A simple way to identify non-viable cells within living plant tissue using confocal microscopy.

BACKGROUND: Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. RESULTS: Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. CONCLUSION: The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

µCube: A Framework for 3D Printable Optomechanics

Scientific instruments often require the integration of mechanics, electronics and optics. While the use of 3D printing techniques and commodity electronics has lowered the cost of instrumentation, the design and prototyping of optical components and light paths can be challenging and expensive. In recent years, attempts have been made to make optical devices more affordable using 3D printing as a method for production of optomechanical components. In this paper we present an assembly standard for the production of 3D printed optical devices. We describe a framework for parametric design of modular mounts, present two modules built using the framework, and demonstrate the potential for generalised design of modular optical devices following the μCube standard.This project was supported by the Biotechnology and Biological Sciences Research Council and Engineering and Physical Sciences Research Council Synthetic Biology Research Centre supported by the Research Councils’ Synthetic Biology for Growth Programme [OpenPlant grant No. BB/L014130/1 to J.H.]; and University of Cambridge BBSRC DTP programme [M.D.

Arabidopsis thaliana outer ovule integument morphogenesis: Ectopic expression of KNAT1 reveals a compensation mechanism

<p>Abstract</p> <p>Background</p> <p>The <it>Arabidopsis </it>outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated.</p> <p>Results</p> <p>To better characterise outer ovule integument morphogenesis, we have isolated some marker lines that show <it>GFP </it>expression in this organ. We have used those lines to identify distinct cell types in the outer integument and to demonstrate similarities between leaves and the outer integument. Using confocal microscopy, we showed that cell sizes and shapes differ between the two cell layers of the outer integument. Expression of <it>KNAT1 </it>in the integuments leads to extra cell divisions specifically in the outer layer of the outer integument. This is being compensated for by a decrease of cell volume in this layer, thus showing that mechanisms exist to control proper ovule integument morphogenesis.</p> <p>Conclusion</p> <p>The <it>Arabidopsis </it>outer ovule integument can be used as a good model system to study the basic principles of plant organ morphogenesis. This work provides new insights into its development and opens new possibilities for the identification of factors involved in the regulation of cell division and elongation during plant organ growth.</p

Sustainable Approaches to Incorporate Plant-Based Biomaterials in Power Generation

Biomass-derived materials have traditionally been used to generate electrical energy through the combustion of their organic components. However, within the past few years, certain common biomass compounds, especially plant-based products such as cellulose and lignin, have drawn attention in the energy field due to their wide availability, low cost, and chemical versatility. In the case of cellulose, the combination of crystalline and amorphous domains, along with the high surface area and abundance of hydroxyl groups, has allowed for its application in multiple devices to harvest energy from the environment. However, to date, there are no reviews focusing on the different approaches that have been developed to implement these sustainable materials in the generation of renewable energies and the desirable material properties for these applications. This manuscript reviews alternative ways that have been developed to exploit biomass compounds in power generation, especially cellulose and lignin. Three different types of energy harvesting are discussed: mechanical, osmotic, and thermal energy. In the case of mechanical energy, the application of plant-derived materials in piezoelectric and triboelectric generators is described. In both cases, approaches where the biomass material has an active role in power generation instead of acting as a mechanical support are reported. For osmotic energy, the performance of inverse electrodialysis systems and the use of plant-derived materials, including the chemical modifications carried out to allow for their use for energy generation, was reviewed. Finally, for thermal energy generation, the reported work on biopolymer-based devices that work using thermoelectricity has been summarised. In each case, the latest advances in the field from the materials science perspective and the reported performance were described. Hybrid approaches involving the combination of biomass materials with other components have also been considered and compared with the performance obtained using biopolymers alone. Current limitations and opportunities are, finally, discussed to offer an overview of the current landscape and indicate future directions of the field

Programmed hierarchical patterning of bacterial populations.

Modern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. Here we report a synthetic genetic system implementing population-based AND-logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations

A simple way to identify non-viable cells within living plant tissue using confocal microscopy

BACKGROUND: Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. RESULTS: Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. CONCLUSION: The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy

A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha.

Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity

Characterization of Intrinsic Properties of Promoters.

Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.TJR was supported by a Microsoft Research studentship and EC FP7 Project No. 612146 (PLASWIRES) awarded to JH, JRB by a Microsoft Research studentship and internship, and FF by CONICYT-PAI/Concurso Nacional de Apoyo al Retorno de Investigadores/as desde el Extranjero Folio 8213002 7, and EPSRC grant EP/H019162/1 awarded to JH. JWA acknowledges the EPSRC and the Wellcome Trust for support.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acssynbio.5b0011

Droplet-based microfluidic analysis and screening of single plant cells.

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.BBSRC and EPSRC OpenPlant BB/L014130/

GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression.

To facilitate the monitoring of guard cells during development and isolation, a population of 704 GAL4 GFP enhancer trap lines was screened and four single insert lines with guard cell GFP expression and one with developmentally-regulated guard cell GFP expression were identified. The location of the T-DNA inserts, the expression of the flanking genes, and the promoter activity of the genomic DNA upstream of the T-DNA were characterized. The results indicated that the GFP expression pattern in at least one of the lines was due to elements in the intergenic DNA immediately upstream of the T-DNA, rather than due to the activity of the promoters of genes flanking the insert, and provide evidence for the involvement of Dof elements in regulating guard cell gene expression. It is shown further that the GAL4 GFP lines can be used to track the contribution of guard cell material in vitro, and this method was used to assess the purity of guard cell samples obtained using two methods of guard cell isolation