Genome-Wide Identification, Expansion Mechanism and Expression Profiling Analysis of GLABROUS1 Enhancer-Binding Protein (GeBP) Gene Family in Gramineae Crops.

The GLABROUS1 enhancer-binding protein (GeBP) gene family encodes a typical transcription factor containing a noncanonical Leucine (Leu-)-zipper motif that plays an essential role in regulating plant growth and development, as well as responding to various stresses. However, limited information on the GeBP gene family is available in the case of the Gramineae crops. Here, 125 GeBP genes from nine Gramineae crops species were phylogenetically classified into four clades using bioinformatics analysis. Evolutionary analyses showed that whole genome duplication (WGD) and segmental duplication play important roles in the expansion of the GeBP gene family. The various gene structures and protein motifs revealed that the GeBP genes play diverse functions in plants. In addition, the expression profile analysis of the GeBP genes showed that 13 genes expressed in all tested organs and stages of development in rice, with especially high levels of expression in the leaf, palea, and lemma. Furthermore, the hormone- and metal-induced expression patterns showed that the expression levels of most genes were affected by various biotic stresses, implying that the GeBP genes had an important function in response to various biotic stresses. Furthermore, we confirmed that OsGeBP11 and OsGeBP12 were localized to the nucleus through transient expression in the rice protoplast, indicating that GeBPs function as transcription factors to regulate the expression of downstream genes. This study provides a comprehensive understanding of the origin and evolutionary history of the GeBP genes family in Gramineae, and will be helpful in a further functional characterization of the GeBP genes.

A metabolome-based core hybridisation strategy for the prediction of rice grain weight across environments.

Marker-based prediction holds great promise for improving current plant and animal breeding efficiencies. However, the predictabilities of complex traits are always severely affected by negative factors, including distant relatedness, environmental discrepancies, unknown population structures, and indeterminate numbers of predictive variables. In this study, we utilised two independent F1 hybrid populations in the years 2012 and 2015 to predict rice thousand grain weight (TGW) using parental untargeted metabolite profiles with a partial least squares regression method. A stable predictive model for TGW was built based on hybrids from the population in 2012 (r = 0.75) but failed to properly predict TGW for hybrids from the population in 2015 (r = 0.27). After integrating hybrids from both populations into the training set, the TGW of hybrids could be predicted but was largely dependent on population structures. Then, core hybrids from each population were determined by principal component analysis and the TGW of hybrids in both environments were successfully predicted (r > 0.60). Moreover, adjusting the population structures and numbers of predictive analytes increased TGW predictability for hybrids in 2015 (r = 0.72). Our study demonstrates that the TGW of F1 hybrids across environments can be accurately predicted based on parental untargeted metabolite profiles with a core hybridisation strategy in rice. Metabolic biomarkers identified from early developmental stage tissues, which are grown under experimental conditions, may represent a workable approach towards the robust prediction of major agronomic traits for climate-adaptive varieties.

The transcription factor OsbHLH138 regulates thermosensitive genic male sterility in rice via activation of TMS5.

Thermosensitive genic male sterile (TGMS) lines favored heterosis exploitation in two-line hybrid rice. TMS5, a member of RNase Z cleavages the UbL40 mRNAs, plays an important role in two-line hybrid rice. Here, we identified a new TGMS mutant 93-11s, which lost two amino acids in the first exon of TMS5 gene and caused thermosensitive genic male sterility in rice. The tms5-2 cannot process mRNAs of the ubiquitin fusion ribosomal protein L40 (UbL40) and hence cause the mRNAs accumulation in restrictive temperature. Further, we identified a nucleus-localized bHLH transcription factor OsbHLH138, which can form the basic helix-loop-helix structure and bind the core region of tms5-2 promoter sequences by bHLH domain, and activate expression of tms5-2 by the acidic amino acid-rich domain. These results indicate a novel mechanism for the tms5-2 regulating thermosensitive male sterility of rice. By altering expression of OsbHLH138, we can regulate the expression level of TMS5 and the accumulation of UbL40 mRNAs to command the male fertility in different temperatures. The identification of OsbHLH138 provides breeders a new choice for development of TGMS rice lines, which will favor the sustainable development of two-line hybrid rice.

Rice PPS1 encodes a DYW motif-containing pentatricopeptide repeat protein required for five consecutive RNA-editing sites of nad3 in mitochondria.

The pentatricopeptide repeat (PPR) protein family is a large family characterized by tandem arrays of a degenerate 35-amino-acid motif whose members function as important regulators of organelle gene expression at the post-transcriptional level. Despite the roles of PPRs in RNA editing in organelles, their editing activities and the underlying mechanism remain obscure. Here, we show that a novel DYW motif-containing PPR protein, PPS1, is associated with five conserved RNA-editing sites of nad3 located in close proximity to each other in mitochondria, all of which involve conversion from proline to leucine in rice. Both pps1 RNAi and heterozygous plants are characterized by delayed development and partial pollen sterility at vegetative stages and reproductive stage. RNA electrophoresis mobility shift assays (REMSAs) and reciprocal competition assays using different versions of nad3 probes confirm that PPS1 can bind to cis-elements near the five affected sites, which is distinct from the existing mode of PPR-RNA binding because of the continuity of the editing sites. Loss of editing at nad3 in pps1 reduces the activity of several complexes in the mitochondrial electron transport chain and affects mitochondrial morphology. Taken together, our results indicate that PPS1 is required for specific editing sites in nad3 in rice.

