The OsWRKY72-OsAAT30/OsGSTU26 module mediates reactive oxygen species scavenging to drive heterosis for salt tolerance in hybrid rice.

Hybrid rice (Oryza sativa) generally outperforms its inbred parents in yield and stress tolerance, a phenomenon termed heterosis, but the underlying mechanism is not completely understood. Here, we combined transcriptome, proteome, physiological, and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000 (CY1000). In addition to surpassing the mean values for its two parents (mid-parent heterosis), CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents (over-parent heterosis or heterobeltiosis). Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance, acting in an overdominant fashion in CY1000. Furthermore, we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30. The salt-sensitive phenotypes were associated with the OsWRKY72paternal genotype or the OsAAT30maternal genotype in core rice germplasm varieties. OsWRKY72paternal specifically repressed the expression of OsGSTU26 under salt stress, leading to salinity sensitivity, while OsWRKY72maternal specifically repressed OsAAT30, resulting in salinity tolerance. These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice, providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.

Research on Physiological Characteristics and Differential Gene Expression of Rice Hybrids and Their Parents under Salt Stress at Seedling Stage.

Soil salinization is one of the most important abiotic stresses which can seriously affect the growth and development of rice, leading to the decrease in or even loss of a rice harvest. Increasing the rice yield of saline soil is a key issue for agricultural production. The utilization of heterosis could significantly increase crop biomass and yield, which might be an effective way to meet the demand for rice cultivation in saline soil. In this study, to elucidate the regulatory mechanisms of rice hybrids and their parents that respond to salt stress, we investigated the phenotypic characteristics, physiological and biochemical indexes, and expression level of salt-related genes at the seedling stage. In this study, two sets of materials, encapsulating the most significant differences between the rice hybrids and their parents, were screened using the salt damage index and a hybrid superiority analysis. Compared with their parents, the rice hybrids Guang-Ba-You-Hua-Zhan (BB1) and Y-Liang-You-900 (GD1) exhibited much better salt tolerance, including an increased fresh weight and higher survival rate, a better scavenging ability towards reactive oxygen species (ROS), better ionic homeostasis with lower content of Na+ in their Na+/K+ ratio, and a higher expression of salt-stress-responsive genes. These results indicated that rice hybrids developed complex regulatory mechanisms involving multiple pathways and genes to adapt to salt stress and provided a physiological basis for the utilization of heterosis for improving the yield of rice under salt stress.

OsPMS1 Mutation Enhances Salt Tolerance by Suppressing ROS Accumulation, Maintaining Na+/K+ Homeostasis, and Promoting ABA Biosynthesis.

World-wide, rice (Oryza sativa L.) is an important food source, and its production is often adversely affected by salinity. Therefore, to ensure stable rice yields for global food security, it is necessary to understand the salt tolerance mechanism of rice. The present study focused on the expression pattern of the rice mismatch repair gene post-meiotic segregation 1 (OsPMS1), studied the physiological properties and performed transcriptome analysis of ospms1 mutant seedlings in response to salt stress. Under normal conditions, the wild-type and ospms1 mutant seedlings showed no significant differences in growth and physiological indexes. However, after exposure to salt stress, compared with wild-type seedlings, the ospms1 mutant seedlings exhibited increased relative water content, relative chlorophyll content, superoxide dismutase (SOD) activity, K+ and abscisic acid (ABA) content, and decreased malondialdehyde (MDA) content, Na+ content, and Na+/K+ ratio, as well as decreased superoxide anion (O2-) and hydrogen peroxide (H2O2) accumulation. Gene ontology (GO) analysis of the differentially expressed genes (DEGs) of ospms1 mutant seedlings treated with 0 mM and 150 mM NaCl showed significant enrichment in biological and cytological processes, such as peroxidase activity and ribosomes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis showed that the DEGs specifically enriched ascorbate and aldarate metabolism, flavone and flavonol biosynthesis, and glutathione metabolism pathways. Further quantitative real-time reverse transcription-PCR (qRT-PCR) analysis revealed significant changes in the transcription levels of genes related to abscisic acid signaling (OsbZIP23, OsSAPK6, OsNCED4, OsbZIP66), reactive oxygen scavenging (OsTZF1, OsDHAR1, SIT1), ion transport (OsHAK5), and osmoregulation (OsLEA3-2). Thus, the study's findings suggest that the ospms1 mutant tolerates salt stress at the seedling stage by inhibiting the accumulation of reactive oxygen species, maintaining Na+ and K+ homeostasis, and promoting ABA biosynthesis.

