Background

Plants consist of multiple organs that play distinctive roles during plant life. Hence, organ-to-organ communication is indispensable for plants to adapt to environmental stresses and maintain homeostasis at the whole-plant level. It is known that various types of macromolecules, such as phytohormones, RNAs, proteins, and peptides mediate long-distance signaling (Okamoto et al., 2015, 2016; Lu et al., 2020). The xylem plays an important role in the translocation of water and micronutrients from roots to shoots. In addition, long-distance signaling molecules, including small secreted peptides and phytohormones, are translocated from roots to shoots through the xylem. Therefore, collecting and analyzing xylem exudates is an effective approach to identify novel long-distance mobile molecules.

To date, several methods have been developed to collect xylem exudates (Buhtz, 2004, Goodger et al., 2005; Dafoe and Constabel, 2009), and the appropriateness of a given method is dependent on plant species and conditions. One method uses a pressure chamber to obtain xylem exudate from woody plants. In this method, roots are placed inside a sealed chamber, and pressurized gas is added to the chamber. This method can be applied to woody plants that have mechanically stronger tissues than herbaceous plants, and a precise protocol was written by Flajšman et al. (2017). Another method depends on root pressure and is usually applied to herbaceous plants whose tissues are soft. Root pressure is one of the driving forces of xylem sap. Accumulation of solutes in root xylem results in low water potential and generates positive hydrostatic pressure in the xylem (Taiz et al., 2015). Although the quantity of xylem exudates obtained with this method depends on plant species and conditions, this method requires no special equipment and has been applied to many herbaceous plants, including Arabidopsis thaliana (Iwai et al., 2003; Buhtz et al., 2004; Kehr et al., 2005; Alvarez et al., 2006; Djordjevic et al., 2007; Ligat et al., 2011; Ko et al., 2014; Tabata et al., 2014; Okamoto et al., 2015; Luo and Zhang, 2019). However, the collection of xylem exudates is likely not familiar to many researchers, and it is difficult to find a step-by-step protocol for this method.

Previously, we collected xylem exudate from soybean to conduct peptidome analysis and identified various endogenous root-to-shoot mobile peptides (Okamoto et al., 2015). In addition, we analyzed xylem exudate from the model plant Arabidopsis thaliana and detected mature CLAVATA3/ESR-related 2 peptides (Okamoto et al., 2022a). In this protocol, we describe step-by-step procedures for the collection of xylem exudates from both the model plant Arabidopsis and the crop plant soybean.

Part I. Collection of xylem exudates from Arabidopsis