Áron Keresztes

Effects of nickel on the chloroplasts of the duckweeds Spirodela polyrhiza and Lemna minor and their possible use in biomonitoring and phytoremediation.

Toxic effects of Ni(2+) on the chloroplasts of the two duckweed species Spirodela polyrhiza, clone SJ and Lemna minor, clone St were investigated according to the ISO 20079 protocol. Ni(2+) induced a transition from chloroplasts to chloro-amyloplasts and amylo-chloroplasts, but not to gerontoplasts, as shown by electron microscopy. The contents of the chlorophylls a and b decreased strongly, whereas that of carotenoids remained approximately constant. Most striking was, however, the accumulation of transitory starch. Bell-shaped dose-response curves showed that Spirodela and Lemna amassed maximum starch contents of approximately 10% and 7%, respectively, on a fresh weight basis. Because Ni(2+) in the concentrations applied does not stimulate photosynthesis, the Ni(2+)-induced starch accumulation indicates that the export of carbohydrates out of the plastids decreased, most probably due to the lower demand of the rest of the cells as a result of the Ni(2+)-dependent inhibition of growth. The half-maximal concentrations for inhibition of the fresh weight increase over the 7-day test period were 3.7 microM and 6.6 microM for Spirodela and Lemna, respectively: Spirodela was thus somewhat more sensitive to the heavy metal. Both species accumulated approximately 3g of Ni(2+) per kg of dry weight after application of 100 microM NiCl(2). Because of their high sensitivity to phytotoxic effects, however, Spirodela and Lemna do not appear to be particularly suitable for phytoremediation of Ni(2+)-contaminated waste water. The high sensitivity to Ni(2+) makes them instead a suitable system for ecotoxicological testing in accordance with the ISO 20079 protocol.

Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings

The effect of some more or less harmful compounds like Cd, Pb, Ni, Ti salts and DCMU at low concentrations on the development of chloroplasts in maize and bean seedlings was investigated. Chlorophyll content, chlorophyll a/b ratio, photosynthetic activity (14CO2 fixation), chlorophyll-protein composition of thylakoid membranes, fluorescence spectra of chloroplasts, fluorescence induction parameters of leaves and electron microscopic structure of maize and bean chloroplasts as well as growth parameters were studied. Stimulation of chlorophyll synthesis and photosynthetic activity was observed at different intervals during all of the treatments, while chlorophyll a/b ratios and fluorescence properties of leaves or chloroplasts did not change considerably except in DCMU treated plants. Heavy metal treatments increased the amount of photosystem I and light-harvesting complex II, while decreased amount of photosystem I and higher amount of light-harvesting complex II was found in DCMU treated thylakoids. Electron microscopy showed only sligth differences in the morphology of chloroplast lamellar system (mostly in DCMU treated plants), while the status of the plasmalemma and tonoplast seemed to be altered as a result of certain metal treatments. Results showed the expression of a cytokinin-like effect on the development of chloroplasts. It is assumed, that these low-dose stressors generate non-specific alarm reactions in plants, which may involve changes of the hormonal balance.

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.

Investigation into the mechanism of stimulation by low-concentration stressors in barley seedlings

Beneficial effects of low-concentration chemical stressors have been investigated previously in different model systems. The symptoms of stimulation are known from earlier studies, but information about the mechanism is at an initial stage. In the present work, the mechanism of stimulation of low-concentration Cd (5 x 10(-8)M) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 10(-7)M) was investigated in barley seedlings. In treated plants, the amount of cytokinins increased in roots and, after being transported to the leaves, they caused stimulation there. To identify the signal transduction pathway(s) involved in the primary stimulation of cytokinin synthesis (and/or activation) in roots, specific phosphatidylinositol-4,5-bisphosphate-inositol-1,4,5-triphosphate/diacylglycerol (PIP(2)-IP(3)/DAG) and mitogen activated protein kinase (MAPK) signaling pathway inhibitors were added to the nutrient solution, and all proved to be effective, eliminating the stimulation by the stressors. Measurements of superoxide dismutase (SOD, EC 1.15.1.1) activity and the amount of malonyldialdehyde (MDA) showed that the increased amount of Cd did not cause oxidative stress in the roots, and no oxidative stress was found in the leaves, where Cd did not even accumulate. DCMU slightly increased the activity of SOD after 1 week in roots, but did not cause lipid peroxidation. In leaves, there was no oxidative stress upon treatment with DCMU. Thus, oxidative stress cannot be responsible for the stimulation with low-concentration stressors, as they changed the activity of SOD differently, while being equally stimulative for the plants.