Utilizing differences in bTH tolerance between the parents of two-line hybrid rice to improve the purity of hybrid rice seed.

Introduction: Two-line hybrid rice based on Photoperiod/thermo-sensitive genic male sterile (P/TGMS) lines has been developed and applied widely in agriculture due to the freedom in making hybrid combinations, less difficulty in breeding sterile lines, and simpler procedures for breeding and producing hybrid seed. However, there are certain risks associated with hybrid seed production; if the temperature during the P/TGMS fertility-sensitive period is lower than the critical temperature, seed production will fail due to self-pollination. In a previous study, we found that the issue of insufficient purity of two-line hybrid rice seed could be initially addressed by using the difference in tolerance to ß-triketone herbicides (bTHs) between the female parent and the hybrid seeds. Methods: In this study, we further investigated the types of applicable herbicides, application methods, application time, and the effects on physiological and biochemical indexes and yield in rice. Results: The results showed that this method could be used for hybrid purification by soaking seeds and spraying plants with the bTH benzobicylon (BBC) at safe concentrations in the range of 37.5-112.5 mg/L, and the seeds could be soaked in BBC at a treatment rate of 75.0 mg/L for 36-55 h without significant negative effects. The safe concentration for spraying in the field is 50.0-400.0 mg/L BBC at the three-leaf stage. Unlike BBC, Mesotrione (MST) can only be sprayed to achieve hybrid purification at concentrations between 10.0 and 70.0 mg/L without affecting yield. The three methods of hybrid seed purification can reach 100% efficiency without compromising the nutritional growth and yield of hybrid rice. Moreover, transcriptome sequencing revealed that 299 up-regulated significant differentially expressed genes (DEGs) in the resistant material (Huazhan) poisoned by BBC, were mainly enriched in phenylalanine metabolism and phenylpropanoid biosynthesis pathway, it may eliminate the toxic effects of herbicides through this way. Discussion: Our study establishes a foundation for the application of the bTH seed purification strategy and the three methods provide an effective mechanism for improving the purity of two-line hybrid rice seeds.

E2Fs co-participate in cadmium stress response through activation of MSHs during the cell cycle.

Cadmium is one of the most common heavy metal contaminants found in agricultural fields. MutSα, MutSß, and MutSγ are three different MutS-associated protein heterodimer complexes consisting of MSH2/MSH6, MSH2/MSH3, and MSH2/MSH7, respectively. These complexes have different mismatch recognition properties and abilities to support MMR. However, changes in mismatch repair genes (OsMSH2, OsMSH3, OsMSH6, and OsMSH7) of the MutS system in rice, one of the most important food crops, under cadmium stress and their association with E2Fs, the key transcription factors affecting cell cycles, are poorly evaluated. In this study, we systematically categorized six rice E2Fs and confirmed that OsMSHs were the downstream target genes of E2F using dual-luciferase reporter assays. In addition, we constructed four msh mutant rice varieties (msh2, msh3, msh6, and msh7) using the CRISPR-Cas9 technology, exposed these mutant rice seedlings to different concentrations of cadmium (0, 2, and 4 mg/L) and observed changes in their phenotype and transcriptomic profiles using RNA-Seq and qRT-PCR. We found that the difference in plant height before and after cadmium stress was more significant in mutant rice seedlings than in wild-type rice seedlings. Transcriptomic profiling and qRT-PCR quantification showed that cadmium stress specifically mobilized cell cycle-related genes ATR, CDKB2;1, MAD2, CycD5;2, CDKA;1, and OsRBR1. Furthermore, we expressed OsE2Fs in yeasts and found that heterologous E2F expression in yeast strains regulated cadmium tolerance by regulating MSHs expression. Further exploration of the underlying mechanisms revealed that cadmium stress may activate the CDKA/CYCD complex, which phosphorylates RBR proteins to release E2F, to regulate downstream MSHs expression and subsequent DNA damage repairment, thereby enhancing the response to cadmium stress.

