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SUMMARYWHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression ofHvWHIRLY1in barley, lines with a 10-and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form indicating that over-accumulating WHIRLY1 exceeded the amount that chloroplasts can sequester. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the efficiency of photosynthesis. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that already in non-stress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance,PR1expression further increased in addition to an increase in the expression ofPR10and of the gene encoding phenylalanine lyase (PAL), the key enzyme of salicylic acid biosynthesis in barley. The activation of defense gene expression in oeW1 plants coincided with an enhanced resistance towards powdery mildew, which in barley is independent of salicylic acid. Taken together, the results show that over-accumulation of WHIRLY1 in barley to levels of 10 or more, amplified the tradeoff between growth and stress resistance.

Parasitic plants live in intimate physical connection with other plants serving as their hosts. These host plants provide the inorganic and organic compounds that the parasites need for their propagation. The uptake of the macromolecular compounds happens through symplasmic connections in the form of plasmodesmata. In contrast to regular plasmodesmata, which connect genetically identical cells of an individual plant, the plasmodesmata that connect the cells of host and parasite join separate individuals belonging to different species and are therefore termed “interspecific”. The existence of such interspecific plasmodesmata was deduced either indirectly using molecular approaches or observed directly by ultrastructural analyses. Most of this evidence concerns shoot parasitic Cuscuta species and root parasitic Orobanchaceae, which can both infect a large range of phylogenetically distant hosts. The existence of an interspecific chimeric symplast is both striking and unique and, with exceptions being observed in closely related grafted plants, exist only in these parasitic relationships. Considering the recent technical advances and upcoming tools for analyzing parasitic plants, interspecific plasmodesmata in parasite/host connections are a promising system for studying secondary plasmodesmata. For open questions like how their formation is induced, how their positioning is controlled and if they are initiated by one or both bordering cells simultaneously, the parasite/host interface with two adjacent distinguishable genetic systems provides valuable advantages. We summarize here what is known about interspecific plasmodesmata between parasitic plants and their hosts and discuss the potential of the intriguing parasite/host system for deepening our insight into plasmodesmatal structure, function, and development.

WHIRLY2 is a single‐stranded DNA binding protein associated with mitochondrial nucleoids. In the why 2‐1 mutant of Arabidopsis thaliana, a major proportion of leaf mitochondria has an aberrant structure characterized by disorganized nucleoids, reduced abundance of cristae, and a low matrix density despite the fact that the macroscopic phenotype during vegetative growth is not different from wild type. These features coincide with an impairment of the functionality and dynamics of mitochondria that have been characterized in detail in wild‐type and why 2‐1 mutant cell cultures. In contrast to the development of the vegetative parts, seed germination is compromised in the why 2‐1 mutant. In line with that, the expression level of why 2 in seeds of wild‐type plants is higher than that of why 3, whereas in adult plant no difference is found. Intriguingly, in early stages of shoots development of the why 2‐1 mutant, although not in seeds, the expression level of why 3 is enhanced. These results suggest that WHIRLY3 is a potential candidate to compensate for the lack of WHIRLY2 in the why 2‐1 mutant. Such compensation is possible only if the two proteins are localized in the same organelle. Indeed, in organello protein transport experiments using intact mitochondria and chloroplasts revealed that WHIRLY3 can be dually targeted into both, chloroplasts and mitochondria. Together, these data indicate that the alterations of mitochondria nucleoids are tightly linked to alterations of mitochondria morphology and functionality. This is even more evident in those phases of plant life when mitochondrial activity is particularly high, such as seed germination. Moreover, our results indicate that the differential expression of why 2 and why 3 predetermines the functional replacement of WHIRLY2 by WHIRLY3, which is restricted though to the vegetative parts of the plant.

As tocopherols are expected to protect PSII against toxic singlet oxygen it is surprising that the null tocopherol mutant vte1 has been reported to show only a weak enhancement of photosystem II photoinhibition under high irradiance. Based on the view that singlet oxygen is formed also in unstressed conditions, such as low light (LL), we hypothesized that some defense strategies are activated in vte1 in these light conditions. In support for that we noted several symptoms of stress at PSII in the mutant under LL, by means of parameters of fast and slow kinetics of chlorophyll fluorescence and of changes in the relative contribution of PSII antenna in comparison to those of PSI. This was associated with a lower extent of phosphorylation of PSII core proteins (D1 and CP43). PSII RCs do not totally recover from stress in vte1 even after the nocturnal phase. As a clear compensation for the impeded performance of PSII in the vte1 we noted an increased quantum efficiency of PSI. A pronounced changes between WT and the vte1 mutant were also related to conformation of LHCII at the beginning of photoperiod, suggesting the absence of LHCII trimers in the mutant. The thylakoids thickness was similar in WT and vte1 under LL, but a pronounced unstacking of thylakoids was evoked by HL only in vte1. In conclusion, we postulate that action of 1O2 on PSII in vte1 leads to some permanent damage at PSII core and at LHCII already under LL.

