Pavement Cells Research Articles

Toxicity of Foroozan crude oil to ornate wrasse (Thalassoma pavo, Osteichthyes, Labridae): ultrastructure and cellular biomarkers

The present study was conducted to assess the toxicity of Foroozan (Iranian crude oil) on the ornate wrasse Thalassoma pavo (Labridae) using three sub-lethal crude oil concentrations. Gills, selected as target organ being the major route of hydrocarbon uptake, were excised after 48, 96 and 192 hours and analyzed by light and both scanning and transmission electron microscopy. Cellular biomarkers of exposure and/or effect were measured in branchial tissues of T. pavo, and severe lesions such as necrosis, aneurysms and disorganization of the lamellae proportional to the Foroozan exposure periods and concentrations were observed. The main alteration was on the lamellar epithelium, which displayed an irregular profile characterized by pavement cell cytoplasmic protrusion and an alteration of the oxygen chemosensing cells, resulting in impairment of various biological activities. Nevertheless, the ability of specimens to regulate calcium homeostasis and neurotransmission, as well as balance cell turnover, suggests that the species tested to not only survive but also recover in such high crude oil dosage regimen. The identification in gills of histological and neurological changes associated with acute crude oil exposure confirms the utility of the sub-lethal toxicity tests.

The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin

Morphology and ultrastructure of the skin of Lissotriton italicus (previously named Triturus italicus) have been described in different phases of its biological cycle: larval stage, metamorphic stage and adult stage with emphasis on modifications occurring between aquatic and terrestrial adults. In the present study, light microscopy and both scanning and transmission electron microscopy were employed to analyze the histological and cytological remodelling that occurs in the skin of L. italicus during metamorphosis. The ultrastructure of the larval epidermis is arranged into three principal layers comprising an external layer of pavement cells, a basal layer and 1–3 intermediate layers consisting of Leydig cells along with accessory cells and mitochondria-rich cells. By the onset of metamorphosis, morphological changes of the skin include stratification and flattening of epidermal layers and disappearance of typical larval cells. In both aquatic and terrestrial adult phases the thin, cornified epidermis shows the same general arrangement as found in other vertebrates with an external stratum corneum and a variable number of intermediate cell layers. During the terrestrial adult phase, the skin is characterized by the presence of numerous tubercles; moreover, the lower epithelium is thicker than in the aquatic phase. Ultrastructural analysis revealed no substantial differences in the cellular composition of the skin between aquatic and terrestrial phases.

Quantitative Changes in Microtubule Distribution Correlate with Guard Cell Function in Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.

ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin-Dependent Endocytosis

Author SummaryFormation of cell polarity is a process of distributing cellular structures or molecules in an asymmetric manner. This process plays an important role in the generation of diverse cell forms and types. In plants, the quintessential hormone auxin is important for diverse physiological functions, including growth and development of cells and organs. To perform these functions, auxin must be transported and localized to specific regions within the plant. This is partially mediated by polar distribution of the PIN-FORMED (PIN) auxin efflux transporters, which transport auxin outside of the cell and allow for the directional short- and long-distance transport of auxin throughout plant tissues and organs. Although auxin itself has been implicated as a signal to regulate PIN polar distribution, how auxin does so remains to be elucidated. We previously showed that auxin promotes the generation of “puzzle-piece” polarity in leaf epidermal pavement cells, which contain interdigitated lobes and indentations, by activating the ROP (Rho-like GTPases from plants) members of the conserved Rho family of small GTPases. Here, we find that auxin-dependent local activation of ROP2 in the lobe region inhibits PIN1 internalization into the endosomal compartments (or endocytosis), leaving higher levels of PIN1 polar distribution in the lobe region. PIN1 internalization is inhibited by altering the actin cytoskeleton through the ROP2 effector protein RIC4, a protein involved in cytoskeletal remodeling. On the basis of our findings, we propose that the Rho GTPase-mediated inhibition of endocytosis of PIN1 provides a self-organizing mechanism for the polar PIN1 distribution. Rho GTPase-based inhibition of endocytosis is also important for the formation of cell polarity in animal cells. Thus, we conclude that Rho GTPase signaling to inhibit endocytosis is a common mechanism for cell polarization in multicellular organisms.

