Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Abstract

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

Figure 1

Tissue-specific expression pattern of Nin88. (A) RNA blot analysis of Nin88 expression in different tobacco tissues: L, leaves; St, stem; R, roots; Sb, small buds; Lb, large buds; F, flowers; Se, sepals; P, petals; O, ovary; A, anther. (B– E) Immunolocalization of Nin88 with cross-sections of anthers at different developmental stages. (B) Tapetum surrounding the pollen mother cells. (Bar = 50 μm.) (C) Tapetum surrounding the pollen mother cells. (Bar = 50 μm.) (D) Microspores in tetrad stage. (Bar = 50 μm.) (E) Tapetum shrunken, pollen grains separated. (Bar = 50 μm.)

Figure 2

GUS staining of Nin88-GUS tobacco plants and wild-type plants. (A) Anther from wild-type control plant (Upper) and anthers from Nin88-GUS plants (Lower) after incubation with X-Glc. (Bar = 1 mm.) (B) Pollen from Nin88-GUS plants. (Bar = 20 μm.) (C) Pollen from wild-type control plants. (Bar = 20 μm.)

Figure 3

Electron microscopic pictures of pollen from wild-type (A and B) and transgenic plants (C–F). (A) SEM picture of wild-type pollen. (Bar = 5 μm.) (B) Transmission EM (TEM) picture of wild-type pollen. (Bar = 5 μm.) (C) SEM picture of pollen from NT23–4. (Bar = 5 μm.) (D) TEM picture of pollen from NT23–4. (Bar = 5 μm.) (E) SEM picture of pollen from NT23–6. (Bar = 5 μm.) (F) TEM picture of pollen from NT23–6. (Bar = 5 μm.) NT23–4 represents one of the plants whose pollen shows germination efficiencies between 25% and 40%; NT23–6 is one of the plants with germination efficiency <2%.

Figure 4

Extracellular invertase activity of pollen at different developmental stages.

Figure 5

GUS staining of Nin88-GUS tomato plants and wild-type plants. (A) Anthers from wild-type control plant. (Bar = 1 mm.) (B) Anthers from Nin88-GUS plants. (Bar = 1 mm.) (C) Pollen from wild-type control plants. (Bar = 20 μm.) (D) Pollen from Nin88-GUS plants. (Bar = 20 μm.)