Ectopic divisions in vascular and ground tissues of Arabidopsis thaliana result in distinct leaf venation defects.

Leaf venation patterns vary considerably between species and between leaves within a species. A mechanism based on canalization of auxin transport has been suggested as the means by which plastic yet organized venation patterns are generated. This study assessed the plasticity of Arabidopsis thaliana leaf venation in response to ectopic ground or procambial cell divisions and auxin transport inhibition (ATI). Ectopic ground cell divisions resulted in vascular fragments between major veins, whereas ectopic procambial cell divisions resulted in additional, abnormal vessels along major veins, with more severely perturbed lines forming incomplete secondary and higher-order venation. These responses imply limited vascular plasticity in response to unscheduled cell divisions. Surprisingly, a combination of ectopic ground cell divisions and ATI resulted in massive vascular overgrowth. It is hypothesized that the vascular overproduction in auxin transport-inhibited wild-type leaves is limited by simultaneous differentiation of ground cells into mesophyll cells. Ectopic ground cell divisions may negate this effect by providing undifferentiated ground cells that respond to accumulated auxin by differentiation into vascular cells.

Dynamic analyses of the expression of the HISTONE::YFP fusion protein in arabidopsis show that syncytial endosperm is divided in mitotic domains.

During early seed development, nuclear divisions in the endosperm are not followed by cell division, leading to the development of a syncytium. The simple organization of the Arabidopsis endosperm provides a model in which to study the regulation of the cell cycle in relation to development. To monitor nuclear divisions, we constructed a HISTONE 2B::YELLOW FLUORESCENT PROTEIN gene fusion (H2B::YFP). To validate its use as a vital marker for chromatin in plants, H2B::YFP was expressed constitutively in Arabidopsis. This enabled the observation of mitoses in living root meristems. H2B::YFP was expressed specifically in Arabidopsis syncytial endosperm by using GAL4 transactivation. Monitoring mitotic activity in living syncytial endosperm showed that the syncytium was organized into three domains in which nuclei divide simultaneously with a specific time course. Each mitotic domain has a distinct spatiotemporal pattern of mitotic CYCLIN B1;1 accumulation. The polar spatial organization of the three mitotic domains suggests interactions between developmental mechanisms and the regulation of the cell cycle.

Technical advance: an aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos.

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.

Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root.

Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.

Hyperpolarisation-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots.

Calcium currents across the plasma membrane of plant cells allow transduction of environmental signals as well as nutritive calcium uptake. Using transgenic Arabidopsis plants with cell-specific expression of green fluorescent protein (GFP), we analyzed whole cell calcium currents in epidermal cells of the rapidly growing root apex, mature epidermal cells, cortical and epidermal cells from the elongation zone, and mature pericycle cells. In cells only from the rapidly growing root apex, a hyperpolarization-activated calcium current was identified. This current was irreversibly inhibited by 10 microM Al3+, as well as being inhibited by 1 mM Co2+ and 100 microM verapamil. In no cells could a depolarisation-activated current be attributed to calcium influx. In the growing root apex, the hyperpolarization-activated calcium current may function to allow constitutive uptake of calcium for rapid cell division and elongation.

Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain.

Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.

Design of highly specific cytotoxins by using trans-splicing ribozymes.

We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.

Trans-splicing ribozymes for targeted gene delivery.

Ribozymes are potential tools for genetic manipulation, and various naturally occurring catalytic RNAs have been dissected and used as the basis for the design of new endoribonuclease activities. While such cleaving ribozymes may work well in vitro, they have not proved to be routinely effective in depleting living cells of the chosen target RNA. Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo. We have designed modified trans-splicing ribozymes with improved biological activity. These allow accurate splicing of a new 3' exon sequence into a chosen site within a target RNA, and in frame fusion of the exon can result in expression of a new gene product. These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition. Both modifications are required for improved biological activity of the ribozymes. Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient trans-splicing can allow the useful expression of a new gene activity, dependent on the presence of a chosen RNA. We have targeted trans-splicing ribozymes against mRNAs of chloramphenicol acetyltransferase, human immunodeficiency virus, and cucumber mosaic virus, and demonstrated trans-splicing and delivery of a marker gene in Escherichia coli cells. The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types.

