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In this study, the cold-tolerance capacity of 133 varieties of weedy rice was evaluated based on the comprehensive evaluation index D, with Kongyu 131 used as a cold-tolerant control. A total of 39.8% of the 133 varieties were considered ‘strong’, indicating that weedy rice populations indeed have relatively strong cold-tolerance capacity as a whole, and the robust cold-tolerant varieties WR29 and WR157 were identified. Regression analysis showed that the metrics including the nitrogen recovery index, superoxide dismutase (SOD) content and malondialdehyde (MDA) content correlated significantly (P < 0.05) with cold tolerance and could be used as indicators of cold tolerance. On the basis of a transcriptome analysis of WR157, a robust cold-tolerant variety identified in this study, a total of 4645 putative DEGs were identified in treated groups compared to the control groups, with 2123 upregulated DEGs and 2522 downregulated DEGs. All upregulated DEGs were enriched on 1388 terms, all downregulated DEGs were enriched on 1566 terms; 911 of the 2123 upregulated DEGs fell into 98 KEGG categories and 1103 of the 2522 downregulated DEGs were in 115 categories. Further analysis showed that GO:0019740 and GO:0006808 are involved in nitrogen utilization; GO:0009269 and GO:0009414 are related to the stress response; and GO:0016491 and GO:0016614 are related to oxidoreductase activity.BackgroundWeedy rice (Oryza) is a related pest species of cultivated rice (Oryza sativa L.) that has strong abiotic stress resistance; however, the comprehensive mechanism governing its cold tolerance is poorly understood.ConclusionOur comprehensive evaluation based on five morphological indices and nine physiological indicators revealed outstanding levels of cold-tolerance capacity among weedy rice varieties from different regions and revealed some terms related to cold tolerance via transcriptome analysis. Our results underscored the reliable evaluation methods for additional cold tolerance studies and revealed several genes related to cold tolerance, which will help researchers breed cultivated rice varieties to increase their cold-tolerance capacity. These traits have the ability to increase seedling survival rate and growth, as well as future yields.

Summary Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H2O2, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.

Precise activation of endogenous genes is a powerful strategy for functional genomics and therapeutic development, but current CRISPR-based transcriptional activation (CRISPRa) systems are limited by the large size of Cas proteins for adeno-associated virus (AAV) delivery. Here, we present a h igh- e fficiency dC a s12f-based transcriptiona l activation system (HEAL), which recruits transactivators through MS2 coat protein binding to MS2 aptamers embedded within the sgRNA scaffold. Engineered to enhance DNA binding, nuclear localization, and transactivator recruitment, HEAL induces over 100,000-fold activation of endogenous genes and outperforms existing CRISPRa systems in vitro and in vivo. We further develop red-light-inducible OptoHEAL and small-molecule-inducible ChemHEAL for remote and precise transcriptional control. AAV-delivered HEAL targeting interleukin 10 alleviates acute kidney injury in mice, while ChemHEAL-mediated activation of thymic stromal lymphopoietin reduces body weight in obese mice. HEAL provides a modular, compact, and controllable platform for endogenous gene activation with strong potential for fundamental research and gene therapy. Precise activation of endogenous genes is a powerful strategy for research and therapy, but existing tools are too large for efficient delivery. Here, authors create a compact and programmable CRISPRa platform that can be remotely controlled and shows therapeutic benefits in mouse disease models.

CHD3 is one of the chromatin-remodeling factors that contribute to controlling the expression of genes associated with plant development. Loss-of-function mutants display morphological and growth defects. However, the molecular mechanisms underlying CHD3 regulation of plant development remain unclear. In this study, a rice CHD3 protein, CHR729, was identified. The corresponding mutant line (t483) exhibited late seed germination, low germination rate, dwarfism, low tiller number, root growth inhibition, adaxial albino leaves, and short and narrow leaves. CHR729 encoded a nuclear protein and was expressed in almost all organs. RNA-sequencing analysis showed that several plant hormone-related genes were up- or down-regulated in t483 compared to wild type. In particular, expression of the gibberellin synthetase gibberellin 20 oxidase 4 gene was elevated in the mutant. Endogenous gibberellin assays demonstrated that the content of bioactive GA3 was reduced in t483 compared to wild type. Moreover, the seedling dwarfism, late seed germination, and short root length phenotypes of t483 were partially rescued by treatment with exogenous GA3. These results suggest that the rice CHD3 protein CHR729 plays an important role in many aspects of seedling development and controls this development via the gibberellin pathway.

