light intensity-RNA silencing cross talk in Nicotiana benthamiana.

Plants have substantially invested in RNA silencing as the central defense mechanism to combat nucleotide 'invaders' such as viruses, trasposable elements and transgenes. The quantity and quality of light perceived by a plant is a constant environmental stimulus refining cell homeostasis and RNA silencing mechanism seems not to be an exception In our recent paper in BMC Plant Biology we documented that light intensity, in physiological ranges, positively affects silencing initiation and spread. (1) Here, we show that virus induced gene silecing under high light conditions results in more frequent systemic silencing events of a transgene and is acompanied by elevated DCL3 and DCL4 mRNA levels. In addition, our results show that DCL3 holds a vital role in systemic silencing spread and the positive effect of light intensity on RNA silencing requires DCL4 function.

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.

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.

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.

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.

Putrescine stimulates chemiosmotic ATP synthesis.

Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a DeltapH and (iv) a pool of putrescine.