Tapetum-specific expression of a cytoplasmic orf507 gene causes semi-male sterility in transgenic peppers

Abstract

Though cytoplasmic male sterility (CMS) in peppers is associated with the orf507 gene, definitive and direct evidence that it directly causes male sterility is still lacking. In this study, differences in histochemical localization of anther cytochrome c oxidase between the pepper CMS line and maintainer line were observed mainly in the tapetal cells and tapetal membrane. Inducible and specific expression of the orf507 gene in the pepper maintainer line found that transformants were morphologically similar to untransformed and transformed control plants, but had shrunken anthers that showed little dehiscence and fewer pollen grains with lower germination rate and higher naturally damaged rate. These characters were different from those of CMS line which does not produce any pollen grains. Meanwhile a pollination test using transformants as the male parent set few fruit and there were few seeds in the limited number of fruits. At the tetrad stage, ablation of the tapetal cell induced by premature programmed cell death (PCD) occurred in the transformants and the microspores were distorted and degraded at the mononuclear stage. Stable transmission of induced semi-male sterility was confirmed by a test cross. In addition, expression of orf507 in the maintainer lines seemed to inhibit expression of atp6-2 to a certain extent, and lead to the increase of the activity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATP synthase. These results introduce the premature PCD caused by orf507 gene in tapetal cells and semi-male sterility, but not complete male sterility.

Keywords: Capsicum annuum L.; cytochrome c oxidase; orf507; tapetum; transgenic semi-male sterility.

Figure 1

Localization of cytochrome c oxidase in anthers at the MMC stage. Purple arrow indicated black particles of cyt c oxidase reaction.

Figure 2

Localization of cytochrome c oxidase in anthers at the tetrad stage. Purple arrow indicated black particles of cyt c oxidase reaction.

Figure 3

Localization of cytochrome c oxidase in anthers at the MNM stage. Purple arrow indicated black particles of cyt c oxidase reaction.

Figure 4

Schematic illustration of pTCON vector construct.

Figure 5

Molecular analysis of T₀ transgenic plants. (A) PCR confirmation of TCON transgenic pepper plants using BamHI coxIVF and SacI orf507R primers that would amplify 693 bp fragment of the cassette in DNA level.(B) RT-PCR analysis of buds from TCON transgenic plants with Kpn I orf507F and SacI orf507R primers that would amplify 507 bp fragment with caubi gene as reference gene. (C) qRT-PCR analysis of buds from TCON transgenic plants. TCON1~8, transgenic TCON plants; WT, transformed control plant; CK, positive control (amplified from plasmid DNA); Leaf, leaves collected from the three transformants; M, Trans2K plus marker. Statistically significant differences between the means were determined using Fisher's LSD test (p < 0.05).

Figure 6

The morphology of the anthers from the T₀ transgenic plant (TCON8). (A) WT. (B) T₀ transgenic plant (TCON8). (C) CMS lines HW203A. Bars = 1 mm.

Figure 7

Cytological analysis of the pollen grains in T₀ transgenic plants. (A,C) Pollen grains of WT. (B,D) Pollen grains of T₀ transgenic plants. (E) Number of pollen grains in each anther. (F) Rate of damaged pollen grains. (G) Pollen germination of WT. (H) Pollen germination of T₀ transgenic plants. (A,B) Bars = 100 μm; (C,D) Bars = 25 μm; (G,H) Bars = 200 μm.

Figure 8

Bright-field photographs of sections of anthers at different stages of development from TCON male sterile transformant and transformed control pepper plants. At the MMC stage, anther development showed no differences in the transformant and control. T, tapetum; MMC, microspore mother cell; Tds, tetrad; MNM, mononuclear microspore; black arrow indicated the degraded mononuclear microspore; ^* nucleus of the tapetum cell. Bars = 25 μm.

Figure 9

Molecular analysis of test cross progeny of transgenic male sterile TCON 8 plant. (A) PCR analysis using BamHI coxIVF and SacI orf507R primers. (B) RT-PCR analysis with Kpn I orf507F and SacI orf507R primers. CK, transformed control plant; M, Trans2K plus marker; Lane1~15, progeny plant numbers; caubi, reference gene.

Figure 10

Morphology of BC₁T₀ anthers (A) and pollen viability of BC₁T₀ plants (B). (A) Bars = 1 cm (B) Bars = 100 μm.

Figure 11

qRT-PCR analysis of orf507 (A) in buds of BC₁T₀, CMS line, Maintainer line, and atp6-2(B) in buds of BC₁T₀, Maintainer line (WT) and ψatp6-2 (B) in CMS line. Statistically significant differences between the means were determined using Fisher's LSD test (p < 0.05).

Figure 12

Analysis of cytochrome c oxidase activity (A) and ATP hydrolysis activity of mitochondrial F₁F_o-ATP synthase (B) in buds of BC₁T₀, CMS line and Maintainer line (WT). Statistically significant differences between the means were determined using Fisher's LSD test (p < 0.05).