miR396b/GRF6 module contributes to salt tolerance in rice.

Salinity, as one of the most challenging environmental factors restraining crop growth and yield, poses a severe threat to global food security. To address the rising food demand, it is urgent to develop crop varieties with enhanced yield and greater salt tolerance by delving into genes associated with salt tolerance and high-yield traits. MiR396b/GRF6 module has previously been demonstrated to increase rice yield by shaping the inflorescence architecture. In this study, we revealed that miR396b/GRF6 module can significantly improve salt tolerance of rice. In comparison with the wild type, the survival rate of MIM396 and OE-GRF6 transgenic lines increased by 48.0% and 74.4%, respectively. Concurrent with the increased salt tolerance, the transgenic plants exhibited reduced H2 O2 accumulation and elevated activities of ROS-scavenging enzymes (CAT, SOD and POD). Furthermore, we identified ZNF9, a negative regulator of rice salt tolerance, as directly binding to the promoter of miR396b to modulate the expression of miR396b/GRF6. Combined transcriptome and ChIP-seq analysis showed that MYB3R serves as the downstream target of miR396b/GRF6 in response to salt tolerance, and overexpression of MYB3R significantly enhanced salt tolerance. In conclusion, this study elucidated the potential mechanism underlying the response of the miR396b/GRF6 network to salt stress in rice. These findings offer a valuable genetic resource for the molecular breeding of high-yield rice varieties endowed with stronger salt tolerance.

Comprehensive identification and analysis of DUF640 genes associated with rice growth.

Protein domains with conserved amino acid sequences and uncharacterized functions are called domains of unknown function (DUF). The DUF640 gene family plays a crucial role in plant growth, particularly in light regulation, floral organ development, and fruit development. However, there exists a lack of systematic understanding of the evolutionary relationships and functional differentiation of DUF640 within the Oryza genus. In this study, 61 DUF640 genes were identified in the Oryza genus. The expression of DUF640s is induced by multiple hormonal stressors including abscisic acid (ABA), cytokinin (CK), ethylene (ETH), and indole-3-acetic acid (IAA). Specifically, OiDUF640-10 expression significantly increased after ETH treatment. Transgenic experiments showed that overexpressing OiDUF640-10 lines were sensitive to ETH, and seedling length was obstructed. Evolutionary analysis revealed differentiation of the OiDUF640-10 gene in O. sativa ssp. indica and japonica varieties, likely driven by natural selection during the domestication of cultivated rice. These results indicate that OiDUF640-10 plays a vital role in the regulation of rice seedling length.

Identification and characterization of BES1 genes involved in grain size development of Oryza sativa L.

BES1 (BRI1-EMS-SUPPRESSOR1) defines a unique class of plant-specific transcription factors that plays an essential role in response to Brassinosteroids (BRs) signal induction pathways. In our study, we conducted genome-wide scanning and comprehensive characterization of the BES1 gene family in rice and other eukaryotes, leading to valuable findings. Molecular docking experiments showed that all OsBES1 genes in rice could directly bind to BR small molecules. Among the identified genes, OsBES1-4 exhibited a remarkable response as it consistently showed induction upon exposure to various phytohormones after treatment. Further functional verification of OsBES1-4 revealed its impact on grain size. Overexpression of OsBES1-4 resulted in increased grain size, as confirmed by cytological observations showing an increase in cell length and cell number. Moreover, we identified that OsBES1-4 plays a role in rice grain size development by binding to the BR response element in the promoter region of the OsBZR1 gene. Evolutionary analysis indicated differentiation of OsBES1-4 between indica and japonica rice varieties, suggesting natural selection during the domestication process of cultivated rice. Therefore, we conclude that OsBES1-4 plays a crucial role in regulating rice grain size and has the potential to be an important target in rice breeding programs, and haplotype analysis found that all OsBES1 genes were associated with grain size development, either thousand-grain weight, grain length, or grain width. Overall, these findings suggest that the BES1 genes are involved in the regulation of grain size development in rice, and the utilization of SNPs in the OsBES1-4 gene promoter could be a favorable option for distinguishing indica and japonica.

A transposon-derived gene family regulates heading date in rice.

As a consequence of transposon domestication, transposon-derived proteins often acquire important biological functions. However, there have been limited studies on transposon-derived proteins in rice, and a systematic analysis of transposon-derived genes is lacking. Here, for the first time, we conducted a comprehensive analysis of the DDE_Tnp_4 (DDE) gene family, which originated from transposons but lost their transpositional ability and acquired new gene functions in Oryza species. A total of 58 DDE family genes, categorized into six groups, were identified in Oryza species, including 13 OsDDE genes in Oryza sativa ssp. japonica. Our analysis indicates that gene duplication events were not the primary mechanism behind the expansion of OsDDE genes in rice. Promoter cis-element analysis combined with haplotype analysis confirmed that OsDDEs regulate the heading date in rice. Specifically, OsDDE9 is a nuclear-localized protein expressed ubiquitously, which promotes heading date by regulating the expression of Ghd7 and Ehd1 under both short-day and long-day conditions. Single-nucleotide polymorphism (SNP) variations in the OsDDE9 promoter leads to changes in promoter activity, resulting in variations in heading dates. This study provides valuable insights into the molecular function and mechanism of the OsDDE genes.

