Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides

Abstract

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.

Fig. 1.

RNA gel blot analysis forVpGAS1 (A) and VpGAS2 (B) in different organs of V. phoeniceum. Ethidium bromide-stained rRNA as loading controls are shown in the lower gels.

Fig. 2.

In situ hybridization of VpGAS1 and VpGAS2. (A and C) VpGAS1 and VpGAS2 antisense probes, respectively, localize to the intermediary cells. Each arrow in A indicates an intermediary cell. Note the arrangement of intermediary cells in paired files along the lengths of the veins. (B and D) VpGAS1 and VpGAS2 sense probes show only background staining. (Scale bar: 50 μm.)

Fig. 3.

Wild type (A) and transgenic plant 8D (B) grown in the greenhouse. (Scale bars: 1 cm.)

Fig. 4.

Carbohydrates in mature leaves. (A) Mono- and disaccharide concentrations. (B) Galactinol and RFO concentrations. Error bars indicate SE (n ≥ 3).

Fig. 5.

Distribution of radiolabel 105 min after photosynthesis in ¹⁴CO₂, calculated as a percentage of the neutral fraction. (A) Radiolabel in the lamina. (B) Radiolabel in petioles. m, monosaccharides include glucose, fructose, and galactose; s, sucrose; g, galactinol; r, raffinose; st, stachyose.

Fig. 6.

Starch retention in plants kept in the dark for 20 h. Hand sections were stained for starch with iodine. (A) Wild-type tissue does not stain, indicating that all starch has been degraded. (B and C) Transgenic plants 8D and 12B, respectively, with heavy starch staining. (Scale bar: 25 μm.)