Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes (VpGAS1 and VpGAS2) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins.plasmodesmata ͉ polymer trap ͉ stachyose ͉ galactinol ͉ RNAi U p to 80% of the carbon fixed in mature leaves by photosynthesis is exported to heterotrophic sinks to enable their growth and development. The first step in the transport pathway, and one that is highly regulated (1, 2), is the transfer of photoassimilate from mesophyll cells to the sieve elements (SEs) and companion cells (CCs) of minor veins (3-6). This process, known as phloem loading, creates the positive hydrostatic pressure difference between source and sink phloem that drives the mass flow of solution.Two loading mechanisms have been proposed. In one, photoassimilate enters the apoplast and is subsequently loaded into the phloem by specific transport proteins (3, 7). The second mechanism appears to be entirely symplastic (by plasmodesmata) (4-6). The first hint that loading might take place symplastically came from the discovery, in certain species, of specialized CCs (intermediary cells) in the minor veins, which are linked to bundle sheath cells by extremely high numbers of asymmetrically branched plasmodesmata (5).In all plants with intermediary cells, the phloem sap contains a substantial amount of raffinose family oligosaccharide (RFO), especially the tri-and tetrasaccharides, raffinose, and stachyose. The consistent association of RFOs with intermediary cells suggests that the synthesis of these sugars is an integral part of the phloem-loading mechanism.This reasoning is the basis of the polymer trap model of symplastic loading (8,9). According to the model, sucrose diffuses from bundle sheath cel...