Ecophysiology, secondary pigments and ultrastructure of Chlainomonas sp. (Chlorophyta) from the European Alps compared with Chlamydomonas nivalis forming red snow.

Red snow is a well-known phenomenon caused by microalgae thriving in alpine and polar regions during the melting season. The ecology and biodiversity of these organisms, which are adapted to low temperatures, high irradiance and freeze-thaw events, are still poorly understood. We compared two different snow habitats containing two different green algal genera in the European Alps, namely algae blooming in seasonal rock-based snowfields (Chlamydomonas nivalis) and algae dominating waterlogged snow bedded over ice (Chlainomonassp.). Despite the morphological similarity of the red spores found at the snow surface, we found differences in intracellular organization investigated by light and transmission electron microscopy and in secondary pigments investigated by chromatographic analysis in combination with mass spectrometry. Spores ofChlainomonassp. show clear differences fromChlamydomonas nivalisin cell wall arrangement and plastid organization. Active photosynthesis at ambient temperatures indicates a high physiological activity, despite no cell division being present. Lipid bodies containing the carotenoid astaxanthin, which produces the red color, dominate cells of both species, but are modified differently. While inChlainomonassp. astaxanthin is mainly esterified with two fatty acids and is more apolar, inChamydomonas nivalis, in contrast, less apolar monoesters prevail.

Subcellular Sequestration and Impact of Heavy Metals on the Ultrastructure and Physiology of the Multicellular Freshwater Alga Desmidium swartzii.

Due to modern life with increasing traffic, industrial production and agricultural practices, high amounts of heavy metals enter ecosystems and pollute soil and water. As a result, metals can be accumulated in plants and particularly in algae inhabiting peat bogs of low pH and high air humidity. In the present study, we investigated the impact and intracellular targets of aluminum, copper, cadmium, chromium VI and zinc on the filamentous green alga Desmidium swartzii, which is an important biomass producer in acid peat bogs. By means of transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) it is shown that all metals examined are taken up into Desmidium readily, where they are sequestered in cell walls and/or intracellular compartments. They cause effects on cell ultrastructure to different degrees and additionally disturb photosynthetic activity and biomass production. Our study shows a clear correlation between toxicity of a metal and the ability of the algae to compartmentalize it intracellularly. Cadmium and chromium, which are not compartmentalized, exert the most toxic effects. In addition, this study shows that the filamentous alga Desmidium reacts more sensitively to aluminum and zinc when compared to its unicellular relative Micrasterias, indicating a severe threat to the ecosystem.

Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions.

Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell wall bound calcium. The antioxidants salicylic acid, ascorbic acid and glutathione were not able to ameliorate any of the investigated metal effects on the green alga Micrasterias when added to the culture medium.

Structures of chlorophyll catabolites in bananas (Musa acuminata) reveal a split path of chlorophyll breakdown in a ripening fruit.

The disappearance of chlorophyll is a visual sign of fruit ripening. Yet, chlorophyll breakdown in fruit has hardly been explored; its non-green degradation products are largely unknown. Here we report the analysis and structure elucidation of colorless tetrapyrrolic chlorophyll breakdown products in commercially available, ripening bananas (Musa acuminata, Cavendish cultivar). In banana peels, chlorophyll catabolites were found in an unprecedented structural richness: a variety of new fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs) were detected. As a rule, FCCs exist only "fleetingly" and are hard to observe. However, in bananas several of the FCCs (named Mc-FCCs) were persistent and carried an ester function at the propionate side-chain. NCCs were less abundant, and exhibited a free propionic acid group, but functional modifications elsewhere. The modifications of NCCs in banana peels were similar to those found in NCCs from senescent leaves. They are presumed to be introduced by enzymatic transformations at the stage of the mostly unobserved, direct FCC-precursors. The observed divergent functional group characteristics of the Mc-FCCs versus those of the Mc-NCCs indicated two major "late" processing lines of chlorophyll breakdown in ripening bananas. The "last common precursor" at the branching point to either the persistent FCCs, or towards the NCCs, was identified as a temporarily abundant "secondary" FCC. The existence of two "downstream" branches of chlorophyll breakdown in banana peels, and the striking accumulation of persistent Mc-FCCs call for attention as to the still-elusive biological roles of the resulting colorless linear tetrapyrroles.

