Comparative Transcriptome Analysis of the Heterosis of Salt Tolerance in Inter-Subspecific Hybrid Rice.

Soil salinity is one of the major abiotic stresses limiting rice growth. Hybrids outperform their parents in salt tolerance in rice, while its mechanism is not completely understood. In this study, a higher seedling survival was observed after salt treatment in an inter-subspecific hybrid rice, Zhegengyou1578 (ZGY1578), compared with its maternal japonica Zhegeng7A (ZG7A) and paternal indica Zhehui1578 (ZH1578). A total of 2584 and 3061 differentially expressed genes (DEGs) with at least twofold changes were identified between ZGY1578 and ZG7A and between ZGY1578 and ZH1578, respectively, in roots under salt stress using the RNA sequencing (RNA-Seq) approach. The expressions of a larger number of DEGs in hybrid were lower or higher than those of both parents. The DEGs associated with transcription factors, hormones, and reactive oxygen species (ROS)-related genes might be involved in the heterosis of salt tolerance. The expressions of the majority of transcription factors and ethylene-, auxin-, and gibberellin-related genes, as well as peroxidase genes, were significantly higher in the hybrid ZGY1578 compared with those of both parents. The identified genes provide valuable clues to elucidate the heterosis of salt tolerance in inter-subspecific hybrid rice.

Glutaredoxin in Rice Growth, Development, and Stress Resistance: Mechanisms and Research Advances.

Rice (Oryza sativa L.) is a staple food for more than half of the global population. Various abiotic and biotic stresses lead to accumulation of reactive oxygen species in rice, which damage macromolecules and signaling pathways. Rice has evolved a variety of antioxidant systems, including glutaredoxin (GRX), that protect against various stressors. A total of 48 GRX gene loci have been identified on 11 of the 12 chromosomes of the rice genome; none were found on chromosome 9. GRX proteins were classified into four categories according to their active sites: CPYC, CGFS, CC, and GRL. In this paper, we summarized the recent research advances regarding the roles of GRX in rice development regulation and response to stresses, and discussed future research perspectives related to rice production. This review could provide information for rice researchers on the current status of the GRX and serve as guidance for breeding superior varieties.

Identification and integrated analysis of glyphosate stress-responsive microRNAs, lncRNAs, and mRNAs in rice using genome-wide high-throughput sequencing.

BACKGROUND: Glyphosate has become the most widely used herbicide in the world. Therefore, the development of new varieties of glyphosate-tolerant crops is a research focus of seed companies and researchers. The glyphosate stress-responsive genes were used for the development of genetically modified crops, while only the EPSPS gene has been used currently in the study on glyphosate-tolerance in rice. Therefore, it is essential and crucial to intensify the exploration of glyphosate stress-responsive genes, to not only acquire other glyphosate stress-responsive genes with clean intellectual property rights but also obtain non-transgenic glyphosate-tolerant rice varieties. This study is expected to elucidate the responses of miRNAs, lncRNAs, and mRNAs to glyphosate applications and the potential regulatory mechanisms in response to glyphosate stress in rice. RESULTS: Leaves of the non-transgenic glyphosate-tolerant germplasm CA21 sprayed with 2 mg·ml- 1 glyphosate (GLY) and CA21 plants with no spray (CK) were collected for high-throughput sequencing analysis. A total of 1197 DEGs, 131 DELs, and 52 DEMs were identified in the GLY samples in relation to CK samples. Genes were significantly enriched for various biological processes involved in detoxification of plant response to stress. A total of 385 known miRNAs from 59 miRNA families and 94 novel miRNAs were identified. Degradome analysis led to the identification of 32 target genes, of which, the squamosa promoter-binding-like protein 12 (SPL12) was identified as a target of osa-miR156a_L + 1. The lncRNA-miRNA-mRNA regulatory network consisted of osa-miR156a_L + 1, two transcripts of SPL12 (LOC_Os06g49010.3 and LOC_Os06g49010.5), and 13 lncRNAs (e.g., MSTRG.244.1 and MSTRG.16577.1). CONCLUSION: Large-scale expression changes in coding and noncoding RNA were observed in rice mainly due to its response to glyphosate. SPL12, osa-miR156, and lncRNAs (e.g., MSTRG.244.1 and MSTRG.16577.1) could be a novel ceRNA mechanism in response to glyphosate in rice by regulating transcription and metal ions binding. These findings provide a theoretical basis for breeding glyphosate-tolerant rice varieties and for further research on the biogenesis of glyphosate- tolerance in rice.

