Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (this protocol), (2) the relative rates of carbon loading and transport through the phloem (Yadav et al., 2017a), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (Yadav et al., 2017b). We propose that conducting all three protocols on experimental and control plants provides 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). In this protocol, Arabidopsis leaf disks isolated from mature rosette leaves are infiltrated with a buffered solution containing [¹⁴C]Suc. Suc transporters (SUCs or SUTs) load Suc into the phloem and excess, unloaded Suc in the leaf disk is then washed away. Loading of labeled Suc into the veins is visualized by autoradiography of lyophilized leaf disks and quantified by scintillation counting. Results are expressed as disintegration per minute per unit of leaf disk fresh weight or area.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (Yadav et al., 2017a), (2) the relative rates of carbon loading and transport through the phloem (Yadav et al., 2017b), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (this protocol). We propose that conducting all three protocols on experimental and control plants provides 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). In this protocol, [¹⁴C]CO₂ is photoassimilated in source leaves and phloem loading and transport of the ¹⁴C label to heterotrophic sink organs, particularly roots, is quantified by scintillation counting. Using this protocol, we demonstrated that overexpression of sucrose transporters and a vacuolar proton pumping pyrophosphatase in the companion cells of Arabidopsis enhanced transport of ¹⁴C label photoassimilates to sink organs (Dasgupta et al., 2014; Khadilkar et al., 2016). This method can be adapted to quantify long-distance transport in other plant species.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (Yadav et al., 2017a), (2) the relative rates of carbon loading and transport through the phloem (this protocol), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (Yadav et al., 2017b), We propose that conducting all three protocols on experimental and control plants provides 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). In this protocol, [¹⁴C]CO₂ is photoassimilated in source leaves and phloem loading and transport of photoassimilate is quantified by collecting phloem exudates into an EDTA solution followed by scintillation counting.