Wenchao Huang

The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162

This work identifies the pentatricopeptide repeat protein RF9 and finds that it restores fertility in cytoplasmic male sterile (CMS) Hong-Lian rice by interacting with a Gly-rich protein as part of a complex that processes the CMS-associated transcript atp6-orfH79, thereby restoring fertility. The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene–mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS–Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration.

Mitochondria and cytoplasmic male sterility in plants.

Mitochondria are essential organelles in cells not only because they supply over 90% of the cells energy but also because their dysfunction is associated with disease. Owing to the importance of mitochondria, there are many questions about mitochondria that must be answered. Cytoplasmic male sterility (CMS) is a mysterious natural phenomenon, and the mechanism of the origin of CMS is unknown. Despite successful utilization of CMS and restoration of fertility (Rf) in practice, the underlying mechanisms of these processes remain elusive. This review summarizes the genes involved in CMS and Rf, with a special focus on recent studies reporting the mechanisms of the CMS and Rf pathways, and concludes with potential working models.

Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility

Significance Plant cytoplasmic male sterility (CMS) and sterility restoration by nuclear restorer-of-fertility (Rf) genes provide a crucial breeding tool to harness hybrid vigor in many crops. Here, we identify RF6 as a novel pentatricopeptide repeat family protein that restores the fertility of Honglian CMS (HL-CMS), a major type of rice CMS used in breeding. We demonstrated that RF6 and hexokinase 6 function together in mitochondria to promote the processing of the aberrant CMS-associated transcript atp6-orfH79, thereby restoring the fertility of HL-CMS rice. This study links CMS restoration to hexokinase, providing novel insights into the mechanisms of CMS restoration, pollen development, mitochondria metabolism, and nuclear-cytoplasmic communication. In addition, the present study offers opportunities to further exploit rice heterosis in hybrid rice production. 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-of-fertility (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 CMS-associated 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.

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F1 plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F2 and BC1F1 derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F1 plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I2-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F1 plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F1 plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.

Protoplast: a more efficient system to study nucleo-cytoplasmic interactions.

Agrobacterium tumefaciens-mediated genetic transformation is a powerful tool for plant research, but it can be labor-intensive and time-consuming. Here, we report a protoplast-based approach to study nucleo-cytoplasmic interactions, such as cytoplasmic male sterility/fertility restoration (CMS/Rf) and organellar RNA editing. To test the system, we transfected the fertility restorer gene Rf5, which is involved in the rice HL-CMS/Rf system, into rice protoplasts prepared from the HL-CMS line. As the Rf5 protein accumulated in the transformed protoplasts, the CMS-associated transcripts were endonucleolytically cleaved. There were much lower levels of the CMS-associated protein ORFH79 in the transfected protoplasts than in the mock-transfected protoplasts. Next, we used a dsRNA-mediated gene silencing approach to down-regulate the pentatricopeptide protein gene MPR25, which participates in RNA editing of the organellar transcript nad5. The editing efficiency of mitochondrial transcripts of nad5 at nucleotide 1580 was much lower in the transfected protoplasts than in the mock-transfected protoplasts. Together, these results show that protoplast is a simple and efficient system to study interactions between the nucleus and organelles.

The mechanism of ORFH79 suppression with the artificial restorer fertility gene Mt-GRP162.

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.

Metabolic prediction of important agronomic traits in hybrid rice (Oryza sativa L.)

Hybrid crops have contributed greatly to improvements in global food and fodder production over the past several decades. Nevertheless, the growing population and changing climate have produced food crises and energy shortages. Breeding new elite hybrid varieties is currently an urgent task, but present breeding procedures are time-consuming and labour-intensive. In this study, parental metabolic information was utilized to predict three polygenic traits in hybrid rice. A complete diallel cross population consisting of eighteen rice inbred lines was constructed, and the hybrids’ plant height, heading date and grain yield per plant were predicted using 525 metabolites. Metabolic prediction models were built using the partial least square regression method, with predictive abilities ranging from 0.858 to 0.977 for the hybrid phenotypes, relative heterosis, and specific combining ability. Only slight changes in predictive ability were observed between hybrid populations, and nearly no changes were detected between reciprocal hybrids. The outcomes of prediction of the three highly polygenic traits demonstrated that metabolic prediction was an accurate (high predictive abilities) and efficient (unaffected by population genetic structures) strategy for screening promising superior hybrid rice. Exploitation of this pre-hybridization strategy may contribute to rice production improvement and accelerate breeding programs.

