Do polyamines alter the sensitivity of lichens to nitrogen stress?

The sensitivity of lichens measuring photosynthetic efficiency and polyamines as modulator of nitrogen stress tolerance was investigated. Two lichen species with a markedly different tolerance to nitrogen compounds, namely Evernia prunastri (L.) Ach. and Xanthoria parietina (L.) Th.Fr., were incubated with deionized water (control) and solutions of KNO(3), NH(4)NO(3) and (NH(4))(2)SO(4) and then exposed to different light conditions. The F(v)/F(m) parameter (maximum quantum efficiency of photosystem II) was used as stress indicator. The results showed that F(v)/F(m) values, in the produced experimental conditions, were independent from the light gradient. Photosynthetic efficiency of E. prunastri was impaired by high ammonium concentrations, while nitrate had no effect; X. parietina was hardly influenced by nitrogen compounds. External supply of polyamines reduced the sensitivity of E. prunastri, while polyamine inhibitors reduced the tolerance of X. parietina to NH(4)(+), suggesting that polyamines play an important role in modulating the sensitivity/tolerance to nitrogen stress.

Characterization of spermidine and spermine synthases in Lotus japonicus: induction and spatial organization of polyamine biosynthesis in nitrogen fixing nodules.

The biosynthesis of the polyamines spermidine (Spd) and spermine (Spm) from putrescine (Put) is catalysed by the consequent action of two aminopropyltransferases, spermidine synthase (SPDS EC: 2.5.1.16) and spermine synthase (SPMS EC: 2.5.1.22). Two cDNA clones coding for SPDS and SPMS homologues in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the encoded polypeptides was confirmed by their ability to complement spermidine and spermine deficiencies in yeast. The temporal and spatial expression pattern of the respective genes was correlated with the accumulation of total polyamines in symbiotic and non-symbiotic organs. Expression of both genes was maximal at early stages of nodule development, while at later stages the levels of both transcripts declined. Both genes were expressed in nodule inner cortical cells, vascular bundles, and central tissue. In contrast to gene expression, increasing amounts of Put, Spd, and Spm were found to accumulate during nodule development and after maturity. Interestingly, nodulated plants exhibited systemic changes in both LjSPDS and LjSPMS transcript levels and polyamine content in roots, stem and leaves, in comparison to uninoculated plants. These results give new insights into the neglected role of polyamines during nodule development and symbiotic nitrogen fixation (SNF).

Influence of the developmental stage on the (proto)-hypericin and (proto)pseudohypericin levels of Hypericum plants from Crete.

The contents of (pseudo)hypericin and their immediate precursors were studied in wild populations of various Hypericum species on the island of Crete, Greece. Therefore, the aerial parts of wild grown H. perforatum, H. triquentrifolium, H. empetrifolium and H. perfoliatum shoots were collected throughout the island and the quantitative variations in (proto)hypericin and (proto)pseudohypericin examined. The plant material was harvested at different stages of the life cycle of the species and the contents in the above-mentioned compounds analyzed discriminating between flowers/fruits and leaves/petioles. HPLC analysis of hypericin, pseudohypericin and their immediate precursors, protohypericin and protopseudohypericin, revealed great differences in the contents of the compounds in dependence on the developmental stage of the plants. In all examined species the highest concentrations of hypericin were found during blossoming whereas the lowest concentrations were present during ripening of the fruits. H. perforatum and H. triquentrifolium show much higher hypericin levels in flowers/fruits compared to leaves/petioles, whereas the species H. empetrifolium and H. perfoliatum show similar concentrations of total hypericins in both flowers/fruits and leaves/petioles. In the different species the levels of (proto)hypericin and (proto)pseudohypericin varied, but in almost all samples from flowers/fruits and leaves/petioles the ratio of (proto)hypericin to (proto)pseudohypericin was higher than one. When the total amount of hypericins per entire aerial part of a plant was calculated for all developmental stages, we found that H. perforatum contained the highest amount of hypericin. This in combination with the comparatively high concentration of hypericins in flowers/fruits and in leaves/petioles in this species, as well as the high ratio of (proto)hypericin to (proto)pseudohypericin, especially during the developmental stage of blossoming, encourages us to think about the possibility of cultivating Hypericum perforatum in Crete as a medicinal plant in the future.

