A rapid and robust colorimetric method for measuring relative abundance of auxins in plant tissues

IntroductionAuxin estimation in plant tissues is a crucial component of auxin signaling studies. Despite the availability of various high-throughput auxin quantification methods like LC-MS, GC-MS, HPLC, biosensors, and DR5-gus/gfp-based assays, auxin quantification remains troublesome because these techniques are very expensive and technology intensive and they mostly involve elaborate sample preparation or require the development of transgenic plants.ObjectivesTo find a solution to these problems, we made use of an old auxin detection system to quantify microbe derived auxins and modified it to effectively measure auxin levels in rice plants.Materials and methodsAuxins from different tissues of rice plants, including root samples of seedlings exposed to IAA/TIBA or subjected to different abiotic stresses, were extracted in ethanol. The total auxin level was measured by the presently described colorimetric assay and counterchecked by other auxin estimation methods like LC-MS or gus staining of DR5-gus overexpressing lines.ResultsThe presented colorimetric method could measure (1) the auxin levels in different tissues of rice plants, thus identifying the regions of higher auxin abundance, (2) the differential accumulation of auxins in rice roots when auxin or its transport inhibitor was supplied exogenously, and (3) the levels of auxin in roots of rice seedlings subjected to various abiotic stresses. The thus obtained auxin levels correlated well with the auxin levels determined by other methods like LC-MS or gus staining and the expression pattern of auxin biosynthesis pathway genes.ConclusionsThe auxin estimation method described here is simple, rapid, cost-effective, and sensitive and allows for the efficient detection of relative auxin abundances in plant tissues. Estimation of plant auxins is a vital component of many auxin-signaling studies. The present study describes a colorimetric method of auxin estimation from plant tissue samples where total auxin is readily extracted in ethanol and determined spectrophotometrically at 540 nm after a brief period of incubation in a solution containing sulfuric acid and ferric chloride. The given colorimetric method was found to be a simple, rapid, sensitive, and cost-effective way of auxin estimation from plant tissues.