GROWTH REGULATING FACTOR 7-mediated arbutin metabolism enhances rice salt tolerance.

Salt stress is an environmental factor that limits plant growth and crop production. With the rapid expansion of salinized arable land worldwide, investigating the molecular mechanisms underlying the salt stress response in plants is urgently needed. Here, we report that GROWTH REGULATING FACTOR 7 (OsGRF7) promotes salt tolerance by regulating arbutin (hydroquinone-ß-D-glucopyranoside) metabolism in rice (Oryza sativa). Overexpression of OsGRF7 increased arbutin content, and exogenous arbutin application rescued the salt-sensitive phenotype of OsGRF7 knockdown and knockout plants. OsGRF7 directly promoted the expression of the arbutin biosynthesis genes URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE 1 (OsUGT1) and OsUGT5, and knockout of OsUGT1 or OsUGT5 reduced rice arbutin content, salt tolerance, and grain size. Furthermore, OsGRF7 degradation through its interaction with F-BOX AND OTHER DOMAINS CONTAINING PROTEIN 13 (OsFBO13) reduced rice salinity tolerance and grain size. These findings highlight an underexplored role of OsGRF7 in modulating rice arbutin metabolism, salt stress response, and grain size, as well as its broad potential use in rice breeding.

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.

Measuring Endogenous GA and IAA.

Plant hormones regulate many physiological processes that largely influence growth, differentiation, and development. Contents of phytohormones were analyzed using a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) system. This protocol describes a detailed procedure to extract and quantify indole-3-acetic acid (IAA) and gibberellin acid (GA) in rice (Oryza sativa) tissues using high-performance liquid chromatography (HPLC)-based method.

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.

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.

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.

Rice GROWTH-REGULATING FACTOR 7 controls tiller number by regulating strigolactone synthesis.

As an important transcription factor family, GROWTH-REGULATING FACTORs (GRFs) are involved in central development processes, including growth regulation, insect and disease resistance, and stress response. The OsGRF7 has recently been shown involving in modulating leaf angle through regulating GA and IAA metabolism. Interestingly, we found that OsGRF7 negatively regulates the tiller number. However, the detailed molecular mechanisms of OsGRF7 underlying the tiller number determination are still not understood. Here, we report that OsGRF7 directly targets the promoter of the NODULATION SIGNALING PATHWAY2 (OsNSP2), a key factor involving in the strigolactone synthesis. Correspondingly, OsGRF7 alters the expression level of OsNSP2 and the endogenous strigolactone content, which rendered repression of the outgrowth of the axillary buds. These findings unveil a novel function of OsGRF7 in rice tillering determination.

Rice GROWTH-REGULATING FACTOR7 Modulates Plant Architecture through Regulating GA and Indole-3-Acetic Acid Metabolism.

Plant-specific GROWTH-REGULATING FACTORs (GRFs) participate in central developmental processes, including leaf and root development; inflorescence, flower, and seed formation; senescence; and tolerance to stresses. In rice (Oryza sativa), there are 12 GRFs, but the role of the miR396-OsGRF7 regulatory module remains unknown. Here, we report that OsGRF7 shapes plant architecture via the regulation of auxin and GA metabolism in rice. OsGRF7 is mainly expressed in lamina joints, nodes, internodes, axillary buds, and young inflorescences. Overexpression of OsGRF7 causes a semidwarf and compact plant architecture with an increased culm wall thickness and narrowed leaf angles mediated by shortened cell length, altered cell arrangement, and increased parenchymal cell layers in the culm and adaxial side of the lamina joints. Knockout and knockdown lines of OsGRF7 exhibit contrasting phenotypes with severe degradation of parenchymal cells in the culm and lamina joints at maturity. Further analysis indicated that OsGRF7 binds the ACRGDA motif in the promoters of a cytochrome P450 gene and AUXIN RESPONSE FACTOR12, which are involved in the GA synthesis and auxin signaling pathways, respectively. Correspondingly, OsGRF7 alters the contents of endogenous GAs and auxins and sensitivity to exogenous phytohormones. These findings establish OsGRF7 as a crucial component in the OsmiR396-OsGRF-plant hormone regulatory network that controls rice plant architecture.