A freshwater green alga under cadmium stress: ameliorating calcium effects on ultrastructure and photosynthesis in the unicellular model Micrasterias.

Cadmium is a highly toxic heavy metal pollutant arising mainly from increasing industrial disposal of electronic components. Due to its high solubility it easily enters soil and aquatic environments. Via its similarity to calcium it may interfere with different kinds of Ca dependent metabolic or developmental processes in biological systems. In the present study we investigate primary cell physiological, morphological and ultrastructural responses of Cd on the unicellular freshwater green alga Micrasterias which has served as a cell biological model system since many years and has proved to be highly sensitive to any kind of abiotic stress. Our results provide evidence that the severe Cd effects in Micrasterias such as unidirectional disintegration of dictyosomes, occurrence of autophagy, decline in photosystem II activity and oxygen production as well as marked structural damage of the chloroplast are based on a disturbance of Ca homeostasis probably by displacement of Ca by Cd. This is indicated by the fact that physiological and structural cadmium effects could be prevented in Micrasterias by pre-treatment with Ca. Additionally, thapsigargin an inhibitor of animal and plant Ca(2+)-ATPase mimicked the adverse Cd induced morphological and functional effects on dictyosomes. Recovery experiments indicated rapid repair mechanisms after Cd stress.

Characterization of an UV- and VIS-absorbing, purpurogallin-derived secondary pigment new to algae and highly abundant in Mesotaenium berggrenii (Zygnematophyceae, Chlorophyta), an extremophyte living on glaciers.

Mesotaenium berggrenii is one of few autotrophs that thrive on bare glacier surfaces in alpine and polar regions. This extremophilic alga produces high amounts of a brownish vacuolar pigment, whose chemical constitution and ecological function is largely unknown until now. Field material was harvested to isolate and characterize this pigment. Its tannin nature was determined by photometric methods, and the structure determination was carried out by means of HPLC-MS and 1D- and 2D-NMR spectroscopy. The main constituent turned out to be purpurogallin carboxylic acid-6-O-ß-d-glucopyranoside. This is the first report of such a phenolic compound in this group of algae. Because of its broad absorption capacities of harmful UV and excessive VIS radiation, this secondary metabolite seems to play an important role for the survival of this alga at exposed sites. Attributes and abundances of the purpurogallins found in M. berggrenii strongly suggest that they are of principal ecophysiological relevance like analogous protective pigments of other extremophilic microorganisms. To prove that M. berggrenii is a true psychrophile, photosynthesis measurements at ambient conditions were carried out. Sequencing of the 18S rRNA gene of this alpine species and of its arctic relative, the filamentous Ancylonema nordenskiöldii, underlined their distinct taxonomic position within the Zygnematophyceae.

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias.

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 µM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased significantly in chromium treated cells, showing that glutathione is playing a major role in intracellular ROS and chromium detoxification.

INTRACELLULAR METAL COMPARTMENTALIZATION IN THE GREEN ALGAL MODEL SYSTEM MICRASTERIAS DENTICULATA (STREPTOPHYTA) MEASURED BY TRANSMISSION ELECTRON MICROSCOPY-COUPLED ELECTRON ENERGY LOSS SPECTROSCOPY1.

Entry of metals in form of aerosols into areas of high air humidity such as peat bogs represents a serious danger for inhabiting organisms such as the unicellular desmid Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zynematophyceae, Streptophyta). To understand cellular detoxification and tolerance mechanisms, detailed intracellular localization of metal pollutants is required. This study localizes the metals aluminum (Al), zinc (Zn), copper (Cu), and cadmium (Cd) in the green algal model system Micrasterias after experimental exposure to sulfate solutions by highly sensitive TEM-coupled electron energy loss spectroscopy (EELS). Concentrations of the metals shown to induce inhibiting effects on cell development and cytomorphogenesis were chosen for these experiments. Long-term exposure to these metal concentrations led to a pronounced impact on cell physiology expressed by a general decrease in apparent photosynthesis. After long-term treatment, Zn, Al, and Cu were detected in the cell walls by EELS. Zn was additionally found in vacuoles and mucilage vesicles, and Cu in starch grains and also in mucilage vesicles. Elevated amounts of oxygen in areas where Zn, Al, and Cu were localized suggest sequestration of these metals as oxides. The study demonstrated that Micrasterias can cope differently with metal pollutants. In low doses and during a limited time period, the cells were able to compartmentalize Cu the best, followed by Zn and Al. Cu and Zn were taken up into intracellular compartments, whereas Al was only bound to the cell wall. Cd was not compartmentalized at all, which explains its strongest impact on growth, cell division rate, and photosynthesis in Micrasterias.

