Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method

Physiological and biochemical studies on the leaf apoplast have been facilitated by the use of the infiltration-centrifugation technique to collect intercellular washing fluid (IWF). However, this technique has been difficult to implement in rice (Oryza sativa L.) for various reasons. We compared the collection efficiency of leaf IWF between two types of rice varieties (Indica and Japonica), as well as between rice and other species (spinach, snap bean and wheat). Although the extraction of IWF in most species took only 2-3 min, it took up to 35 min in rice. The difficulty in infiltration with rice was ascribed to the small stomatal aperture and hydrophobicity of the leaves. In this study, we have established an improved method for collecting IWF and determining the apoplastic air and water volumes in rice leaves. We have shortened the infiltration time to 8 min via the following improvements: (i) infiltration under outdoor shade in the daytime to prevent stomatal closure and a rise in temperature of the infiltration medium; (ii) soaking of leaves in a surfactant solution to decrease the leaf hydrophobicity; and (iii) continuous pressurization using a sealant injector to facilitate the infiltration. The rapid collection of IWF achieved using this technique will facilitate study of the leaf apoplast in rice.