Knockout of OsNRAMP5 enhances rice tolerance to cadmium toxicity in response to varying external cadmium concentrations via distinct mechanisms.

OsNRAMP5 is a transporter responsible for cadmium (Cd) and manganese (Mn) uptake and root-to-shoot translocation of Mn in rice plants. Knockout of OsNRAMP5 is regarded as an effective approach to minimize Cd uptake and accumulation in rice. It is vital to evaluate the effects of knocking out OsNRAMP5 on Cd and Mn accumulation, as well as Cd tolerance of rice plants in response to varying environmental Cd concentrations, and to uncover the underlying mechanism, which until now, has remained largely unexplored. This study showed that knockout of OsNRAMP5 decreased Cd uptake, but simultaneously facilitated Cd translocation from roots to shoots. The effect of OsNRAMP5 knockout on reducing root Cd uptake weakened, however its effect on improving root-to-shoot Cd translocation was constant with increasing environmental Cd concentrations. As a result, its mutation dramatically reduced Cd accumulation in shoots under low and moderate Cd stress, but inversely increased that under high Cd conditions. Interestingly, Cd tolerance of its knockout mutants was persistently enhanced, irrespective of lower or higher Cd concentrations in shoots, compared with that of wild-type plants. Knockout of OsNRAMP5 mitigated Cd toxicity by dramatically diminishing Cd uptake at low or moderate external Cd concentrations. Remarkably, its knockout effectively complemented deficient mineral nutrients in shoots, thereby indirectly enhancing rice tolerance to severe Cd stress. Additionally, its mutation conferred preferential delivery of Mn to young leaves and grains. These results have important implications for the application of the OsNRAMP5 mutation in mitigating Cd toxicity and lowering the risk of excessive Cd accumulation in rice grains.

Overexpression of OsLCT2, a Low-Affinity Cation Transporter Gene, Reduces Cadmium Accumulation in Shoots and Grains of Rice.

Cadmium (Cd)-contaminated rice is a serious issue affecting food safety. Understanding the molecular regulatory mechanisms of Cd accumulation in rice grains is crucial to minimizing Cd concentrations in grains. We identified a member of the low-affinity cation transporter family, OsLCT2 in rice. It was a membrane protein. OsLCT2 was expressed in all tissues of the elongation and maturation zones in roots, with the strongest expression in pericycle and stele cells adjacent to the xylem. When grown in Cd-contaminated paddy soils, rice plants overexpressing OsLCT2 significantly reduced Cd concentrations in the straw and grains. Hydroponic experiment demonstrated its overexpression decreased the rate of Cd translocation from roots to shoots, and reduced Cd concentrations in xylem sap and in shoots of rice. Moreover, its overexpression increased Zn concentrations in roots by up-regulating the expression of OsZIP9, a gene responsible for Zn uptake. Overexpression of OsLCT2 reduces Cd accumulation in rice shoots and grains by limiting the amounts of Cd loaded into the xylem and restricting Cd translocation from roots to shoots of rice. Thus, OsLCT2 is a promising genetic resource to be engineered to reduce Cd accumulation in rice grains.

OsPDCD5 negatively regulates plant architecture and grain yield in rice.

Plant architecture is an important agronomic trait that affects crop yield. Here, we report that a gene involved in programmed cell death, OsPDCD5, negatively regulates plant architecture and grain yield in rice. We used the CRISPR/Cas9 system to introduce loss-of-function mutations into OsPDCD5 in 11 rice cultivars. Targeted mutagenesis of OsPDCD5 enhanced grain yield and improved plant architecture by increasing plant height and optimizing panicle type and grain shape. Transcriptome analysis showed that OsPDCD5 knockout affected auxin biosynthesis, as well as the gibberellin and cytokinin biosynthesis and signaling pathways. OsPDCD5 interacted directly with OsAGAP, and OsAGAP positively regulated plant architecture and grain yield in rice. Collectively, these findings demonstrate that OsPDCD5 is a promising candidate gene for breeding super rice cultivars with increased yield potential and superior quality.

