Etioplasts are more susceptible to salinity stress than chloroplasts and photosynthetically active etio-chloroplasts of wheat (Triticum aestivum L.).

High soil salinity is a global problem in agriculture that directly affects seed germination and the development of the seedlings sown deep in the soil. To study how salinity affected plastid ultrastructure, leaf segments of 11-day-old light- and dark-grown (etiolated) wheat (Triticum aestivum L. cv. Mv Béres) seedlings were floated on Hoagland solution, 600 mM KCl:NaCl (1:1) salt or isosmotic polyethylene glycol solution for 4 h in the dark. Light-grown seedlings were also treated in the light. The same treatments were also performed on etio-chloroplasts of etiolated seedlings greened for different time periods. Salt stress induced slight to strong changes in the relative chlorophyll content, photosynthetic activity, and organization of thylakoid complexes. Measurements of malondialdehyde contents and high-temperature thermoluminescence indicated significantly increased oxidative stress and lipid peroxidation under salt treatment, except for light-grown leaves treated in the dark. In chloroplasts of leaf segments treated in the light, slight shrinkage of grana (determined by transmission electron microscopy and small-angle neutron scattering) was observed, while a swelling of the (pro)thylakoid lumen was observed in etioplasts. Salt-induced swelling disappeared after the onset of photosynthesis after 4 h of greening. Osmotic stress caused no significant alterations in plastid structure and only mild changes in their activities, indicating that the swelling of the (pro)thylakoid lumen and the physiological effects of salinity are rather associated with the ionic component of salt stress. Our data indicate that etioplasts of dark-germinated wheat seedlings are the most sensitive to salt stress, especially at the early stages of their greening.

Ionic, not the osmotic component, is responsible for the salinity-induced inhibition of greening in etiolated wheat (Triticum aestivum L. cv. Mv Béres) leaves: a comparative study.

MAIN CONCLUSION: Greening was partially (in 300 mM NaCl, CaCl2, 600 mM KNO3 or KCl) or fully inhibited (in 600 mM NaCl, NaNO3 or NaCl:KCl) by the ionic and not the osmotic component of salinity. Although high soil salinity is an increasing global problem, not much is known about how direct exposure to salinity affects etiolated leaves of seedlings germinating in the soil and then reaching the surface. We investigated the effect of various salt treatments on the greening process of leaves in 8- to 11-day-old etiolated wheat (Triticum aestivum L. Mv. Béres) seedlings. Etiolated leaf segments pre-treated on different salt (600 mM NaCl:KCl 1:1, 600 mM NaCl, 600 mM KCl, 600 mM NaNO3, 600 mM KNO3, 300 mM KCl, 300 mM NaCl or 300 mM CaCl2) or isosmotic polyethylene glycol 6000 (PEG) solutions for 1.5 h in the dark and then greened for 16 h on the same solutions were studied. Leaf segments greened on PEG (osmotic stress) or on 300 mM KCl had similar chloroplasts compared to control samples greened on Hoagland solution. Slightly slower development of chloroplast structure and function (photosynthetic activity) was observed in segments greened on 300 mM NaCl or CaCl2, 600 mM KNO3 or KCl. However, etioplast-to-chloroplast transformation and chlorophyll accumulation were fully inhibited and peculiar prothylakoid swelling occurred in segments greened on 600 mM NaCl, NaNO3 or NaCl:KCl (1:1) solutions. The data indicate that not the high osmolarity of the used salt solution, but its ions, especially Na+, had the strongest negative impact on these processes.

Iron uptake of etioplasts is independent from photosynthesis but applies the reduction-based strategy.

