ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three‐dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam‐scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam‐scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.     

MORPHOLOGICAL CHANGES IN MITOCHONDRIAL AND CHLOROPLAST NUCLEOIDS AND MITOCHONDRIA DURING THE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELL CYCLE1

Morphological changes in the organellar nucleoids and mitochondria of living Chlamydomonas reinhardtii Dang were examined during the cell cycle under conditions of 12:12 light:dark. The nucleoids were stained with SYBR‐Green I, and the mitochondria were stained with 3,3‐dihexyloxacarbocyanine iodide. An mocG33 mutant, which contains one large chloroplast nucleoid throughout the cell cycle, was used to distinguish between the mitochondrial and chloroplast nucleoids. Changes in the total levels of organellar DNA levels were assessed by real‐time PCR. Each of the G₁, S, M, and Smt,cp phases was estimated. At the start of the light period, the new daughter cells were in G₁ and contained about 30 mitochondrial and 10 chloroplast nucleoids, which were dispersed and had diameters of 0.1 and 0.2 μm, respectively. During the G₁ phase of the light period, and at the start of the S phase, both nucleoids formed short thread‐like or bead‐like structures, probably divided, and increased continuously in number, concomitantly with DNA synthesis. The nucleoids probably became smaller due to the decrease in DNA of each particle and were indistinguishable. The cells in the S and M phases contained extremely high numbers of scattered nucleoids. However, in the G₁ phase of the dark period, the nucleoids again formed short thread‐like or bead‐like structures, probably fused, and decreased in number. The mitochondria appeared as tangled sinuous structures that extended throughout the cytoplasm and resembled a single large mitochondrion. During the cell cycle, the numbers of mitochondrial nucleoids and sinuous structures varied relative to one another.     

Inheritance pattern of chloroplast DNA is correlated with gamete types based on sex-specific arrangement of the cell fusion site in Caulerpa (Ulvophyceae, Chlorophyta)

Using field emission scanning electron microscopy (FE‐SEM) and fluorescence microscopy, the respective relationships between the arrangement of the gamete cell‐fusion site and the inheritance pattern of chloroplast DNA (cp‐DNA) were studied for Caulerpa brachypus Harvey, C. okamurae Weber‐van Bosse, C. racemosa (Forsskål) J. Agardh var. laete‐virens (Montagne) Weber‐van Bosse, and C. serrulata (Forsskål) J. Agardh var. serrulata f. lata (Weber‐van Bosse) Tseng. The eyespot of the biflagellate gamete was visualized using FE‐SEM. The female gamete, but not the male, has one eyespot on the cell body posterior. In most mating pairs, the female gamete is fused at the anterior left side of the eyespot and the male gamete at a cell surface that is perpendicular to the plane of the flagellar beat when both gametes are mixed. Then, the inheritance pattern of cp‐DNA was observed using fluorescence microscopy after staining with 4′6‐diamidino‐2‐phenylindole. Male and female gametes have one cell nucleus and one chloroplast each. Chloroplasts of the female gamete usually contain 1–11 spherical or rod‐shaped nucleoids. In contrast, nucleoids are not usually detected in the male gamete’s chloroplast. After mixing male and female gametes, the male gamete without nucleoids and female gametes with nucleoids are always associated at the lateral side and become planozygotes. Such a correlation between the arrangement of the cell fusion site and the inheritance pattern of cp‐DNA was found in another member of Caulerpales, Bryopsis maxima Okamura. These results suggest the possibility that the arrangement of the cell fusion site in the gamete is not determined randomly regardless of sex, but is rather correlated with specific mating types. The relation of these results to those for Chlamydomonas is discussed.     

Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.     

