Quantifying the Capacity of Phloem Loading in Leaf Disks with [¹⁴C]Sucrose

Background

Transport of photoassimilates from source to sink organs is essential for normal growth and maintenance of whole plants. Phloem loading in leaves is the delivery of photoassimilate synthesized in mesophyll cells to the companion cells (CC) and sieve elements (SE) of the phloem vasculature system. Three distinct loading mechanisms are recognized. Two of these expend energy to accumulate high concentrations of sugar in the CC and SEs and generate a high hydrostatic pressure in source-leaf phloem. The first is apoplastic phloem loading, in which Suc (and/or sugar alcohols in some species) is loaded across the plasma membrane from the cell wall space (i.e., the apoplast) into the CCs at the expense of the proton motive force (Giaquinta, 1983). The second is polymer trapping, in which Suc diffuses into the phloem through specialized plasmodesmata and is converted to oligosaccharides that are too large to diffuse back out (Turgeon, 1996). The third mechanism is passive loading, in which the highest solute concentrations are in the mesophyll cells and plasmodesmata provide an open path for passive movement into the CCs and SEs (Rennie and Turgeon, 2009).

Due to the central role of phloem loading and transport to plant physiology and productivity, it is desirable to have reliable methods to identify and quantify the contents of the phloem and the rates of transport. In addition, phloem loading and transport are targets for biotechnology and metabolic engineering to enhance productivity (Ainsworth and Bush, 2011; Cao et al., 2013; Dasgupta et al., 2014; Zhang et al., 2015; Yadav et al., 2015). Quantitatively assessing the rates and capacity of phloem loading and transport in natural and engineered systems is difficult for several reasons. As examples, CCs and SEs are imbedded in surrounding tissues and are very narrow relative to surrounding non-phloem cells; the phloem is under high pressure and seals rapidly when damaged; collected phloem sap is usually contaminated with the content of other cells; because transport is a dynamic process, estimates of phloem content do not indicate rates of transport (Turgeon and Wolf, 2009; Dinant and Kehr, 2013; Tetyuk et al., 2013). C isotope ¹¹C, ¹³C and ¹⁴C, in CO₂ or labeled sugars, have provided, and continue to provide, critical information on phloem loading mechanisms (Sovonick et al., 1974; Turgeon and Gowan, 1990; Turgeon and Medville, 1998), as well as quantitative information on the rates of loading and transport (Thorpe and Minchin, 1988; Karve et al., 2015; Dersch et al., 2016).

Arabidopsis loads Suc from the apoplast with the Suc transporter (SUT) encoded by AtSUC2 (Truernit and Sauer, 1995; Gottwald et al., 2000; Srivastava et al., 2008). AtSUC2 and other transporters have been assessed in Saccharomyces cerevisiae and Xenopus laevis oocytes as heterologous systems to establish Michaelis Menten kinetic parameters (reviewed in [Ayre, 2011]). While valuable for comparing the activities and affinities among transporters, these do not inform on the activity of the transporters in planta, or on the overall impact on plant growth and productivity. As an example of this, SUTs from Solanaceae species with roughly the same kinetic properties as AtSUC2 did not rescue an Arabidopsis Atsuc2 mutant, while an AtSUC2 cDNA and ZmSUT1 cDNA from Zea mays did rescue the mutant (Dasgupta et al., 2014). In this protocol, and two that follow (Yadav et al., 2017a and 2017b), we detail the use of [¹⁴C]CO₂ and [¹⁴C]Suc to quantitatively assess phloem loading and carbon transport in living explants and intact plants. These methods are used routinely in our laboratory, and recently contributed to our demonstration that plants over expressing certain SUTs in CCs showed increased phloem loading and transport, despite having stunted growth (Dasgupta et al., 2014) and, in a separate study, over expression of a H⁺-pumping pyrophosphatase enhanced Suc loading and transport without a corresponding increase in SUT expression levels (Khadilkar et al., 2016).

In the procedure described here (Figure 1), Arabidopsis leaf disks isolated from mature rosette leaves are vacuum infiltrated with a buffered solution containing [¹⁴C]Suc. SUTs load the Suc into CCs, and excess Suc is washed away. The disks are quickly frozen in powdered dry ice, lyophilized, and autoradiography is used to visualize loading into the veins. Rapid freezing and lyophilization prevent the highly soluble labeled sugar from diffusing away from the sites of loading. Scintillation counting then quantifies the loaded label. We generally pool four leaf disks into one replicate, and perform 6 biological replicates for each control and experimental system. This protocol was adapted for Arabidopsis (Dasgupta et al., 2014; Khadilkar et al., 2016) from procedures described by Turgeon and colleagues (Turgeon and Wimmers, 1988; Turgeon and Gowan, 1992; Turgeon and Medville, 1998; Haritatos et al., 2000; Goggin et al., 2001), and can be further adapted for other plants. Typical autoradiography results for Arabidopsis, tobacco, and wheat are shown in Figure 2; representative quantitative results of loading in different transgenic Arabidopsis plants are published (Dasgupta et al., 2014; Khadilkar et al., 2016).