The Alpha Subunit of Mitochondrial Processing Peptidase Participated in Fertility Restoration in Honglian-CMS Rice.

The cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration system is a favorable tool for the utilization of heterosis in plant hybrid breeding. Many restorer-of-fertility (Rf) genes have been characterized in various species over the decades, but more detailed work is needed to investigate the fertility restoration mechanism. Here, we identified an alpha subunit of mitochondrial processing peptidase (MPPA) that is involved in the fertility restoration process in Honglian-CMS rice. MPPA is a mitochondrial localized protein and interacted with the RF6 protein encoded by the Rf6. MPPA indirectly interacted with hexokinase 6, namely another partner of RF6, to form a protein complex with the same molecular weight as the mitochondrial F1F0-ATP synthase in processing the CMS transcript. Loss-of-function of MPPA resulted in a defect in pollen fertility, the mppa+/- heterozygotes showed semi-sterility phenotype and the accumulation of CMS-associated protein ORFH79, showing restrained processing of the CMS-associated atp6-OrfH79 in the mutant plant. Taken together, these results threw new light on the process of fertility restoration by investigating the RF6 fertility restoration complex. They also reveal the connections between signal peptide cleavage and the fertility restoration process in Honglian-CMS rice.

OsGRP3 Enhances Drought Resistance by Altering Phenylpropanoid Biosynthesis Pathway in Rice (Oryza sativa L.).

As a sessile organism, rice often faces various kinds of abiotic stresses, such as drought stress. Drought stress seriously harms plant growth and damages crop yield every year. Therefore, it is urgent to elucidate the mechanisms of drought resistance in rice. In this study, we identified a glycine-rich RNA-binding protein, OsGRP3, in rice. Evolutionary analysis showed that it was closely related to OsGR-RBP4, which was involved in various abiotic stresses. The expression of OsGRP3 was shown to be induced by several abiotic stress treatments and phytohormone treatments. Then, the drought tolerance tests of transgenic plants confirmed that OsGRP3 enhanced drought resistance in rice. Meanwhile, the yeast two-hybrid assay, bimolecular luminescence complementation assay and bimolecular fluorescence complementation assay demonstrated that OsGRP3 bound with itself may affect the RNA chaperone function. Subsequently, the RNA-seq analysis, physiological experiments and histochemical staining showed that OsGRP3 influenced the phenylpropanoid biosynthesis pathway and further modulated lignin accumulation. Herein, our findings suggested that OsGRP3 enhanced drought resistance in rice by altering the phenylpropanoid biosynthesis pathway and further increasing lignin accumulation.

Genome-wide identification and comprehensive analysis of tubby-like protein gene family in multiple crops.

Introduction: The highly conserved tubby-like proteins (TLPs) play key roles in animal neuronal development and plant growth. The abiotic stress tolerance function of TLPs has been widely explored in plants, however, little is known about comparative studies of TLPs within crops. Methods: Bioinformatic identification, phylogenetic analysis, Cis-element analysis, expression analysis, Cis-element analysis, expression analysis and so on were explored to analysis the TLP gene family of multiple crops. Results: In this study, a comprehensive analysis of TLP genes were carried out in seven crops to explore whether similar function of TLPs in rice could be achieved in other crops. We identified 20, 9, 14, 11, 12, 35, 14 and 13 TLP genes in Glycine max, Hordeum vulgare, Sorghum bicolor, Arabidopsis thaliana, Oryza sativa Japonica, Triticum aestivum, Setaria italic and Zea mays, respectively. All of them were divided into two groups and ten orthogroups (Ors) based on amino acids. A majority of TLP genes had two domains, tubby-like domain and F-box domain, while members of Or5 only had tubby-like domain. In addition, Or5 had more exons and shorter DNA sequences, showing that characteristics of different Ors reflected the differentiated function and feature of TLP genes in evolutionary process, and Or5 was the most different from the other Ors. Besides, we recognized 25 cis-elements in the promoter of TLP genes and explored multiple new regulation pathway of TLPs including light and hormone response. The bioinformatic and transcriptomic analysis implied the stresses induced expression and possible functional redundancy of TLP genes. We detected the expression level of 6 OsTLP genes at 1 to 6 days after seed germination in rice, and the most obvious changes in these days were appeared in OsTLP10 and OsTLP12. Discussion: Combined yeast two-hybrid system and pull down assay, we suggested that the TLP genes of Or1 may have similar function during seed germination in different species. In general, the results of comprehensive analysis of TLP gene family in multiple species provide valuable evolutionary and functional information of TLP gene family which are useful for further application and study of TLP genes.

Genic male sterility increases rice drought tolerance.

Plant fertility and resistance to stress environments are antagonistic to each other. At booting stage, fertility is often sacrificed for survive in rice under abiotic stress. However, the relationship between fertility and resistance at molecular level remains elusive. Here, we identified a transcription factor, OsAlfin like 5, which regulates the OsTMS5 and links both the drought stress response and thermosensitive genic male sterility. The OsAL5 overexpression plants (OE-OsAL5) became sensitive to temperature owning to the OsTMS5 that the OE-OsAL5 plants were fertile under low temperature (23 °C) and sterile under high temperature (28 °C). Significantly, the survival rate of OE-OsAL5 lines was higher than that of the wide-type (WT) under drought stress. Further experiments confirmed that the OsAL5 regulated both of the OsTMS5 and the down-stream drought-related genes by binding to the 'GTGGAG' element in vivo, revealing that the OsAL5 participated both in the drought stress response and thermosensitive genic male sterility in rice. These findings open up the possibility of breeding elite TGMS lines with strong drought tolerance by manipulating the expression of OsAL5.

The metabolomic landscape of rice heterosis highlights pathway biomarkers for predicting complex phenotypes.

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.

Crystal structures of rice hexokinase 6 with a series of substrates shed light on its enzymatic mechanism.

Hexokinases (HXKs) have determined to be multifaceted proteins, and they are the only ones able to phosphorylate glucose in plants. However, the binding mode for ATP to plant HXKs remains unclear. Here, we report the crystal structures of rice hexokinase 6 (OsHXK6) in four different forms: (i) apo-form, (ii) binary complex with D-Glc, (iii) quaternary complex with ADP, PO4 and Mg2+, and (iv) pentanary complex with D-Glc, ADP, PO4, and Mg2+. The apo form is in the open state conformation, and the three others are in the closed state, indicating that glucose and ADP-PO4 binding induces a large conformational change by domain rearrangement. The quaternary complex is a novel intermediate during the catalytic reaction we trapped for the first time, which provides a new evidence for the enzymatic mechanism of HXKs. In addition, the latter two complexes reveal the binding mode for ADP-PO4 to plant HXKs, which provide the structural explanation for the dual-function of OsHXK6. In addition, we identified that residues Gly112, Thr261, Gly262, and Gly450 are essential to the binding between ADP-PO4 and OsHXK6 by a series of single mutations and enzymatic assays. Our study provide structural basis for the other functional studies of OsHXK6 in rice.

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.

Rational Improvement of Rice Yield and Cold Tolerance by Editing the Three Genes OsPIN5b, GS3, and OsMYB30 With the CRISPR-Cas9 System.

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 repeats-associated 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 T2 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.

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

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.

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.