Environmental science


Protocols in Current Issue
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0 Q&A 357 Views Jun 20, 2023

Mandelonitrile is a nitrogen-containing compound, considered an essential secondary metabolite. Chemically, it is a cyanohydrin derivative of benzaldehyde, with relevant functions in different physiological processes including defense against phytophagous arthropods. So far, procedures for detecting mandelonitrile have been effectively applied in cyanogenic plant species such as Prunus spp. Nevertheless, its presence in Arabidopsis thaliana, considered a non-cyanogenic species, has never been determined. Here, we report the development of an accurate protocol for mandelonitrile quantification in A. thaliana within the context of A. thaliana–spider mite interaction. First, mandelonitrile was isolated from Arabidopsis rosettes using methanol; then, it was derivatized by silylation to enhance detection and, finally, it was quantified using gas chromatography–mass spectrometry. The selectivity and sensitivity of this method make it possible to detect low levels of mandelonitrile (LOD 3 ppm) in a plant species considered non-cyanogenic that, therefore, will have little to no cyanogenic compounds, using a small quantity of starting material (≥100 mg).

0 Q&A 489 Views Apr 5, 2023

Paraquat is a cost-effective herbicide, widely used in many countries, that can induce severe oxidative stress in photosynthetic tissues. Studying plant herbicide resistance or antioxidant stress mechanisms requires determining the cellular paraquat level when plants are treated by paraquat. The traditional isotopic labeling method has the potential risk to cause problems to both human health and the environment. For radioisotope manipulation, special operation spaces and strict environmental inspection are also required. In addition, the radiolabeled paraquat is increasingly hard to buy due to the extended production cycle. Here, we describe a nonradioactive method to determine the paraquat level in a small number of Arabidopsis tissues or protoplasts, using a high resolution ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)/MS method. This method is highly selective and sensitive, and more environmentally compatible and technically feasible than the isotope detection method.

0 Q&A 2451 Views Mar 5, 2022

Iron (Fe) is an indispensable micronutrient for plant growth and development. Since both deficiency, as well as a surplus of Fe, can be detrimental to plant health, plants need to constantly tune uptake rates to maintain an optimum level of Fe. Quantification of Fe serves as an important parameter for analyzing the fitness of plants from different accessions, or mutants and transgenic lines with altered expression of specific genes. To quantify metals in plant samples, methods based on inductively coupled plasma-optical emission spectrometry (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS) have been widely employed. Although these methods are highly accurate, these methodologies rely on sophisticated equipment which is not always available. Moreover, ICP-OES and ICP-MS allow for surveying several metals in the same sample, which may not be necessary if only the Fe status is to be determined. Here, we outline a simple and cost-efficient protocol to quantify Fe concentrations in roots and shoots of Arabidopsis seedlings, by using a spectroscopy-based assay to quantify Fe2+-BPDS3 complexes against a set of standards. This protocol provides a fast and reproducible method to determine Fe levels in plant samples with high precision and low costs, which does not depend on expensive equipment and expertise to operate such equipment.

0 Q&A 2491 Views Oct 5, 2021

Dark respiration refers to experimental measures of leaf respiration in the absence of light, done to distinguish it from the photorespiration that occurs during photosynthesis. Dark aerobic respiration reactions occur solely in the mitochondria and convert glucose molecules from cytoplasmatic glycolysis and oxygen into carbon dioxide and water, with the generation of ATP molecules. Previous methods typically use oxygen sensors to measure oxygen depletion or complicated and expensive photosynthesis instruments to measure CO2 accumulation. Here, we provide a detailed, step-by-step approach to measure dark respiration in plants by recording CO2 fluxes of Arabidopsis shoot and root tissues. Briefly, plants are dark acclimated for 1 hour, leaves and roots are excised and placed separately in airtight chambers, and CO2 accumulation is measured over time with standard infrared gas analyzers. The time-series data is processed with R scripts to produce dark respiration rates, which can be standardized by fresh or dry tissue mass. The current method requires inexpensive infrared gas analyzers, off-the-shelf parts for chambers, and publicly available data analysis scripts.

0 Q&A 3830 Views Sep 20, 2021

Identification of novel genes and their functions in rice is a critical step to improve economic traits. Agrobacterium tumefaciens-mediated transformation is a proven method in many laboratories and widely adopted for genetic engineering in rice. However, the efficiency of gene transfer by Agrobacterium in rice is low, particularly among japonica and indica varieties. In this protocol, we elucidate a rapid and highly efficient protocol to transform and regenerate transgenic rice plants through important key features of Agrobacterium transformation and standard regeneration media, especially enhancing culture conditions, timing, and growth hormones. With this protocol, transformed plantlets from the embryogenetic callus of the japonica cultivar ‘Taichung 65’ may be obtained within 90 days. This protocol may be used with other japonica rice varieties.

0 Q&A 5385 Views Jul 20, 2019
Symbiotic interactions between arbuscular mycorrhizal fungi (AMF) and plants are widespread among land plants and can be beneficial for both partners. The plant is provided with mineral nutrients such as nitrogen and phosphorous, whereas it provides carbon resources for the fungus in return. Due to the large economic and environmental impact, efficient characterization methods are required to monitor and quantify plant-AMF colonization. Existing methods, based on destructive sampling and elaborate root tissue analysis, are of limited value for high-throughput (HTP) screening. Here we describe a detailed protocol for the HTP quantification of blumenol derivatives in leaves by a simple extraction procedure and sensitive liquid chromatography mass spectrometry (LC/MS) analysis as accurate proxies of root AMF-associations in both model plants and economically relevant crops.
0 Q&A 5779 Views May 5, 2019
One of the most remarkable metabolic features of plant roots is their ability to secrete a wide range of compounds into the rhizosphere, defined as the volume of soil around living roots. Around 5%-21% of total photosynthetically fixed carbon is transferred into the rhizosphere through root exudates. Until recently, studies on the quantity and quality of root exudates were conducted mostly under axenic or monoxenic in vitro conditions. Today, in situ assays are required to provide a better understanding of root exudates dynamics and role in plant-microbe interactions. By incubating plants with 13CO2 in situ for one week and quantifying 13C enrichment from the root-adhering soil using mass spectrometry, we were able to determine root exudate levels. Indeed, labeled substrate 13CO2 is converted into organic carbon via plant photosynthesis and transferred into the soil through root exudation. We assume that all 13C increases above natural abundance are mainly derived from exudates produced by 13C-labeled plants.
0 Q&A 5184 Views Aug 20, 2018
Insect pollinators, herbivores and their natural enemies use olfactory cues emitted by their host plants to locate them. In insect-plant ecology, understanding the mechanisms underlying these interactions are of critical importance, as this bio-communication has both ecological and agricultural applications. However, the first step in such research is to identify and quantify the insect community associated with the plant/s species of interest. Traditionally, this has been accomplished by a variety of insect trapping methods, either using pitfall traps, or sticky traps, or sweep nets in field. The data collected from these traps tend to be incomplete, and also damage the specimens, making them unusable for any taxonomic purposes. This protocol derives ideas from these traditional traps and use a combination of three easily made inexpensive modified traps that conceals the host plant, but allows the plant volatiles to pass through as olfactory cues. These traps are economical, can be made to fit with most plant sizes, and are also reusable. Collectively, these traps will provide a solid estimate (quantifiable) of all associated community of arthropods that can also be stored for future studies.

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