Influence of physicochemical properties and starch fine structure on the eating quality of hybrid rice with similar apparent amylose content.

In this study, we compared the physicochemical properties and starch structures of hybrid rice varieties with similar apparent amylose content but different taste values. In addition to the apparent amylose content, gel permeation chromatography analysis showed that the higher proportions of amylopectin short chains and relatively lower proportions of amylopectin long chains, which could lead to higher peak viscosity and breakdown value, as well as a softer and stickier texture of cooked rice, were the key factors in determining the eating quality of hybrid rice. High-performance anion-exchange chromatography analyses showed that the proportion of amylopectin short chains (degree of polymerization 6-10) and intermediate chains (degree of polymerization 13-24), which might affect the gelatinisation enthalpy and crystallinity, also contributed greatly to the eating quality of hybrid rice. Moreover, this study indicated that a greater diversity of forms and sizes of starch granules might influence the eating quality of hybrid rice.

Corrigendum: Correlations Between Parental Lines and Indica Hybrid Rice in Terms of Eating Quality Traits.

[This corrects the article DOI: 10.3389/fnut.2020.583997.].

Rice MutLγ, the MLH1-MLH3 heterodimer, participates in the formation of type I crossovers and regulation of embryo sac fertility.

The development of embryo sacs is crucial for seed production in plants, but the genetic basis regulating the meiotic crossover formation in the macrospore and microspore mother cells remains largely unclear. Here, we report the characterization of a spontaneous rice female sterile variation 1 mutant (fsv1) that showed severe embryo sacs abortion with low seed-setting rate. Through map-based cloning and functional analyses, we isolated the causal gene of fsv1, OsMLH3 encoding a MutL-homolog 3 protein, an ortholog of HvMLH3 in barley and AtMLH3 in Arabidopsis. OsMLH3 and OsMLH1 (MutL-homolog 1) interact to form a heterodimer (MutLγ) to promote crossover formation in the macrospore and microspore mother cells and development of functional megaspore during meiosis, defective OsMLH3 or OsMLH1 in fsv1 and CRISPR/Cas9-based knockout lines results in reduced type I crossover and bivalent frequency. The fsv1 and OsMLH3-knockout lines are valuable germplasms for development of female sterile restorer lines for mechanized seed production of hybrid rice.

Exploring Natural Allelic Variations of the ß-Triketone Herbicide Resistance Gene HIS1 for Application in indica Rice and Particularly in Two-Line Hybrid Rice.

BACKGROUND: Benzobicyclon (BBC) is a ß-triketone herbicide (bTH) used in rice paddy fields. It has the advantages of high efficiency, low toxicity, high crop safety, and good environmental compatibility, and shows efficacy against paddy weeds resistant to other types of herbicides. However, as some important indica rice varieties are susceptible to BBC, BBC is currently only registered and applied in japonica rice cultivation areas. RESULTS: By analyzing haplotypes of the bTHs broad-spectrum resistance gene HIS1 and phenotypes for BBC in 493 major indica rice accessions in China, we identified a novel non-functional allelic variant of HIS1 in addition to the previously reported 28-bp deletion. Through detection with markers specific to the two non-functional mutations, it was clear that 25.4% of indica conventional varieties, 59.9% of fertility restorers, and 15.9% of sterile lines were susceptible to BBC. In addition, due to natural allelic variations of the HIS1 gene in the sterile and restorer lines, some two-line hybrid sterile lines were sensitive to bTHs, and the corresponding restorers were resistant. We showed the potential effectiveness of using bTHs to address the issue of two-line hybrid rice seed purity stemming from the self-crossing of sterile lines during hybrid rice seed production. Finally, allelic variations of the HIS1 gene may also play an important role in the mechanized seed production of hybrid rice. CONCLUSIONS: Our findings offer guidance for the application of BBC in indica rice areas and provide a non-transgenic approach to address the seed purity issue of two-line hybrid rice.

