植物科学

The ascorbate peroxidase (APX) is a widely distributed antioxidant enzyme. It differs from catalase and other peroxidases in that it scavenges/reduces reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) to water using reduced ascorbate as the electron donor. It is advantageous over other similar antioxidant enzymes in scavenging ROS since ascorbate may react with superoxide, singlet oxygen, and hydroxyl radical, in addition to reacting with H₂O₂. The estimation of its activity is helpful to analyze the level of oxidative stress in living systems under stressful conditions. The present protocol was performed to analyze the impact of heavy metal chromium (Cr) toxicity on sorghum plants in the form of APX enzyme activity under the application of glycine betaine (GB) and arbuscular mycorrhizal fungi (AMF) as stress ameliorators. Plant defense strategies against heavy metals toxicity involve the utilization of APX and the instigation of AMF symbiotic system, as well as their possible collaboration with one another or with the plant antioxidant system; this has been examined and discussed in literature. In this protocol, an increased APX activity was observed on underlying functions and detoxification capabilities of GB and AMF that are typically used by plants to enhance tolerance to Cr toxicity.

Graphical abstract:

Flow chart of standardized or calibrated enzyme assay with leaf samples of sorghum

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor of numerous enzymatic reactions found in all living cells. Pyridine nucleotides (NAD⁺ and NADH) are also key players in signaling through reactive oxygen species (ROS), being crucial in the regulation of both ROS-producing and ROS-consuming systems in plants. NAD content is a powerful modulator of metabolic integration, protein de-acetylation, and DNA repair. The balance between NAD oxidized and reduced forms, i.e., the NADH/NAD⁺ ratio, indicates the redox state of a cell, and it is a measurement that reflects the metabolic health of cells. Here we present an easy method to estimate the NAD⁺ and NADH content enzymatically, using alcohol dehydrogenase (ADH), an oxido-reductase enzyme, and with MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as the substrate and 1-methoxy PMS (1-Methoxy-5-methylphenazinium methyl sulfate) as the electron carrier. MTT is reduced to a purple formazan, which is then detected. We used Arabidopsis leaf samples exposed to aluminum toxicity and under untreated control conditions. NADH/NAD⁺ connects many aspects of metabolism and plays vital roles in plant developmental processes and stress responses. Therefore, it is fundamental to determine the status of NADH/NAD⁺ under stress.

We describe a method to test the preference of insects in response to (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). We use a device that includes a horizontal glass tube, two grooves (with activated carbon), air flow, rubber stoppers/tubes, transparent glass containers (optional), and a holder for the glass tube (optional). Equal amounts of activated carbon in the groove (removable) are placed at both ends to avoid air contamination. The air flow is generated by an air pump. In the closed device, different samples are placed at each end of the glass tube. The air pump at the top of the glass tube forms an air flow that converges to the middle site of the glass tube. In each test, insect larvae are located in the middle of the glass test tube. If the test samples release DMNT that can be sensed by insects, the insects will selectively move to one specific end of the glass tube. The number of insects that move to each end will be recorded for further studies. This method can also be used to test the preference of insects in response to other volatile compounds.

We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols). To construct a nanocarrier that is more clearly defined than GDNP-2, we isolated lipids from GDNP-2 and demonstrated that they could self-assemble into ginger lipid-derived nanoparticles (GLDNP) in an aqueous solution. GLDNP can be used as a nanocarrier to deliver drug candidates such as 6-shogaol or its metabolites (M2 and M13) to the colon. To characterize the nanostructure of GLDNP, our lab extensively used atomic force microscopy (AFM) technique as a tool for visualizing the morphology of the drug-loaded GLDNP. Herein, we provide a detailed protocol for demonstrating such a process.

Targeted metabolomics is a useful approach to evaluate crop breeding studies. Antioxidant and flavor-related traits are of increasing interest and are considered quality traits in tomato breeding. The present study presents chromatographic methods to study antioxidants (carotenoids, vitamin C, vitamin E, phenolic compounds, and glutathione) and flavor-related characters (sugars and organic acids) in tomato. Two different extraction methods (for polar and apolar entities) were applied to isolate the targeted compounds. The extraction methods developed in this work were time and cost-effective since no further purification was needed. Carotenoids, vitamin C, glutathione, and phenolic acids were analyzed by HPLC-PDA using a RP C18 column at an appropriate wavelength for each compound. Vitamin E and sugars were analyzed by HPLC with RP C18 and NH2 columns and detected by FLD and RI detectors, respectively. In addition, organic acids were analyzed with GC-FID using a Rtx 5DA column after derivatization with MSTFA. As a result, sensitive analytical methods to quantify important plant metabolites were developed and are described herein. These methods are not only applicable in tomato but are also useful to characterize other species for flavor-related and antioxidant compounds. Thus, these protocols can be used to guide selection in crop breeding.

Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technological advances have been reviewed (Allen et al., 2015). The present protocol describes the use of ¹⁴C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism (Allen et al., 2015), including the role of autophagy in membrane lipid turnover (Fan et al., 2019) and the interplay between lipid and starch metabolism pathways (Yu et al., 2018).

Plant lipid metabolism is a dynamic network where synthesis of essential membrane lipids overlaps with synthesis of valuable storage lipids (e.g., vegetable oils). Monogalactosyldiacylglycerol (MGDG) is a key component of the chloroplast membrane system required for photosynthesis and is produced by multiple pathways within the lipid metabolic network. The bioengineering of plants to enhance oil production can alter lipid metabolism in unexpected ways which may not be apparent by static quantification of lipids, but changes to lipid metabolic flux can be traced with isotopic labeling commonly with [¹⁴C]acetate. Because lipid classes such as MGDG are composed of many different molecular species, full analysis of metabolically labeled lipids requires separation and quantification of the individually labeled molecular species which is traditionally performed by thin layer chromatography. Here we present a reverse phase HPLC method for the separation of MGDG molecular species from tobacco leaves in under 35 min. The quantification of each ¹⁴C-labeled molecular species was accomplished by an in-line flow radio detector. This method of analysis for [¹⁴C]Acetate labeled MGDG molecular species by radio-HPLC provides a rapid, high throughput, and reliable analytical approach to identify changes in MGDG metabolism due to bioengineering or other perturbations of metabolism.

Pipecolic acid (Pip), a non-proteinacious product of lysine catabolism, is an important regulator of immunity in plants and humans alike. For instance, Pip accumulation is associated with the genetic disorder Zellweger syndrome, chronic liver diseases, and pyridoxine-dependent epilepsy in humans. In plants, Pip accumulates upon pathogen infection and is required for plant defense. The aminotransferase ALD1 catalyzes biosynthesis of Pip precursor piperideine-2-carboxylic acid, which is converted to Pip via ornithine cyclodeaminase. A variety of methods are used to quantify Pip, and some of these involve use of expensive amino acid analysis kits. Here, we describe a simplified procedure for quantitative analysis of Pip from plant tissues. Pipecolic acid was extracted from leaf tissues along with an internal standard norvaline, derivatized with propyl chloroformate and analyzed by gas chromatography-coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient and does not involve isotopic internal standards or multiple-step derivatizations.

Flavonols are a subclass of flavonoids of the group of plant secondary metabolites. In planta, flavonols play various functions such as antioxidant and natural regulator of auxin polar transport. Many lines of evidence have shown that flavonols also contribute to human health in anti-oxidation, anti-inflammation, and even prevention some types of cancer. Several methods have been utilized to measure flavonols such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and diphenylboric acid-2-aminoethyl ester (DPBA) staining. While HPLC or LC-MS can quantitatively determine the level of flavonols, DPBA staining can provide an in-situ view of flavonols accumulation in the plants. In this protocol, a detailed procedure for staining the flavonols in Arabidopsis root tips is described. Five-day-old Arabidopsis seedlings are soaked in a solution containing DPBA and latterly the flavonols (kaempferol and quercetin) can be observed under a confocal microscope.

The ureides allantoin and allantoate are the main organic nitrogen compounds transported in several legumes, predominantly from N₂ fixation. Moreover, recent studies point out a remarkable role for allantoin during several stress responses of plants other than legumes. The goal of this protocol is to determine ureides concentration in different plant tissues. Ureides are extracted from plant material by boiling it in phosphate buffer. The allantoin and allantoate present in the supernatants are subjected to alkaline-acidic hydrolysis to glyoxylate. The glyoxylate is converted into glycoxylic acid phenylhydrazone, that is then oxidized to red-colored 1,5-diphenylformazan. The absorbance of supernatants is measured using a spectrophotometer at 520 nm. Ureides concentration can be inferred by using a glyoxylate calibration curve. Ureide quantification of different tissues of Arabidopsis thaliana and soybean plants were carried out following this protocol.