Xylem Sap Extraction Method from Hop Plants

Background

Extraction of xylem sap from plants is mostly used for studies of xylem sap proteome and various methods have been used to extract sap from plant xylem tissues. Buhtz et al. (2004) used hand-held pipettes to collect xylem sap from cut plant stems for comparing xylem proteomes in different plants (broccoli, oilseed rape, pumpkin and cucumber). The same method was used for collecting xylem sap from Brassica napus (Kehr et al., 2005), Brassica oleracea (Ligat et al., 2011) and soybean (Subramanian et al., 2009). Alvarez et al. (2006) extracted the maize xylem sap proteome using ‘root pressure’, as described by Goodger et al. (2005). Dafoe and Constabel (2009) used a Tygon tube, which was fitted over the wood, to collect xylem sap from hybrid poplar and no additional pressure was applied. Information on the protein content of xylem sap is also available for apple, pear and peach (Biles and Abeles, 1991).

Because vascular plant fungal pathogens spread inside host plants through their xylem, this fluid is the most appropriate medium to search for in planta secreted virulence factors from the fungal pathogen. Rep et al. (2002) used a simple xylem sap extraction method by Satoh et al. (1992) whereby cut stems of Fusarium oxysporum f. sp. lycopersici-infected tomato were placed in a horizontal position and sap was dripped from the cut surface. A Scholander pressure chamber (Scholander et al., 1965) was used to obtain xylem sap from Verticillium longisporum-infected oilseed rape (Floerl et al., 2008).

It is hard to draw any conclusion as to which method works best for what kind of plant since no comparative studies have been performed on different methods on the same plant species. It appears that plant species with soft and juicy stems need no pressure added to obtain xylem sap and simple methods are efficient for xylem sap extraction. This may be related to the root pressure, which causes xylem sap to rise through a plant stem from the roots towards the leaves due to osmosis in the roots (Taiz and Zeiger, 2010). However, some species never generate any root pressure (Kramer and Boyer, 1995) so some external force (e.g., a pressure chamber) must be provided in order to extract xylem sap from such plants. So far, there is no specific extraction method available for collecting xylem sap from hop plant infected with Verticillium nonalfalfae. Hop plants have woody roots and exposure of roots to pressure causes no harm to root tissue and no contamination (e.g., with cytosol liquids, cell membranes and other parts). We therefore used a Scholander pressure chamber on hops. This is the first protocol for sampling xylem sap from hop plants.