Introduction: Iron (Fe) is one of themost important cofactors in the photosynthetic apparatus, and its uptake by chloroplasts has also been associated with the operation of the photosynthetic electron transport chain during reduction-based plastidial Fe uptake. Therefore, plastidial Fe uptake was considered not to be operational in the absence of the photosynthetic activity. Nevertheless, Fe is also required for enzymatic functions unrelated to photosynthesis, highlighting the importance of Fe acquisition by non-photosynthetic plastids. Yet, it remains unclear how these plastids acquire Fe in the absence of photosynthetic function. Furthermore, plastids of etiolated tissues should already possess the ability to acquire Fe, since the biosynthesis of thylakoid membrane complexes requires a massive amount of readily available Fe. Thus, we aimed to investigate whether the reduction-based plastidial Fe uptake solely relies on the functioning photosynthetic apparatus. Methods: In our combined structure, iron content and transcript amount analysis studies, we used Savoy cabbage plant as a model, which develops natural etiolation in the inner leaves of the heads due to the shading of the outer leaf layers. Results: Foliar and plastidial Fe content of Savoy cabbage leaves decreased towards the inner leaf layers. The leaves of the innermost leaf layers proved to be etiolated, containing etioplasts that lacked the photosynthetic machinery and thus were photosynthetically inactive. However, we discovered that these etioplasts contained, and were able to take up, Fe. Although the relative transcript abundance of genes associated with plastidial Fe uptake and homeostasis decreased towards the inner leaf layers, both ferric chelate reductase FRO7 transcripts and activity were detected in the innermost leaf layer. Additionally, a significant NADP(H) pool and NAD(P)H dehydrogenase activity was detected in the etioplasts of the innermost leaf layer, indicating the presence of the reducing capacity that likely supports the reduction-based Fe uptake of etioplasts. Discussion: Based on these findings, the reduction-based plastidial Fe acquisition should not be considered exclusively dependent on the photosynthetic functions.

Protein Changes in Shade and Sun Haberlea rhodopensis Leaves during Dehydration at Optimal and Low Temperatures.

Haberlea rhodopensis is a unique resurrection plant of high phenotypic plasticity, colonizing both shady habitats and sun-exposed rock clefts. H. rhodopensis also survives freezing winter temperatures in temperate climates. Although survival in conditions of desiccation and survival in conditions of frost share high morphological and physiological similarities, proteomic changes lying behind these mechanisms are hardly studied. Thus, we aimed to reveal ecotype-level and temperature-dependent variations in the protective mechanisms by applying both targeted and untargeted proteomic approaches. Drought-induced desiccation enhanced superoxide dismutase (SOD) activity, but FeSOD and Cu/ZnSOD-III were significantly better triggered in sun plants. Desiccation resulted in the accumulation of enzymes involved in carbohydrate/phenylpropanoid metabolism (enolase, triosephosphate isomerase, UDP-D-apiose/UDP-D-xylose synthase 2, 81E8-like cytochrome P450 monooxygenase) and protective proteins such as vicinal oxygen chelate metalloenzyme superfamily and early light-induced proteins, dehydrins, and small heat shock proteins, the latter two typically being found in the latest phases of dehydration and being more pronounced in sun plants. Although low temperature and drought stress-induced desiccation trigger similar responses, the natural variation of these responses in shade and sun plants calls for attention to the pre-conditioning/priming effects that have high importance both in the desiccation responses and successful stress recovery.

Reactivation of the Photosynthetic Apparatus of Resurrection Plant Haberlea rhodopensis during the Early Phase of Recovery from Drought- and Freezing-Induced Desiccation.

Haberlea rhodopensis is a unique desiccation-tolerant angiosperm that also survives winter frost. As, upon freezing temperatures, H. rhodopensis desiccates, the taxon is proposed to survive low temperature stress using its desiccation tolerance mechanisms. To reveal the validity of this hypothesis, we analyzed the structural alterations and organization of photosynthetic apparatus during the first hours of recovery after drought- and freezing-induced desiccation. The dynamics of the ultrastructure remodeling in the mesophyll cells and the restoration of the thylakoid membranes shared similarities independent of the reason for desiccation. Among the most obvious changes in thylakoid complexes, the proportion of the PSI-LHCII complex strongly increased around 70% relative water content (RWC), whereas the proportion of Lhc monomers decreased from the beginning of rehydration. We identified enhanced levels of cyt b6f complex proteins that contributed to the enhanced electron flow. The high abundance of proteins related to excitation energy dissipation, PsbS, Lhcb5, Lhcb6 and ELIPs, together with the increased content of dehydrins contributed to the preservation of cellular integrity. ELIP expression was maintained at high levels up to 9 h into recovery. Although the recovery processes from drought- and freezing-induced desiccation were found to be similar in progress and time scale, slight variations indicate that they are not identical.

Qualitative and quantitative evaluation of thylakoid complexes separated by Blue Native PAGE.

