A critical comparison of two high-throughput ascorbate analyses methods for plant samples

Abstract

Ascorbate (AsA) is an important metabolite involved in stress response and development of plants. Therefore it is necessary to quantify the AsA content in many fields of plant science, including high throughput and critical applications. In this study we compared two different microplate-based AsA assays, which are suitable for high throughput applications: an ascorbate oxidase (AO)-based assay and a dipyridyl (DPD)-based assay. These methods were compared in critical applications, i.e. (i) when AsA concentrations were very low such as in apoplastic extracts, (ii) when plants contained pigments interfering with the spectrometric measurements, and (iii) when plants contained high iron concentration interfering with the color reactions. The precision of measurements was higher with the DPD method, as illustrated by higher recovery rates of internal AsA standards. On the other hand, the AO method was more sensitive to low levels of AsA. This was an advantage in determining apoplastic AsA concentration in rice, which was substantially lower than that of whole tissues. The AO method also had the advantage that plant pigments and high iron concentrations in plants tissues did not interfere with the analysis, as opposed to the DPD assay. In conclusion, both assays had advantages and the choice of a suitable method depends on the specific application.

Keywords: 2,2′-dipyridyl; AO; Apoplast; AsA; Ascorbate; DHA; DPD; DTT; High-throughput analysis; IWF; Iron toxicity; MPA; N-ethylmaleimide; NEM; Oryza sativa; Pigments; ROS; TCA; ascorbate; ascorbate oxidase; dehydroascorbate; dithiothreitol; intercellular washing fluid; metaphosphoric acid; reactive oxygen species; trichloroacetic acid.