Ammonia and cortisol are transcriptional regulators of the ammonia transporter Rhcg2 in trout gill pavement cells

Ammonia is the primary nitrogenous waste excreted across the gills of fish, but the mechanisms involved have never been clearly defined. Several very recent studies have now revealed that Rh glycoproteins have a role as ammonia transporters in fish gills. Based on gene expression studies, Rhcg2 appears to have a main role in this process in the pavement cells of the freshwater trout gill. Little is known however, about how these Rh genes are regulated. Using cultured trout pavement cells we have examined some of the possible transcriptional regulatory mechanisms of Rhcg2. We show that ammonia alone increases the level of Rhcg2 transcripts in the pavement cells while cortisol and 11‐deoxycorticosterone have a potentiating effect on this transcriptional response. We focus on cAMP and mitogen‐activated protein kinase pathways as potential signaling pathways involved in the regulation of Rhcg2 (NSERC Discovery).

The tomato SlIAA15 is involved in trichome formation and axillary shoot development

The Aux/IAA genes encode a large family of short-lived proteins known to regulate auxin signalling in plants. Functional characterization of SlIAA15, a member of the tomato (Solanum lycopersicum) Aux/IAA family, shows that the encoded protein acts as a strong repressor of auxin-dependent transcription. The physiological significance of SlIAA15 was addressed by a reverse genetics approach, revealing that SlIAA15 plays multiple roles in plant developmental processes. The SlIAA15 down-regulated lines display lower trichome number, reduced apical dominance with associated modified pattern of axillary shoot development, increased lateral root formation and decreased fruit set. Moreover, the leaves of SlIAA15-inhibited plants are dark green and thick, with larger pavement cells, longer palisade cells and larger intercellular space of spongy mesophyll cells. The SlIAA15-suppressed plants exhibit a strong reduction in type I, V and VI trichome formation, suggesting that auxin-dependent transcriptional regulation is required for trichome initiation. Concomitant with reduced trichome formation, the expression of some R2R3 MYB genes, putatively involved in the control of trichome differentiation, is altered. These phenotypes uncover novel and specialized roles for Aux/IAAs in plant developmental processes, clearly indicating that members of the Aux/IAA gene family in tomato perform both overlapping and specific functions.

Dynamic analysis of epidermal cell divisions identifies specific roles for COP10 in Arabidopsis stomatal lineage development

Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.

Molecular diagnostics of Lemon Myrtle (Backhousia citriodora versus Leptospermum citratum)

‘Lemon Myrtle’ is becoming increasingly popular in Europe both for use in cuisine and phytotherapy. However, this common name covers two completely different species, Backhousia citriodora F. Muell. and Leptospermum citratum Challinor, Cheel & A.R.Penfold. These species differ with respect to secondary compounds and even can cause, if mixed up and applied in high dose, toxic effects. We describe how the two species can be discriminated microscopically making use of differences in the morphology of leaf pavement cells and the relative size of palisade parenchyma. Based on the large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL) as molecular marker, the phylogenetic position of the two species within the Myrtaceae could be clarified. This sequence information was used to develop a simple assay to discriminate the two species even in dried and highly fragmented mixtures as typically occurring in commercial samples. This assay utilises the occurrence of single-nucleotide exchanges between those species that produce different fragments when the rbcL amplificates are restricted with Sac II.

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

BackgroundThe positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport.ResultsSimultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils.ConclusionsWe conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes.

Exit from Proliferation during Leaf Development in Arabidopsis thaliana: A Not-So-Gradual Process

Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana, we showed that the cell-cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.