DESICCATION STRESS CAUSES STRUCTURAL AND ULTRASTRUCTURAL ALTERATIONS IN THE AEROTERRESTRIAL GREEN ALGA KLEBSORMIDIUM CRENULATUM (KLEBSORMIDIOPHYCEAE, STREPTOPHYTA) ISOLATED FROM AN ALPINE SOIL CRUST1.

Klebsormidium crenulatum (Kütz.) Lokhorst (Klebsormidiophyceae, Streptophyta) isolated from an alpine soil in Tyrol, Austria, was experimentally exposed to desiccation under various relative air humidities (RH 5, 75, and >95%, ambient air 55%-60%). The effects on the structure and ultrastructure of K. crenulatum after 1, 4, or 7 d of desiccation at 5, 75, and >95% RH were investigated. The cross walls were deformed to an undulated shape, and the cell diameter was reduced to ∼60% of the control. Regardless of the RH applied, in all cases the cytoplasm appeared denser compared to that of liquid-culture-grown cells. Electron-dense particles with diameters of 0.4 µm-0.8 µm were observed in the cytoplasm, likely representing lipid droplets. The chloroplasts of desiccated samples contained a large number of plastoglobules. The number and appearance of mitochondria were not visibly altered, as also verified by 3,3' dihexyloxacarbocyanine iodine (DIOC6 ) staining. The amphiphilic styryl dye FM 1-43 resulted in staining of the plasma membrane in cells from liquid culture. In 7 d desiccated samples, a marked fluorescence is seen in ∼40%-50% of the cells, which were dead. Actin microfilaments (MFs) were drastically disrupted after desiccation; only dotlike actin batches remained. These results demonstrate that flexibility of the cell walls and maintenance of the key organelles play a key role in the tolerance of desiccation stress in K. crenulatum.

A novel blue fluorescent chlorophyll catabolite accumulates in senescent leaves of the peace lily and indicates a split path of chlorophyll breakdown.

Colorless, non-fluorescent Chl-catabolites (NCCs) are the typical, ubiquitous products of chlorophyll (Chl)-breakdown in senescent leaves. However, a fluorescent Chl-catabolite (FCC) accumulated in de-greened leaves of Spathiphyllum wallisii (Peace Lily), which showed a weak blue luminescence. The FCC, named Sw-FCC-62, was 'hypermodified' with an unprecedented 6-(2-[3,4-dihydroxy-phenyl]-ethyl)-ß-glucopyranosidyl ester at the propionyl group. Such esters stabilize FCCs against their typical and rapid, spontaneous isomerization to NCCs. Chl-breakdown in Sp. wallisii thus branches off from the 'common' path in leaves, and furnishes unique and 'persistent' FCCs. Our findings on 'hypermodified' FCCs also call for attention as to possible physiological roles of Chl-catabolites in plants.

Cell physiology of plants growing in cold environments.

