Collection and Analysis of <em>Arabidopsis</em> Phloem Exudates Using the EDTA-facilitated Method

Abstract

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines

“…We acknowledge that the EDTA-based method employed here for the collection of phloem sap has welldocumented pitfalls (Turgeon and Wolf, 2009), including that the procedure requires the cutting of a surface and extended exposure of cells to EDTA causes damage, leakage from which may contaminate the sap. Therefore, we chose the EDTA-facilitated phloem exudation method described by Tetyuk et al that has been optimized for Arabidopsis (Tetyuk et al, 2013), in which the samples are exposed to EDTA for 1 h and washed with water, followed by collection of the phloem exudate into water. This strives to minimize cell damage and downstream interference from EDTA, while removing compounds that have accumulated from damaged or cut cells.…”

Regulation of Sucrose Transporters and Phloem Loading in Response to Environmental Cues

“…Phloem exudates were obtained using the EDTA-facilitated exudation method (Tetyuk et al, 2013) and adapted for all the plants tested in this experiment. For Arabidopsis, about 20 leaves from the control and plants subjected to different stimuli were cut with a razor blade at the base of the petiole, close to the center of the rosette, and immediately placed in petri dishes containing 20 mM EDTA solution.…”

Constitutive and Companion Cell-Specific Overexpression of AVP1, Encoding a Proton-Pumping Pyrophosphatase, Enhances Biomass Accumulation, Phloem Loading, and Long-Distance Transport

“…4C), 14 to 16 mature rosette source leaves were harvested from 40-d-old adult plants for each time point. Phloem exudation was done as reported by Tetyuk et al (2013). In brief, leaves were cut and immediately placed in 20 mM K 2 -EDTA.…”