HvPAP14 is a cysteine protease found in association with thylakoid membranes. Among its putative substrates are proteins such as LHCB1, LHCB5, PSBO, and RbcL, as revealed in overexpressing barley plants., Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

Cysteine proteases are known to be associated with programmed cell death, developmental senescence and some types of pathogen and stress-induced responses. In the present study, we have characterized the cysteine protease Tr-cp 14 in white clover (Trifolium repens). Tr-cp 14 belongs to the C1A family of cysteine proteases with homology to XCP1 and XCP2 from Arabidopsis thaliana and p48h-17 from Zinnia elegans, which previously have been reported to be associated with tracheary element differentiation. The proform as well as the processed form of the protein was detected in petioles, flowers and leaves, but the processed form was more abundant in leaves and petioles than in flowers. The Tr-cp 14 protein was localized to differentiating tracheary elements within the xylem, indicating that the cysteine protease is involved in protein re-mobilization during tracheary element differentiation. Immunogold studies suggest that the protease prior to the burst of the vacuole was associated to the ER cisternae. After disruption of the tonoplast, it was found in the cytoplasm, and, in later stages, associated with disintegrating material dispersed throughout the cell.

Crassulacean acid metabolism (CAM) is an intriguing metabolic strategy to maintain photosynthesis under conditions of closed stomata. A shift from C(3) photosynthesis to CAM in Mesembryanthemum crystallinum plants was induced by high salinity (0.4 M NaCl). In CAM-performing plants, the quantum efficiencies of photosystem II and I were observed to undergo distinct diurnal fluctuations that were characterized by a strong decline at the onset of the day, midday recovery, and an evening drop. The temporal recovery of both photosystems' efficiency at midday was associated with a more rapid induction of the electron transport rate at PSII. This recovery of the photosynthetic apparatus at midday was observed to be accompanied by extreme swelling of thylakoids. Despite these fluctuations, a persistent effect of CAM was the acceptor side limitation of PSI during the day, which was accompanied by a strongly decreased level of Rubisco protein. Diurnal changes in the efficiency of photosystems were parallel to corresponding changes in the levels of mRNAs for proteins of PSII and PSI reaction centers and for rbcL, reaching a maximum in CAM plants at midday. This might reflect a high demand for new protein synthesis at this time of the day. Hybridization of run-on transcripts with specific probes for plastid genes of M. crystallinum revealed that the changes in plastidic mRNA levels were regulated at the level of transcription.

Stromuli sind bewegliche Fortsatze der Plastiden, die Brucken zwischen den Plastiden bilden oder Kontakte zwischen Plastiden und anderen Raumen der Zelle herstellen konnen. Damit konnten sie eine Funktion bei der Koordination von Prozessen in verschiedenen Plastiden haben und Signale zwischen Plastiden und anderen Teilen der Zelle austauschen. Stromuli wurden vor mehr als 100 Jahren zuerst in Zellen des Wasserfarns Selaginella beobachtet und spater mit verbesserten lichtmikroskopischen Verfahren auch in Hoheren Pflanzen nachgewiesen. Als es vor ca. 15 Jahren moglich wurde, das Stroma der Plastiden durch das grun fluoreszierende Protein (GFP) sichtbar zu machen, wurden sie neu entdeckt. Ihre Bildung wird vom Gewebe, Entwicklungszustand und von externen Faktoren wie der Temperatur beeinflusst wird. Ein intaktes System von Aktinfasern ist Voraussetzung zur Bildung der Stromuli. An ihrer Bewegung ist das Motorprotein Myosin beteiligt. Plastidic bridges in the plant cell network: Stromules Stromules are mobile protrusions emanating from plastids. They might form bridges between plastids and connect them also with other compartments of the plant cell. They could be involved in coordination of plastid activities and in signalling. Stromules have been first observed in the water fern Selaginella more than 100 years ago. Later improved light microscopy enabled the visualization of stromules in higher plant plastids. 15 years ago, since plants accumulating the green fluorescing proteins (GPF) in the stroma became available they have been newly detected and are now studied intensively. Formation of stromules differs among plant tissues, developmental stages and environmental situations. Actin and myosin are required for the formation of stromules.

