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: 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.

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.

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.

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.

Lichen as Micro-Ecosystem: Extremophilic Behavior with Astrobiotechnological Applications.

This work demonstrates the tolerance of lichen Pleurosticta acetabulum under extreme conditions similar to those encountered in extraterrestrial environments. Specifically, the impact of three extreme Mars-like conditions-complete dehydration, extremely low temperature (-196°C/77K), and oxygen depletion-on lichens was investigated. The symbiosis of mycobiont and photobiont partners creates a micro-ecosystem that ensures viability of both symbiotic partners under prolonged desiccation and extremely low temperatures without any cultivation care. Changes in the molecular structure and function of the photosynthetic apparatus, in the level of chlorophylls, polyamines, fatty acids, carbohydrates, ergosterol, efflux of K+, and DNA methylation ensure the ecological integrity of the system and offer resistance of lichens to above-mentioned extreme environmental conditions. For the first time, we also demonstrate that the unprecedented polyextremophilic characteristic of lichens could be linked to biotechnological applications, following exposure to these extreme conditions, such that their ability to produce a high yield of hydrogen was unchanged. All these support that lichens are (a) ideal model systems for a space mission to inhabit other planets, supporting also the aspect that the panspermia theory could be extended to incorporate in the traveling entities not only single organisms but micro-ecosystems like lichens, and (b) ideal model systems for astrobiotechnological applications (hydrogen production), such as in the development of bioregeneration systems for extraterrestrial environments.

Exogenous induction of thermogenesis in Arum concinnatum by salicylic acid.

Arum concinnatum Schott is a highly thermogenic species, with the temperature of the appendix exceeding ~10.9°C above the ambient temperature during thermogenesis, whereas the rates of respiration of the male florets in intact inflorescences peak at 0.92µmol s-1 g-1, which is the highest rate so far measured among the plants. Here, we attempt the ex situ exogenous induction of thermogenesis in whole inflorescences and in separate appendices of the spadix, and explore the thermogenic patterns under controlled laboratory conditions of light and temperature. Mature but unopened inflorescences and appendices showed thermogenic responses when treated with salicylic acid (SA), but not when treated with distilled water (control). With regard to light conditions, the responses revealed only one significant difference for inflorescences, which concerns the higher maximum temperature in the continuous light treatment compared with continuous dark. Along the ambient temperature gradient, at the lowest temperature edge individuals remained stable close to ambient temperature and to control. These findings suggest that, in general, ex situ exogenous induction of thermogenesis can be achieved in whole inflorescences and in separate appendices of spadix of A. concinnatum using SA. This study also indicates that SA acts independently of light conditions, while exogenous induction of thermogenesis takes place within an ambient temperature range.

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.

The Over-expression of the Plastidial Transglutaminase from Maize in Arabidopsis Increases the Activation Threshold of Photoprotection.

Plastidial transglutaminase is one of the most promising enzymes in chloroplast bioenergetics due to its link with polyamine pathways and the cross talk with signals such as Ca(2+) and GTP. Here, we show the effect of the increase of transglutaminase activity in Arabidopsis by using genetic transformation techniques. These lines fulfill their biological cycle normally (normal growth in soil, production of viable seeds) and show a relatively mild increase in transglutaminase activity (127%). These overexpressors of transglutaminase (OE TGase) have an extended stroma thylakoid network (71% higher number of PSIIß centers), similar chlorophyll content (-4%), higher linear electron flow (+13%), and higher threshold of photoprotection activation (∼100%). On the other hand OE TGase showed a reduced maximum photochemistry of PSII (-6.5%), a smaller antenna per photosystem II (-25%), a lower photoprotective "energization" quenching or qE (-77% at 490 µmol photons m(-2) s(-1)) due to a higher threshold of qE activation and slightly lower light induced proton motive force (-17%). The role of the polyamines and of the transglutaminase in the regulation of chemiosmosis and photoprotection in chloroplasts is discussed.

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.

Spermine and lutein quench chlorophyll fluorescence in isolated PSII antenna complexes.

Non photochemical quenching is a spontaneous mechanism that protects plants and algae from photodamage. In the last two decades, carotenoids through the xanthophylls cycle have been proposed to play a key role in quenching of chlorophyll. More recently, the involvement of endogenous polyamines in energy-dependent component of non photochemical quenching has been suggested by several research groups. In the present contribution, the combined effect of spermine and the xanthophylls, zeaxanthin and lutein on the fluorescence of antenna complexes of photosystem II was tested in vitro. Lutein caused significant quenching on trimeric and monomeric antenna complexes, whereas zeaxanthin under our experimental conditions had negligible effect. Spermine has been shown to allow fluorescence quenching to be induced in isolated antenna in the absence of ΔpH and to accelerate quenching formation. The simultaneous treatment of spermine and lutein maximizes quenching even at relatively low concentrations.

Lichen symbiosis: nature's high yielding machines for induced hydrogen production.

Hydrogen is a promising future energy source. Although the ability of green algae to produce hydrogen has long been recognized (since 1939) and several biotechnological applications have been attempted, the greatest obstacle, being the O2-sensitivity of the hydrogenase enzyme, has not yet been overcome. In the present contribution, 75 years after the first report on algal hydrogen production, taking advantage of a natural mechanism of oxygen balance, we demonstrate high hydrogen yields by lichens. Lichens have been selected as the ideal organisms in nature for hydrogen production, since they consist of a mycobiont and a photobiont in symbiosis. It has been hypothesized that the mycobiont's and photobiont's consumption of oxygen (increase of COX and AOX proteins of mitochondrial respiratory pathways and PTOX protein of chrolorespiration) establishes the required anoxic conditions for the activation of the phycobiont's hydrogenase in a closed system. Our results clearly supported the above hypothesis, showing that lichens have the ability to activate appropriate bioenergetic pathways depending on the specific incubation conditions. Under light conditions, they successfully use the PSII-dependent and the PSII-independent pathways (decrease of D1 protein and parallel increase of PSaA protein) to transfer electrons to hydrogenase, while under dark conditions, lichens use the PFOR enzyme and the dark fermentative pathway to supply electrons to hydrogenase. These advantages of lichen symbiosis in combination with their ability to survive in extreme environments (while in a dry state) constitute them as unique and valuable hydrogen producing natural factories and pave the way for future biotechnological applications.

Spermine is a potent modulator of proton transport through LHCII.

The effect of spermine on proton transport across large unilamellar liposomes containing incorporated complexes of the PSII antenna has been studied with the application of a pH-sensitive dye entrapped inside the vesicles. Both monomeric LHCbs and trimeric LHCII increased the permeability of proteoliposomes to protons when in a partly aggregated state within the lipid membrane. We have previously shown that a spermine-induced conformational change in LHCII results in its aggregation and ultimately in the enhancement of excitation energy as heat (qE). In this paper, spermine-induced aggregation of LHCII was found to facilitate proton transport across the proteoliposomes, indicating that a second protective mechanism (other than qE) might exist and might be regulated in vivo by polyamines when photosynthesis is saturated in excess light.

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.

Polyamines in chemiosmosis in vivo: A cunning mechanism for the regulation of ATP synthesis during growth and stress.

Polyamines (PAs) are low molecular weight amines that occur in every living organism. The three main PAs (putrescine, spermidine, and spermine) are involved in several important biochemical processes covered in recent reviews. As rule of thumb, increase of the cellular titer of PAs in plants is related to cell growth and cell tolerance to abiotic and biotic stress. In the present contribution, we describe recent findings from plant bioenergetics that bring to light a previously unrecognized dynamic behavior of the PA pool. Traditionally, PAs are described by many authors as organic polycations, when in fact they are bases that can be found in a charged or uncharged form. Although uncharged forms represent less than 0.1% of the total pool, we propose that their physiological role could be crucial in chemiosmosis. This process describes the formation of a PA gradient across membranes within seconds and is difficult to be tested in vivo in plants due to the relatively small molecular weight of PAs and the speed of the process. We tested the hypothesis that PAs act as permeable buffers in intact leaves by using recent advances in vivo probing. We found that an increase of PAs increases the electric component (Δψ) and decreases the ΔpH component of the proton motive force. These findings reveal an important modulation of the energy production process and photoprotection of the chloroplast by PAs. We explain in detail the theory behind PA pumping and ion trapping in acidic compartments (such as the lumen in chloroplasts) and how this regulatory process could improve either the photochemical efficiency of the photosynthetic apparatus and increase the synthesis of ATP or fine tune antenna regulation and make the plant more tolerant to stress.

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.