BACKGROUND: Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging. RESULTS: Using a detergent mixture of 1% (w/V) n-dodecyl-ß-D-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3-8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed. CONCLUSIONS: Here we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes.

The developmental and iron nutritional pattern of PIC1 and NiCo does not support their interdependent and exclusive collaboration in chloroplast iron transport in Brassica napus.

MAIN CONCLUSION: The accumulation of NiCo following the termination of the accumulation of iron in chloroplast suggests that NiCo is not solely involved in iron uptake processes of chloroplasts. Chloroplast iron (Fe) uptake is thought to be operated by a complex containing permease in chloroplast 1 (PIC1) and nickel-cobalt transporter (NiCo) proteins, whereas the role of other Fe homeostasis-related transporters such as multiple antibiotic resistance protein 1 (MAR1) is less characterized. Although pieces of information exist on the regulation of chloroplast Fe uptake, including the effect of plant Fe homeostasis, the whole system has not been revealed in detail yet. Thus, we aimed to follow leaf development-scale changes in the chloroplast Fe uptake components PIC1, NiCo and MAR1 under deficient, optimal and supraoptimal Fe nutrition using Brassica napus as model. Fe deficiency decreased both the photosynthetic activity and the Fe content of plastids. Supraoptimal Fe nutrition caused neither Fe accumulation in chloroplasts nor any toxic effects, thus only fully saturated the need for Fe in the leaves. In parallel with the increasing Fe supply of plants and ageing of the leaves, the expression of BnPIC1 was tendentiously repressed. Though transcript and protein amount of BnNiCo tendentiously increased during leaf development, it was even markedly upregulated in ageing leaves. The relative transcript amount of BnMAR1 increased mainly in ageing leaves facing Fe deficiency. Taken together chloroplast physiology, Fe content and transcript amount data, the exclusive participation of NiCo in the chloroplast Fe uptake is not supported. Saturation of the Fe requirement of chloroplasts seems to be linked to the delay of decomposing the photosynthetic apparatus and keeping chloroplast Fe homeostasis in a rather constant status together with a supressed Fe uptake machinery.

Incorporation of iron into chloroplasts triggers the restoration of cadmium induced inhibition of photosynthesis.

Photosynthetic symptoms of acute Cd stress can be remedied by elevated Fe supply. To shed more light on the most important aspects of this recovery, the detailed Fe trafficking and accumulation processes as well as the changes in the status of the photosynthetic apparatus were investigated in recovering poplar plants. The Cd-free, Fe-enriched nutrient solution induced an immediate intensive Fe uptake. The increased Fe/Cd ratio in the roots initiated the translocation of Fe to the leaf with a short delay that ultimately led to the accumulation of Fe in the chloroplasts. The chloroplast Fe uptake was directly proportional to the Fe translocation to leaves. The accumulation of PSI reaction centers and the recovery of PSII function studied by Blue-Native PAGE and chlorophyll a fluorescence induction measurements, respectively, began in parallel to the increase in the Fe content of chloroplasts. The initial reorganization of PSII was accompanied by a peak in the antennae-based non-photochemical quenching. In conclusion, Fe accumulation of the chloroplasts is a process of prime importance in the recovery of photosynthesis from acute Cd stress.

Stress hardening under long-term cadmium treatment is correlated with the activation of antioxidative defence and iron acquisition of chloroplasts in Populus.

Cadmium (Cd), a highly toxic heavy metal affects growth and metabolic pathways in plants, including photosynthesis. Though Cd is a transition metal with no redox capacity, it generates reactive oxygen species (ROS) indirectly and causes oxidative stress. Nevertheless, the mechanisms involved in long-term Cd tolerance of poplar, candidate for Cd phytoremediation, are not well known. Hydroponically cultured poplar (Populus jacquemontiana var. glauca cv. 'Kopeczkii') plants were treated with 10 µM Cd for 4 weeks. Following a period of functional decline, the plants performed acclimation to the Cd induced oxidative stress as indicated by the decreased leaf malondialdehyde (MDA) content and the recovery of most photosynthetic parameters. The increased activity of peroxidases (PODs) could have a great impact on the elimination of hydrogen peroxide, and thus the recovery of photosynthesis, while the function of superoxide dismutase (SOD) isoforms seemed to be less important. Re-distribution of the iron content of leaf mesophyll cells into the chloroplasts contributed to the biosynthesis of the photosynthetic apparatus and some antioxidative enzymes. The delayed increase in photosynthetic activity in relation to the decline in the level of lipid peroxidation indicates that elimination of oxidative stress damage by acclimation mechanisms is required for the restoration of the photosynthetic apparatus during long-term Cd treatment.

