Search

Abstract Background Understanding thiamin (thiamine; vitamin B1) metabolism in plants is crucial, as it impacts plant nutritional value as well as stress tolerance. Studies aimed at elucidating novel aspects of thiamin in plants rely on adequate assessment of thiamin content. Mass spectrometry-based methods provide reliable quantification of thiamin as well as closely related biomolecules. However, these techniques require expensive equipment and expertise. Microbiological turbidimetric assays can evaluate the presence of thiamin in a given sample, only requiring low-cost, standard lab equipment. Although these microbiological assays do not reach the accuracy provided by mass spectrometry-based methods, the ease with which they can be deployed in an inexpensive and high-throughput manner, makes them a favorable method in many circumstances. However, the thiamin research field could benefit from a detailed step-by-step protocol to perform such assays as well as a further assessment of its potential and limitations. Results Here, we show that the Saccharomyces cerevisiae thiamin biosynthesis mutant thi6 is an ideal candidate to be implemented in a turbidimetric assay aimed at assessing the content of thiamin and its phosphorylated equivalents (total vitamer B1). An optimized protocol was generated, adapted from a previously established microbiological assay using the thi4 mutant. A step-by-step guidance for this protocol is presented. Furthermore, the applicability of the assay is illustrated by assessment of different samples, including plant as well as non-plant materials. In doing so, our work provides an extension of the applicability of the microbiological assay on top of providing important considerations upon implementing the protocol. Conclusions An inexpensive, user-friendly protocol, including step-by-step guidance, which allows adequate estimation of vitamer B1 content of samples, is provided. The method is well-suited to screen materials to identify altered vitamer B1 content, such as in metabolic engineering or screening of germplasm.

Background: Understanding thiamin (thiamine; vitamin B1) metabolism in plants is crucial, as it impacts plant nutritional value as well as stress tolerance. Studies aimed at elucidating novel aspects of thiamin in plants rely on adequate assessment of thiamin content. Mass spectrometry-based methods provide reliable quantification of thiamin as well as closely related biomolecules. However, these techniques require expensive equipment and expertise. Microbiological turbidimetric assays can evaluate the presence of thiamin in a given sample, only requiring low-cost, standard lab equipment. Although these microbiological assays do not reach the accuracy provided by mass spectrometry-based methods, the ease with which they can be deployed in an inexpensive and high-throughput manner, makes them a favorable method in many circumstances. However, the thiamin research field could benefit from a detailed step-by-step protocol to perform such assays as well as a further assessment of its potential and limitations. Results: Here, we show that the Saccharomyces cerevisiae thiamin biosynthesis mutant thi6 is an ideal candidate to be implemented in a turbidimetric assay aimed at assessing the content of thiamin and its phosphorylated equivalents (total vitamer B1). An optimized protocol was generated, adapted from a previously established microbiological assay using the thi4 mutant. A step-by-step guidance for this protocol is presented. Furthermore, the applicability of the assay is illustrated by assessment of different samples, including plant as well as non-plant materials. In doing so, our work provides an extension of the applicability of the microbiological assay on top of providing important considerations upon implementing the protocol. Conclusions: An inexpensive, user-friendly protocol, including step-by-step guidance, which allows adequate estimation of vitamer B1 content of samples, is provided. The method is well-suited to screen materials to identify altered vitamer B1 content, such as in metabolic engineering or screening of germplasm. [ABSTRACT FROM AUTHOR]

Summary: Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter‐mining metabolite levels in plant material and provide an affordable alternative to MS‐based analysis.Here, we evaluate, by comparison to the LC–MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples.The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild‐type and thiamin biofortified Arabidopsis plant samples.The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species. [ABSTRACT FROM AUTHOR]