Primary hybrid zone formation in Tephroseris helenitis (Asteraceae), following postglacial range expansion along the central Northern Alps.

Distinguishing between secondary versus primary hybrid zone formation remains a challenging task as, for instance, the time window in which these historical (vicariant) versus contemporary (environmental-selective) processes are distinguishable may be relatively narrow. Here, we examine the origin and structure of a transition zone between two subspecies of Tephroseris helenitis along the central Northern Alps, using molecular (AFLP) and morphological (achene type) data in combination with ecological niche models (ENMs) to hindcast ranges at the Last Glacial Maximum (LGM) and mid-Holocene. Samples were collected over a c. 350 km long transect, largely covered by ice during the LGM. Genetically nonadmixed individuals of subspp. helenitis versus salisburgensis dominated the westernmost versus eastern transect areas, with admixed individuals occurring in between. Clines for achene morphology and outlier loci potentially under climate-driven selection were steep, largely noncoincidental, and displaced to the east of the cline centre for neutral AFLPs. During the LGM, ssp. helenitis should have been able to persist in a refugium southwest of the transect, while suitable habitat for ssp. salisburgensis was apparently absent at this time. Together with patterns of genetic and clinal variation, our ENM data are suggestive of a primary hybrid zone that originated after the species' postglacial, eastward expansion. The observed clinal changes may thus reflect random/nonadaptive processes during expansion and selection on particular loci, and possibly achene type, in response to a long-term, west-to-east climate gradient in the direction of more stressful (e.g., wetter/cooler) conditions. Overall, this study adds to the vast hybrid zone literature a rare example of a hybrid zone caused by primary differentiation within a plant species, underlaid by historical range expansion.

Genome-wide nuclear data confirm two species in the Alpine endemic land snail Noricella oreinos s.l. (Gastropoda, Hygromiidae).

The Austrian endemic land snail species Noricella oreinos (formerly Trochulus oreinos) occurs in the Northeastern Calcareous Alps at high elevations. Two morphologically highly similar subspecies N. o. oreinos and N. o. scheerpeltzi have been described. First analyses of mitochondrial and nuclear marker sequences indicated a high genetic divergence between them. In the present study, we aimed to assess gene flow between the two subspecies which should help to re-evaluate their taxonomic status. Sequence data and amplified fragment length polymorphism (AFLP) markers of 255 Noricella specimens covering the whole distribution range were analyzed. A clear geographic separation was found within the potential contact zone, the Haller Mauern mountain range. Samples of all western sites were part of the clade representing N. o. scheerpeltzi and almost all samples from the eastern sites clustered with N. o. oreinos. However, within two sampling sites of the eastern Haller Mauern, a few individuals possessed a COI sequence matching the N. o. oreinos clade whereas at the ITS2 locus they were heterozygous possessing the alleles of both taxa. Contrary to the ITS2 results indicating historical and/or ongoing hybridization, AFLP analyses of 202 individuals confirmed a clear separation of the two taxa congruent with the mitochondrial data. Although they occur on the same mountain range without any physical barrier, no indication of ongoing gene flow between the two taxa was found. Thus, we conclude that the two taxa are separate species N. oreinos and N. scheerpeltzi.

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.

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.

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.

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism.

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato . The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.

Plant aquaporins.

Aquaporins are ubiquitous membrane channel proteins that facilitate and regulate the permeation of water across biological membranes. Aquaporins are members of the MIP family and some of them seem to be also able to transport other molecules such as urea or glycerol. In the plant kingdom, a single plant expresses a considerably large number of MIP homologues. These homologues can be subdivided into four groups (PIP, TIP, NIP, SIP) with highly conserved amino acid sequences and intron positions in each group. Since their discovery, advancing knowledge of their structure led to an understanding of the basic features of the water transport mechanism. An optimal water balance is essential to the homeostasis of most organisms, and aquaporins may be one of the mechanisms involved under changing environmental and developmental conditions. In fact, this may be one reason for the abundance and diversity of aquaporins, in particular in plants. In addition, exposure to different types of stress alters water relations and thus, aquaporins may be involved in stress responses as well. The transcriptional and/or post-translational regulation of aquaporins would determine changes in membrane water permeability. Both phosphorylation and translocation to/from vesicles have been reported as post-translational mechanisms. However, translocation in plants has not yet been shown. Although significant advances have been achieved, complete understanding of aquaporin function and regulation remains elusive.