Detection of Ascorbate Peroxidase (APX) Activity in Plant Tissues: Using Non-denaturing PAGE and Spectrophotometric Assay.

At the cellular level, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), due to different abiotic or biotic stress, causes oxidative stress that induces an imbalance in the metabolism. Among the different H2O2-scavenging enzymatic antioxidants, ascorbate peroxidase (APX) is a heme-peroxidase that plays an important role in the ascorbate-glutathione pathway using ascorbate to reduce H2O2 to water. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a spectrophotometric assay for APX activity, the protocol allows identifying diverse APX isozymes present in different organs and plant species.

Analysis of Plant L-Cysteine Desulfhydrase (LCD) Isozymes by Non-denaturing Polyacrylamide Gel Electrophoresis.

Hydrogen sulfide (H2S) is a signaling molecule that achieves different regulatory functions in animal and plant cells. The cytosolic enzyme L-cysteine desulfhydrase (LCD; EC 4.4.1.28) catalyzes the conversion of cysteine (L-Cys) to pyruvate and ammonium with the concomitant generation of H2S, this enzyme being considered one of the main sources of H2S in higher plants. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a specific assay for LCD activity, the present protocol allows identifying diverse LCD isozymes present in different organs (roots, shoots, leaves, and fruits) and plant species including pea, garlic, Arabidopsis, and pepper.

Extraction and Purification of Viroids from Herbaceous Hosts.

Protocols for extraction and purification of viroid RNAs from the tissues of infected herbaceous plant hosts are numerous. They range from lengthy, traditional protocols that require large amounts of starting tissue and take several days to perform to those based on column chromatography which is more efficient and can be performed with smaller amounts of infected tissue. The goal of all protocols is to enrich for RNA fractions that contain viroid RNAs, and the RNA extraction procedure is chosen and adjusted for the downstream method used for detection and characterization. Removal of inhibitors/impurities is generally not an issue for herbaceous hosts unless they contain and inordinate amounts of polysaccharides, tannins, and phenols. Subsequent purification of viroid circular and linear RNAs is performed using denaturing polyacrylamide gel electrophoresis. In this chapter, a specific method routinely used for viroid purification from herbaceous hosts and problems that may be encountered is described and is intended as a reference for beginners in the field.

RNase Protection Assay.

Unbound, single-stranded RNA can be digested by RNase (A or T1) to ribonucleotides, whereas double-stranded RNA is not digested by RNase. Based on this principle, the RNase Protection Assay (RPA) is used to validate chimeric RNAs. Importantly, this assay does not employ reverse transcription (RT), thus avoiding potential false-positive results which could occur during RT such as template-switching. We first generate RNA probes with 32phosphate (P) or biotin that are complementary to the predicted nucleotide sequence of the chimeric RNA, then hybridize them to RNA samples. The labeled RNA probes can bind specifically with the target chimeric RNA in order to form double-stranded RNA. This newly formed RNA is resistant to digestion by RNase and therefore can be identified by high-resolution, denaturing polyacrylamide gel electrophoresis.

Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis.

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, mitochondria and peroxisomes ( Corpas et al., 1998 ; Krepinsky et al., 2001 ; Mateos et al., 2009 ; Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016 ). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Assessment of direct interaction between CD36 and an oxidized glycerophospholipid species.

Cluster of differentiation 36 (CD36) is a transmembrane protein that recognizes multiple diverse ligands. It is believed that (i) oxidized glycerophosphatidylcholine species having a terminal γ-hydroxyl(or oxo)-α,ß-unsaturated carbonyl on the sn-2 acyl group (oxGPCCD36), which can occur on the surface of lipoprotein particles, serve as high-affinity ligands for CD36, and (ii) the amino acid 150-168 of CD36 (CD36150-168) is responsible for recognizing oxGPCCD36. However, it remains uncertain whether CD36150-168 directly interacts with oxGPCCD36 alone. In this study, we addressed this issue by investigating and comparing the banding pattern by non-denaturing polyacrylamide gel electrophoresis of a glutathione S-transferase (GST) fusion protein containing CD36150-168 (GST-CD36150-168), in the presence and absence of an oxGPCCD36 species, 1-(palmitoyl)-2-(5-keto-6-octenedioyl)phosphatidylcholine (KOdiA-PC). It was shown that GST-CD36150-168 pre-incubated with KOdiA-PC produced bands at upper positions than did the fusion protein alone. Further analyses revealed that the bands produced by the loading of GST-CD36150-168/KOdiA-PC mixture represent complexes consisting of the fusion protein and lipid. To our knowledge, this is the first evidence for direct interaction between CD36150-168 and oxGPCCD36 alone. It is also notable that the electrophoresis-based technique provides a convenient means to evaluate protein-lipid interactions.