Changes in the LHCII-mediated energy utilization and dissipation adjust the methanol-induced biomass increase.

Considerably low methanol concentrations of 0.5% (v/v), induce an immense increase in biomass production in cultures of the unicellular green alga Scenedesmus obliquus compared to controls without additional methanol. The effect is light-regulated and it mimics high-CO2 induced changes of the molecular structure and function of the photosynthetic apparatus. There is evidence that methanol enhances under high light conditions by molecular changes in the LHCII--a decrease of the functional antenna-size per active reaction center--the photochemical effectiveness of the absorbed energy. This means that the non-photochemical quenching (NPQ) is minimized and thereby the overall dissipation energy. Experiments with mutants of Scenedesmus Wt produced evidence that the LHCII is the locus of the mechanism which regulates the methanol effect. The employed mutants were Wt-LHC, lacking a functioning LHCII, the light-dependent greening mutant C-2A', and the double mutant C-2A'-LHC, combining both mutations.

Simulated solar irradiation with enhanced UV-B adjust plastid- and thylakoid-associated polyamine changes for UV-B protection.

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.

A cell-based model for the photoacclimation and CO(2)-acclimation of the photosynthetic apparatus.

We have developed a mathematical model based on the underlying mechanisms concerning the responses of the photosynthetic apparatus of a microalga cell which grows under constant incident light intensity and ambient CO(2) concentration. Photosynthesis involves light and carbon-fixation reactions which are mutually dependent and affect each other, but existing models for photosynthesis don't account for both reactions at once. Our modeling approach allows us to derive distinct equations for the rates of oxygen production, NADPH production, carbon dioxide fixation, carbohydrate production, and rejected energy, which are generally different. The production rates of the photosynthesis products are hyperbolic functions of light and CO(2) concentration. The model predicts that in the absence of photoinhibition, CO(2)-inhibition, photorespiration, and chlororespiration, a cell acclimated to high light and/or CO(2) concentration has higher photosynthetic capacity and lower photosynthetic efficiency than does a cell acclimated to low conditions. This results in crossing between the two curves which represent the oxygen production rates and carbon fixation rates in low and high conditions. Finally, in the absence of photoinhibition and CO(2)-inhibition, the model predicts the carbohydrate production rate in terms of both light intensity and CO(2) concentration.

Ozone impact on the photosynthetic apparatus and the protective role of polyamines.

One of the primary plant mechanisms protecting leaf cells against enhanced atmospheric ozone is the accumulation of polyamines, generally observed as an increase in putrescine level, and in particular its bound form to thylakoid membranes. Ozone-sensitive plants of tobacco (cultivar Bel W3) in contrast to ozone-tolerant Bel B, are not able to increase their endogenous thylakoid membrane-bound putrescine when they are exposed to an atmosphere with enhanced ozone concentration, resulting in reduction of their photosynthetic rates and consequently reduction in plant biomass formation. In comparison to the tolerant cultivar Bel B, a prolongation of ozone exposure thus can lead to typical visible symptoms (necrotic spots) in leaves of the sensitive plant. Exogenously manipulated increase of the cellular putrescine levels of the ozone-sensitive Bel W3 is sufficient to revert these effects, whereas a reduction in endogenous putrescine levels of the tolerant cultivar Bel B renders them sensitive to ozone treatment. The results of this work reveal a regulator role for polyamines in adaptation of the photosynthetic apparatus and consequently to its protection in an environment polluted by ozone.

