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In CMS­WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion.

Annotated Information


Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS­WA) has been exploited in the majority of 'three­line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown.

Plant mitochondrial genomic transformation is currently infeasible, but CMS gene function can be tested by nuclear transformation of candidate gene(s) fused with a mitochondrial transit signal (MTS)[1,2].

The suppression of the WA352-interacting gene O. sativa COX11(OsCOX11) can produce male sterility (Fig.1).

"Figure 1 OsCOX11 RNAi was driven by the rice RTStapetum-specific promoter"

COX11 proteins are conserved in eukaryotes and function in the assembly of cytochrome c(Cyt c) oxidase[3] (Fig.2).

"Figure 2 Multiple alignments of COX11 protein sequences of five plant species and yeast. Accession numbers of the protein sequences are given in parenthesis: OsCOX11 of O. sativa (ABF98570), ZmCOX11 of Zea mays (ACG32461), SbCOX11 of Sorghum bicolor"

In yeast, Saccharomyces cerevisiaeCOX11 (ScCOX11) has a role in hydrogen peroxide degradation[4]. COX11 proteins also function in peroxide metabolism and may act as negative regulators of PCD[5].


OsCOX11 is constitutively expressed (Fig. 3). A. thaliana AtCOX11 (287 residues), which shares 80% identity with OsCOX11, also interacted with WA352 (Figure 4a). Y2H deletion assays identified two regions (residues 218–292 and 294–352) of WA352 that interact with OsCOX11 (Fig. 4a). Similarly, a 37-residue sequence (184–220) in the highly conserved region of OsCOX11 confers the WA352 binding (Fig. 4b,c). A bimolecular fluorescence complementation (BiFC) assay confirmed the mitochondrial localization of OsCOX11 and its in vivo interaction with WA352 (Fig. 4d and Fig. 5)[5].

"Figure 3 Expression of OsCOX11 in various tissues of ZS97B. The expression of OsCOX11(Os03g0718600, up panel) was assayed by semi-qRT-PCR, with 32 cycles for OsCOX11and 27 cycles for OsActin1."
"Figure 4 WA352 interacts with the nuclear-encoded mitochondrial protein COX11. (a) Interaction of WA352 with COX11 proteins of rice and A. thalianaby Y2H assay and mapping of WA352 regions (shaded) for the interaction. The combination of WA352 with the empty prey vector pGADT7, and that of the empty bait vector pGBKT7 with OsCOX11, served as negative controls. The yeast cells were grown on SD/-Leu/-Trp/-His/-Ade medium. TM, predicted transmembrane segment. aa, amino acids. (b) Y2H assay mapping of the OsCOX11 region (shaded) interacting with WA352. (c) The WA352-interacting domain in OsCOX11 and the conserved sequence in A. thalianaCOX11 (AtCOX11). (d) Rice protoplasts were co-transformed with four constructs that expressed, respectively, MTS-mOrange (a red fluorescent protein variant) for marking mitochondria, OsCOX11-CFP (cyan fluorescent protein) for mitochondrial localization of OsCOX11, and MTS-SPYNE-WA352218–352and MTSSPYCE-OsCOX11 for a BiFC assay of the in vivoWA352-OsCOX11 interaction. Scale bars, 10 µm."
"Figure 5 Excitation/emission spectrum analysis of the BiFC signal and fluorescent proteins expressed in the transformed rice protoplasts. (a) The emission signal peak of MTS-mOrange, excited by 543-nm laser, was detected at 565 nm. (b) OsCOX11-CFP was excited by 454-nm laser and the emission signal peaks were detected at 480 nm and 505 nm. (c) When excited by 488-nm laser, the emission signal peak of the reconstituted YFP (yellow fluorescent protein) by BiFC of MTS-SPYNE-WA352218-352and MTS-SPYCE-OsCOX11 wasdetected at 522 nm, indicating that the BiFC signal did notoverlap with those of MTS-mOrange and OsCOX11-CFP."


CMS-­related cytoplasmic-­nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene.

Labs working on this gene

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, College of Life Sciences, South China Agricultural University, Guangzhou, China.

College of Forestry, Guangxi University, Nanning, China.

State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Plant Resources, Sun Yat-sen University, Guangzhou, China.


1. Wang, Z. et al. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell 18, 676–687 (2006).

2. He, S., Abad, A.R., Gelvin, S.B. & Mackenzie, S.A. A cytoplasmic male sterilityassociated mitochondrial protein causes pollen disruption in transgenic tobacco. Proc. Natl. Acad. Sci. USA 93, 11763–11768 (1996).

3. Banting, G.S. & Glerum, D.M. Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p. Eukaryot. Cell 5, 568–578 (2006).

4. Veniamin, S., Sawatzky, L.G., Banting, G.S. & Glerum, D.M. Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins. Free Radic. Biol. Med. 51, 1589–1600 (2011).

5. Luo D.P., Xu H., Liu Z.L. et al. A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice. Nature Genetics, 45(5): 573-577 (2013).

Structured Information