Permeability properties of the teleost gill epithelium under ion-poor conditions

Permeability properties of the goldfish gill epithelium were examined in vivo and in vitro following exposure to ion-poor water (IPW) conditions. In gill tissue of IPW-acclimated goldfish, transcript abundance of tight junction (TJ) proteins occludin, claudin-b, -d, -e, -h, -7, and -8d increased, whereas ZO-1 and claudin 12 mRNA decreased and claudin-c was unaltered. In association with these changes, TJ depth increased among gill pavement cells (PVCs) and gill PVCs and mitochondria-rich cells (MRCs). PVC and MRC gill cell fractions were isolated using Percoll. Transcripts encoding for occludin, claudin-b, -c, -d, -e, -h, -7, -8d, -12, and ZO-1 were present in both fractions. After IPW acclimation, occludin, claudin-b and -e, and ZO-1 mRNA abundance increased in both fractions. In contrast, claudin-8d mRNA abundance increased in PVCs only while claudin-h decreased in MRCs. Gill permeability was examined using primary cultured goldfish PVC epithelia supplemented with serum derived from IPW-acclimated goldfish. IPW serum supplementation increased transepithelial resistance, reduced [(3)H]PEG-4000 permeability, and enhanced epithelial integrity during in vitro IPW exposure. IPW serum increased mRNA abundance of occludin, claudin-8d and -e in vitro. Using small interfering RNA, we found that occludin abundance was decreased in cultured gill epithelia, resulting in an increase in [(3)H]PEG-4000 flux. As occludin increased in the gills of IPW-acclimated fish as well as cultured gill epithelia exposed to IPW serum, results suggest that occludin is a barrier-forming TJ protein in fish gill epithelia. These studies support the idea that TJ proteins play an important role in regulating gill permeability in IPW.

細胞膜におけるオーキシン受容と PIN1 局在制御を介したフィードバック機構によるシロイヌナズナ表皮細胞形態形成の制御機構の解析

細胞膜におけるオーキシン受容と PIN1 局在制御を介したフィードバック機構によるシロイヌナズナ表皮細胞形態形成の制御機構の解析

Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis

Arabidopsis NSN1 encodes a nucleolar GTP-binding protein and is required for flower development. Defective flowers were formed in heterozygous nsn1/+plants. Homozygous nsn1 plants were dwarf and exhibited severe defects in reproduction. Arrests in embryo development in nsn1 could occur at any stage of embryogenesis. Cotyledon initiation and development during embryogenesis were distorted in nsn1 plants. At the seedling stage, cotyledons and leaves of nsn1 formed upward curls. The curled leaves developed meristem-like outgrowths or hyperplasia tissues in the adaxial epidermis. Long and enlarged pavement cells, characteristic of the abaxial epidermis of wild type plants, were found in the adaxial epidermis in nsn1 leaves, suggesting a disoriented leaf polarity in the mutant. The important role of NSN1 in embryo development and leaf differentiation was consistent with the high level expression of the NSN1 gene in the developing embryos and the primordia of cotyledons and leaves. The CLAVATA 3 (CLV3) gene, a stem cell marker in the Arabidopsis shoot apical meristem (SAM), was expressed in expanded regions surrounding the SAM of nsn1 plants, and induced ectopically in the meristem-like outgrowths in cotyledons and leaves. The nsn1 mutation up-regulated the expression levels of several genes implicated in the meristem identity and the abaxial cell fate, and repressed the expression of other genes related to the specification of cotyledon boundary and abaxial identity. These results demonstrate that NSN1 represents a novel GTPase required for embryogenesis, leaf development and leaf polarity establishment in Arabidopsis.

Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon.

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.

Molecular Profiling of Stomatal Meristemoids Reveals New Component of Asymmetric Cell Division and Commonalities among Stem Cell Populations inArabidopsis

The balance between maintenance and differentiation of stem cells is a central question in developmental biology. Development of stomata in Arabidopsis thaliana begins with de novo asymmetric divisions producing meristemoids, proliferating precursor cells with stem cell-like properties. The transient and asynchronous nature of the meristemoid has made it difficult to study its molecular characteristics. Synthetic combination of stomatal differentiation mutants due to loss- or gain-of-function mutations in SPEECHLESS, MUTE, and SCREAM create seedlings with an epidermis overwhelmingly composed of pavement cells, meristemoids, or stomata, respectively. Through transcriptome analysis, we define and characterize the molecular signatures of meristemoids. The reporter localization studies of meristemoid-enriched proteins reveals pathways not previously associated with stomatal development. We identified a novel protein, POLAR, and demonstrate through time-lapse live imaging that it exhibits transient polar localization and segregates unevenly during meristemoid asymmetric divisions. The polar localization of POLAR requires BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE. Comparative bioinformatic analysis of the transcriptional profiles of a meristemoid with shoot and root apical meristems highlighted cytokinin signaling and the ERECTA family receptor-like kinases in the broad regulation of stem cell populations. Our work reveals molecular constituents of stomatal stem cells and illuminates a common theme among stem cell populations in plants.

Model-Based Analysis of Arabidopsis Leaf Epidermal Cells Reveals Distinct Division and Expansion Patterns for Pavement and Guard Cells

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.

