Background

The allocation of reduced carbon and other compounds from photoautotrophic source tissues to heterotrophic sink tissues through the phloem is a crucial physiological process influencing growth and yield of plants. Because of this central role, there is interest in analyzing and quantifying phloem content from many areas of plant biology. However, collecting authentic phloem sap is difficult because the translocation stream is generally under high hydrostatic pressure and sieve elements have a rapid self-sealing mechanisms to prevent loss when damaged. Several collection techniques have emerged, but there is not currently a single or combination of methods that provide a complete and artifact-free measure of translocating phloem sap. Here, we briefly describe alternative techniques before detailing our approach to collecting phloem exudates into solutions containing low concentrations of ethylenediaminetetraacetic acid (EDTA) after photosynthetically labeling shoots with [¹⁴C]CO₂. Turgeon and Wolf provide a comprehensive review of alternative techniques and their limitations (Turgeon and Wolf, 2009).

Phloem feeding insects, including aphids, scale insects, and planthoppers, evolved mechanisms to feed by directly drawing phloem sap from a plant’s vascular system and evade the self-sealing mechanism. Severing the feeding insect from a stylet penetrated into the phloem–referred to as stylectomy–can provide nanoliter to microliter quantities of sap that may most accurately reflect phloem content. Limitations of the technique are that it is technically challenging, works with only specific insect/plant combinations, the insects are selective for phloem with desired content, and insect saliva injected into the plant influences phloem content (Will et al., 2007; Hewer et al., 2011).

Another technique is to use plants that exude solution from cut stems without apparent sealing. Cucurbits, legumes, Ricinus communis, and some trees are well known for this and have become model systems for studying metabolites and signaling compounds in the phloem. However, the sap collected by this technique generally has low sugar concentrations, suggesting significant dilution and contamination from non-phloem sources and, particularly in the case of cucurbits, may be derived from specialized extrafascicular phloem elements rather than canonical fascicular phloem within vascular bundles (Zhang et al., 2010 and 2012).

The most common technique to sample phloem contents, and the one described in this protocol, is to collect phloem exudates from cut stems or petioles into solutions containing low concentrations of chelating agents, such as EDTA. Cations, particularly calcium (Ca²⁺), are involved in the rapid self-sealing mechanism of sieve tubes. Therefore, application of chelating agents to the cut ends of phloem-containing tissues limits sealing and permits phloem exudations for long periods from most, if not all, plants (King and Zeevaart, 1974; van Bel and Hess, 2008; Liu et al., 2012; Tetyuk et al., 2013). Although this method is most commonly used, it has important limitations and is not without controversy. Exudates are not collected directly, but are diluted into the EDTA solution during the collection period. This method therefore does not provide a measure of concentration, but rather a rate of exudation (i.e., unit of quantity per unit of time). As with the use of plants that naturally continue to exude, it is not clear how much the exudate is diluted by other sources in the plant or how much of the exudate is derived from the fascicular phloem involved in long distance translocation of photoassimilate in phloem sap. Enzymes, such as invertase from damaged cells, can enter the exudate solution and alter the profile of molecules exuded, as discussed at length in several articles (van Bel and Hess, 2008; Liu et al., 2012). A particularly important pitfall of this approach is that EDTA is toxic to cells and promotes membrane leakage which exacerbates contamination of the exudates with the contents of damaged cells and alteration of metabolite composition by leaked enzymes (Turgeon and Wolf, 2009). To minimize these impacts, we use the lowest EDTA concentration that still prevents sieve element sealing and as little tissue as possible is submersed in the EDTA solution (van Bel and Hess, 2008). In addition, we limit EDTA uptake via the xylem by conducting exudations in darkness and high humidity to promote stomatal closure and limit transpiration. Importantly, this procedure uses photosynthetic labeling of rosette leaves with [¹⁴C]CO₂, and exudates are collected from cut petioles that have negligible photosynthetic activity and are well shaded by the mature rosette above. Therefore, counting ¹⁴C in the exudate solution is a quantitative representation of photoassimilate translocated from the leaves. When combined with other protocols (Yadav et al., 2017a and 2017b), this contributes to a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation (Dasgupta et al., 2014; Khadilkar et al., 2016).