Nikolaos E. Ioannidis

Thylakoid-associated polyamines adjust the UV-B sensitivity of the photosynthetic apparatus by means of light-harvesting complex II changes.

Abstract The sensitivity of the photosynthetic apparatus to ultraviolet-B (UV-B) irradiation was studied in cultures of unicellular green alga Scenedesmus obliquus incubated in low light (low photosynthetically active radiation intensity [LL]) and high light (high photosynthetically active radiation intensity [HL]) conditions, treated or not with exogenous polyamines. Biochemical and physicochemical measurements showed that UV-B radiation induces a decrease in the thylakoid-associated putrescine (Put) and an increase in spermine (Spm), so that the reduction of Put/Spm ratio leads to the increase of light-harvesting complex II (LHCII) size per active reaction center and, consequently, the amplification of UV-B effects on the photosynthetic apparatus. The separation of oligomeric and monomeric forms of LHCII from isolated thylakoids showed that UV-B induces an increase in the oligomeric forms of LHCII, which was more intense in LL than in HL. By manipulating the LHCII size with exogenous polyamines, the sensitivity degree of the photosynthetic apparatus to UV-B changed significantly. Specifically, the addition of Put decreased highly the sensitivity of LL culture to UV-B because of the inhibitory effect of Put on the LHCII size increasing, whereas the addition of Spm enhanced the UV-B injury induced in HL culture because of the increasing of LHCII size. The ability of the photosynthetic apparatus to recover the UV-B induced changes was also investigated.

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.

Fast and reversible response of thylakoid-associated polyamines during and after UV-B stress: a comparative study of the wild type and a mutant lacking chlorophyll b of unicellular green alga Scenedesmus obliquus.

The functional and biochemical aspects of the photosynthetic apparatus in response to UV-B radiation were examined in unicellular oxygenic algae Scenedesmus obliquus. The wild type (Wt) and a chlorophyll b-less mutant (Wt-lhc) were used as a specific tool for the understanding of antenna role. Photosynthesis was monitored during and after UV-B stress by time resolved fluorescence spectroscopy and polarography. Carotenoids, such as neoxanthin, loroxanthin, lutein, violaxanthin, antheraxanthin, zeaxanthin, α- and β-carotene, cellular and thylakoid-associated putrescine, spermidine, spermine and subcomplexes of light-harvesting complex (LHCII) of photosystem II (PSII) were investigated to assess their possible involvement in response to UV-B. Oxygen evolution depression by UV-B was higher in the Wt-lhc mutant than in the Wt. Photosynthesis recovery occurred in the Wt, but not in the mutant. The dissipation of excess excitation energy during UV-B stress was accompanied by changes in the thylakoid-associated polyamines which were much higher than changes in xanthophylls. We conclude that, at least in the unicellular green alga S. obliquus, mutants lacking chlorophyll b have significant lower capacity for recovery after UV-B stress. In addition, the comparison of xanthophylls and thylakoid-associated polyamines reveals that the latter are more responsive to UV-B stress and in a reversible manner.

High yields of hydrogen production induced by meta-substituted dichlorophenols biodegradation from the green alga Scenedesmus obliquus.

Hydrogen is a highly promising energy source with important social and economic implications. The ability of green algae to produce photosynthetic hydrogen under anaerobic conditions has been known for years. However, until today the yield of production has been very low, limiting an industrial scale use. In the present paper, 73 years after the first report on H2-production from green algae, we present a combinational biological system where the biodegradation procedure of one meta-substituted dichlorophenol (m-dcp) is the key element for maintaining continuous and high rate H2-production (>100 times higher than previously reported) in chloroplasts and mitochondria of the green alga Scenedesmus obliquus. In particular, we report that reduced m-dcps (biodegradation intermediates) mimic endogenous electron and proton carriers in chloroplasts and mitochondria, inhibit Photosystem II (PSII) activity (and therefore O2 production) and enhance Photosystem I (PSI) and hydrogenase activity. In addition, we show that there are some indications for hydrogen production from sources other than chloroplasts in Scenedesmus obliquus. The regulation of these multistage and highly evolved redox pathways leads to high yields of hydrogen production and paves the way for an efficient application to industrial scale use, utilizing simple energy sources and one meta-substituted dichlorophenol as regulating elements.

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.

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.

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.

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.

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 Ca2+ 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 PSIIβ 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.

Chemical Bonding of Chlorophylls and Plant Aminic Axial Ligands Impact Harvesting of Visible Light and Quenching of Fluorescence

In the present work, we tested the mode of interaction of all three polyamines (putrescine, spermidine and spermine) with chlorophyll a and b, as well as pheophytin a and b. The results showed that all three polyamines bind to the Mg ion of chlorophyll ring as probed by Raman spectroscopy. The coordination of spermine with Chl b has the most interesting features from all pigments tested. Spermine induces reversible increases and decreases of the fluorescence yield of Chl b at about 661 nm. Interestingly, equilibrium between a high‐fluorescence yield conformation and a low yield is feasible by the interaction of chlorophyll b and aminic ligands. Furthermore, absorption data for the diagnostic regions of 518 and 535 nm are provided for all combinations of pigments and ligands. The significance and consistence of these results with respect to photochemical and bioenergetic principles are discussed.