Quantification of Plasmodesmatal Endoplasmic Reticulum Coupling between Sieve Elements and Companion Cells Using Fluorescence Redistribution after Photobleaching

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Abstract
Transgenic tobacco (Nicotiana tabacum) was studied to localize the activity of phloem loading during development and to establish whether the endoplasmic reticulum (ER) of the companion cell (CC) and the sieve element (SE) reticulum is continuous by using a SUC2 promoter-green fluorescent protein (GFP) construct targeted to the CC-ER. Expression of GFP marked the collection phloem in source leaves and cotyledons as expected, but also the transport phloem in stems, petioles, midveins of sink leaves, nonphotosynthetic flower parts, roots, and newly germinated seedlings, suggesting that sucrose retrieval along the pathway is an integral component of phloem function. GFP fluorescence was limited to CCs where it was visualized as a well-developed ER network in close proximity to the plasma membrane. ER coupling between CC and SEs was tested in wild-type tobacco using an ER-specific fluorochrome and fluorescence redistribution after photobleaching (FRAP), and showed that the ER is continuous via pore-plasmodesma units. ER coupling between CC and SE was quantified by determining the mobile fraction and half-life of fluorescence redistribution and compared with that of other cell types. In all tissues, fluorescence recovered slowly when it was rate limited by plasmodesmata, contrasting with fast intracellular FRAP. FRAP was unaffected by treatment with cytochalasin D. The highest degree of ER coupling was measured between CC and SE. Intimate ER coupling is consistent with a possible role for ER in membrane protein and signal exchange between CC and SE. However, a complete lack of GFP transfer between CC and SE indicated that the intraluminal pore-plasmodesma contact has a size exclusion limit below 27 kD.