Mapping and application of the twin-grain1 gene in rice.

MAIN CONCLUSION: The map-based cloning and application of a flower organ number gene twin - grain1 provide great potential for improving seed production in hybrid rice. A new germplasm for high-yield rice breeding, the twin-grain1 (tg1) mutant with more than one grain in a glume, was obtained from the Zhejing 22 rice variety via physical mutagenesis. The mapping results showed that TG1 is allelic to FLORAL ORGAN NUMBER2 (FON2)/FLORAL ORGAN NUMBER4 (FON4), a flower organ number gene located at 88.7 cM on chromosome 11. The novel tg1 gene allele was introgressed into the cytoplasmic male sterility (CMS) line Zhejing 22A, giving rise to a new CMS line Zhejing 22-tg1A. The Zhejing 22-tg1A line showed enhanced glume opening and stigma exsertion, which increased the outcrossing rate in hybrid rice. A small-scale hybrid rice seed production test demonstrated that the grain yield of the Zhejing 22-tg1A/Zhejinghui 5 line was significantly increased compared to that of the Zhejing 22A/Zhejinghui 5 line. The plot yield evaluation of the F1 hybrid lines showed a higher yield for the Zhejing 22-tg1A/Zhejinghui 5 line than that of the Zhejing 22A/Zhejinghui 5 line. The results implied great potentials for the tg1 gene in hybrid rice breeding.

Curved chimeric palea 1 encoding an EMF1-like protein maintains epigenetic repression of OsMADS58 in rice palea development.

Floral organ specification is controlled by various MADS-box genes in both dicots and monocots, whose expression is often subjected to both genetic and epigenetic regulation in Arabidopsis thaliana. However, little information is known about the role of epigenetic modification of MADS-box genes during rice flower development. Here, we report the characterization of a rice gene, curved chimeric palea 1 (CCP1) that functions in palea development. Mutation in CCP1 resulted in abnormal palea with ectopic stigmatic tissues and other pleiotropic phenotypes. We found that OsMADS58, a C-class gene responsible for carpel morphogenesis, was ectopically expressed in the ccp1 palea, indicating that the ccp1 palea was misspecified and partially acquired carpel-like identity. Constitutive expression of OsMADS58 in the wild-type rice plants caused morphological abnormality of palea similar to that of ccp1, whereas OsMADS58 knockdown by RNAi in ccp1 could rescue the abnormal phenotype of mutant palea, suggesting that the repression of OsMADS58 expression by CCP1 is critical for palea development. Map-based cloning revealed that CCP1 encodes a putative plant-specific emBRYONIC flower1 (EMF1)-like protein. Chromatin immunoprecipitation assay showed that the level of the H3K27me3 at the OsMADS58 locus was greatly reduced in ccp1 compared with that in the wild-type. Taken together, our results show that CCP1 plays an important role in palea development through maintaining H3K27me3-mediated epigenetic silence of the carpel identity-specifying gene OsMADS58, shedding light on the epigenetic mechanism in floral organ development.

BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development.