Salinity is one of the most widespread abiotic stresses affecting rice productivity worldwide. Understanding the genetic basis of salt tolerance is key for breeding salt-tolerant rice varieties. Numerous QTLs have been identified to help dissect rice salt-tolerance genetic mechanisms, yet only rare genes located in significant QTLs have been thoroughly studied or fine-mapped. Here, a combination of linkage mapping and transcriptome profiling analysis was used to identify salt tolerance-related functional candidate genes underlying stable QTLs. A recombinant inbred line (RIL) population derived from a cross between Jileng 1 (salt-sensitive) and Milyang 23 (salt-tolerant) was constructed. Subsequently, a high-density genetic map was constructed by using 2921 recombination bin markers developed from whole genome resequencing. A total of twelve QTLs controlling the standard evaluation score under salt stress were identified by linkage analysis and distributed on chromosomes 2, 3, 4, 6, 8 and 11. Notably, five QTL intervals were detected as environmentally stable QTLs in this study, and their functions were verified by comparative transcriptome analysis. By comparing the transcriptome profiles of the two parents and two bulks, we found 551 salt stress-specific differentially expressed genes. Among them, fifteen DEGs located in stable QTL intervals were considered promising candidate genes for salt tolerance. According to gene annotations, the gene OsRCI2-8 ( Os06g0184800 ) was the most promising, as it is known to be associated with salt stress, and its differential expression between the tolerant and sensitive RIL bulks highlights its important role in salt stress response pathways. Our findings provide five stable salt tolerance-related QTLs and one promising candidate gene, which will facilitate breeding for improved salt tolerance in rice varieties and promote the exploration of salt stress tolerance mechanisms in rice.

Low temperature affects the rice plants at all stages of growth. It can cause severe seedling injury and male sterility resulting in severe yield losses. Using a mini core collection of 174 Chinese rice accessions and 273 SSR markers we investigated cold tolerance at the germination and booting stages, as well as the underlying genetic bases, by association mapping. Two distinct populations, corresponding to subspecies indica and japonica showed evident differences in cold tolerance and its genetic basis. Both subspecies were sensitive to cold stress at both growth stages. However, japonica was more tolerant than indica at all stages as measured by seedling survival and seed setting. There was a low correlation in cold tolerance between the germination and booting stages. Fifty one quantitative trait loci (QTLs) for cold tolerance were dispersed across all 12 chromosomes; 22 detected at the germination stage and 33 at the booting stage. Eight QTLs were identified by at least two of four measures. About 46% of the QTLs represented new loci. The only QTL shared between indica and japonica for the same measure was qLTSSvR6-2 for SSvR. This implied a complicated mechanism of old tolerance between the two subspecies. According to the relative genotypic effect (RGE) of each genotype for each QTL, we detected 18 positive genotypes and 21 negative genotypes in indica, and 19 positive genotypes and 24 negative genotypes in japonica. In general, the negative effects were much stronger than the positive effects in both subspecies. Markers for QTL with positive effects in one subspecies were shown to be effective for selection of cold tolerance in that subspecies, but not in the other subspecies. QTL with strong negative effects on cold tolerance should be avoided during MAS breeding so as to not cancel the effect of favorable QTL at other loci.

BackgroundUnderstanding and identifying the factors responsible for genetic differentiation is of fundamental importance for efficient utilization and conservation of traditional rice landraces. In this study, we examined the spatial genetic differentiation of 594 individuals sampled from 28 locations in Yunnan Province, China, covering a wide geographic distribution and diverse growing conditions. All 594 accessions were studied using ten unlinked target genes and 48 microsatellite loci, and the representative 108 accessions from the whole collection were sampled for resequencing.ResultsThe genetic diversity of rice landraces was quite different geographically and exhibited a geographical decline from south to north in Yunnan, China. Population structure revealed that the rice landraces could be clearly differentiated into japonica and indica groups, respectively. In each group, the rice accessions could be further differentiated corresponded to their geographic locations, including three subgroups from northern, southern and middle locations. We found more obvious internal geographic structure in the japonica group than in the indica group. In the japonica group, we found that genetic and phenotypic differentiation were strongly related to geographical distance, suggesting a pattern of isolation by distance (IBD); this relationship remained highly significant when we controlled for environmental effects, where the likelihood of gene flow is inversely proportional to the distance between locations. Moreover, the gene flow also followed patterns of isolation by environment (IBE) whereby gene flow rates are higher in similar environments. We detected 314 and 216 regions had been differentially selected between Jap-N and Jap-S, Ind-N and Ind-S, respectively, and thus referred to as selection signatures for different geographic subgroups. We also observed a number of significant and interesting associations between loci and environmental factors, which implies adaptation to local environment.ConclusionsOur findings highlight the influence of geographical isolation and environmental heterogeneity on the pattern of the gene flow, and demonstrate that both geographical isolation and environment drives adaptive divergence play dominant roles in the genetic differentiation of the rice landraces in Yunnan, China as a result of limited dispersal.

