Engineering cytoplasmic male sterility via the chloroplast genome by expression of {beta}-ketothiolase

Abstract

While investigating expression of the polydroxybutyrate pathway in transgenic chloroplasts, we addressed the specific role of beta-ketothiolase. Therefore, we expressed the phaA gene via the chloroplast genome. Prior attempts to express the phaA gene in transgenic plants were unsuccessful. We studied the effect of light regulation of the phaA gene using the psbA promoter and 5' untranslated region, and evaluated expression under different photoperiods. Stable transgene integration into the chloroplast genome and homoplasmy were confirmed by Southern analysis. The phaA gene was efficiently transcribed in all tissue types examined, including leaves, flowers, and anthers. Coomassie-stained gel and western blots confirmed hyperexpression of beta-ketothiolase in leaves and anthers, with proportionately high levels of enzyme activity. The transgenic lines were normal except for the male-sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Floral developmental studies revealed that transgenic lines showed an accelerated pattern of anther development, affecting their maturation, and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium, and vacuolation affected pollen grains and resulted in the irregular shape or collapsed phenotype. Reversibility of the male-sterile phenotype was observed under continuous illumination, resulting in viable pollen and copious amount of seeds. This study results in the first engineered cytoplasmic male-sterility system in plants, offers a new tool for transgene containment for both nuclear and organelle genomes, and provides an expedient mechanism for F(1) hybrid seed production.

Figure 1.

Molecular characterization of transgenic lines. A, Schematic representation of the transformed chloroplast genome and the pLDR-5′UTR-phaA cassette. Annealing sites for the primer pairs and expected sizes of PCR products are shown. BamHI restriction sites, DNA fragment produced after restriction digestion, and the phaA probe used in Southern-blot analyses are shown. B, The map shows the wild-type chloroplast genome, restriction enzyme sites used for Southern-blot analysis, expected fragment sizes after digestion, and probing with the flanking sequence.

Figure 2.

A, PCR analysis of untransformed plants and putative transformants. Top section is transgenic line 4B; bottom section is transgenic line 4A. A, Lane 1, Untransformed plant; 2, 3P-3M (1.65 kb); 3, 4P-4M (1.65 kb); 4, 5P-2M (3.56 kb); 5, 5P-3′phaA (2.0 kb); 6, 5P-phaA internal (1.5 kb); 7, positive control (pLD-5′UTR-phbA plasmid DNA) 5P-phbA internal (1.5 kb); M, marker. B and D, Southern-blot analysis of T₀ and T₁ generation transgenic lines, respectively, with the chloroplast flanking sequence probe; 10-kb fragment shows integration of transgenes; 7.1-kb fragment shows wild-type fragment. C and E, Transgenic T₀ and T₁ plants, respectively, and untransformed plant probed with the phaA gene. The 10-kb fragment is observed in transgenic lines but not in the untransformed plant. F and G, Northern-blot analysis using the phaA and aadA probes, respectively. Monocistron (a, 5′UTR-phaA, 1,384 nt) and polycistrons (b, aadA-phaA, 2,255 nt; c, from native 16S Prrn, 4,723 nt) containing the phaA gene are observed in the transgenic lines when the phaA probe is used. Only polycistrons are observed with the aadA probe. In B to G: 1, untransformed plant; 2 to 4, chloroplast transgenic lines 4A, 4B, and 4C, respectively; B, blank lane.

Figure 3.

β-Ketothiolase characterization in transgenic lines. A, Coomassie-stained gel showing abundant β-ketothiolase expression in transgenic lines; 15 μg of total plant protein was loaded per lane (a, D1 protein; b, β-ketothiolase, 40.8 kD; c, Rubisco large subunit, based on molecular range). M, Marker; 1 to 3, transgenic lines 4A, 4B, and 4C, respectively; 4, 4A T₁ generation; 5, untransformed plant; 6, bacterial purified β-ketothiolase. B, Western-blot analysis; 10 μg of total plant protein from transgenic lines and wild type was loaded per lane; anti-β-ketothiolase antibody was used. Bands corresponding to the 40.8-kD monomers (a), a possible modified version of the monomer (b), trimer (c), or tetramer (d) are observed in transgenic lines. 1, Untransformed plant; 2 to 4, transgenic lines 4A, 4B, and 4C, respectively; 5 and 6, 4A and 4B T₁ generation, respectively.

Figure 4.

Characterization of the male-sterile phenotype. A to C, Flowers from transgenic lines; note the absence of fruit capsules and fallen flowers. D and E, Untransformed tobacco flowers and fruit capsules. Comparison of stamens and stigma: note shorter stamens in transgenic lines (F) compared to untransformed (G). Comparison of mature anthers: note abundant pollen in untransformed anther (I) and the lack of pollen in transformed anther (H). J, Transgenic fruit capsule with seeds after pollination of transgenic stigma with untransformed pollen. K, Germination and growth of T₁ seedlings on Murashige and Skoog medium with 500 μg/mL spectinomycin. wt, Untransformed; 4A, T₁ transgenic line 4A; 4B, T₁ transgenic line 4B, obtained after pollination with untransformed pollen.

Figure 5.

Comparison of growth and development. A, Untransformed plant (WT) and T₀ generation transgenic (T) line (4A) grown for 2 months in soil. B, Untransformed plant (WT) and T₁ generation independent transgenic lines 4A, 4B, 4C, and 4D, 1.5 months after germination.

Figure 6.

SEM pictures of pollen grains in anthers of untransformed (A–C) and transgenic (D–F) plants at different magnifications. A and D, ×500; B and E, ×1,000; C, ×3,500; F, ×3,000.

Figure 7.

β-Ketothiolase expression in anthers. A, Pigmentation of anthers during flower development in transgenic plants. Stage 1 of flower development is shown. B, Northern-blot analysis of flowers and anthers. Three micrograms of total plant RNA was loaded per lane, and the phaA probe was used. M, Marker; 1, transgenic flower; 2 and 3, transgenic anthers from line 4A and 4B, respectively; 4, untransformed flower; 5, transgenic leaf; 6, untransformed leaf. C, β-Ketothiolase expression in transgenic flowers and anthers detected by western-blot analysis in lanes 2 and 3, respectively. RNA and protein samples used per lane were the product of the combined extraction from flowers or anthers from stages 1 and 3.

Figure 8.

Studies on anther development. Bright-field photographs of untransformed (wt) and transgenic anthers at different developmental stages (S). Anthers at the designated stages were fixed, embedded with paraffin, and sliced into 5- and 10-μm transverse sections. The fixed sections were stained with toluidine blue and visualized under the light microscope at a magnification of ×100. C, Connective tissue; E, epidermis; En, endothecium; MMC, microspore mother cells; Msp, microspores; PS, pollen sac; S, stomium; T, tapetum; TDS, tetrads.

Figure 9.

Reversibility of male sterility after 10 d under continuous illumination. A, Transgenic flower after 9 d in continuous light; note normal length of the anther filaments and pollen grains. B, Fully developed fruit capsules containing seeds from reversed transgenic lines. C, Abundant seeds from a transgenic fruit capsule. D, Seedlings produced via the reversibility to male fertility. Transgenic seeds germinated in Murashige and Skoog medium supplied were with 500 μg/mL spectinomycin. E, Bleached wild-type tobacco seedlings.