Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea

Abstract

Background: The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.

Results: It was shown that the ORF220 protein can be guided to the mitochondria using the mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic stem mustard plants expressed the chimeric gene containing the orf220 gene and a mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic plants were male-sterile, most being unable to produce pollen while some could only produce non-vigorous pollen. The transgenic stem mustard plants also showed aberrant floral development identical to that observed in the CMS stem mustard phenotype. Results obtained from oligooarray analysis showed that some genes related to mitochondrial energy metabolism were down-regulated, indicating a weakening of mitochondrial function in transgenic stem mustard. Some genes related to pollen development were shown to be down-regulated in transgenic stem mustard and the expression of some transcription factor genes was also altered.

Conclusion: The work presented furthers our understanding of how the mitochondrially-targeted expression of CMS-associated orf220 gene causes male sterility through retrograde regulation of nuclear gene expression in Brassica juncea.

Figure 1

Construction of chimeric orf220 gene, transformation into wild type stem mustard and its sub-localization in mitochondria. Orf220, a candidate cytoplasmic male gene associated with sterility in Brassica juncea, was isolated from cytoplasmic male-sterile stem mustard. A mitochondrial targeted peptide (atp2-1) was isolated from Nicotiana encoding the β subunit of F1-ATPase (Figure 1-A). Figure 1-B shows the transgenic regenerations from proximal portions of hypocotyls of cotyledons in stem mustard. Figure 1-C shows the identification of transgenic stem mustard by checking for the presence of the orf220 gene using PCR and RT-PCR. Figure 1-D shows that the mitochondrial targeted peptide (atp2-1) can guide the ORF220 protein to the mitochondria target, while the ORF220 protein is only found in the nucleus in the absence of the mitochondrial targeted peptide (atp2-1). Mt-RFP was used as the mitochondrially-targeted control.

Figure 2

Phenotypes of reproductive development in transgenic plants. Delayed flowering of transgenic plants is shown in Figure 2-A. The appearance of novel petal-like floral structures in transgenic plants is shown by an arrow with a hand in Figure 2-B. The time of seed-set of transgenic plants is shown by an arrow without a hand in Figure 2-C. Transgenic plant stamens with pollen are shown by an arrow with a hand, and stamens without by an arrow without a hand in Figure 2-D. WT denotes wild type; mt-ex-orf220 denotes mitochondrial targeted expression of orf220 gene; SP denotes self-pollination; CP denotes cross-pollination with normal pollen.

Figure 3

Morphology and activity of pollen produced from transgenic and wild type plants. mt-ex-orf220 denotes the mitochondrial targeted expression of orf220 gene. TTC denotes 2,3,5-triphenyl-2h-tetrazolium chloride.

Figure 4

Classification of genes expressed according to their cellular components, molecular function and biological processes in transgenic plants. Up-regulated and down-regulated genes expressed in transgenic stem mustard plants.

Figure 5

Identical down-regulated genes in transgenic and CMS stem mustard. WT indicates wild type.