Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
26 Related JoVE Articles!
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis
Institutions: University of Arizona, Michigan State University, The Institute for Advanced Learning and Research, Michigan State University.
To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1
) via a double stranded RNA interference technique. The ZmCCR1_RNAi
construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.
Bioengineering, Issue 89, Zea mays, cinnamoyl-CoA reductase (CCR), dsRNAi, Klason lignin measurement, cell wall carbohydrate analysis, gas chromatography (GC)
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition
Institutions: University of Twente.
Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and reduction half reactions, and fast electron (hole) diffusion and charge separation. Nanowires present suitable architectures to meet these requirements. Axially segmented Ag|ZnO and radially segmented (coaxial) TiO2
-Ag nanowires with a diameter of 200 nm and a length of 6-20 µm were made by templated electrodeposition within the pores of polycarbonate track-etched (PCTE) or anodized aluminum oxide (AAO) membranes, respectively. In the photocatalytic experiments, the ZnO and TiO2
phases acted as photoanodes, and Ag as cathode. No external circuit is needed to connect both electrodes, which is a key advantage over conventional photo-electrochemical cells. For making segmented Ag|ZnO nanowires, the Ag salt electrolyte was replaced after formation of the Ag segment to form a ZnO segment attached to the Ag segment. For making coaxial TiO2
-Ag nanowires, a TiO2
gel was first formed by the electrochemically induced sol-gel method. Drying and thermal annealing of the as-formed TiO2
gel resulted in the formation of crystalline TiO2
nanotubes. A subsequent Ag electrodeposition step inside the TiO2
nanotubes resulted in formation of coaxial TiO2
-Ag nanowires. Due to the combination of an n
-type semiconductor (ZnO or TiO2
) and a metal (Ag) within the same nanowire, a Schottky barrier was created at the interface between the phases. To demonstrate the photocatalytic activity of these nanowires, the Ag|ZnO nanowires were used in a photocatalytic experiment in which H2
gas was detected upon UV illumination of the nanowires dispersed in a methanol/water mixture. After 17 min of illumination, approximately 0.2 vol% H2
gas was detected from a suspension of ~0.1 g of Ag|ZnO nanowires in a 50 ml 80 vol% aqueous methanol solution.
Physics, Issue 87, Multicomponent nanowires, electrochemistry, sol-gel processes, photocatalysis, photochemistry, H2 evolution
Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity
Institutions: Case Western Reserve University.
Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.
Environmental Sciences, Issue 85, Coexistence, community assembly, environmental drivers, plant-soil feedback, soil heterogeneity, soil microbial communities, soil patch
Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants
Institutions: RWTH Aachen University, Fraunhofer Institute for Molecular Biology and Applied Ecology.
Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei
Cel5A, in Nicotiana tabacum
are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei
Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.
Environmental Sciences, Issue 88, heterologous expression, endoplasmic reticulum, endoglucanase, cellulose, glycosyl-hydrolase, fluorescence, cellulase, Trichoderma reesei, tobacco plants
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate
Institutions: Case Western Reserve University.
-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum
) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium
and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium
carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana
and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.
Plant Biology, Issue 94, Flax (Linum usitatissimum), Floral-dip, Plant transformation, Transgenic, Agrobacterium tumefaciens, Plant binary vector, Direct PCR testing
A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems
Institutions: University of Verona, Verona, Italy, University of Perugia, Perugia, Italy.
Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.
Plant Biology, Issue 97, Plant biotechnology, transient expression, stable expression, transgenic plant, Nicotiana tabacum, Nicotiana benthamiana, Baculovirus/insect cells, recombinant protein
Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity
Institutions: New York Medical College.
the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.
Immunology, Issue 97, Toxoplasma, macrophages, innate immunity, intracellular pathogen, immune evasion, infectious disease, forward genetics, parasite
Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
Institutions: University of Alabama.
Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster.
Here, we have used the conserved innate immune response in Drosophila
to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster,
and this assay can be modified to be applicable to other Drosophila
tissues or even other small, whole-organism inflammation models.
Immunology, Issue 82, biology (general), environmental effects (biological, animal and plant), immunology, animal models, Immune System Diseases, Pathological Conditions, Signs and Symptoms, Life Sciences (General), Neuroinflammation, inflammation, nitric oxide, nitric oxide synthase, Drosophila, neurodegeneration, brain, Griess assay, nitrite detection, innate immunity, Parkinson disease, tissue culture
Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins
Institutions: Arizona State University .
Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium
into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium
into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis
. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.
Plant Biology, Issue 77, Genetics, Molecular Biology, Cellular Biology, Virology, Microbiology, Bioengineering, Plant Viruses, Antibodies, Monoclonal, Green Fluorescent Proteins, Plant Proteins, Recombinant Proteins, Vaccines, Synthetic, Virus-Like Particle, Gene Transfer Techniques, Gene Expression, Agroinfiltration, plant infiltration, plant-made pharmaceuticals, syringe agroinfiltration, vacuum agroinfiltration, monoclonal antibody, Agrobacterium tumefaciens, Nicotiana benthamiana, GFP, DsRed, geminiviral vectors, imaging, plant model
Analytical Techniques for Assaying Nitric Oxide Bioactivity
Institutions: University of Texas Health Science Center at Houston , Baylor College of Medicine .
Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1
. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo
NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo
. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-
) and nitrate (NO3-
) may only represent part of NO's effects in vivo
. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo
experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro
ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo
with organ bath myography.
Medicine, Issue 64, Molecular Biology, Nitric oxide, nitrite, nitrate, endothelium derived relaxing factor, HPLC, chemiluminscence
Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
Institutions: Cornell University, Cornell University.
Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.
Plant Biology, Issue 15, Annual Review, Aphids, Plant Virus, Potato Leaf Roll Virus, Microinjection Technique
Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment
Institutions: Cornell University.
Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.
Plant Biology, Issue 15, Annual Review, Nicotine, Aphids, Plant Feeding Resistance, Tobacco
Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis
Institutions: University of British Columbia - UBC, University of British Columbia - UBC.
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 pathogenic microorganisms. The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface. The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax. Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.
Plant Biology, Issue 16, Annual Review, Cuticle, Arabidopsis, Eceriferum Mutants, Cryso-SEM, Gas Chromatography
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Institutions: Uppsala University, Swedish University of Agricultural Sciences.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ
hybridization, a technique used to localize cell specific mRNA expression. The in situ
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies
). Here we present a modified DIG in situ
hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana
and Brassica napus
. The protocol worked equally well for the species and genes studied. AtAP3
were observed in second and third whorl floral organs in A. thaliana
and B. napus
and DAL13 in microsporophylls of male cones from P. abies
. For P. abies
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Plant Biology, Issue 26, RNA, expression analysis, Norway spruce, Arabidopsis, rapeseed, conifers
Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana
Institutions: Michigan State University (MSU), Michigan State University (MSU).
Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1
. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2
. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43
. The biliverdin reductase (BVR
) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4
. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR
gene in cells marked by GFP
. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7
. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR
gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP
expression. Photobiological and molecular genetic analyses of BVR
transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8
. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR
-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.
Plant Biology, Issue 39, Arabidopsis thaliana, confocal microscopy, expression profiling, microarray, fluorescence, FACS, photomorphogenesis, phytochrome, protoplasting
Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging
Institutions: University of Texas at Arlington .
Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis
plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al.
2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.
Plant Biology, Issue 44, Plant innate immunity, propidium iodide staining, biotic and abiotic stress, leaf microscopy, guard cell, stomatal defense, plant defense, Arabidopsis, Pseudomonas syringae
Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens
Institutions: Worcester Polytechnic Institute- WPI.
A simple and efficient method to transform Physcomitrella pantens
protoplasts is described. This method is adapted from protocols for Physocmitrella
protonemal protoplast and Arabidopsis
mesophyll protoplast transformation1
. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens
has emerged as an important model system in recent years2
. This capacity allows high frequencies of gene targeting3-9
, which is not seen in other model plants such as Arabidopsis
. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10
and PEG-mediated DNA uptake11
. Although particle bombardment can produce a high transformation efficiency12
, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13
. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.
Plant Biology, Issue 50, Transformation, Physcomitrella patens, polyethylene glycol, protoplasts
Generation of Composite Plants in Medicago truncatula used for Nodulation Assays
Institutions: St. Louis, Missouri.
Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes
can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes
can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes
can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes
infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula
, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula
Plant Biology, Issue 49, hairy root, composite plants, Medicago truncatula, rhizobia, GFP
Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton
Institutions: Texas A&M University, Texas A&M University.
Cotton (Gossypium hirsutum
) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1
. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3
. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis
and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4
. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1
) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1
gene is involved in chloroplast development6
, and previous studies have shown that loss-of-function of AtCLA1
resulted in an albino phenotype on true leaves7
, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium
infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.
Plant Biology, Issue 54, Agrobacterium, Cotton, Functional Genomics, Virus-Induced Gene Silencing
Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example
Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1
. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2
. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1
.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1
. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria
spp. Several species of African grasses of the genus Brachiaria
are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4
.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5
Plant Biology, Issue 52, host plant resistance, antibiosis, antixenosis, tolerance, Brachiaria, spittlebugs
Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
Institutions: Duke University Medical Center, Duke University , University of Pennsylvania , Duke University Medical Center.
The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1
Our flow chamber design and flow circuit (Fig. 1
) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B
), at various time points during flow (Fig. 11C
), and after flow (Fig. 11D
). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E
), or scanning electron microscopy5
. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12
Bioengineering, Issue 59, Fluid Shear Stress, Shear Stress, Shear Force, Endothelium, Endothelial Progenitor Cells, Flow Chamber, Laminar Flow, Flow Circuit, Continuous Flow, Cell Adhesion
Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae
Institutions: Utah State University.
Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina
growth. Microalgae were cultivated in photobioreactors illuminated at 984 µmol m-2
and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina
is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.
Environmental Sciences, Issue 101, algae, heavy metals, Nannochloropsis salina, photobioreactor, flue gas, inductively coupled plasma mass spectrometry, ICPMS, cold vapor atomic absorption spectrometry, CVAAS