The nucleic acid binding protein Whirly1 of barley has been located to both chloroplasts and the nucleus of the same cell. Immunogold labelling furthermore showed that in vivo Whirly1 does not strictly co-localize with DNA in chloroplasts, while it is closely associated with DNA in the nucleus. High-resolution imaging of Whirly1-GFP and PEND-RFP fusion proteins revealed that only a minor part of Whirly1 co-localizes with nucleoids. The co-localization with nucleoids is in accordance with the detection of Whirly1 in a conventionally prepared fraction of the transcriptionally active chromosome (TAC). By further purification and enrichment of transcriptional activity Whirly1, however, was lost from the TAC fraction. Knockdown of Whirly1 in transgenic barley plants had neither impact on transcription of selected protein coding genes nor on genes coding for ribosomal RNAs or tRNAs. The results of RIP-chip experiments showed that barley Whirly1 as its maize orthologue associates with a set of intron containing plastid RNAs. Taken together, the results suggest that plastid-located Whirly1 functions primarily in RNA metabolism rather than as a DNA binding protein.

This article concerns the intriguing protein Whirly1 (Why1) that belongs to a small family of single-stranded DNA-binding proteins and has been described to have functions in the nucleus ([Desveaux et al., 2002][1]; [Yoo et al., 2007][2]). In contrast, in vitro import assays with isolated organelles

Die meisten biologischen und medizinischen Untersuchungsobjekte sind zu gros und/oder zu dick, um sie direkt zu mikroskopieren. Normalerweise muss man die Zellen oder Gewebeteile, die von Interesse sind, zunachst aus ihrem Umfeld isolieren. Dabei sollen die morphologischen und biochemischen Eigenschaften moglichst erhalten bleiben. Zellen lassen sich direkt durch verschiedene Abstrich- und Abtupfverfahren gewinnen oder durch Mazerationsmethoden aus einem Gewebeverband losen. Aus Suspensionen konnen sie durch Filtration oder Zentrifugation angereichet werden. Ihre spezifischen Eigenschaften werden genutzt, um sie zu sortieren und in getrennten Fraktionen anzureichern. Organ- oder Gewebeproben werden mit feinen Scheren, speziellen Nadeln, Rasierklingen oder scharfem Skalpell entnommen. Durch verschiedene Schnitttechniken lassen sie sich direkt oder nach Fixierung (7 Kap. 5) weiter verkleinern und fur die direkte Beobachtung, fur Farbungen und Lokalisationsstudien oder fur die weitere Praparation (z. B. fur die TEM) vorbereiten. Die Gewinnung von Proben fur biochemische Untersuchungen kann die mikroskopische Beobachtung und Kontrolle erfordern. Dazu werden hier ebenfalls einige Techniken (Zellfraktionierung, Lasermikrodissektion) vorgestellt, auch wenn die Mikro

Praparate fur die konventionelle Transmissionselektronenmikroskopie (TEM) mussen vakuumfest, also wasserfrei sein. Sie mussen dem Elektronenstrahl standhalten, durfen also unter der Belastung nicht schmelzen oder verdampfen. Sie mussen elektronendurchlassig sein, durfen also eine gewisse Dicke und Dichte nicht uberschreiten. Und sie mussen Kontraste aufweisen, damit man Strukturen erkennen kann. Um diese Anforderungen zu erfullen, werden normalerweise Ultradunnschnitte angefertigt. Nur sehr kleine und dunne Proben (z. B. Viren, Organellen, Elemente des Cytoskeletts) konnen direkt kontrastiert und betrachtet werden. Wie in der histologischen Technik gibt es kein starres Schema fur die Vorgehensweise, sondern die einzelnen Schritte mussen dem Praparat und dem Ziel der Untersuchung jeweils angepasst werden. Die folgenden Seiten sollen dabei eine Einfuhrung und Hilfestellung geben. Ausfuhrlichere Anleitungen und Rezepte zu speziellen Fragestellungen finden sich in der Fachliteratur und in Buchern zur elektronenmikroskopischen Praparationstechnik (z. B. Dashek 2000, Hayat 1989).