The life of plants growing in cold extreme environments has been well investigated in terms of morphological, anatomical, and ecophysiological adaptations. In contrast, long-term cellular or metabolic studies have been performed by only a few groups. Moreover, a number of single reports exist, which often represent just a glimpse of plant behavior. The review draws together the literature which has focused on tissue and cellular adaptations mainly to low temperatures and high light. Most studies have been done with European alpine plants; comparably well studied are only two phanerogams found in the coastal Antarctic. Plant adaptation in northern polar regions has always been of interest in terms of ecophysiology and plant propagation, but nowadays, this interest extends to the effects of global warming. More recently, metabolic and cellular investigations have included cold and UV resistance mechanisms. Low-temperature stress resistance in plants from cold environments reflects the climate conditions at the growth sites. It is now a matter of molecular analyses to find the induced genes and their products such as chaperones or dehydrins responsible for this resistance. Development of plants under snow or pollen tube growth at 0 degrees C shows that cell biology is needed to explain the stability and function of the cytoskeleton. Many results in this field are based on laboratory studies, but several publications show that it is not difficult to study cellular mechanisms with the plants adapted to a natural stress. Studies on high light and UV loads may be split in two parts. Many reports describe natural UV as harmful for the plants, but these studies were mainly conducted by shielding off natural UV (as controls). Other experiments apply additional UV in the field and have had practically no negative impact on metabolism. The latter group is supported by the observations that green overwintering plants increase their flavonoids under snow even in the absence of UV. Thus, their defense and antioxidant role dominates. Ultrastructural comparisons were unable to find special light adaptations in plants taken from polar regions vs. high alpine species. The only adaptation found at the subcellular level for most alpine and polar plants are protrusions of the chloroplast envelopes. They are seen as a demand for fast membrane transport requiring additional membrane surface area, whereby the increase in stroma volume may help to support carbohydrate formation. Plants forming such protrusions have to cope with a short vegetation time. These observations are connected to the question as to how photosynthesis works quite well even at or under zero temperatures. The interplay between plastids, mitochondria, and peroxisomes, known as photorespiration, seems to be more intense than in lowland plants. This organelle cooperation serves as a valve for a surplus in solar energy input under cold conditions. Additional metabolic acclimations are under investigation, such as the role of an alternative plastid terminal oxidase. Plants from cold environments may also be seen as ideal objects for studying the combined effects of high light plus cold resistance-from the molecular level to the whole plant adaptation. Modern instrumentation makes it possible to perform vital metabolic measurements under outdoor conditions, and research stations in remote polar and alpine areas provide support for scientists in the preparation of samples for later cellular studies in the home laboratory.

Physiological and morphological processes in the Alpine snow alga Chloromonas nivalis (Chlorophyceae) during cyst formation.

Amongst a specialised group of psychrophilic microalgae that have adapted to thrive exclusively in summer snow fields, Chloromonas nivalis has been reported as a species causing green, orange or pink blooms in many alpine and polar regions worldwide. Nevertheless, the cytology, ecophysiology and taxonomy of this species are still unresolved. Intracellular processes during cyst formation, which is the dominant stage on snow fields, were examined with samples from the European Alps to better understand the cellular strategies of a green alga living in this harsh habitat. We show with two different methods, i.e. oxygen optode fluorometry and by chlorophyll fluorescence, that the cysts are photosynthetically highly active, although they do not divide, and that Chloromonas nivalis can cope with low as well as high light conditions. During cyst formation, the chloroplast is fragmented into several smaller parts, enlarging the surface to volume ratio. The pool of xanthophyll-cycle pigments is significantly enlarged, which is different from other snow algae. The cytoplasm is filled with lipid bodies containing astaxanthin, a secondary carotenoid that causes the typical orange colour. The cyst wall surface possesses characteristic elongate flanges, which are assembled extracellulary by accumulation of material in the periplasmatic interspace. Comparison of Chloromonas nivalis samples from different locations (Austrian Alps, Spitsbergen) by molecular methods indicates genetic variations due to spatial isolation, while a North American strain has no close relationship to the taxon.

Physiological, biochemical, and ultrastructural responses of the green macroalga Urospora penicilliformis from Arctic Spitsbergen to UV radiation.

Exposure of the filamentous turf green alga Urospora penicilliformis to ambient and artificial ultraviolet radiation (UVR) revealed a considerable resilient species. This explains the ability of this alga to thrive in the middle-upper intertidal zones of the Arctic sea where it is periodically exposed to environmental extremes. A transient UVR effect on photosynthesis under photosynthetically active radiation (PAR) + UV-A and PAR + UV-A + UV-B was found, but dynamic recovery of photoinhibition was observed immediately after reduction of the photon fluence rate of PAR in the absence or presence of background UVR under laboratory and natural solar radiation, respectively. Chlorophylls, carotenoids, and xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) concentrations were not significantly different between freshly collected samples and filaments exposed to additional laboratory radiation treatment. The ultrastructure of the U. penicilliformis gametophytes showed that the cells are well adapted to UVR. No significant ultrastructural alterations were observed in filaments exposed to different spectral irradiance in the laboratory compared to in situ acclimated specimen. The antioxidant alpha-tocopherol was detected in minute quantity while the search for flavonoid-like compounds was negative. Other UV screening strategies or certain genetically fixed physiological protective mechanism could be operating in this species responsible for their occurrence in higher shoreline and ecological success. Further molecular and biochemical studies are needed to elucidate the stress resistance in this turf alga. There is an indication that the extremely thick cell wall of U. penicilliformis gametophytes covered with mucilage sheath and dense layer of mineral depositions may provide a shield against unfavorable environmental conditions in general and against UVR in particular.

