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.

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.

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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.

Polyamines have been described to protect against numerous oxidative stresses in plants. Increasing UV-B radiation (280-315 nm) in the biosphere may also induce an increase in radical formation in tissues. This study employed the tobacco cultivars Bel B and Bel W3 to describe possible protective functions of polyamines against UV-B radiation in sun light simulators (GSF/Munich) with natural diurnal fluctuations of simulated UV-B. Polyamine measurements on a whole leaf basis in isolated chloroplasts and thylakoids were paralleled to photosynthetic and respiration rates, photosynthetic efficiency, leaf thickness and photosynthetic pigment compositions. The study revealed that an increase of polyamines, and especially of putrescine level in thylakoid membranes upon elevated UV-B exposure comprises one of the primary protective mechanisms in the photosynthetic apparatus of the tobacco variety Bel B against UV-B radiation. The tobacco cultivar Bel W3, sensitive to ozone, was also proved to be sensitive to UV-B. This sensitivity is attributed to its incapability to enhance putrescine level in thylakoid membranes. After prolongation of UV-B exposure, when endogenous plant balances are being gradually restored, due to secondary responses, (e.g., biosynthesis of carotenoids and of additional flavonoids) and the plant is adapting to the altered environmental conditions, then the polyamine level is being reduced. Thus, we can discriminate the UV-B induced stress period from a UV-B acclimation period.

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 qE mechanism, and governing electron transfer at the cytochrome b6f 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.

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.

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