Spike-Stalk Injection Method Causes Extensive Phenotypic and Genotypic Variations for Rice Germplasm.

Genetic diversities or favorable genes within distantly related species are the important resources for crop genetic improvement and germplasm innovation. Spike-Stalk injection method (SSI) has long been applied in rice genetic improvement by directly introducing genetic materials from non-mating donor species, while its inheritance patterns and the underlying mechanisms are poorly elucidated. In this study, a rice variant ERV1 with improved yield-related traits was screened out in the way of introducing genomic DNA of Oryza eichingeri (2n=24, CC genome) into RH78 (Oryza sativa L. 2n=24, AA genome) using SSI method. Genome-wide comparison revealed that the genomic heterozygosity of ERV1 was approximately 8-fold higher than RH78. Restriction-site associated DNA sequencing technology (RAD-seq) and association analysis of the ERV1 inbred F2 population identified 5 quantitative trait loci (QTLs) regions responsible for these yield-related traits, and found that genomic heterozygosity of ERV1 inbred lines was significantly lower than ERV1, while spontaneous mutation rate of the ERV1 inbred lines was significantly higher than ERV1. Our results preliminarily uncovered the inheritance patterns of SSI variant rice, and the potential genomic regions for traits changes, which yielded novel insights into the mechanisms of SSI method, and may accelerate our understanding of plant genome evolution, domestication, and speciation in nature.

Allelic variations of the Wx locus in cultivated rice and their use in the development of hybrid rice in China.

To make better use of global germplasm resources for improving the eating quality of hybrid rice, using the resequencing data from the 3,000 rice genomes project (3K RGP), the allelic variations of the rice Wx locus were analysed. With the exception of five rare alleles discovered for the first time in our study, most of these alleles were known alleles of Wx. Furthermore, a set of Kompetitive allele-specific PCR (KASP) markers based on these Wx alleles have been developed, and thirty-six main parents of hybrid rice from 1976 to 2018 were selected for Wx genotyping. The results showed that only three Wx alleles existed in the main parents of hybrids, and the allelic combination of the hybrids changed from Wxa/Wxb and Wxlv/Wxb to Wxb/Wxb with the development of hybrid rice. Wxb is widely used in the male parents of hybrid rice. Wxa and Wxlv were used in the female parents of early hybrid rice, and they were gradually replaced by Wxb. In the future, more favourable Wx alleles from cultivated rice should be identified, introduced, and effectively used to improve hybrid rice quality.

Correlations Between Parental Lines and Indica Hybrid Rice in Terms of Eating Quality Traits.

In this study, by analyzing the relationship between hybrid combinations and parental lines, we found that the eating quality traits of hybrid combinations were determined by both parents. The sterile lines determined the overall eating quality characteristics of the hybrid combinations. For the same sterile line, there were some correlations between the hybrid combinations and restorer lines in terms of taste value, rapid visco analyzer breakdown and setback values, apparent amylose content, and cooked rice hardness and stickiness. Analysis of the starch fine structure between hybrid combinations and their restorer lines demonstrated positive correlations between them in terms of short-branch amylopectin chains and amylose. Moreover, different allelic combinations of the Wx gene showed different genetic effects on the eating quality traits of hybrid rice. Overall, this study provides a framework for the development of hybrid rice with superior eating quality.

The bZIP73 transcription factor controls rice cold tolerance at the reproductive stage.

Cold temperature during the reproductive stage often causes great yield loss of grain crops in subtropical and temperate regions. Previously we showed that the rice transcription factor bZIP73Jap plays an important role in cold adaptation at the seedling stage. Here we further demonstrate that bZIP73Jap also confers cold stress tolerance at the reproductive stage. bZIP73Jap was up-regulated under cold treatment and predominately expressed in panicles at the early binucleate and flowering stages. bZIP73Jap forms heterodimers with bZIP71, and co-expression of bZIP73Jap and bZIP71 transgenic lines significantly increased seed-setting rate and grain yield under natural cold stress conditions. bZIP73Jap :bZIP71 not only repressed ABA level in anthers, but also enhanced soluble sugar transport from anthers to pollens and improved pollen grain fertility, seed-setting rate, and grain yield. Interestingly, bZIP73Jap :bZIP71 also regulated the expression of qLTG3-1Nip , and qLTG3-1Nip overexpression lines greatly improved rice tolerance to cold stress during the reproductive stage. Therefore, our work establishes a framework for rice cold stress tolerance through the bZIP71-bZIP73Jap -qLTG3-1Nip -sugar transport pathway. Together with our previous work, our results provide a powerful tool for improving rice cold stress tolerance at both the seedling and the reproductive stages.

