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Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method
Many rhizobacteria isolated from plant rhizosphere produce various phytohormones in the form of secondary metabolites, the most common of which is Indole-3-acetic acid (IAA). Here, we detail analytical protocols of IAA detection and quantification, in vitro and in situ, as recently applied to Klebsiella SGM 81, a rhizobacterium isolated from the rhizosphere of Dianthus caryophyllus (a commercially important flower across the globe). Specifically, we describe a detailed protocol for a colorimetric assay using the Salkowski reagent method, which can be used to screen for the presence of Indole compounds. To further detect and quantify IAA, a highly accurate analytical approach of LC-MS/MS is used. To detect the presence of IAA around the root system of Dianthus caryophyllus, in situ staining of plant roots is done using Salkowski reagent.
Quantification of Salicylic Acid (SA) and SA-glucosides in Arabidopsis thaliana
Homeostasis between the cytoplasmic plant hormone salicylic acid (SA) and its’ inactive, vacuolar storage forms, SA-2-O-β-D-glucoside (SAG) and SA-β-D-Glucose Ester (SGE), regulates the fine-tuning of defense responses to biotrophic pathogens in Arabidopsis thaliana. This protocol describes a simplified, optimized procedure to extract and quantify free SA and total hydrolyzable SA in plant tissues using a classical HPLC-based method.
Rice Lamina Joint Inclination Assay
Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied the protocol presented below. The protocol focuses on: (1) how to prepare rice samples for the assay, and (2) how to treat BL exogenously. Finally, we have added a result showing lamina inclination between WT and osbul1 in BL solutions of various concentrations.
Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae
Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance (Kobayashi et al., 2016). This report describes the detailed method for the extraction and quantification of ABA in the model red alga C. merolae.
Quantification of Ethylene Production in Tomato Leaves Infected by Xanthomonas euvesicatoria
Ethylene is a gaseous plant hormone controlling fruit ripening, flower opening, leaf senescence as well as abscission, and disease symptom development. Ethylene plays a critical role in the bacterial pathogen Xanthomonas euvesicatoria (X. euvesicatoria)-elicited symptom development in tomato. This protocol describes the measurement of ethylene gas produced by tomato leaves infected with X. euvesicatoria. Infected leaflets are placed in a glass tube for 30 min without sealing. The glass tubes are then capped with a septa stopper, and incubated for an hour. A 1 ml gas sample is removed from the tube using a syringe and then injected into a gas chromatograph to quantify ethylene gas levels. This protocol will be applicable for other plants with other pathogens with modifications.
Indole Derivative Feeding Test and Detection of TRP and Indole derivatives by Thin Layer Chromatography
The mutualistic root endophyte Piriformospora indica colonizes a wide range of plants and the colonization of root cells by this fungus is very often associated with beneficial effects to its host, such as growth promotion and increased biotic and abiotic stress tolerance. These traits could be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. It is known, that many mutualistic microorganisms are able to synthesize and secrete phytohormones during the interaction with their host plants. This protocol has been successfully utilized to analyze tryptophan (TRP)-dependent biosynthesis of indole-3-acetic acid (IAA) and its indole derivatives by P. indica (Hilbert et al., 2012).
Growth Assay and Detection of TRP and Indole Derivatives in Piriformospora indica Culture Supernatant by LC-MS/MS
The mutualistic root endophyte Piriformospora indica colonizes a wide range of plants and the colonization of root cells by this fungus is very often associated with beneficial effects to its host, such as growth promotion and increased biotic and abiotic stress tolerance. These traits may be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. It is known, that many mutualistic microorganisms are able to synthesize and secrete phytohormones during the interaction with their host plants. This protocol has been successfully utilized to analyze tryptophan (TRP)-dependent biosynthesis of indole-3-acetic acid (IAA) and its indole derivatives by P. indica as well as their influence on the growth of this fungus (Hilbert et al., 2012).