Overcoming the Difficulties in Collecting Apoplastic Fluid from Rice Leaves by the Infiltration–Centrifugation method

Abstract

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.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using <em>Phaseolus vulgaris</em> as an Example

“…In well-performed extractions of P. vulgaris AWF, G6PDH was undetectable by a standard enzyme assay. This is consistent with other reports where G6PDH activity becomes detectable only above a threshold centrifugal force 12 . In contrast, a baseline level of malate dehydrogenase activity (2.5 U/ ml) was detectable in all P. vulgaris AWF samples using a standard metabolic assay.…”

“…Importantly, the source of plant tissue must be standardized, including leaf type, leaf age, growth/ environmental conditions and time of day ( Table 1). Large differences exist in the ease with which different leaves are infiltrated and the AWF subsequently recovered by centrifugation; these differences are correlated with stomata number, aperture size and mesophyll resistance 12,14 . Even among different cultivars of P. vulgaris there are large differences in the ease and yield of the AWF extraction procedure; for example leaves of the Tendergreen variety used here are more amenable to AWF extraction using this method than the Canadian Wonder cultivar.…”

“…Within P. vulgaris the first true leaves are the biggest and the easiest to infiltrate, making them the obvious choice for apoplastic extractions. The apoplastic air and water volume has been shown to vary with leaf age in several species leading to differences in AWF extractability 12,14 . In P. vulgaris, older leaves become substantially more difficult to infiltrate and yield less AWF upon centrifugation; therefore leaves were harvested when they reached full expansion.…”

“…This technique has been evaluated previously [11][12][13] and Lohaus et al 2001 14 provide a thorough examination of many of the method's technical parameters. As implied by the name, the infiltration-centrifugation technique is a two-step method that essentially involves replacement of the apoplastic air space with an aqueous infiltration fluid, which mixes with the native apoplastic fluid, followed by recovery of the infiltration/apoplastic fluid mixture by gentle centrifugation of the leaves.…”

Altered Expression of a Malate-Permeable Anion Channel, OsALMT4, Disrupts Mineral Nutrition

“…Concentrations of Mn, B, and Ca in leaf apoplast and symplast were measured following the method of Nouchi et al (2012). Plants were grown in flooded soil for 8 weeks, and the most recently expanded leaves were selected from four separate plants for each genotype.…”