GFP variants for multispectral imaging of living cells.

Unlike enzyme markers, green fluorescent protein can be visualized at high resolution in living cells using confocal microscopy. The images are not prone to fixation or staining artifacts, and can be of exceptional clarity. Moreover, the activities of living cells, such as cytoplasmic streaming, are clearly evident during microscopy. Ordinarily, movement within a sample is a nuisance, placing constraints on the use of sometimes lengthy techniques for noise reduction during confocal microscopy, such as frame averaging. However, it is possible to monitor dynamic events by time-lapse confocal microscopy, and this combination of a vital fluorescent reporter with high-resolution optical techniques shows much promise for use in cell biological and physiological experiments. Genetic systems such as that of Arabidopsis provide a large resource of potentially informative mutants, and there has been much recent improvement in techniques for determining the molecular basis of a particular phenotype. The use of fluorescent proteins will provide further tools for examining the biology of mutant cells. The precision with which particular cellular structures can be decorated with GFP and the ease with which subcellular traffic can be monitored indicate that this approach will be very useful for cell biological and physiological observations, particularly for detailed examination of plant mutant phenotypes.

Miranda mediates asymmetric protein and RNA localization in the developing nervous system.

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR. Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.

Positional information in root epidermis is defined during embryogenesis and acts in domains with strict boundaries.

BACKGROUND: Cell position rather than cell lineage governs most aspects of development in plants. However, the nature and the origin of positional information remains elusive. Animal epidermal patterning relies in many cases on positional information provided by cell-cell communication. The epidermal layer of the Arabidopsis root is made of alternating files of two cell types and thus presents a simple pattern to study positional mechanisms. RESULTS: Clonal analysis of the root epidermis in combination with molecular and morphological markers has shown that cell fate is determined by position relative to the underlying cell layer, the cortex. The epidermal pattern appears to be organised during embryogenesis. Fate is not fixed in the developing root, though, as cells that move into a position previously occupied by neighbour cells ablated using laser microsurgery change fate. In contrast, cell fate is not altered when communication with living neighbour cells is impaired. Precise mapping of the influence of the position of extracellular cues on cell fate has shown that domains of positional information are organised with well-defined boundaries. CONCLUSIONS: Cell-fate specification in the root epidermis relies on positional information that is organised in stable domains with sharp boundaries. The epidermal pattern is defined during embryogenesis and positional information remains active in the root until the initiation of cell morphogenesis. The origin of some positional cues might be extracellular.

Stomata patterning on the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning.

Stomata complexes are epidermal specialized structures typical of the upper aerial part of plants (shoot). In the model plant Arabidopsis thaliana, we show that in the hypocotyl (the) junction between the shoot and the root), stomata are organized according to a clear pattern reminiscent of the root epidermis pattern. Although stomata complexes are typical of the shoot epidermis, their pattern on the hypocotyl is under the control of genes involved in root epidermis patterning. Moreover, we have isolated a GFP marker line for the hypocotyl epidermal cells which do not differentiate stomata complexes. In this line the root and the hypocotyl epidermal patterns are similar. Our data support the existence of interactions between developmental mechanisms involved in the control of the apical/basal polarity and the radial symmetry of the plant body.

Following cell fate in the living mouse embryo.

It has been difficult to follow many of the dramatic changes in cell fate and cell migration during mouse development. This is because there has been no enduring marker that would allow cells to be recognised in the living embryo. We believe that we have overcome this problem by developing a novel form of green fluorescent protein, named MmGFP, that proves to be easily visible and non toxic to mouse cells and does not perturb embryogenesis. We show that synthetic mRNA encoding MmGFP can be injected into blastomeres to follow the fate of their progeny during preimplantation development. We have made a stable embryonic stem cell line that expresses MmGFP and introduced these fluorescent cells into mouse embryos. For the first time, we have been able to follow the fate of embryonic stem cells in living embryos and to observe directly the contribution of these cells to distinct lineages of the postimplantation embryo. This approach should lead to a more complete description of the dynamics of cell fate in the mouse.

Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of fluorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly fluorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly fluorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.

Mutations that suppress the thermosensitivity of green fluorescent protein.

BACKGROUND: The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently attracted great interest as the first example of a cloned reporter protein that is intrinsically fluorescent. Although successful in some organisms, heterologous expression of GFP has not always been straight forward. In particular, expression of GFP in cells that require incubation temperatures around 37 degrees C has been problematic. RESULTS: We have carried out a screen for mutant forms of GFP that fluoresce more intensely than the wild-type protein when expressed in E. coli at 37 degrees C. We have characterized a bright mutant (GFPA) with reduced sensitivity to temperature in both bacteria and yeast, and have shown that the amino acids substituted in GFPA act by preventing temperature-dependent misfolding of the GFP apoprotein. We have shown that the excitation and emission spectra of GFPA can be manipulated by site-directed mutagenesis without disturbing its improved folding characteristics, and have produced a thermostable folding mutant (GFP5) that can be efficiently excited using either long-wavelength ultraviolet or blue light. Expression of GFP5 results in greatly improved levels of fluorescence in both microbial and mammalian cells cultured at 37 degrees C. CONCLUSIONS: The thermotolerant mutants of GFP greatly improve the sensitivity of the protein as a visible reporter molecule in bacterial, yeast and mammalian cells. The fluorescence spectra of these mutants can be manipulated by further mutagenesis without deleteriously affecting their improved folding characteristics, so it may be possible to engineer a range of spectral variants with improved tolerance to temperature. Such a range of sensitive reporter proteins will greatly improve the prospects for GFP-based applications in cells that require relatively high incubation temperatures.

An indelible lineage marker for Xenopus using a mutated green fluorescent protein.

We describe the use of a DNA construct (named GFP.RN3) encoding green fluorescent protein as a lineage marker for Xenopus embryos. This offers the following advantages over other lineage markers so far used in Xenopus. When injected as synthetic mRNA, its protein emits intense fluorescence in living embryos. It is non-toxic, and the fluorescence does not bleach when viewed under 480 nm light. It is surprisingly stable, being strongly visible up to the feeding tadpole stage (5 days), and in some tissues for several weeks after mRNA injection. We also describe a construct that encodes a blue fluorescent protein. We exemplify the use of this GFP.RN3 construct for marking the lineage of individual blastomeres at the 32- to 64-cell stage, and as a marker for single transplanted blastula cells. Both procedures have revealed that the descendants of one embryonic cell can contribute single muscle cells to nearly all segmental myotomes rather than predominantly to any one myotome. An independent aim of our work has been to follow the fate of cells in which an early regulatory gene has been temporarily overexpressed. For this purpose, we co-injected GFP.RN3 mRNA and mRNA for the early Xenopus gene Eomes, and found that a high concentration of Eomes results in ectopic muscle gene activation in only the injected cells. This marker may therefore be of general value in providing long term identification of those cells in which an early gene with ephemeral expression has been overexpressed.

Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy.

The cDNA for the green fluorescent protein (GFP) of Aequorea victoria has been expressed in transformed cells of Saccharomyces cerevisiae and the recombinant GFP isolated. Protonation and deprotonation of the cloned and purified GFP produced major effects on its spectral absorption characteristics with an increase in pH enhancing the fluorescence emission of the GFP more than twofold. Finally, molecular characterisation of GFP by fluorescence correlation microscopy in a minimal target volume of 1 fL yielded a translational diffusion coefficient (DT) of 8.7 x 10(-7) cm2.sec-1, equivalent to a Stokes radius of 2.82nm for a monodisperse globular protein of 27kDa.

Evolution and replication of tobacco ringspot virus satellite RNA mutants.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.