Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-offertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMSassociated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.cytoplasmic male sterility | pentatricopeptide repeat | restorer-of-fertility | RF6 | hexokinase 6 P lant cytoplasmic male sterility (CMS) is a trait characterized by a lack of functional pollen grains in plants, although female gametes are still viable (1). CMS has been widely used to produce hybrid seeds to improve plant yield, improve resistance to diseases or stresses, or enhance taste (2). Plant CMS results from genomic incompatibility between mitochondria and nuclei and is typically associated with an aberrant chimeric gene in the mitochondria (3-6). A specific set of nuclear genes called restorer-of-fertility (Rf) genes, which primarily belong to the pentatricopeptide repeat (PPR) family, repress CMS through the promotion of the cleavage, degradation, or editing of CMS-associated aberrant transcripts or the inhibition of the translation of these genes (7-9). PPR proteins regulate RNA metabolism at multiple levels, from RNA editing, stability, processing, and splicing to translation (10, 11). PPR proteins can be classified into PLS and P families based on the structure of PPR motifs (11). PLS family members generally exhibit direct RNA binding (12)(13)(14) or editing activity (15-17), whereas P-family proteins often lack sites for binding to RNA, particularly mitochondrial CMS-related RNA (18,19). Most of the known PPR proteins implicated in restoration of fertility (RF) are P-family members. Thus, RF-related PPR proteins may require additional partners, such as RNA-binding or splicing factors to process CMSrelated transcripts.Rice CMS is categorized into three major types: Wild Abortive (WA), originating from...

Significant increases in rice yield and stress resistance are constant demands for breeders. However, high yield and high stress resistance are often antagonistic to each other. Here, we report several new rice mutants with high yield and excellent cold tolerance that were generated by simultaneously editing three genes, OsPIN5b (a panicle length gene), GS3 (a grain size gene) and OsMYB30 (a cold tolerance gene) with the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeatsassociated protein 9) system. We edited two target sites of each gene with high efficiency: 53% for OsPIN5b-site1, 42% for OsPIN5b-site2, 66% for GS3-site1, 63% for GS3-site2, 63% for OsMYB30-site1, and 58% for OsMYB30-site2. Consequently, the ospin5b mutants, the gs3 mutants, and the osmyb30 mutants exhibited increased panicle length, enlarged grain size and increased cold tolerance, respectively. Then nine transgenic lines of the ospin5b/gs3, six lines of ospin5b/osmyb30 and six lines of gs3/ osmyb30 were also acquired, and their yield related traits and cold tolerance corresponded to the genes being edited. Additionally, we obtained eight ospin5b/gs3/ osmyb30 triple mutants by editing all three genes simultaneously. Aside from the ospin5b/ gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25 mutants, the remaining six mutants had off-target events at the putative off-target site of OsMYB30-site1. The results also showed that the T 2 generations of these two mutants exhibited higher yield and better cold tolerance compared with the wild type. Together, these results demonstrated that new and excellent rice varieties with improved yield and abiotic stress resistance can be generated through gene editing techniques and may be applied to rice breeding. Furthermore, our study proved that the comprehensive agronomic traits of rice can be improved with the CRISPR-Cas9 system.

Understanding the molecular mechanisms underlying complex phenotypes requires systematic analyses of complicated metabolic networks and contributes to improvements in the breeding efficiency of staple cereal crops and diagnostic accuracy for human diseases. Here, we selected rice (Oryza sativa) heterosis as a complex phenotype and investigated the mechanisms of both vegetative and reproductive traits using an untargeted metabolomics strategy. Heterosis-associated analytes were identified, and the overlapping analytes were shown to underlie the association patterns for six agronomic traits. The heterosis-associated analytes of four yield components and plant height collectively contributed to yield heterosis, and the degree of contribution differed among the five traits. We performed dysregulated network analyses of the high- and low-better-parent heterosis hybrids and found multiple types of metabolic pathways involved in heterosis. The metabolite levels of the significantly enriched pathways (especially those from amino acid and carbohydrate metabolism) were predictive of yield heterosis (area under the curve = 0.907 with 10 features), and the predictability of these pathway biomarkers was validated with hybrids across environments and populations. Our findings elucidate the metabolomic landscape of rice heterosis and highlight the potential application of pathway biomarkers in achieving accurate predictions of complex phenotypes.