Background The Dong people mainly live in Hunan, Guangxi and Guizhou provinces, China, with a long history of glutinous rice cultivation, among which Kam Sweet Rice (KSR) is a group of rice landraces that has been domesticated for thousands of years by the Dong people. The core distribution area of KSR is Liping, Congjiang and Rongjiang County of southeast, Guizhou Province. Paddy fields, forests, livestock and cottages have formed a special artificial wetland ecosystem in local area, and the Dong people have also formed a set of traditional farming systems of KSR for variety breeding, field management, and soil and water conservation. However, this traditional agricultural management has not been reported at multiple levels based on landraces, species and ecosystems. Methods Fieldwork was conducted in ten villages in southeast Guizhou from 2019 to 2021. A total of 229 informants were interviewed from the villages. Semi-structured and key informant interviews were administered to collect ethnoecological data on the characteristics and traditional utilization of KSR, traditional farming systems and agricultural management of the Dong people. Results (1): A total of 57 KSR landraces were recorded as used by the Dong people in southeast Guizhou. We analyzed the cultural importance index (CII) of all KSRs. KSR with high CII often has a pleasant taste, special biological characteristics of cold resistance, disease and insect resistance and high utilization in the traditional culture of Dong people. (2) There is a clear division of labor between men and women in the breeding, seed retention, field management and grain storage management of different landraces of KSR in Dong communities. In order to resist natural disasters and insect pests, the cultivation of KSR is usually managed by multi-variety mixed planting. These agricultural management modes are the result of Dong people’s understanding and adaptation to the local natural geographical environment, as well as the experience and wisdom crystallization of Dong people’s long-term practice. (3) The traditional farmland of Dong People is a typical artificial wetland ecosystem that is planted with mixed KSR landraces with rich traditional wisdom. In addition, the economic benefit of the rice–fish–duck symbiotic system was 3.07 times that of hybrid rice alone; therefore, the rice–fish–duck system not only has the function of maintaining soil, water and ecological balance but also improves the income of Dong people. Conclusion KSR is a special kind of rice that has been domesticated and cultivated by Dong people for thousands of years. Dong people have also formed traditional agriculture dominated by KSR cultivation. The traditional agricultural management of Dong people provides suitable habitats for flora and fauna with biodiversity protection, and convenient conditions for rational utilization and distribution of water resources were also provided. This traditional management mode is of great significance for environmental protection, climate change response, community resource management, sustainable utilization and agricultural transformation in modern society. Therefore, we call for interdisciplinary research in natural and social sciences, in-depth study of the ecological culture of ethnic areas, and sort out treasures conducive to the development of all mankind.

Salt stress affects rice seed germination and seedling formation, seriously restricting rice production. Screening salt-tolerant rice varieties and analyzing the genetic mechanisms underlying salt tolerance are therefore very important to ensure rice production. In this study, 313 Oryza sativa ssp. japonica germplasm were used to conduct a genome-wide association study (GWAS) using 1% NaCl as a salt stress treatment during germination stage. The germination potential (GP) on different days and the germination index (GI) under salt stress were used as salt tolerance indicators. The results of population structure analysis showed that the 313 germplasm studied could be divided into two subpopulations, consistent with the geographical origins of the materials. There were 52 loci significantly related to salt tolerance during germination, and the phenotypic contribution rate of 29 loci was > 10%. A region on chromosome 11 (17049672–17249672 bp) was repeatedly located, and the candidate gene LOC_Os11g29490 , which encodes a plasma membrane ATPase, was identified in this locus. Further haplotype analysis showed the GP of germplasm with different haplotypes at that locus significantly differed under salt stress ( p < 0.05), and germplasm carrying Hap2 displayed strong salt tolerance during the germination stage. Two other promising candidate genes for salt tolerance were identified: LOC_Os01g27170 ( OsHAK3 ), which encodes a potassium transporter, and LOC_Os10g42550 ( OsITPK5 ), which encodes an inositol 1, 3, 4-trisphosphate 5/6-kinase. The results of this study provide a theoretical basis for salt-tolerant gene cloning and molecular design breeding in rice.

Drought is a complicated abiotic stress factor with severe effects on rice growth and production. Weedy rice is a valuable genetic resource that possesses a strong capacity for drought tolerance, cold tolerance, and salt tolerance, and is an excellent material for studying rice tolerance. Here, according to comprehensive tolerance to drought index D, accession WR16 was selected based on strong drought tolerance among 133 studied weedy red rice germplasms. WR16 was compared with Oryza sativa ssp. Japonica . cv. IAPAR-9, a reference genotype originating from Brazil. In addition, accession WR24 was classified as moderately tolerant to drought accessions. Transcriptomic and proteomic analyses were combined to identify 38 co-upregulated proteins related to drought tolerance, and targeted parallel reaction monitoring (PRM) was used to precisely quantify and verify nine proteins in the complex backgrounds. Result showed that six proteins were significantly (Fisher's exact P value < 0.05) related to drought tolerance in accessions WR16 and WR24. Among them, OS09T0478300-01, OS09T0530300-01, and OS01T0800500-01 formed a combined defense system to respond to drought stress in weedy rice. Results of these studies provide comprehensive information for precisely identifying and verifying tolerance to drought proteins and lay a solid theoretical foundation for research on drought tolerance mechanisms.