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The restoration fertility complex ( RFC) was previously identified in Honglian ( HL)-cytoplasmic male sterility ( CMS) rice ( Oryza sativa), and glycine-rich protein 162 ( GRP162) is responsible for binding to the CMS-associated transcript atp6-orfH79., Here, we engineered a recombinant GRP162 containing the mitochondrial transit peptide, termed Mt- GRP162, as an artificial restorer of fertility ( Rf) gene. Mt- GRP162 was confirmed to bind to CMS-associated RNA and to localize to the mitochondria. The transgenic plants showed restored fertility with partially functional pollen., We found that the expression of ORFH79 decreased in transgenic plants, while the expression of atp6-orfH79 was not changed. These findings indicate that Mt- GRP162 restores fertility by suppressing the expression of the cytotoxic protein ORFH79 at the post-transcriptional level rather than via the cleavage of atp6-orfH79 in the presence of RFC., These findings contribute to our understanding of the mechanisms of restoration through diverse pathways. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility ( CMS) has attracted great interest because of its application in crop breeding. Despite increasing knowledge of CMS, not much is understood about its molecular mechanisms. Previously, orf H79 was cloned and identified as the CMS gene in Honglian rice, but how the ORFH79 protein causes pollen abortion is still unknown., Through bacterial two-hybrid library screening, P61, a subunit of the mitochondrial electron transport chain ( ETC) complex III, was selected as a candidate that interacts with ORFH79. Bimolecular fluorescence complementation ( Bi FC) and coimmunoprecipitation (co IP) assays verified their interaction inside mitochondria. Blue native polyacrylamide gel electrophoresis ( BN- PAGE) and western blotting showed ORF79 and P61 colocalized in mitochondrial ETC complex III of CMS lines., Compared with the maintainer line, Yuetai B ( YB), a significant decrease of enzyme activity was detected in mitochondrial complex III of the CMS line, Yuetai A ( YA), which resulted in decreased ATP concentrations and an increase in the reactive oxygen species ( ROS) content., We propose that the CMS protein, ORFH79, can bind to complex III and decrease its enzyme activity through interaction with P61. This defect results in energy production dysfunction and oxidative stress in mitochondria, which may work as retrograde signals that lead to abnormal pollen development. [ABSTRACT FROM AUTHOR]