Assessing Long-distance Transport from Photosynthetic Source Leaves to Heterotrophic Sink Organs with [¹⁴C]CO₂

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.