Light piping driven photosynthesis in the soil: Low-light adapted active photosynthetic apparatus in the under-soil hypocotyl segments of bean (Phaseolus vulgaris).

Photosynthetic activity was identified in the under-soil hypocotyl part of 14-day-old soil-grown bean plants (Phaseolus vulgaris L. cv. Magnum) cultivated in pots under natural light-dark cycles. Electron microscopic, proteomic and fluorescence kinetic and imaging methods were used to study the photosynthetic apparatus and its activity. Under-soil shoots at 0-2cm soil depth featured chloroplasts with low grana and starch grains and with pigment-protein compositions similar to those of the above-soil green shoot parts. However, the relative amounts of photosystem II (PSII) supercomplexes were higher; in addition a PIP-type aquaporin protein was identified in the under-soil thylakoids. Chlorophyll-a fluorescence induction measurements showed that the above- and under-soil hypocotyl segments had similar photochemical yields at low (10-55µmolphotonsm(-2)s(-1)) light intensities. However, at higher photon flux densities the electron transport rate decreased in the under-soil shoot parts due to inactivation of the PSII reaction centers. These properties show the development of a low-light adapted photosynthetic apparatus driven by light piping of the above-soil shoot. The results of this paper demonstrate that the classic model assigning source and sink functions to above- and under-soil tissues is to be refined, and a low-light adapted photosynthetic apparatus in under-soil bean hypocotyls is capable of contributing to its own carbon supply.

Comparison of thylakoid structure and organization in sun and shade Haberlea rhodopensis populations under desiccation and rehydration.

The resurrection plant, Haberlea rhodopensis can survive nearly total desiccation only in its usual low irradiation environment. However, populations with similar capacity to recover were discovered recently in several sunny habitats. To reveal what kind of morphological, structural and thylakoid-level alterations play a role in the acclimation of this low-light adapted species to high-light environment and how do they contribute to the desiccation tolerance mechanisms, the structure of the photosynthetic apparatus, the most sensitive component of the chlorophyll-retaining resurrection plants, was analyzed by transmission electron microscopy, steady state low-temperature fluorescence and two-dimensional Blue-Native/SDS PAGE under desiccation and rehydration. In contrast to the great differences in the morphology of plants, the ultrastructure and the organization of thylakoids were surprisingly similar in well-hydrated shade and sun populations. A high ratio of photosystem (PS)I binding light harvesting complex (LHC)II, important in low- and fluctuating light environment, was characteristic to both shade and sun plant, and the ratios of the main chlorophyll-protein complexes were also similar. The intensive protective mechanisms, such as shading by steep leaf angle and accumulation of protective substances, probably reduced the light intensity at the chloroplast level. The significantly increased ratio of monomer to oligomer antennae in well-hydrated sun plants may be connected with the temporary high light exposure of chloroplasts. During desiccation, LHCII was removed from PSI and part of PSII supercomplexes disassembled with some loss of PSII core and LHCII. The different reorganization of antennae, possibly connected with different quenching mechanisms, involved an increased amount of monomers in shade plants but unchanged proportion of oligomers in sun plants. Desiccation-induced responses were more pronounced in sun plants which also had a greater capacity to recover due to their stress-acclimated attitude.

Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

Uptake and incorporation of iron in sugar beet chloroplasts.

Chloroplasts contain 80-90% of iron taken up by plant cells. Though some iron transport-related envelope proteins were identified recently, the mechanism of iron uptake into chloroplasts remained unresolved. To shed more light on the process of chloroplast iron uptake, trials were performed with isolated intact chloroplasts of sugar beet (Beta vulgaris). Iron uptake was followed by measuring the iron content of chloroplasts in the form of ferrous-bathophenantroline-disulphonate complex after solubilising the chloroplasts in reducing environment. Ferric citrate was preferred to ferrous citrate as substrate for chloroplasts. Strong dependency of ferric citrate uptake on photosynthetic electron transport activity suggests that ferric chelate reductase uses NADPH, and is localised in the inner envelope membrane. The K(m) for iron uptake from ferric-citrate pool was 14.65 ± 3.13 µM Fe((III))-citrate. The relatively fast incorporation of (57)Fe isotope into Fe-S clusters/heme, detected by Mössbauer spectroscopy, showed the efficiency of the biosynthetic machinery of these cofactors in isolated chloroplasts. The negative correlation between the chloroplast iron concentration and the rate of iron uptake refers to a strong feedback regulation of the uptake.