Structure of Lamellae and Chloride Cell in the Gill of <i>Alosa Caspio Caspio</i> (Clupeidae, Teleostei)

As a surface for respiratory gas diffusion, ion regulation and waste excretion, gill epithelium of Alosa Caspio Caspio has been investigated. Problem statement: Surely, morphological study of gill cells is the main route for giving the basic data which can be inductive for physiological and pathological investigations. Approach: Gills were removed from Alosa Caspio which is from euryhaline species and fixed in freshly prepared glutaraldehyde and osmium tetroxide. Thin sections were stained in Uranyl acetate-lead citrate and examined under LEO transmission electron microscope. Results: Flattened respiratory lamellae extend in two rows from the sides of the filaments. The pavement cell is the major cell covering the thin epithelia of lamellae, but mucous cell and chloride cell also can be seen on lamellar epithelia, however, most of them are observed in filamental epithelia. The other cell forms the wall of capillaries in lamellae is Pillar cell. Chloride cells which are as single cells without complex junction, are surrounded by the pavement cells, have high number of large mitochondria and extensive membranous tubular system, with a free and smooth surface. There is one type of chloride cell with a light cytoplasm. Conclusion: The main ultrastructure aspects of the major cells are similar in teleosts, although some features such as the localization of chloride cell on lamellae, one type of cell, small crypts and the smooth surface are special features of this cell in Alosa Caspio that resemble this cell to freshwater species.

Impact of Sewage Effluents on Osmoregulation in a Freshwater Teleost, Anabas testudineus

This study investigated the impact of sewage effluents of the polluted river, Parvathyputhenar in Trivandrum city, Kerala, India, on the activities of osmoregulatory enzymes such as Na + /K + and Ca 2+ ATPases, the concentration of sodium and potassium ion content in the gill and on the chloride cells (CCs) and pavement cells (PCs) that regulate ions transport in the gill epithelium of a freshwater fish. The results indicate a significant (P<0.05) decrease in the activity of branchial Na + /K + ATPase and Ca 2+ ATPase in the fish exposed to sewage effluents for 7, 14 and 28 days and the fish caught from the polluted river. When the fish caught from the polluted river were kept in normal pond water in the laboratory for 30, 60 and 90 days, the enzyme's activities were gradually increased and almost restored to the control level. Scanning electron microscopic analysis of gill epithelium showed noticeable changes in the surface area morphology of CCs and PCs in the fish exposed to sewage effluents. Exposure to the sewage effluents drastically altered the size and characteristic finger print pattern of PCs and also reduced number of CCs in the gill epithelium. As the ATPases play an important role in maintenance of functional integrity of gill epithelium it is suggested that measurement of the activities of ATPases may be used as a biomarker of exposure to sewage effluents. This work is highly pertinent in the context of increased level and effect of endocrine disrupting chemicals present in the aquatic systems, which are increasing day by day.

Changes in the cellular composition of the gill epithelium during the life cycle of a nonparasitic lamprey: functional and evolutionary implications

Several lamprey species form pairs, comprising an anadromous parasitic species and a derivative nonparasitic species that neither leaves fresh water nor feeds as an adult. This paper provides the first description of the radical changes undergone by the cellular composition of the gill epithelium during the major phases in the life cycle of a nonparasitic lamprey (American brook lamprey, Lethenteron appendix (DeKay, 1842) (= Lampetra appendix (DeKay, 1842)) and discusses their potential functional and evolutionary significance. The gill epithelium of the larva of L. appendix contains ammocoete mitochondrion-rich cells (AMRCs), intercalated mitochondrion-rich cells, and pavement cells, as does that of the larva of anadromous parasitic species which likewise lives in fresh water. By the completion of metamorphosis, the AMRCs have disappeared and well-developed chloride cells have been produced, the latter cell type being essential for osmoregulation by its closely related anadromous species in hypertonic environments. By the attainment of sexual maturity, the chloride cells have been lost. Such changes in the timing of chloride cell representation could help account for the ability of some metamorphosing, but not mature individuals of another nonparasitic species ( Lampetra planeri (Bloch, 1784)), to osmoregulate in up to 70% of seawater. The well-developed chloride cells in the nonparasitic L. appendix represent the retention of an ancestral character.

Bellis perennis: a useful tool for protein localization studies

Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar "Galaxy red") which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.