Moving pictures of DNA released upon lysis from bacteria,chloroplasts, and mitochondria

Summary Cells and organelles suspended in gelled agarose agarose were lysed with detergent and protease, stained with ethidium bromide and their DNA was observed by fluorescence microscopy. The migration of individual DNA molecules during electrophoresis on a microscope slide was recorded on video tape so that moving pictures could be analyzed. The DNA from lysed bacteria (Escherichia coli andAgrobacterium tumefaciens) appeared as a rosette of at least twenty loops of varying size, whereas that from bacterial spheroplasts (E. coli andPseudomonas aeruginosa) appeared as circular forms or rods with many fine filaments of RNA extending toward the anode. The DNA from chloroplasts of watermelon (Citrullus vulgaris) and pea (Pisum sativum) did not appear as a rosette of loops. Many or most of the chloroplast DNA molecules per lysed chloroplast were immobile in the electric field, as if in circular form hooked on agarose fibers. The amount of DNA-fluorescence per watermelon mitochondrial particle was much less than that found for either chloroplasts or bacteria. The appearance of the mitochondrial DNA during electrophoresis was that of linear molecules, no obviously circular forms were evident and no rosette structures were observed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - kb kilobase pairs - mtDNA mitochondrial DNA - PFGE pulsed-field gel electrophoresis     

Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse-labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several-hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane-bound structures, which then interacted with Golgi-derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.     

Discovery of a new RNA-containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy). Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ‐H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ‐H2AX a round, electron‐dense nuclear structure was found, which was hitherto not identified in UV‐stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy‐loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV‐induced nuclear structure contains a high amount of RNA. Conclusions. Because the UV‐induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well‐known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV‐induced apoptotic pathway.     

MULTIPLE FtsZ RING FORMATION AND REDUPLICATED CHLOROPLAST DNA IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) UNDER PHOSPHATE‐ENRICHED CULTURE1

We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate‐limited (no β‐glycerophosphate) and phosphate‐enriched (2 mM β‐glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote‐derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate‐enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate‐enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate‐enriched than in phosphate‐limited culture and decreased in cells cultured in phosphate‐enriched medium containing 5‐fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate‐enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.     

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane,is involved in thylakoid biogenesis

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T‐DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale‐green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.     

Effect of reddening of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) leaves on the ultrastructure of mesophyll cells

The ultrastructure of cotton leaves, exhibiting reddening as symptom of physiological disorder, was examined by means of transmission electron microscopy. Osmiophilisation of the membrane compartment was established. Massive agglomerations on the tonoplast in the vacuole of cells under the adaxial epidermis were observed, and were referred to as electron-dense osmiophilic substance, most probably of anthocyanin nature. In chloroplast stroma a zone of low electron density enclosing numerous osmiophilic aggregations of unclear chemical character was differentiated. Fragmentation and severe destruction of thylakoids in chloroplasts of reddening cotton leaves was not detected.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

Localization of HPV-16 DNA sequence in CaSki cells by electron microscopic hybridocytochemistry.

We investigated the HPV-16 DNA sequence in the CaSki cervical carcinoma cell line by electron microscopic hybridocytochemistry using biotinylated HPV-16/18 probes. At the light microscopic level, reaction product of hybridized HPV-16 DNA sequence was not seen in the cytoplasm but appeared as spots or rods randomly distributed in the nuclei. By electron microscopy, reaction product was seen aggregated in several regions in the nuclei. Most of the stained areas did not reveal particular architecture but showed part of the chromatin structure. In other nuclei, reaction product was observed to be associated with strings of loop-like structure, and some stained loops were seen to be connected directly to the nuclear filamentous chromatin structure. The skeletonized images of hybridized HPV-16 DNA in the nuclei were illustrated by computerized image analysis. In conclusion, we have demonstrated the HPV-16 DNA sequence in the nuclei of CaSki cells by electron microscopy. The identification of stained areas localized only in the chromatin suggests an integrated form of HPV-16 DNA sequence in the cells. This method could be used to identify an integrated or episomal form of viral DNA in the virus-containing cells.     