Die REM dient insbesondere zur Darstellung von Oberflachen. Dies konnen naturliche oder kunstlich geschaffene Oberflachen sein. Mit einer Auflosung von 0,1 μm bis einige Millimeter und einer hohen Tiefenscharfe (7 Kapitel 1) konnen ganze Tiere (z. B. Insekten) sehr viel detailreicher und ubersichtlicher als im Lichtmikroskop abgebildet werden. Aber auch Gewebe- oder Organteile, pflanzliche und tierische Strukturelemente, bis hin zu Zellen, Organellen oder Partikeln und Aggregaten eignen sich zur Untersuchung (. Abb. 8.1). Daruber hinaus werden Abdrucke von Oberflachen im REM abgebildet.

Summary By differential screening of a cDNA library from the feeding stage (trophont) of the marine ciliate Eufolliculina uhligi we have isolated and sequenced the DNA of a clone, EuT2, which is preferentially expressed in the trophont. The amino acid sequence deduced from the reading frame characterizes it clearly as belonging to the aconitase family. Sequence comparisons to a number of cytoplasmic and mitochondrial aconitases of diverse organisms reveal high similarity to vertebrate iron regulatory proteins (IRPs), but less similarity to mitochondrial aconitases. The cDNA clone of E. uhligi has a length of 1960 nt; the translated sequence corresponds to the C-terminal part of an open reading frame starting at aa 256 when compared to human IRP-1. The size of the transcript is 3050 nt. As revealed by Northern blotting analyses, the aconitase gene in E. uhligi is differentially expressed during the life cycle; it is strongly expressed in the trophont, but at a low level during most developmental stages. Our results indicate that ciliates have an iron regulatory system similar to that in animals.

Immunmarkierungen konnen an Zellkulturen, ganzen Organismen (z. B. Protozoen, Nematoden), kleinen Gewebestucken oder dunnen Gewebeschnitten (Kryostatschnitte, Vibratomschnitte) durchgefuhrt und dann direkt betrachtet werden (whole mount-Immunmarkierung). Oder sie werden danach fur die hoher auflosende licht- oder die elektronenmikroskopische Untersuchung fixiert, eingebettet und geschnitten (preembedding-Technik). Alternativ erfolgt die Immunmarkierung erst am Ende des Praparationsganges an Schnitten des Paraffin- oder Kunststoff-eingebetteten Materials (Schnittmarkierung oder postembedding-Technik). Jede Technik hat, je nach Objekt und Fragestellung, bestimmte Vor- und Nachteile und erfordert eigene Praparationsschritte.

Die Bindung von Antikorpern an Antigene in Zellen und Geweben kann durch verschiedene Techniken sichtbar gemacht werden. Welche Technik anzuwenden ist, um ein jeweils optimales Ergebnis zu erhalten, hangt von verschiedenen Parametern ab (zum Beispiel Praparationstechnik, Gewebe, Antigenkonzentration, Fragestellung etc.).

Falsch-positive Signale nach einer Immunmarkierung lassen sich durch sorgfaltige Kontrollen ausschliesen. Bei indirekten Methoden sollte zum Vergleich z. B. stets mindestens ein Praparat mitgefuhrt werden, bei dem in Praimmunserum oder nur in Sekundarantikorper inkubiert wird. Daruber hinaus kann das Antikorperserum gegen homologe und heterologe Antigene praadsorbiert werden. Aus einer Vielzahl von Grunden kann es zu einer zu schwachen oder unspezifischen Markierung kommen. Tissue-Prints sowie Dot-Spot-Verfahren helfen dabei, den Einfluss der Praparationsschritte und der Inkubationsbedingungen auf die Markierbarkeit der Antigene zu uberprufen. Haufige Fehlerquellen und mogliche Abhilfen sind tabellarisch zusammengefasst.

Zarte und weiche Gewebe, wie zum Beispiel Bluten oder tierische und menschliche Gewebeproben, lassen sich am besten als Kryostatschnitte (siehe Romeis, 18. Aufl., 2010) fur Tissue-Prints verwenden.

Die Tissue-Prints werden mit Coomassie-Blau gefarbt, um die Struktur des gedruckten Gewebes erkennen zu konnen.

Durch verschiedene Verfahren konnen die ubertragenen Molekule (z. B. Proteine und Nukleinsauren aber auch kleine, diffusible Molekule wie H2O2) und Enzymaktivitaten auf den Membranen sichtbar gemacht werden. Durch Vergleiche mit der Anatomie von Schnittpraparaten ist es moglich, sie mit hoher Sensitivitat zu identifizieren und zu lokalisieren.