Identification of rice Di19 family reveals OsDi19-4 involved in drought resistance

Key messageTheOsDi19 proteins functioned as transcription factors and played crucial roles in response to abiotic stress. Overexpression ofOsDi19-4in rice increased drought tolerance by enhancing ROS-scavenging activity.AbstractMany transcription factors play crucial roles in plant responses to abiotic stress. Here, comprehensive sequence analysis suggested that the drought-induced 19 (Di19) gene family in rice genome contain seven members, and these proteins contained a well-conserved zinc-finger Di19 domain. Most OsDi19 proteins were mainly targeted to the nucleus and have transactivation activity in yeast. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that most OsDi19 proteins could form protein dimers. Expression analysis demonstrated that the OsDi19 genes were differentially and abundantly expressed in vegetative tissues, but expressed little in reproductive tissues and some of the OsDi19 genes were markedly induced by abiotic stresses and hormones in qRT-PCR analysis and microarray data. Overexpression of one stress-responsive gene, OsDi19-4, in rice resulted in significantly increased tolerance to drought stress compared with the wild type plants. Moreover, obviously increased ROS-scavenging ability was detected in the OsDi19-4-overexpressing plants under normal and drought stress conditions. These results suggested that the increased stress tolerance of OsDi19-4-overexpressing plants may be attributable to the enhanced ROS-scavenging activity. Taken together, these studies provide a detailed overview of the rice Di19 gene family, and suggest that the OsDi19 family may play crucial roles in the plant response to abiotic stress.

OsDi19-4 acts downstream of OsCDPK14 to positively regulate ABA response in rice.

The drought-induced 19 protein family consists of several atypical Cys2/His2-type zinc finger proteins in plants and plays an important role in abiotic stress. In this study, we found that overexpressing OsDi19-4 in rice altered the expression of a series of abscisic acid (ABA)-responsive genes, resulting in strong ABA-hypersensitive phenotypes including ABA-induced seed germination inhibition, early seedling growth inhibition and stomatal closure. On the contrary, OsDi19-4 knockdown lines were less sensitive to ABA. Additionally, OsCDPK14 was identified to interact with OsDi19-4 and be responsible for the phosphorylation of OsDi19-4, and the phosphorylation of OsDi19-4 was further enhanced after the treatment of ABA. Apart from these, OsDi19-4 was shown to directly bind to the promoters of OsASPG1 and OsNAC18 genes, two ABA-responsive genes, and regulate their expression. Transient expression assays confirmed the direct regulation role of OsDi19-4, and the regulation was further enhanced by the increased phosphorylation of OsDi19-4 after the treatment of ABA. Taken together, these data demonstrate that OsDi19-4 acts downstream of OsCDPK14 to positively regulate ABA response by modulating the expression of ABA-responsive genes in rice.

Mitochondrial ORFH79 is Essential for Drought and Salt Tolerance in Rice

The mitochondrion is deemed to be one of the most important organelles, and plays an essential role in various biological processes. Nonetheless, the role of mitochondria in response to abiotic stress remains unclear. Here, we report that accumulation of the cytoplasmic male sterility (CMS) protein ORFH79 in the vegetative tissues resulted in the dysfunction of mitochondria with decreased enzymatic activities of respiratory chain complexes, reduced ATP content and even a morphological change of the mitochondria. However, the suppression of orfH79 by overexpressing a fertility restorer gene Rf5, which is targeted to mitochondria and induced an endonucleolytic cleavage on the atp6-orfH79 transcripts, could recover the function of mitochondria and further significantly improved the tolerance to drought and salt stress. The above evidence suggests that the mitochondrion plays a pivotal role in tolerance to drought and salt stress in rice.