Development of an infiltration-based RNA preservation method for cryogen-free storage of leaves for gene expression analyses in field-grown plants.

Abstract

Background: Gene expression is a fundamental process for plants to express their phenotype, and its analysis is the basis of molecular studies. However, the instability of RNA often poses an obstacle to analyzing plants grown in fields or remote locations where the availability of liquid nitrogen or dry ice is limited. To deepen our understanding of plant phenotypes and tolerance to field-specific stresses, it is crucial to develop methodologies to maintain plant RNA intact and safely transfer it for downstream analyses such as qPCR and RNA-seq.

Results: In this study, the author developed a novel tissue preservation method that involved the infiltration of RNA preservation solution into the leaf apoplast using a syringe and subsequent storage at 4 °C. RNA-seq using samples stored for 5 d and principal component analyses showed that rice leaves treated with the infiltration method maintained the original transcriptome pattern better than those treated with the traditional method when the leaves were simply immersed in the solution. Additionally, it was also found that extracted RNA can be transported with minimum risk of degradation when it is bound to the membrane of RNA extraction kits. The developed infiltration method was applied to rice plants grown in a local farmer's field in northern Madagascar to analyze the expression of nutrient-responsive genes, suggesting nutrient imbalances in some of the fields examined.

Conclusions: This study showed that the developed infiltration method was effective in preserving the transcriptome status of rice and sorghum leaves when liquid nitrogen or a deep freezer is not available. The developed method was useful for diagnosing plants in the field based on the expression of nutrient-responsive marker genes. Moreover, the method used to protect RNA samples from degradation during transportation offers the possibility to use them for RNA-seq. This novel technique could pave the way for revealing the molecular basis of plant phenotypes by accelerating gene expression analyses using plant samples that are unique in the field.