Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants.

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Biomineralization of strontium and barium contributes to detoxification in the freshwater alga Micrasterias.

The unicellular model alga Micrasterias denticulata inhabits acid peat bogs that are highly endangered by pollutants due to their high humidity. As it was known from earlier studies that algae like Micrasterias are capable of storing barium naturally in form of BaSO4 crystals, it was interesting to experimentally investigate distribution and sequestration of barium and the chemically similar alkaline earth metal strontium. Additionally, we intended to analyze whether biomineralization by crystal formation contributes to diminution of the generally toxic effects of these minerals to physiology and structure of this alga which is closely related to higher plants. The results show that depending on the treatment differently shaped crystals are formed in BaCl2 and Cl2Sr exposed Micrasterias cells. Modern microscopic techniques such as analytical TEM by electron energy loss spectroscopy and Raman microscopy provide evidence for the chemical composition of these crystals. It is shown that barium treatment results in the formation of insoluble BaSO4 crystals that develop within distinct compartments. During strontium exposure long rod-like crystals are formed and are surrounded by membranes. Based on the Raman signature of these crystals their composition is attributed to strontium citrate. These crystals are instable and are dissolved during cell death. During strontium as well as barium treatment cell division rates and photosynthetic oxygen production decreased in dependence of the concentration, whereas cell vitality was reduced only slightly. Together with the fact that TEM analyses revealed only minor ultrastructural alterations as consequence of relatively high concentrated BaCl2 and Cl2Sr exposure, this indicates that biomineralization of Sr and Ba protects the cells from severe damage or cell death at least within a particular concentration range and time period. In the case of Sr treatment where ROS levels were found to be elevated, hallmarks for autophagy of single organelles were observed by TEM, indicating beginning degradation processes.

Carbon starvation induces lipid degradation via autophagy in the model alga Micrasterias.

Autophagy is regarded as crucial intracellular process in plant development but also in intracellular stress response. It is known to be controlled by the energy level of the cell and consequently can be triggered by energy deprivation. In this study carbon starvation evoked in different ways was investigated in the freshwater algae model system Micrasterias denticulata (Streptophyta) which is closely related to higher plants. Cells exposed to the photosynthesis inhibiting herbicide DCMU, to the glycolysis inhibitor 2-Deoxy-d-glucose and to complete darkness over up to 9 weeks for preventing metabolism downstream of glucose supply, were investigated by means of Nile red staining and analyses in CLSM, and TEM after cryo-preparation. Our results show that lipid bodies containing both neutral and polar lipids are evenly distributed inside the chloroplast in control cells. During carbon starvation they are displaced into the cytoplasm and are either degraded via autophagy and/or excreted from the cell. Upon discharge from the chloroplast lipid bodies become engulfed by double membranes probably deriving from the ER, thus forming autophagosomes which later fuse with vacuoles. Coincidently indications for autophagy of other organelles and cytoplasmic portions were found during starvation and particularly in DCMU treated cells the number of starch grains decreased and pyrenoids disintegrated. Additionally our molecular data provide first evidence for the existence of a single ATG8 isoform in Micrasterias. ATG8 is known as main regulator of both bulk and selective autophagy in eucaryotes. Our study indicates that lipid degradation during carbon starvation is achieved via "classical" autophagy in the alga Micrasterias. This process has so far only been very rarely observed in plant cells and seems to allow recruitment of lipids for energy supply on the one hand and elimination of unusable or toxicated lipids on the other hand.

Structural stress responses and degradation of dictyosomes in algae analysed by TEM and FIB-SEM tomography.

Stress-induced physiological deficiencies in cells are reflected in structural, morphological and functional reactions of organelles. Although numerous investigations have focused on chloroplasts and mitochondria as main targets of different stressors in plant cells, there is insufficient information on the plant Golgi apparatus as stress sensor. By using the advantages of field emission scanning electron microscopy tomography in combination with classical ultrathin sectioning and transmission electron microscopic analyses, we provide structural evidence for common stress responses of the large and highly stable dictyosomes in the algal model system Micrasterias. Stress is induced by different metals such as manganese and lead, by starvation in 9 weeks of darkness or by inhibiting photosynthesis or glycolysis and by disturbing ionic homeostasis via KCl. For the first time a stress-induced degradation pathway of dictyosomes is described that does not follow "classical" autophagy but occurs by disintegration of cisternae into single membrane balls that seem to be finally absorbed by the endoplasmic reticulum (ER). Comparison of the morphological features that accompany dictyosomal degradation in Micrasterias to similar reactions observed during the same stress application in Nitella indicates an ubiquitous degradation process at least in algae. As the algae investigated belong to the closest relatives of higher land plants these results may also be relevant for understanding dictyosomal stress and degradation responses in the latter phylogenetic group. In addition, this study shows that two-dimensional transmission electron microscopy is insufficient for elucidating complex processes such as organelle degradation, and that information from three-dimensional reconstructions as provided by field emission scanning electron microscopy tomography is absolutely required for a comprehensive understanding of the phenomenon.

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.

3-D analysis of dictyosomes and multivesicular bodies in the green alga Micrasterias denticulata by FIB/SEM tomography.

In the present study we employ FIB/SEM tomography for analyzing 3-D architecture of dictyosomes and formation of multivesicular bodies (MVB) in high pressure frozen and cryo-substituted interphase cells of the green algal model system Micrasterias denticulata. The ability of FIB/SEM of milling very thin 'slices' (5-10 nm), viewing the block face and of capturing cytoplasmic volumes of several hundred µm(3) provides new insight into the close spatial connection of the ER-Golgi machinery in an algal cell particularly in z-direction, complementary to informations obtained by TEM serial sectioning or electron tomography. Our FIB/SEM series and 3-D reconstructions show that interphase dictyosomes of Micrasterias are not only closely associated to an ER system at their cis-side which is common in various plant cells, but are surrounded by a huge "trans-ER" sheath leading to an almost complete enwrapping of dictyosomes by the ER. This is particularly interesting as the presence of a trans-dictyosomal ER system is well known from mammalian secretory cells but not from cells of higher plants to which the alga Micrasterias is closely related. In contrast to findings in plant storage tissue indicating that MVBs originate from the trans-Golgi network or its derivatives our investigations show that MVBs in Micrasterias are in direct spatial contact with both, trans-Golgi cisternae and the trans-ER sheath which provides evidence that both endomembrane compartments are involved in their formation.

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.