Cd, Fe, and light sensitivity: interrelationships in Cd-treated populus.

Cadmium is a toxic heavy metal causing iron deficiency in the shoot and light sensitivity of photosynthetic tissues that leads to decreased photosynthetic performance and biomass production. Light intensity had strong impact on both photosynthetic activity and metal accumulation of cadmium-treated plants. At elevated irradiation, cadmium accumulation increased due to the higher dry mass of plants, but its allocation hardly changed. A considerable amount of iron accumulated in the roots, and iron concentration was higher in leaves developed at moderate rather than low irradiation. At the same time, the higher the irradiation the lower the maximal photochemical quantum efficiency. The decreased photochemical efficiency, however, started to recover after a week of Cd treatment at moderate light without substantial change in metal concentrations but following the accumulation of green fluorescent compounds. Both cadmium treatment and higher light caused the accumulation of flavonoids in leaf mesophyll vacuoles/chloroplasts, but accumulation of flavonols, fluorescing at 510 nm, was characteristic to cadmium stress. Therefore, flavonoids, which may act by scavenging reactive radicals, chelating Cd, and shielding against excess irradiation, play an important part in Cd stress tolerance of Populus, and may have special impact on its phytoremediation capacity.

Impact of moderate Fe excess under Cd stress on the photosynthetic performance of poplar (Populus jacquemontiana var. glauca cv. Kopeczkii).

Cadmium interference with Fe nutrition has a strong impact on the development and efficiency of the photosynthetic apparatus. To shed more light on the interaction between Fe and Cd, it was studied how iron given in moderate excess under Cd stress affects the development and functioning of chlorophyll-protein complexes. Poplar plants grown in hydroponics up to four-leaf stage were treated with 10 µM Cd(NO3)2 in the presence of 50 µM Fe([III])-citrate as iron supply (5xFe + Cad) for two weeks. Though leaf area growth was inhibited similarly to that of Cad (10 µM Cd(NO3)2 + 10 µM Fe([III])-citrate) plants, chlorophyll content, ¹4CO2 fixation and quenching parameters calculated from PAM fluorescence induction measurements were control-like in 5xFe+Cad leaves. Increased chloroplast iron content (measured photometrically by the bathophenanthroline disulfonate method) without changes in the iron and cadmium content of leaves (determined by inductively coupled plasma mass spectrometry) pointed out that a key factor in the observed protection of photosynthesis is the iron-excess-induced redistribution of iron in the leaf. However, the chlorophyll a/b ratio and the chlorophyll-protein pattern obtained by Deriphat PAGE remained similar to that of Cad leaves. The decreased amount of PSII core and PSI in mature and developing leaves, respectively, refers to developmental stage-dependent remodelling of thylakoids in the presence of Cd. The results underline not only the beneficial effect of iron excess under Cd stress, but also refer to the importance of a proper Fe/Cd ratio and light environment to avoid its possible harmful effects.

Cd affects the translocation of some metals either Fe-like or Ca-like way in poplar.

In plants, Cd causes perturbation of root metal uptake and is known to interfere with the metal translocation to the shoot. The most significant effect is the strongly reduced transport of Fe. Fe accumulation in roots under Cd stress revealed that it is not the Fe acquisition but the Fe loading to xylem elements that is blocked by Cd, which can be a result of competition between Fe and Cd for the transporters. However, in animal cells as well as in plant stomata guard cells, Cd was shown to move through Ca channels. To clarify whether the perturbation of metal translocation/xylem loading caused by Cd show any regularity, translocation ability was tested by the determination of the metal content in leaves of hydroponically cultured (» Hoagland nutrient solution, Fe source: 10 µM Fe((III))-citrate) poplar plants grown for three weeks with or without 10 µM Cd(NO3)2 treatment. Metals could be classified into two groups according to the behavior of their translocation under Cd treatment: alkaline earth metals (except Mg), Zn and Mn were influenced similarly to Ca, but other transition metals (together with alkali metals and Al) behaved like the Fe. Based on the translocation pattern, Cd seems to inhibit the transport of Ca-like metals competitively, but a different type of inhibition is exerted on the transition metal transport, with which Cd can share a common translocation system. The strongly decreased translocation of chelator-dependent transition metals may indicate Cd related disturbances in signalling pathways and gene expression of xylem transporters or chelators.

