Root sampling method for aquaporin gene expression analysis in rice

Abstract

A rice root system consists of main roots, and lateral roots which account for a large portion of the total length and surface area of the whole root system, indicating that lateral roots greatly contribute to its hydraulic conductance. It is therefore worthwhile measuring aquaporin activities of lateral roots in comparison with main roots to evaluate their contribution to hydraulic conductivity. However, the sampling of fine lateral roots is time-consuming, and its method can be critical because of the difficulties of handling them for sampling. In addition, root has been reported to be sensitive to the surrounding environment and affected by destructive sampling procedures in which lateral roots are removed from main roots. Therefore, in this study, we attempted to establish a method for collecting samples of lateral and main roots for aquaporin analysis while minimizing the effect of separation of roots from the shoot, and the removable of lateral roots from main roots on the transcript abundance. We conducted three experiments and measured the expression levels of four aquaporin genes including OsPIP2;1, OsPIP2;4, OsPIP2;5 and OsTIP2;1 with different sampling time length (experiment 1), with or without shoot removal (experiment 2) and light and dark periods samplings with different light conditions (experiment 3). Our results revealed that the aquaporin expression levels in separated roots did not change compared to those of the roots which were immediately preserved after sampling from the shoot, when root separation was conducted within 10 minutes without shoot removal under fluorescent light during light period of growth, as well as under indirect lighting during dark period of growth.