A rice dual-localized pentatricopeptide repeat protein is involved in organellar RNA editing together with OsMORFs.

In flowering plants, various RNA editing events occur in the mitochondria and chloroplasts as part of post-transcriptional processes. Although several pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factors (MORFs) have been identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among these proteins are still obscure. Here, we identified a rice dual-localized PPR protein, OsPGL1. The loss of function of OsPGL1 resulted in defects in both chloroplast RNA editing of ndhD-878 and mitochondrial RNA editing of ccmFc-543, both of which could be restored in transgenic complementation lines. Despite synonymous editing of ccmFc-543, the loss of editing of ndhD-878 caused a failed conversion of serine to leucine, leading to chloroplast dysfunction and defects in the photosynthetic complex; the results of additional experiments demonstrated that OsPGL1 directly binds to both transcripts. Interactions between three OsMORFs (OsMORF2/8/9) and OsPGL1 both in vitro and in vivo were confirmed, implying that OsPGL1 functions in RNA editing via an editosome. These findings also suggested that OsMORFs assist with and contribute to a flexible PPR-RNA recognition model during RNA editing. These results indicate that, in cooperation with PPRs, OsPGL1 is required for RNA editing. In addition, our study provides new insights into the relationship between RNA editing and plant development.

The rice DUF1620-containing and WD40-like repeat protein is required for the assembly of the restoration of fertility complex.

Cytoplasmic male sterility (CMS) and restoration of fertility (Rf) are widely distributed in plant species utilized by humans. RF5 and GRP162 are subunits of the restoration of fertility complex (RFC) in Hong-Lian rice. Despite the fact that the RFC is 400-500 kDa in size, the other proteins or factors in the complex still remain unknown. Here, we identified RFC subunit 3, which encodes a DUF1620-containing and WD40-like repeat protein (RFC3) that is present in all tissues but highly expressed in leaves. We established that RFC3 interacts with both RF5 and GRP162 in vitro and in vivo, and is transported into the mitochondria as a membrane protein. Furthermore, CMS RNA (atp6-orfH79) and CMS cytotoxic protein (ORFH79) accumulate when RFC3 is silenced in restorer lines. We presented the analysis with blue-native polyacrylamide gel electrophoresis, indicating that RFC is disrupted in the RNAi line. We concluded that RCF3 is indispensable as a scaffold protein for the assembly of the RFC complex. We unveil a new molecular player of the RFC in the Rf pathway in rice and propose the model of RFC based on these data.

Silencing of rice PPR gene PPS1 exhibited enhanced sensibility to abiotic stress and remarkable accumulation of ROS.

Abiotic stresses widely constrain the development and reproduction of plant, especially impaired the yield of crops greatly. Recent researches presented pentatricopeptide repeat (PPR) proteins play crucial role in response to abiotic stress. However, the underlying mechanism of PPR genes in regulation of abiotic stress is still obscures. In our recent study, we found that the knockout of rice PPS1 causes pleiotropic growth disorders, including growth retardation, dwarf and sterile pollen, and finally leads to impaired C-U RNA editing at five consecutive sites on the mitochondrial nad3. In this study, we further investigate the roles of PPS1 in abiotic stress tolerance, we confirmed that pss1-RNAi line exhibited enhanced sensitivity to salinity and ABA stress at vegetative stage, specifically. While reactive oxygen species (ROS) accumulate significantly only at reproductive stage, which further activated the expression of several ROS-scavenging system related genes. These results implied that PPS1 functioned on ROS signaling network to contribute for the flexibility to abiotic stresses. Our research emphasizes the stress adaptability mediated by the PPR protein, and also provides new insight into the understanding of the interaction between cytoplasm and nucleus and signal transduction involved in RNA editing.

A simplified method to isolate rice mitochondria.

BACKGROUND: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies. RESULTS: To isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies. CONCLUSION: The combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

The Rice Pentatricopeptide Repeat Protein PPR756 Is Involved in Pollen Development by Affecting Multiple RNA Editing in Mitochondria.

In land plants, the pentatricopeptide repeat (PPR) proteins form a large family involved in post-transcriptional processing of RNA in mitochondria and chloroplasts, which is critical for plant development and evolutionary adaption. Although studies showed a number of PPR proteins generally influence the editing of organellar genes, few of them were characterized in detail in rice. Here, we report a PLS-E subclass PPR protein in rice, PPR756, loss of function of which led to the abolishment of RNA editing events among three mitochondrial genes including atp6, ccmC, and nad7. Their defective C-to-U transformation then resulted in improper amino acid retention which could cause abortive pollen development. Furthermore, PPR756 could bind to the three target genes directly and interact with three OsMORFs (multiple organellar RNA editing factors): OsMORF1, OsMORF8-1, and OsMORF8-2. The knock-out plants of PPR756 exhibited retarded growth and greener leaves during the early vegetative stages, along with sterile pollen and lower seed setting at the reproductive stage. These results established a role for PPR756 in rice development, participating in RNA editing of three various transcripts and cooperating with OsMORFs via an editosome manner in rice.