Jim Haseloff

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.

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal.

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.

Shoot Na+ Exclusion and Increased Salinity Tolerance Engineered by Cell Type-Specific Alteration of Na+ Transport in Arabidopsis

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.

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.

Gibberellin Signaling in the Endodermis Controls Arabidopsis Root Meristem Size

Plant growth is driven by cell proliferation and elongation. The hormone gibberellin (GA) regulates Arabidopsis root growth by controlling cell elongation, but it is currently unknown whether GA also controls root cell proliferation. Here we show that GA biosynthetic mutants are unable to increase their cell production rate and meristem size after germination. GA signals the degradation of the DELLA growth repressor proteins GAI and RGA, promoting root cell production. Targeting the expression of gai (a non-GA-degradable mutant form of GAI) in the root meristem disrupts cell proliferation. Moreover, expressing gai in dividing endodermal cells was sufficient to block root meristem enlargement. We report a novel function for GA regulating cell proliferation where this signal acts by removing DELLA in a subset of, rather than all, meristem cells. We suggest that the GA-regulated rate of expansion of dividing endodermal cells dictates the equivalent rate in other root tissues. Cells must double in size prior to dividing but cannot do so independently, because they are physically restrained by adjacent tissues with which they share cell walls. Our study highlights the importance of probing regulatory mechanisms linking molecular- and cellular-scale processes with tissue and organ growth responses.

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.

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.

The NAC Domain Transcription Factors FEZ and SOMBRERO Control the Orientation of Cell Division Plane in Arabidopsis Root Stem Cells

Because plant cells do not migrate, cell division planes are crucial determinants of plant cellular architecture. In Arabidopsis roots, stringent control of cell divisions leads to a virtually invariant division pattern, including those that create new tissue layers. However, the mechanisms that control oriented cell divisions are hitherto poorly understood. Here, we reveal one such mechanism in which FEZ and SOMBRERO (SMB), two plant-specific NAC-domain transcription factors, control the delicately tuned reorientation and timing of cell division in a subset of stem cells. FEZ is expressed in root cap stem cells, where it promotes periclinal, root cap-forming cell divisions. In contrast, SMB negatively regulates FEZ activity, repressing stem cell-like divisions in the root cap daughter cells. FEZ becomes expressed in predivision stem cells, induces oriented cell division, and activates expression of its negative regulator, SMB, thus generating a feedback loop for controlled switches in cell division plane.

Diarch Symmetry of the Vascular Bundle in Arabidopsis Root Encompasses the Pericycle and Is Reflected in Distich Lateral Root Initiation

The outer tissues of dicotyledonous plant roots (i.e. epidermis, cortex, and endodermis) are clearly organized in distinct concentric layers in contrast to the diarch to polyarch vascular tissues of the central stele. Up to now, the outermost layer of the stele, the pericycle, has always been regarded, in accordance with the outer tissue layers, as one uniform concentric layer. However, considering its lateral root-forming competence, the pericycle is composed of two different cell types, with one subset of cells being associated with the xylem, showing strong competence to initiate cell division, whereas another group of cells, associated with the phloem, appears to remain quiescent. Here, we established, using detailed microscopy and specific Arabidopsis thaliana reporter lines, the existence of two distinct pericycle cell types. Analysis of two enhancer trap reporter lines further suggests that the specification between these two subsets takes place early during development, in relation with the determination of the vascular tissues. A genetic screen resulted in the isolation of mutants perturbed in pericycle differentiation. Detailed phenotypical analyses of two of these mutants, combined with observations made in known vascular mutants, revealed an intimate correlation between vascular organization, pericycle fate, and lateral root initiation potency, and illustrated the independence of pericycle differentiation and lateral root initiation from protoxylem differentiation. Taken together, our data show that the pericycle is a heterogeneous cell layer with two groups of cells set up in the root meristem by the same genetic pathway controlling the diarch organization of the vasculature.

High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy

Most plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales.