I. Visuelle Screens und genetische Ansätze zur Identifizierung Cuticle Mutanten. Cuticle Mutanten in der Modellpflanze Arabidopsis thaliana werden als &quot;eceriferum&quot; Mutanten, was bedeutet, &quot;die nicht mit Wachs&quot;. Sie können in Sichtschutz sein, das heißt, indem man sich einfach die Mutante, können Sie sehen, dass der Blütenstand Stamm von Wildtyp-Pflanzen weißlich wird, während die Mutanten dunkelgrün glänzende Stängel haben. Einer der Mutanten, die in dieser Art von Sichtschutz isoliert wurde, ist dieser, als eceriferum4 oder cer4. Eine weitere Möglichkeit der Entdeckung interessanter Nagelhaut Mutanten ist mit einem reversen Genetik Ansatz: Auswahl Kandidatengene von Interesse und dann studieren Anlage Zeilen mit, dass Gen unterbrochen. Diese Mutanten haben tDNA Störung des Gens von Interesse, in diesem Fall ein Cytochrom P450, dass wir heute MAH1. PCR-Ergebnisse (wie in dem Video zu sehen) kann bestätigen, dass die tDNA Insertion in das Gen von Interesse ist. Wenn zwei Gen-spezifische Primer verwendet werden, um Wildtyp-DNA verstärken, sehen wir ein Produkt, aber wenn die tDNA unterbricht sie, wie in dieser Mutante, gibt es kein Produkt gibt. Umgekehrt unter Verwendung von Primern spezifisch für die tDNA und Gen ist ein Produkt nur in der Mutante zu sehen. Wir suchen homozygot mutierten Linien, bei denen sowohl die mütterlichen und väterlichen Kopien des Gens gestört sind. Wenn eine Kopie des Gens durch tDNA gestört ist und die andere Kopie ist immer noch wild-type, werden die PCR-Ergebnisse gemischt. II. Mittels Gaschromatographie auf die chemische Zusammensetzung der Cutlicle Wax Bestimmen. Wir analysieren die chemische Zusammensetzung des Wachses mit Hilfe der Gaschromatographie. Zunächst werden die löslichen Wachsverbindungen aus der Anlage Oberfläche durch Eintauchen in Chloroform entfernt. Dann werden die Proben in die Gas-Chromatographie-Säule injiziert, wo sie erhitzt und geschickt durch auf einem Gasstrom. Verschiedene Verbindungen stick mehr oder weniger an den Wänden der Säule, die Trennung der Verbindungen, und sie kommen aus einer nach dem anderen am anderen Ende der Spalte. Dann, als die Verbindungen über den Flammenionisationsdetektor passieren, sehen wir die Gipfel. Die Verweildauer der einzelnen Peaks ist charakteristisch für die verschiedenen Verbindungen und von Massenspektrometrie, haben wir jede der Komponenten von Arabidopsis Wachs identifiziert. Die wichtigsten Peaks vorhanden sind, dass der C29-Alkan, das C29-Keton und die C29 sekundärer Alkohol. Der kleinere Gipfel sind die sekundären Alkohole, Fettsäuren, Aldehyde und Ester. Die Fläche unter den Peaks, bezogen auf Standard, erzählt uns, wie viel von jeder Verbindung vorhanden ist. Bei der Betrachtung Gaschromatogramme von diesen Nagelhaut Mutanten, kann man feststellen, wie cer4 die primären Alkohole und Ester fehlt, während MAH1 fehlt sekundäre Alkohole und Ketone. Diese Phänotypen können uns viel über die Funktion des Gens von Interesse in der Anlage. Zum Beispiel, bevor wir wussten, welche Gen wurde in die cer4 Linie mutiert, sagte der Phänotyp uns, dass es ein Problem in der primären Alkohol Zweig der Biosyntheseweg. Nach der molekularen Identität des Gens in der Kunst-Labor untersucht wurde, wurde festgestellt, dass es Teil einer Genfamilie von Fettacyl Reduktasen, die gut mit dieser chemischen Phänotyp ist. III. Mit Cryo-SEM, um die Struktur der Kutikula Kristalle bestimmen Wenn wir das Arabidopsis-Mutanten Nagelhaut studieren, können wir sehen, ob sie den dunkelgrünen, glänzenden Phänotyp oder der Wildtyp-weißliches Aussehen auf dem Vorbau zu haben. Wir verwenden das Rasterelektronenmikroskop an der Kutikula auf der Oberfläche der Pflanze suchen und identifizieren anhand der visuellen Phänotyp. Wir verwenden Kryo-SEM, das Einfrieren der Pflanzen und betrachten bei der Temperatur flüssigen Stickstoffs in Ordnung zu halten die Schuppenschicht intakt, weil die SEM erfordert ein Vakuum zu bedienen und ohne halten sie gefroren, die Proben würden austrocknen und zerfallen. Hier cer4 und MAH1 Proben, zusammen mit dem Wildtyp-Kontrollen sind präpariert und eingefroren auf dem SEM Stummel. Als die Proben zu den kalten Stufen des Mikroskops übertragen werden, können Sie sehen, dass die Oberfläche des Wildtyp-Arabidopsis Stammzellen Nagelhaut mit Kristallen bedeckt ist. Es ist das Licht, Streuung an ist die Kristalle, die die Stammzellen gibt seinen weißliches Aussehen. Hier ist die cer4 Stammzellen; erinnern sah es glänzend? Es fehlt die Kristalle auf der Oberfläche. Die Menschen haben Experimente gemacht, wo sie ein reines Wachs Verbindung aus der Kutikula einer Pflanze und zu isolieren, wenn sie es rekristallisieren im Labor bilden die Kristalle die gleiche Form wie die auf den Pflanzen gefunden. In Arabidopsis, glauben wir, dass die Kristalle bestehen aus einer Mischung von Verbindungen hergestellt und die Situation ist komplizierter. Zum Beispiel zeigt die chemische Analyse uns, dass in cer4, nur die kleinere Komponente des primären Alkohole und ihre Derivate Ester fehlen, doch die Kristalle auf der cer4 Stammzellen sind weg. Im Falle von MAH1, ist der Effekt der Mutation in das Cytochrom-P450-Enzym, das die mutierte Pflanzen zwei Hauptkomponenten des Wachses, sekundäre Alkohole und Ketone Mangel, doch die Kristalle auf der MAH1 Pflanzen bilden in der Regel. Wenn Sie mehr über Arabidopsis-Mutanten Kutikula finden Sie unter <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.59.103006.093219" target="_blank">Annual Review of Plant Biology</a> .

use of arabidopsis eceriferum mutants to explore plant cuticle biosynthesis

Die Kutikula ist eine wachsartige Außenhaut auf Pflanzen, die eine primäre Rolle in Wasserschutzgebieten hat, ist aber auch eine wichtige Barriere gegen das Eindringen von pathogenen Mikroorganismen. Die Kutikula besteht aus einem harten vernetzten Polymer namens &quot;Cutin&quot; und einer schützenden Wachsschicht, die Dichtungen der Anlage Oberfläche. Die Wachsschicht der Cuticula ist offensichtlich bei vielen Pflanzen, erscheint als glänzender Film auf der Efeublatt oder als eine staubige äußere Hülle auf der Oberfläche von einer Traube oder ein Kohlblatt dank Streuung Kristalle im Wachs Licht. Da die Kutikula ist eine wesentliche Anpassung der Anlagen an eine terrestrische Umwelt, das Verständnis der Gene in Kutikula-Bildung beteiligt hat Anwendungen in Land-und Forstwirtschaft. Heute zeigen wir die Analyse der Kutikula-Mutanten durch Forward-und Reverse Genetik Ansätze identifiziert.

Watch this Scientific Journal Video about Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis at JoVE.com

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

Die Kutikula ist eine wachsartige Außenhaut auf Pflanzen, die eine primäre Rolle in Wasserschutzgebieten hat aber auch eine wichtige Barriere gegen das Eindringen von pathogenen Mikroorganismen. In diesem Video zeigen wir die Analyse der Kutikula-Mutanten durch Forward-und Reverse Genetik Ansätze identifiziert.

Verwendung von Arabidopsis-Mutanten zu eceriferum Kutikula Biosynthese entdecken

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of ...

use-arabidopsis-eceriferum-mutants-to-explore-plant-cuticle

Research

JoVE Journal

Biology

12.8K Aufrufe.  University of British Columbia - UBC. Die Kutikula ist eine wachsartige Außenhaut auf Pflanzen, die eine primäre Rolle in Wasserschutzgebieten hat aber auch eine wichtige Barriere gegen das Eindringen von pathogenen Mikroorganismen. In diesem Video zeigen wir die Analyse der Kutikula-Mutanten durch Forward-und Reverse Genetik Ansätze identifiziert.

Serie: Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Effiziente und schnelle Isolierung von Frühphasen-Embryonen aus<em&gt; Arabidopsis thaliana</em&gt; Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Forschung

Biologie

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolische Markierung und Membran Fraktionierung für Vergleichende Proteomanalyse von<em&gt; Arabidopsis thaliana</em&gt; Fahrwerk Zellkulturen

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Ein Mantel Bettwäsche Seed-Test zur Genetisch erkunden<em&gt; In-vitro-</em&gt; Wie das Endosperm Kontrollen Keimung der Samen in<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Eine effiziente Methode zur quantitativen, Einzel-Zell-Analyse von Chromatin Modifikation und Reaktor Architektur in Whole-Mount-Samenanlagen in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Mess räumliche und zeitliche Ca<sup&gt; 2 +</sup&gt; Signale in Arabidopsis-Pflanzen

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Isolierung und Analyse der Zusammensetzung von Pflanzen Cuticle Lipid Polyester Monomere

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Seitenwurzel induzierbaren Systems in<em&gt; Arabidopsis</em&gt; Und Mais

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Hohe Auflösung Quantifizierung kristalliner Cellulose Akkumulation in<em&gt; Arabidopsis</em&gt; Roots Gewebespezifische Zellwand Änderungen überwachen

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Schnelle Analyse von Circadian Phänotypen in Arabidopsis Protoplasten Transfizierte mit einem lumineszenten Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Standardisierte Methode für Hochdurchsatz-Sterilisation von Arabidopsis Samen

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...