Identification of lncRNAs involved in rice ovule development and female gametophyte abortion by genome-wide screening and functional analysis.

BACKGROUND: As important female reproductive tissues, the rice (Oryza sativa L.) ovule and female gametophyte is significant in terms of their fertility. Long noncoding RNAs (lncRNAs) play important and wide-ranging roles in the growth and development of plants and have become a major research focus in recent years. Therefore, we explored the characterization and expression change of lncRNAs during ovule development and female gametophytic abortion. RESULTS: In our study, whole-transcriptome strand-specific RNA sequencing (ssRNA-seq) was performed in the ovules of a high-frequency female-sterile rice line (fsv1) and a wild-type rice line (Gui99) at the megaspore mother cell meiosis stage (stage 1), functional megaspore mitosis stage (stage 2) and female gametophyte mature stage (stage 3). By comparing two rice lines, we identified 152, 233, and 197 differentially expressed lncRNAs at the three ovule developmental stages. Functional analysis of the coherent target genes of these differentially expressed lncRNAs indicated that many lncRNAs participate in multiple pathways such as hormone and cellular metabolism and signal transduction. Moreover, there were many differentially expressed lncRNAs acting as the precursors of some miRNAs that are involved in the development of ovules and female gametophytes. In addition, we have found that lncRNAs can act as decoys, competing with mRNAs for binding to miRNAs to maintain the normal expression of genes related to ovule and female gametophyte development. CONCLUSION: These results provide important clues for elucidating the female gametophyte abortion mechanism in rice. This study also expands our understanding about the biological functions of lncRNAs and the annotation of the rice genome.

Early selection of bZIP73 facilitated adaptation of japonica rice to cold climates.

Cold stress is a major factor limiting production and geographic distribution of rice (Oryza sativa). Although the growth range of japonica subspecies has expanded northward compared to modern wild rice (O. rufipogon), the molecular basis of the adaptation remains unclear. Here we report bZIP73, a bZIP transcription factor-coding gene with only one functional polymorphism (+511 G>A) between the two subspecies japonica and indica, may have facilitated japonica adaptation to cold climates. We show the japonica version of bZIP73 (bZIP73Jap) interacts with bZIP71 and modulates ABA levels and ROS homeostasis. Evolutionary and population genetic analyses suggest bZIP73 has undergone balancing selection; the bZIP73Jap allele has firstly selected from standing variations in wild rice and likely facilitated cold climate adaptation during initial japonica domestication, while the indica allele bZIP73Ind was subsequently selected for reasons that remain unclear. Our findings reveal early selection of bZIP73Jap may have facilitated climate adaptation of primitive rice germplasms.

Correction to: OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice.

Due to an error in combining the figure, an incorrect version of Fig. 9e was presented in the original publication.

Cold stress tolerance in rice: physiological changes, molecular mechanism, and future prospects.

Low temperature is a major factor affecting rice geographical distribution growth, development, and productivity. Cold stress mediates a series of physiological and metabolite changes, such as alterations in chlorophyll fluorescence, electrolyte leakage, reactive oxygen species (ROS), malondialdehyde (MAD), sucrose, lipid peroxides, proline, and other metabolites, plant endogenous hormones abscisic acid (ABA) and gibberellin (GA) also changes. In this review, we summarize the recent research progress on physiological and metabolic changes under low temperature, cold stress related loci and QTL reported by map-based cloning and genome-wide association analysis (GWAS), and some molecular mechanisms in response to low temperature in rice. We also discuss the future prospects on breeding cold tolerance varieties of rice.