Light-dependent induction of strongly increased microalgal growth by methanol.

Low methanol concentrations (about 0.5% v/v) induce biomass production in cultures of the unicellular green alga Scenedesmus obliquus by more than 300%, compared to controls without this solvent. This effect on the microalgal growth was found to be dependent on the solvent concentration, the packed cell volume (PCV), light intensity and light quality. It could be shown that methanol addition leads to a decrease in size of the light harvesting complex (LHC) on the basis of chlorophylls and proteins, and thus to changes in structure and functioning of the photosynthetic apparatus. These alterations lead to enhanced photosynthesis and respiration rates. The action of methanol on the photosynthetic apparatus is thus comparable to the effect of enhanced CO(2) concentrations. These findings support the previously proposed pathway for methanol metabolization with CO(2) as the final product. We conclude that the subsequent assimilation of the increased CO(2) amounts by the Calvin-Benson cycle is a possible explanation for the methanol-mediated increase in biomass production in terms of PCV. The methanol effect is observed only in the light and in the presence of a functioning photosynthetic apparatus. Preliminary action spectra suggest that the primary photoreceptor is a chlorophyll-protein complex with two absorption maxima at 680 and 430 nm, which may possibly be attributed to the reaction center of photosystem II (PSII).

Blue light signaling chains in Phycomyces: phototransduction of carotenogenesis and morphogenesis involves distinct protein kinase/phosphatase elements.

Carotenogenesis and morphogenesis represent two of the several responses sensitive to blue light which characterize the lower eukaryote Phycomyces blakesleeanus. Speculating that reversible phosphorylation may be an intracellular event beyond the photoperception step, we resorted to the use of first-choice inhibitors of protein phosphatases and protein kinases. The mycelial beta-carotene content of dark-grown cultures was induced by all agents administered, while the morphogenic output showed the typical trend effected by light only with one of the protein kinase inhibitors. Our data provide convincing evidence that protein phosphorylation plays a regulatory role in photocarotenogenesis and photomorphogenesis of Phycomyces. According to the model we propose, the putative signaling elements involved are anticipated to have a repressive function in the dark so that the responses are maintained in the "off" mode until the moment photon information has to flow through the regulatory circuit.

Free, conjugated and bound polyamines during the cell cycle in synchronized cultures of Scenedesmus obliquus.

The levels of free, conjugated and bound polyamines (PA) were analyzed during the cell cycle of the synchronized unicellular green alga Scenedesmus obliquus. The polyamines putrescine (PUT) and spermidine (SPD) in their free and conjugated forms accumulated per cell to a maximum in the cell cycle at about the 16th hour after onset of illumination. The polyamines bound to macromolecules and membrane systems showed an additional peak around the 8-10th hour of the cell cycle. The possible role of the different forms of polyamines in DNA replication, mitosis, cell division and development of the photosynthetic apparatus is discussed.

A narrow-bore HPLC method for the identification and quantitation of free, conjugated, and bound polyamines.

A modified sensitive high-pressure liquid chromatography (HPLC) method, using a narrow-bore column (2.1 x 200 mm, C-18, 5-microns particle size) with a methanol:water gradient (55-84%, v/v), is described for direct analysis of benzoylated free (S), soluble-conjugated (SH), and insoluble-bound (PH) polyamines in plant tissues. Regression curves for each fraction of polyamines allow a precise quantitative determination of putrescine, cadaverine, spermidine, spermine, and agmatine in all fractions (S, SH, PH) of a crude extract. A comparison of the results to the corresponding values for dansylated polyamines separated by thin-layer chromatography and quantitated by fluorometry is also presented. S, SH, and PH polyamines as benzoylated derivatives from leaves of Vitis vinifera L. cv Dogridge were characterized and quantitatively determined with this improved sensitive HPLC method.

Polyamines in the photosynthetic apparatus : Photosystem II highly resolved subcomplexes are enriched in spermine.

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.