During leaf senescence, chloroplasts are transformed into gerontoplasts involving typical structural changes that have been revealed by electron microscopy for more than 30 years. The structural changes involved in chloroplast-to-gerontoplast transition affect the organization of the thylakoid membrane system which is progressively degraded. In parallel, the number and size of plastoglobules was observed to increase. The internal changes in the structure of chloroplasts occurring during leaf senescence are accompanied by a change from an ellipsoid to a round shape and by a reduction in volume. Recent results on Rubisco degradation involving modern cell biology approaches suggest that plastids during senescence release material including Rubisco and other stromal proteins for degradation outside the organelle. In order to get further insight into the structural changes associated with chloroplast dismantling, we have revisited the pertinent literature and furthermore analyzed the ultrastructure of chloroplasts at different stages of barley leaf senescence and under different conditions leading to yellowing of the leaves. Specific changes at the periphery of chloroplasts at certain stages during aging might be related to an exchange of material between chloroplasts and the endoplasmic reticulum. Electron microscopy cannot, however, discriminate between anterograde and retrograde vesicle movements. Electron lucent areas in the matrix of chloroplasts indicate that protein degradation occurs not only outside but also inside the organelle.

The marine, loricate ciliate Eufolliculina uhligi (Heterotrichida) has a complex life-cycle which includes a motile swarmer. The swarmer settles on the substrate, secretes a lorica and transforms into the trophic stage. This is metamorphosis. Swarmers have been induced to settle by treatment with EGTA to synchronize metamorphosis. Stomatogenesis was observed in protargol-stained cells. It can be characterized as follows: Stomatogenesis of E. uhligi (suborder Coliphorina) is compared with that of members of the suborder Heterotrichina. It is concluded that folliculinids are closely related to the Stentoridae. The separation of the Folliculinidae from the other Heterotrichida should be reconsidered.

Changes in function and composition of the photosynthetic apparatus as well as the ultrastructure of chloroplasts in mesophyll cells were analyzed in flag leaves of the high-yield barley (Hordeum vulgare) variety cv. Lomerit during senescence under field conditions in two successive years. In contrast to previous results obtained with the elder variety cv. Carina photosystem II efficiency measured by F(v)/F(m) was found to be rather stable until a very late stage of senescence. Chlorophyll a fluorescence and P700 absorbance measurements revealed that the activities of the two photosystems declined in parallel. An increase in the chlorophyll a/b ratio at a late stage of senescence was observed to coincide with a decline in the level of the Lhcb1 apoprotein of the light harvesting complex (LHC) and the level of the corresponding transcript. Ultrastructural investigations revealed the presence of gerontoplasts that have long, single or pairwise thylakoids and lack large grana stacks. It is hypothesized that the early degradation of grana thylakoids harboring the major LHC reduced the risk of photoinhibition and might be causally related to the high yield of the barley variety cv. Lomerit.

Whirly1 was shown to be dually located in chloroplasts and nucleus of the same cell. To investigate whether the protein translocates from chloroplasts to the nucleus, we inserted a construct encoding an HA-tagged Whirly1 into the plastid genome of tobacco. Although the tagged protein was synthesized in plastids, it was detected in nuclei. Dual location of the protein was confirmed by immunocytological analyses. These results indicate that the plastidial Whirly1 is translocated from the plastid to the nucleus where it affects expression of target genes such as PR1. Our results support a role of Whirly1 in plastid-nucleus communication.

Summary The morphology, behavior, trophont ultrastructure, and life cycle of the folliculinid species, Lagotia minor, is described. Lagotia minor has a simple lorica, a rounded macronucleus, broad and stiff peristomial wings, and a deep buccal cavity. The behavior of the swarmer and its mode of lorica secretion differ from these events in Eufolliculina and Metafolliculina. The ultrastructural investigations reveal further differences between the genera, e.g., in Lagotia minor the somatic dikinetids are associated with a cross-striated fiber which runs along the postciliary ribbon. The peristome of L. minor is supported from ladder-like nemadesmata which originate from opposite sides of the wings. Based on these results and on light microscopical observations on other folliculinid genera, new characters for folliculinid classification are discussed.

Der Arbeitsschutz und somit auch die Sicherheit am Arbeitsplatz besitzen heute zu Recht einen hohen Stellenwert und sollten schon im eigenen Interesse beachtet und befolgt werden.