Abstract
Key messageP-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3.In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.
Highlights
Cytoplasmic male sterility (CMS), a maternally inherited trait characterized by the inability of a plant to produce viable pollen grains, is caused by incompatibility between mitochondrial and nuclear genes
The ATP levels in osppr939-4 and osppr939-5 were only 55.9 and 65.6% that of the wild type, respectively (Fig. 6a). These results suggest that the lower level of mature nad5 in the osppr939 mutants due to the reduced splicing efficiency of nad5 introns 1–3 leads to reduced levels of complex I, which might stimulate the accumulation of other mitochondrial complexes via unknown retrograde signaling
Because plant mitochondrial introns cannot undergo self-splicing, they require the help of nucleus-encoded splicing cofactors, which interact with intron RNA to catalyze the formation of its active conformation (Brown et al 2014)
Summary
Cytoplasmic male sterility (CMS), a maternally inherited trait characterized by the inability of a plant to produce viable pollen grains, is caused by incompatibility between mitochondrial and nuclear genes. The sterile phenotype of CMS lines can be restored by nuclear genes called restorer-of-fertility genes (Rf genes), which encode highly diverse proteins. Rf2, the first plant restorer gene isolated in maize CMS-T, encodes an aldehyde dehydrogenase (Cui et al 1996). Rf17 in rice CMS-CW (Chinese wild rice) encodes a mitochondrial sorting protein containing an acyl-carrier protein synthase-like domain (Fujii and Toriyama 2009). The candidate gene for Rf1 in sugar beet (Beta vulgaris) CMS-Owen, bvORF20, encodes a putative peptidase of the M48 family (Hagihara et al 2005). Most plant Rf genes encode pentatricopeptide repeat (PPR) proteins
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