The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants. However, the molecular mechanisms linking the actin cytoskeleton to these processes remain largely unknown. Here, we report the functional analysis of rice (Oryza sativa) FH5/BENT UPPERMOST INTERNODE1 (BUI1), which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice. The bui1 mutant displayed pleiotropic phenotypes, including bent uppermost internode, dwarfism, wavy panicle rachis, and enhanced gravitropic response. Cytological observation indicated that the growth defects of bui1 were caused mainly by inhibition of cell expansion. Map-based cloning revealed that BUI1 encodes the class II formin FH5. FH5 contains a phosphatase tensin-like domain at its amino terminus and two highly conserved formin-homology domains, FH1 and FH2. In vitro biochemical analyses indicated that FH5 is capable of nucleating actin assembly from free or profilin-bound monomeric actin. FH5 also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end. Interestingly, the FH2 domain of FH5 could bundle actin filaments directly and stabilize actin filaments in vitro. Consistent with these in vitro biochemical activities of FH5/BUI1, the amount of filamentous actin decreased, and the longitudinal actin cables almost disappeared in bui1 cells. The FH2 or FH1FH2 domains of FH5 could also bind to and bundle microtubules in vitro. Thus, our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments, which are important for proper cell expansion and rice morphogenesis.

A mitochondrion-associated PPR protein, WBG1, regulates grain chalkiness in rice.

Rice kernel quality has vital commercial value. Grain chalkiness deteriorates rice's appearance and palatability. However, the molecular mechanisms that govern grain chalkiness remain unclear and may be regulated by many factors. In this study, we identified a stable hereditary mutant, white belly grain 1 (wbg1), which has a white belly in its mature grains. The grain filling rate of wbg1 was lower than that of the wild type across the whole filling period, and the starch granules in the chalky part were oval or round and loosely arranged. Map-based cloning showed that wbg1 was an allelic mutant of FLO10, which encodes a mitochondrion-targeted P-type pentatricopeptide repeat protein. Amino acid sequence analysis found that two PPR motifs present in the C-terminal of WBG1 were lost in wbg1. This deletion reduced the splicing efficiency of nad1 intron 1 to approximately 50% in wbg1, thereby partially reducing the activity of complex I and affecting ATP production in wbg1 grains. Furthermore, haplotype analysis showed that WBG1 was associated with grain width between indica and japonica rice varieties. These results suggested that WBG1 influences rice grain chalkiness and grain width by regulating the splicing efficiency of nad1 intron 1. This deepens understanding of the molecular mechanisms governing rice grain quality and provides theoretical support for molecular breeding to improve rice quality.

Whole-Transcriptome Analysis Reveals Autophagy Is Involved in Early Senescence of zj-es Mutant Rice.

Senescence is a necessary stage of plant growth and development, and the early senescence of rice will lead to yield reduction and quality decline. However, the mechanisms of rice senescence remain obscure. In this study, we characterized an early-senescence rice mutant, designated zj-es (ZheJing-early senescence), which was derived from the japonica rice cultivar Zhejing22. The mutant zj-es exhibited obvious early-senescence phenotype, such as collapsed chloroplast, lesions in leaves, declined fertility, plant dwarf, and decreased agronomic traits. The ZJ-ES gene was mapped in a 458 kb-interval between the molecular markers RM5992 and RM5813 on Chromosome 3, and analysis suggested that ZJ-ES is a novel gene controlling rice early senescence. Subsequently, whole-transcriptome RNA sequencing was performed on zj-es and its wild-type rice to dissect the underlying molecular mechanism for early senescence. Totally, 10,085 differentially expressed mRNAs (DEmRNAs), 1,253 differentially expressed lncRNAs (DElncRNAs), and 614 differentially expressed miRNAs (DEmiRNAs) were identified, respectively, in different comparison groups. Based on the weighted gene co-expression network analysis (WGCNA), the co-expression turquoise module was found to be the key for the occurrence of rice early senescence. Furthermore, analysis on the competing endogenous RNA (CeRNA) network revealed that 14 lncRNAs possibly regulated 16 co-expressed mRNAs through 8 miRNAs, and enrichment analysis showed that most of the DEmRNAs and the targets of DElncRNAs and DEmiRNAs were involved in reactive oxygen species (ROS)-triggered autophagy-related pathways. Further analysis showed that, in zj-es, ROS-related enzyme activities were markedly changed, ROS were largely accumulated, autophagosomes were obviously observed, cell death was significantly detected, and lesions were notably appeared in leaves. Totally, combining our results here and the remaining research, we infer that ROS-triggered autophagy induces the programmed cell death (PCD) and its coupled early senescence in zj-es mutant rice.