Metabolome-based prediction of yield heterosis contributes to the breeding of elite rice.

Improvement of the breeding efficiencies of heterotic crops adaptive to different conditions can mitigate the food shortage crisis due to overpopulation and climate change. To date, diverse molecular markers have been used to guide field phenotypic selection, whereas accurate predictions of complex heterotic traits are rarely reported. Here, we present a practical metabolome-based strategy for predicting yield heterosis in rice. The dissection of population structure based on untargeted metabolite profiles as the initial critical step in multivariate modeling performed better than the screening of predictive variables. Then the assessment of each predictive variable's contribution to predictive models according to all latent factors was more precise than the conventional first one. Metabolites belonging to specific pathways were closely associated with yield heterosis, and the up-regulation of galactose metabolism promoted robust yield heterosis in hybrids under different growth conditions. Our study demonstrates that metabolome-based predictive models with correctly dissected population structure and screened predictive variables can facilitate accurate predictions of yield heterosis and have great potential for establishing molecular marker-based precision breeding programs.

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.

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.

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.

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.

Hierarchical additive effects on heterosis in rice (Oryza sativa L.).

Exploitation of heterosis in crops has contributed greatly to improvement in global food and energy production. In spite of the pervasive importance of heterosis, a complete understanding of its mechanisms has remained elusive. In this study, a small test-crossed rice population was constructed to investigate the formation mechanism of heterosis for 13 traits. The results of the relative mid-parent heterosis and modes of inheritance of all investigated traits demonstrated that additive effects were the foundation of heterosis for complex traits in a hierarchical structure, and multiplicative interactions among the component traits were the framework of heterosis in complex traits. Furthermore, new balances between unit traits and related component traits provided hybrids with the opportunity to achieve an optimal degree of heterosis for complex traits. This study dissected heterosis of both reproductive and vegetative traits from the perspective of hierarchical structure for the first time. Additive multiplicative interactions of component traits were proven to be the origin of heterosis in complex traits. Meanwhile, more attention should be paid to component traits, rather than complex traits, in the process of revealing the mechanism of heterosis.

Balance between a higher degree of heterosis and increased reproductive isolation: a strategic design for breeding inter-subspecific hybrid rice.

The application of heterosis (hybrid vigor) has brought great success to plant breeding, particularly of hybrid rice, achieving significant yield increases. Attempts to explore the heterosis of inter-subspecific hybrids between indica and japonica rice, which result in even greater yield increases, have greatly increased in the past decades. However, because of the reduced seed setting rate in F1 hybrids as a result of increased reproductive isolation, the application of inter-subspecific hybrids in rice has slowed. Understanding the balance between heterosis and the reproductive isolation of inter-subspecific hybrids will facilitate the strategic design of inter-subspecific hybrid breeding. In this study, five indica and seven japonica rice varieties were chosen as the parental lines of a complete diallel mating design. Data from six group traits from all of the hybrids and inbred lines were collected. We found that the grain weight per plant, grain number per panicle, tiller per plant, thousand grain weight and plant height, which reflected increased heterosis, were associated with the genetic divergence index (GDI) of the parents. Meanwhile, owing to the reduced seed setting rate, which was also associated with the parents' GDI, the grain production of the hybrids was negatively affected. After analyzing the relationships between the GDI of indica-japonica parents and the grain weight per plant of the F1 hybrids, an ideal GDI value (0.37) for the two indica-japonica parents that could provide an optimal balance between the inter-subspecific heterosis and reproductive isolation was proposed. Our findings will help in the strategic design of an inter-subspecific hybrid rice breeding program by identifying the ideal indica and japonica parents for a hybrid combination to achieve hybrid rice with an optimal yield. This strategic design of an inter-subspecific hybrid rice breeding program will be time saving and cost effective.

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.

ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice.

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, orfH79 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 (BiFC) and coimmunoprecipitation (coIP) 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.