A Carrot Proliferation Disease Associated with Rickettsia-like Organisms in the Czech Republic

Carrot plants showing severe proliferation symptoms were observed in East Bohemia in 1996. Blue‐while fluorescent areas were observed under fluorescence microscope in sieve tube cells of symptomatic plants, but not in healthy ones. Scanning electron microscopy revealed the presence of elongated structures in the phloem sieve elements of diseased carrots. Transmission electron microscopy revealed rickettsia‐like organisms present in sieve tube elements of symptomatic plants. The diameter of the bodies ranged from about 0.2‐0.3μm. and the length of the elongated forms reached 2.2 μm. No other organisms were detected. This is the first report of infection by single rickettsia‐like organisms on carrot plants.     

Re-examination of ultrastructures of the stellate chloroplast organization in brown algae: Structure and development of pyrenoids

Some taxa of brown algae have a so‐called ‘stellate’ chloroplast arrangement composed of multiple chloroplasts arranged in a stellate configuration, or else a single chloroplast with radiating lobes. The fine structures of chloroplasts and pyrenoids have been studied, but the details of their membrane configurations as well as pyrenoid ontogeny have not been well understood. The ultrastructure of the single stellate chloroplast in Splachnidium rugosum and Scytothamnus australis were re‐examined in the present study, as well as the stellate arrangement of chloroplasts in Asteronema ferruginea and Asterocladon interjectum, using freeze‐substitution fixation. It was confirmed that the chloroplast envelope invaginated into the pyrenoid in Splachnidium rugosum, Scytothamnus australis and Asteronema ferruginea, but chloroplast endoplasmic reticulum (CER) remained on the surface of the chloroplast. The space between the invaginated chloroplast envelope and CER was filled with electron‐dense material. In Asteronema ferruginea, CER surrounding each pyrenoid was closely appressed to the neighboring CER over the pyrenoids, so that the chloroplasts formed a stellate configuration; however, in the apical cells chloroplasts formed two or more loose groups, or were completely dispersed. The pyrenoids of Asterocladon interjectum did not have any invagination of the chloroplast envelope, but a unique membranous sac surrounded the pyrenoid complex and occasionally other organelles (e.g. mitochondria). Immunolocalization of β‐1,3‐glucans showed that the membranous sac in Asterocladon interjectum did not contain photosynthetic products such as chrysolaminaran. Observations in the dividing cells of Splachnidium rugosum and Scytothamnus australis indicated that the pyrenoid in the center of the chloroplast enlarged and divided into two before or during chloroplast division.     

HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co‐culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel‐cultured cardiomyocytes, areas of high co‐localization between HSP90 and eNOS and co‐localization coefficients, which indicated the highest correlation with respect to the other substrates. Conclusions. Our three‐dimensional adult cardiomyocyte cultures are suitable for both analysing cell—ECM interactions at electron and confocal microscopy levels and monitoring micro‐environment impact on cardiomyocyte phenotype.     

Nutlet micromorphology of Iranian Nepeta (Lamiaceae) species

To evaluate nutlets characteristics for systematic relationships, a comprehensive morphological and micro‐morphological study of the nutlets of 16 Iranian taxa of Nepeta (Lamiaceae) was conducted using scanning electron microscopy (SEM). Differences in surface ornamentation, size, shape and color were observed between the species. The studied taxa were categorized in two basic types based on surface ornamentation: non‐sculptured (smooth‐type) and sculptured nutlets. The non‐sculptured nutlets type could be further divided into four sub‐types, including smooth, granulate, undulate‐cellular and reticulate. In the sculptured nutlets, three subtypes of tuberculate, tuberculate‐cellular and verrucate were recognized. The shape of nutlets were described as oblong, ovoid‐oblong, oblong‐linear, to elliptic‐oblong and their size range are 1.2–2.0 mm in length and 0.5–2.0 mm in width. Based on micrographs, the areole form and location were defined as bi‐lobed, straight, basal, sub‐basal or lateral. Nutlet micromorphological characteristics such as surface ornamentation can be useful for classification and identification of e.g. medicinal species of Nepeta in Iran.