Fluorescent chlorophyll catabolites in bananas light up blue halos of cell death.

Breakdown of chlorophyll is a major contributor to the diagnostic color changes in fall leaves, and in ripening apples and pears, where it commonly provides colorless, nonfluorescent tetrapyrroles. In contrast, in ripening bananas (Musa acuminata) chlorophylls fade to give unique fluorescent catabolites (FCCs), causing yellow bananas to glow blue, when observed under UV light. Here, we demonstrate the capacity of the blue fluorescent chlorophyll catabolites to signal symptoms of programmed cell death in a plant. We report on studies of bright blue luminescent rings on the peel of very ripe bananas, which arise as halos around necrotic areas in 'senescence associated' dark spots. These dark spots appear naturally on the peel of ripe bananas and occur in the vicinity of stomata. Wavelength, space, and time resolved fluorescence measurements allowed the luminescent areas to be monitored on whole bananas. Our studies revealed an accumulation of FCCs in luminescent rings, within senescing cells undergoing the transition to dead tissue, as was observable by morphological textural cellular changes. FCCs typically are short lived intermediates of chlorophyll breakdown. In some plants, FCCs are uniquely persistent, as is seen in bananas, and can thus be used as luminescent in vivo markers in tissue undergoing senescence. While FCCs still remain to be tested for their own hypothetical physiological role in plants, they may help fill the demand for specific endogenous molecular reporters in noninvasive assays of plant senescence. Thus, they allow for in vivo studies, which provide insights into critical stages preceding cell death.

The vegetative arctic freshwater green alga Zygnema is insensitive to experimental UV exposure.

The physiological performance and ultrastructural integrity of the vegetative freshwater green alga Zygnema sp., growing under ambient polar day solar radiation and after exposure to experimentally low radiation, but with high UVR:PAR ratio were investigated. In the laboratory, algae were exposed to low photosynthetic active radiation (PAR=P, 400-700 nm, 20 micromol m(-2) s(-1)), PAR + UV-A = PA (320-400 nm, 4.00 W m(-2) = UV-A) and PAR + UV-A + UV-B = PAB (280-320 nm, 0.42 W m(-2) = UV-B) for 24 h at 7 degrees C. Photosynthetic performance and ultrastructure of ambient solar radiation-exposed (field control) and experimentally treated Zygnema samples were assessed using chlorophyll fluorescence, and transmission electron microscopy (TEM). No significant treatment effect was observed in the photosynthesis-irradiance curve parameters. Exclusion of the UV-B spectrum in the laboratory treatment caused significantly lower effective photosynthetic quantum yield compared to samples exposed to the whole radiation spectrum. TEM revealed no obvious differences in the ultrastructure of field control and laboratory P-, PA- and PAB-exposed samples. Substantial amounts of lipid bodies, visualized by Sudan IV staining, were observed in all samples. Chloroplasts contained numerous plastoglobules. Organelles like mitochondria, Golgi bodies and the nucleus remained unaffected by the radiation exposures. Zygnema is well adapted to ambient solar radiation, enabling the alga to cope with experimental UV exposure and it is expected to persist in a scenario with enhanced UV radiation caused by stratospheric ozone depletion.

PCD and autophagy in the unicellular green alga Micrasterias denticulata.

Programmed cell death (PCD) plays a central role in normal plant development and is also induced by various biotic and abiotic stress factors. In the unicellular freshwater green alga Micrasterias denticulata morphological and biochemical hallmarks such as the appearance of autophagosomes, increased production of ROS and degradation of genomic DNA into small fragments ("DNA laddering") indicate PCD. Our data not only demonstrate that Micrasterias is capable of performing PCD under salt stress, but also that it is triggered by the ionic and not osmotic component of salinity. Additionally, results from the present and previous studies suggest that different inducers may lead to different cell death pathways in one and the same organism.

Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata.

Programmed cell death (PCD) is a key element in normal plant growth and development which may also be induced by various abiotic and biotic stress factors including salt stress. In the present study, morphological, biochemical, and physiological responses of the theoretically immortal unicellular freshwater green alga Micrasterias denticulata were examined after salt (200 mM NaCl or 200 mM KCl) and osmotic stress induced by iso-osmotic sorbitol. KCl caused morphological changes such as cytoplasmic vacuolization, extreme deformation of mitochondria, and ultrastructural changes of Golgi and ER. However, prolonged salt stress (24 h) led to the degradation of organelles by autophagy, a special form of PCD, both in NaCl- and KCl-treated cells. This was indicated by the enclosure of organelles by ER-derived double membranes. DNA of NaCl- and KCl-stressed cells but not of sorbitol-treated cells showed a ladder-like pattern on agarose gel, which means that the ionic rather than the osmotic component of salt stress leads to the activation of the responsible endonuclease. DNA laddering during salt stress could be abrogated by addition of Zn(2+). Neither cytochrome c release from mitochondria nor increase in caspase-3-like activity occurred after salt stress. Reactive oxygen species could be detected within 5 min after the onset of salt and osmotic stress. Respiration, photosynthetic activity, and pigment composition indicated an active metabolism which supports programmed rather than necrotic cell death in Micrasterias after salt stress.

Response of arctic snow and permafrost algae to high light and nitrogen stress by changes in pigment composition and applied aspects for biotechnology.

Ten algal strains from snow and permafrost substrates were tested for their ability to produce secondary carotenoids and alpha-tocopherol in response to high light and decreased nitrogen levels. The Culture Collection of Cryophilic Algae at Fraunhofer IBMT in Potsdam served as the bioresource for this study. Eight of the strains belong to the Chlorophyceae and two strains are affiliated to the Trebouxiophyceae. While under low light, all 10 strains produced the normal spectrum of primary pigments known to be present in Chlorophyta, only the eight chlorophyceaen strains were able to synthesize secondary carotenoids under stress conditions, namely canthaxanthin, echinenone and astaxanthin; seven of them were also able to synthesize minor amounts of adonixanthin and an unidentified hydroxyechinenone. The two trebouxiophyceaen species of Raphidonema exhibited an unusually high pool of primary xanthophyll cycle pigments, possibly serving as a buffering reservoir against excessive irradiation. They also proved to be good alpha-tocopherol producers, which might also support the deactivation of reactive oxygen species. This study showed that some strains might be interesting novel candidates for biotechnological applications. Cold-adapted, snow and permafrost algae might serve as valuable production strains still exhibiting acceptable growth rates during the cold season in temperate regions.

Effects of temperature and light on the formation of chloroplast protrusions in leaf mesophyll cells of high alpine plants.

Chloroplasts of many alpine plants have the ability to form marked, stroma-filled protrusions that do not contain thylakoids. Effects of temperature and light intensity on the frequency of chloroplasts with such protrusions in leaf mesophyll cells of nine different alpine plant species (Carex curvula All., Leontodon helveticus Merat., Oxyria digyna (L.) Hill., Poa alpina L. ssp. vivipara, Polygonum viviparum L., Ranunculus glacialis L., Ranunculus alpestris L., Silene acaulis L. and Soldanella pusilla Baumg.) covering seven different families were studied. Leaves were exposed to either darkness and a stepwise increase in temperature (10-38 degrees C) or to different light intensities (500 and 2000 micromol photons m(-2) s(-1)) and a constant temperature of 10 or 30 degrees C in a special temperature-regulated chamber. A chloroplast protrusions index characterising the relative proportion of chloroplasts with protrusions was defined. Seven of the nine species showed a significant increase in chloroplast protrusions when temperature was elevated to over 20 degrees C. In contrast, the light level did not generally affect the abundance of chloroplasts with protrusions. Chloroplast protrusions lead to a dynamic enlargement of the chloroplast surface area. They do not appear to be directly connected to a distinct photosystem II (PSII) (F(v)/F(m)) status and thus seem to be involved in secondary, not primary, photosynthetic processes.