Sorbitol-6-phosphate dehydrogenase expression in transgenic tobacco. High amounts of sorbitol lead to necrotic lesions

Abstract

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 &mgr;mol g-1 fresh weight were found. Plants that accumulated up to 2 to 3 &mgr;mol g-1 fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 &mgr;mol g-1 fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 &mgr;mol g-1 fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.

Figure 1

Schematic presentation of the gene construction leading to the expression of sorbitol-6-phosphate dehydrogenase (S6PDH) in transgenic tobacco. The gene cassette was subcloned into pBIN19 and introduced into plants by A. tumefaciens-based transformation.

Figure 2

Phenotype of S5C plant and examples of leaves with necrotic lesions. The top panel shows the habitus of a 10-week-old plant typical of plants with a sorbitol concentration of more than approximately 15 μmol g⁻¹ fresh weight. The bottom panel shows mature leaves typical of plants that accumulated sorbitol to 5 to 10 μmol g⁻¹ fresh weight in their young, expanding leaves.

Figure 3

Relationship between myo-inositol, Glc, Fru, and sorbitol amounts in S5C plants. Plants were grown for 10 weeks in a growth room (see Methods). The plant material was collected 2 h after the beginning of illumination. The same relationship was found when starch content and sorbitol amounts were compared. gfw, Grams fresh weight.

Figure 4

Seedling growth supplemented with myo-inositol. Seedlings of SR1 (A and B) and S5C (C and D) plants were grown in Murashige and Skoog medium with (B and D) and without myo-inositol (A and C).