Assessment of the Influence of Different Sample Processing and Cold Storage Duration on Plant Free Proline Content Analyses

Introduction - A method which is widely accepted for the analysis of free proline content in plant tissues is based on the use of 3% sulfosalicylic acid as an extractant, followed by spectrophotometric quantification of a proline-ninhydrin complex in toluene. However, sample preparation and storage may influence the proline actually measured. This may give misleading or difficult to compare data. Objective and Methodology - To evaluate free proline levels fresh and frozen strawberry (Fragaria x ananassa Duch.) leaves and soybean [Glycine max (L.) Mem] hypocotyl tissues were used. These were ground with or without liquid nitrogen and proline extracted with sulfosalicylic acid. A particular focus was the influence of plant sample cold storage duration (1, 4 and 12 weeks at -20 degrees C) on tissue proline levels measured. Results - The free proline content analyses, carried out in leaves of Fragaria x ananassa Duch. as well as in hypocotyls of Glycine max (L.) Mem, showed a significant influence of the sample preparation method and cold storage period. Long-term storage of up to 12 weeks at -20 degrees C led to a significant increase in the measured proline in all samples analysed. Conclusion - The observed changes in proline content in plant tissue samples stored at -20 degrees C indicate the likelihood of the over-estimation of the proline content if the proline analyses are delayed. Plant sample processing and cold storage duration seem to have an important influence on results of proline analyses. Therefore it is recommended that samples should be ground fresh and analysed immediately. Copyright (C) 2010 John Wiley & Sons, Ltd.