Phytotoxicity of cobalt ions on the duckweed Lemna minor – Morphology, ion uptake, and starch accumulation

Cobalt (Co2+) inhibits vegetative growth of Lemna minor gradually from 1 μM to 100 μM. Fronds accumulated up to 21 mg Co2+ g(-1) dry weight at 10 μM external Co2+ indicating hyperaccumulation. Interestingly, accumulation of Co2+ did not decrease the iron (Fe) content in fronds, highlighting L. minor as a suitable system for studying effects of Co2+ undisturbed by Fe deficiency symptoms unlike most other plants. Digital image analysis revealed the size distribution of fronds after Co2+ treatment and also a reduction in pigmentation of newly formed daughter fronds unlike the mother fronds during the 7-day treatment. Neither chlorophyll nor photosystem II fluorescence changed significantly during the initial 4d, indicating effective photosynthesis. During the later phase of the 7-day treatment, however, chlorophyll content and photosynthetic efficiency decreased in the Co2+-treated daughter fronds, indicating that Co2+ inhibits the biosynthesis of chlorophyll rather than leading to the destruction of pre-existing pigment molecules. In addition, during the first 4d of Co2+ treatment starch accumulated in the fronds and led to the transition of chloroplasts to chloro-amyloplasts and amylo-chloroplasts, while starch levels strongly decreased thereafter.

Plastid structure, diversification and interconversions II. land plants

In the course of endosymbiogenesis, the photosynthetic prokaryotes engulfed and retained by different het- erotrophic organisms have partially lost their independence during evolution and became semi-autonomous organelles, the chloroplasts. The chloroplast represents the most ancestral form of plastids that has parallelly evolved in several algal groups (reviewed in (1)) and in land plants. After briefly discussing plastid morphology, we review the most important ul- trastructural features of the plastids of land plants. Then we discuss how plastids got gradually specialized in parallel with the increasing developmental and/or organizational complexity of the plant body. The plastids of non-photosynthetic tis- sues and cells do not need to produce and maintain a photosynthetic apparatus, but have adjusted their metabolism to the major function of the host cell (and tissue). This way, different plastid forms specialized for other functions such as stor- age (e.g. starch storing leucoplasts called amyloplast) or carotenoid synthesis (chromoplasts) have developed. However, the classical ultrastructural characterization and classification of plastids is often problematic. First of all, the term plastid refers to the extremely high plasticity of this organelle, and its capacity to be readily transformed from one type into an- other one upon different environmental or developmental stimuli. Therefore, transitional (or if persistent, intermediate) plastids with morphological features characteristic for two different plastid types can be often observed. Sometimes plas- tids with similar ultrastructure can have different specific functions and basically different metabolism, and should be therefore treated separately. After having recalled the different plastid types of land plants we present a dynamic model about their interconversions.

Light-Induced Degradation of Starch Granules in Turions of Spirodela polyrhiza Studied by Electron Microscopy

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.

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.

Mycorrhiza Symbiosis Increases the Surface for Sunlight Capture in Medicago truncatula for Better Photosynthetic Production

Arbuscular mycorrhizal (AM) fungi play a prominent role in plant nutrition by supplying mineral nutrients, particularly inorganic phosphate (Pi), and also constitute an important carbon sink. AM stimulates plant growth and development, but the underlying mechanisms are not well understood. In this study, Medicago truncatula plants were grown with Rhizophagus irregularis BEG141 inoculum (AM), mock inoculum (control) or with Pi fertilization. We hypothesized that AM stimulates plant growth through either modifications of leaf anatomy or photosynthetic activity per leaf area. We investigated whether these effects are shared with Pi fertilization, and also assessed the relationship between levels of AM colonization and these effects. We found that increased Pi supply by either mycorrhization or fertilization led to improved shoot growth associated with increased nitrogen uptake and carbon assimilation. Both mycorrhized and Pi-fertilized plants had more and longer branches with larger and thicker leaves than the control plants, resulting in an increased photosynthetically active area. AM-specific effects were earlier appearance of the first growth axes and increased number of chloroplasts per cell section, since they were not induced by Pi fertilization. Photosynthetic activity per leaf area remained the same regardless of type of treatment. In conclusion, the increase in growth of mycorrhized and Pi-fertilized Medicago truncatula plants is linked to an increase in the surface for sunlight capture, hence increasing their photosynthetic production, rather than to an increase in the photosynthetic activity per leaf area.

On the mechanism of rejuvenation of ageing detached bean leaves by low-concentration stressors.

The effect of low concentrations of some stress-inducing compounds of different toxicity and chemical nature, such as Cd and Pb salts or DCMU, was investigated on the senescence of chloroplasts in detached primary leaves of bean (Phaseolus vulgaris L.). After 1 week of senescence followed by root development from the petiole, these agents stimulated chlorophyll accumulation and photosynthetic activity ((14)CO(2) fixation) as compared to the control, thus inducing rejuvenation. Low-concentration stressors increased the level of active cytokinins in roots and leaves during the treatment, as monitored by the Amaranthus betacyanin bioassay and high-pressure liquid chromatography. The lithium ion, an inhibitor of the PIP(2)-IP(3)/DAG signal transduction pathway, abolished the stimulating effect of stressors, both in roots (retarding cytokinin synthesis) and consequently also in leaves (reducing cytokinin-dependent chlorophyll accumulation). This suggests the involvement of the PIP(2)-IP(3)/DAG signal transduction pathway in generation of these consecutive organ-specific responses.