AbstractUptake of photoassimilates into the leaf phloem is the key step in carbon partitioning and phloem transport. Symplasmic and apoplasmic loading strategies have been defined in different plant taxa based on the abundance of plasmodesmata between mesophyll and phloem. For apoplasmic loading to occur, an absence of plasmodesmata is a sufficient but not a necessary criterion, as passage of molecules through plasmodesmata might well be blocked or restricted. Here, we present a noninvasive, whole-plant approach to test symplasmic coupling and quantify the intercellular flux of small molecules using photoactivation microscopy. Quantification of coupling between all cells along the prephloem pathways of the apoplasmic loader Vicia faba and Nicotiana tabacum showed, to our knowledge for the first time in vivo, that small solutes like sucrose can diffuse through plasmodesmata up to the phloem sieve element companion cell complex (SECCC). As expected, the SECCC was found to be symplasmically isolated for small solutes. In contrast, the prephloem pathway of the symplasmic loader Cucurbita maxima was found to be well coupled with the SECCC. Phloem loading in gymnosperms is not well understood, due to a profoundly different leaf anatomy and a scarcity of molecular data compared with angiosperms. A cell-coupling analysis for Pinus sylvestris showed high symplasmic coupling along the entire prephloem pathway, comprising at least seven cell border interfaces between mesophyll and sieve elements. Cell coupling together with measurements of leaf sap osmolality indicate a passive symplasmic loading type. Similarities and differences of this loading type with that of angiosperm trees are discussed.