Differential Physiological Responses to Salt Stress between Salt-Sensitive and Salt-Tolerant japonica Rice Cultivars at the Post-Germination and Seedling Stages.

Soil salinity is a key source of abiotic stress in the cultivation of rice. In this study, two currently cultivated japonica rice species-Zhegeng 78 (salt-tolerant) and Zhegeng 99 (salt-sensitive)-with similar backgrounds were identified and used to investigate their differential responses to salt stress at the post-germination and seedling stages. Quantitative RT-PCR analysis demonstrated that the expression of OsSOS1, OsHAK1, and OsHAK5 at the post-germination stage, and the expression of OsHKT1,1, OsHTK2,1, and OsHAK1 at the seedling stage, were significantly higher in the salt-tolerant Zhegeng 78 compared with those of the salt-sensitive Zhegeng 99 under salt stress. The significantly lower Na+ net uptake rate at the post-germination and higher K+ net uptake rates at the post-germination and seedling stages were observed in the salt-tolerant Zhegeng 78 compared with those of the salt-sensitive Zhegeng 99 under salt stress. Significantly higher activity of peroxidase (POD) and the lower hydrogen peroxide (H2O2) accumulation were observed in the salt-tolerant Zhegeng 78 compared with those of salt-sensitive Zhegeng 99 under salt stress at the seeding stage. The salt-tolerant Zhegeng 78 might be valuable in future cultivation in salinity soils.

Beak-shaped grain 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice.

Grain shape and size both determine grain weight and therefore crop yield. However, the molecular mechanisms controlling grain shape and size are still largely unknown. Here, we isolated a rice mutant, beak-shaped grain1 (bsg1), which produced beak-shaped grains of decreased width, thickness and weight with a loosely interlocked lemma and palea that were unable to close tightly. Starch granules were also irregularly packaged in the bsg1 grains. Consistent with the lemma and palea shapes, the outer parenchyma cell layers of these bsg1 tissues developed fewer cells with decreased size. Map-based cloning revealed that BSG1 encoded a DUF640 domain protein, TRIANGULAR HULL 1, of unknown function. Quantitative PCR and GUS fusion reporter assays showed that BSG1 was expressed mainly in the young panicle and elongating stem. The BSG1 mutation affected the expression of genes potentially involved in the cell cycle and GW2, an important regulator of grain size in rice. Our results suggest that BSG1 determines grain shape and size probably by modifying cell division and expansion in the grain hull.

Characterization and mapping of a novel mutant sms1 (senescence and male sterility 1) in rice.

Plant senescence plays diverse important roles in development and environmental responses. However, the molecular basis of plant senescence is remained largely unknown. A rice spontaneous mutant with the character of early senescence and male sterility (sms) was found in the breeding line NT10-748. In order to identify the gene SMS1 and the underlying mechanism, we preliminarily analyzed physiological and biochemical phenotypes of the mutant. The mutant contained lower chlorophyll content compared with the wild type control and was severe male sterile with lower pollen viability. Genetic analysis showed that the mutant was controlled by a single recessive gene. By the map-based cloning approach, we fine-mapped SMS1 to a 67 kb region between the markers Z3-4 and Z1-1 on chromo-some 8 using 1,074 F(2) recessive plants derived from the cross between the mutant sms1 (japonica) x Zhenshan 97 (indica), where no known gene involved in senescence or male sterility has been identified. Therefore the SMS1 gene will be a novel gene that regulates the two developmental processes. The further cloning and functional analysis of the SMS1 gene is under way.