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.

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.

Identification of phytochelatins in the cadmium-stressed conjugating green alga Micrasterias denticulata.

Aquatic environments like peat bogs are affected by anthropogenic metal input into the environment. These ecosystems are inhabited by unicellular green algae of the class Zygnematophyceae. In this study the desmid Micrasterias denticulata was stressed with 600 nM Cd, 10 µM Cr and 300 nM Cu for 3 weeks. GSH levels were measured with HPLC and did not differ between the different treatments or the control. According to the metallo-thiolomics concept, mass spectrometry was used as a method for unambiguous thiol peptide identification. PC2, PC3 and PC4 were clearly identified in the Cd stressed sample with UPLC-MS by their MS spectrum and molecular masses. PC2 and PC3 were determined to be the main thiol compounds, while PC4 was only abundant in traces in Micrasterias. In addition, the identity of PC2 and PC3 was confirmed by MS/MS. No PCs were detected in the Cu stressed algae sample. However, in the Cr stressed sample traces of PC2 were indicated by a peak in UPLC-MS at the retention time of the PC2 standard, but the intensity was too low to acquire reliable MS and MS/MS spectra. In this study PCs have been detected for the first time in a green alga of the division Streptophyta, a close relative to higher plants.

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.