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching.

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 micromol m(-2) s(-1) photon flux density (PFD) white light and subsequently incubated in total darkness for 4-24 h at 24 degrees C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form-dominating in epicotyls of dark-grown seedling-required 18-24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4-7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.

Protection of thylakoids against combined light and drought by a lumenal substance in the resurrection plant Haberlea rhodopensis.

BACKGROUND AND AIMS: Haberlea rhodopensis is a perennial, herbaceous, saxicolous, poikilohydric flowering plant that is able to survive desiccation to air-dried state under irradiance below 30 micromol m-2 s-1. However, desiccation at irradiance of 350 micromol m-2 s-1 induced irreversible changes in the photosynthetic apparatus, and mature leaves did not recover after rehydration. The aim here was to establish the causes and mechanisms of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance, and to elucidate the mechanisms determining recovery. METHODS: Changes in chloroplast structure, CO2 assimilation, chlorophyll fluorescence parameters, fluorescence imaging and the polypeptide patterns during desiccation of Haberlea under medium (100 micromol m-2 s-1; ML) irradiance were compared with those under low (30 micromol m-2 s-1; LL) irradiance. KEY RESULTS: Well-watered plants (control) at 100 micromol m-2 s-1 were not damaged. Plants desiccated at LL or ML had similar rates of water loss. Dehydration at ML decreased the quantum efficiency of photosystem II photochemistry, and particularly the CO2 assimilation rate, more rapidly than at LL. Dehydration induced accumulation of stress proteins in leaves under both LL and ML. Photosynthetic activity and polypeptide composition were completely restored in LL plants after 1 week of rehydration, but changes persisted under ML conditions. Electron microscopy of structural changes in the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense luminal substance, DLS), while the thylakoid membranes are lightly stained. Upon dehydration and rehydration the DLS thinned and disappeared, the time course largely depending on the illumination: whereas DLS persisted during desiccation and started to disappear during late recovery under LL, it disappeared from the onset of dehydration and later was completely lost under ML. CONCLUSIONS: Accumulation of DLS (possibly phenolics) in the thylakoid lumen is demonstrated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis during desiccation and recovery under LL. Disappearance of DLS during desiccation in ML could leave the thylakoid membranes without protection, allowing oxidative damage during dehydration and the initial rehydration, thus preventing recovery of photosynthesis.

Cd-Fe interactions: comparison of the effects of iron deficiency and cadmium on growth and photosynthetic performance in poplar.

To check the importance of Cd-induced iron deficiency in Cd stress, symptoms of Cd stress were compared with those of iron deficiency or the combination of these two stresses. Poplar plants grown in hydroponics with Fe-EDTA (e) or Fe-citrate (c) up to four-leaf stage were treated for two weeks either by the withdrawal of iron (Fedef), or supplying 10 µM Cd(NO3)2 in the presence (Cad) or absence of an iron source (Fedef + Cad). Cadmium and iron content of leaves developing under the stress was in the order of cCad > eCad > cFedef + Cad and cCad ≈ eFedef ≈ cFedef + Cad < eCad < cFedef, respectively. Growth inhibition was much stronger in Cad than Fedef plants. The inhibitory effects on CO2 fixation, maximal and actual efficiency of PSII, chlorophyll synthesis, as well as the stimulation of the accumulation of violaxanthin cycle components and increase in non-photochemical quenching were the strongest in cFedef+Cad plants, otherwise these parameters changed parallel to the iron deficiency of leaves. Tendency of changes in thylakoid composition were similar under Cad treatments and strong iron deficiency: particularly PSI and LHCII decreased. Therefore, the development of the photosynthetic apparatus under Cd stress was mainly influenced by the Cd-induced strong iron deficiency, while leaf growth was affected primarily by the presence of Cd.

Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation.

Real-time RT-PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin ß-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.