Corrigendum: Comprehensive evolutionary analysis of growth-regulating factor gene family revealing the potential molecular basis under multiple hormonal stress in Gramineae crops.

[This corrects the article DOI: 10.3389/fpls.2023.1174955.].

Comprehensive evolutionary analysis of growth-regulating factor gene family revealing the potential molecular basis under multiple hormonal stress in Gramineae crops.

Growth-regulating factors (GRFs) are plant-specific transcription factors that contain two highly conserved QLQ and WRC domains, which control a range of biological functions, including leaf growth, floral organ development, and phytohormone signaling. However, knowledge of the evolutionary patterns and driving forces of GRFs in Gramineae crops is limited and poorly characterized. In this study, a total of 96 GRFs were identified from eight crops of Brachypodium distachyon, Hordeum vulgare, Oryza sativa L. ssp. indica, Oryza rufipogon, Oryza sativa L. ssp. japonica, Setaria italic, Sorghum bicolor and Zea mays. Based on their protein sequences, the GRFs were classified into three groups. Evolutionary analysis indicated that the whole-genome or segmental duplication plays an essential role in the GRFs expansion, and the GRFs were negatively selected during the evolution of Gramineae crops. The GRFs protein function as transcriptional activators with distinctive structural motifs in different groups. In addition, the expression of GRFs was induced under multiple hormonal stress, including IAA, BR, GA3, 6BA, ABA, and MeJ treatments. Specifically, OjGRF11 was significantly induced by IAA at 6 h after phytohormone treatment. Transgenic experiments showed that roots overexpressing OjGRF11 were more sensitive to IAA and affect root elongation. This study will broaden our insights into the origin and evolution of the GRF family in Gramineae crops and will facilitate further research on GRF function.

The Anti-Glioma Effect of Juglone Derivatives through ROS Generation.

Juglone has been extensively reported as a natural antitumor pigment. However, it is easy to be oxidized due to active hydroxy in the quinone. Here, we designed some new juglone derivatives, as the hydroxy was replaced by methyl (D1), allyl (D2), butyl (D3), and benzyl (D4) groups. Nuclear magnetic resonance spectra and mass spectrometry were applied to confirm the derivatives and oxidative products of juglone. U87 and U251 cell lines were used for tests in vitro, and primary human glioblastoma cells were applied for in vivo experiments. The CCK8 and EdU assay demonstrated the anti-tumor effect of the four derivatives, and IC50 for U87 was 3.99, 3.28, 7.60, and 11.84 µM, respectively. In U251, IC50 was 7.00, 5.43, 8.64, and 18.05 µM, respectively. D2 and D3 were further selected, and flow cytometry showed that apoptosis rates were increased after D2 or D3 treatment via ROS generation. Potential targets were predicted by network pharmacology analysis, most of which were associated with apoptosis, cell cycle, and metabolism pathway. CDC25B and DUSP1 were two of the most likely candidates for targets. The orthotopic glioblastoma model was established to evaluate the anti-glioma effect and side-effect of juglone derivatives, and the in vivo experiments confirmed the anti-glioma effects of juglone derivatives. In conclusion, new derivatives of juglone were created via chemical group substitution and could inhibit glioma cell viability and proliferation and induce apoptosis rate via ROS generation.

Identification and validation of a novel 16-gene prognostic signature for patients with breast cancer.

Despite increased early diagnosis and improved treatment in breast cancer (BRCA) patients, prognosis prediction is still a challenging task due to the disease heterogeneity. This study was to identify a novel gene signature that can accurately evaluate BRCA patient survival. The gene expression and clinical data of BRCA patients were collected from The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of BRCA International Consortium (METABRIC) databases. Genes associated with prognosis were determined by Kaplan-Meier survival analysis and multivariate Cox regression analysis. A prognostic 16-gene score was established with linear combination of 16 genes. The prognostic value of the signature was validated in the METABRIC and GSE202203 datasets. Gene expression analysis was performed to investigate the diagnostic values of 16 genes. The 16-gene score was associated with shortened overall survival in BRCA patients independently of clinicopathological characteristics. The signalling pathways of cell cycle, oocyte meiosis, RNA degradation, progesterone mediated oocyte maturation and DNA replication were the top five most enriched pathways in the high 16-gene score group. The 16-gene nomogram incorporating the survival-related clinical factors showed improved prediction accuracies for 1-year, 3-year and 5-year survival (area under curve [AUC] = 0.91, 0.79 and 0.77 respectively). MORN3, IGJ, DERL1 exhibited high accuracy in differentiating BRCA tissues from normal breast tissues (AUC > 0.80 for all cases). The 16-gene profile provides novel insights into the identification of BRCA with a high risk of death, which eventually guides treatment decision making.

Favorable QTLs from Oryza longistaminata improve rice drought resistance.

BACKGROUND: Drought is the major abiotic stress to rice grain production under unpredictable changing climatic environments. Wild rice of O. longistaminata show diverse responses and strong tolerance to stress environments. In order to identify whether the O. longistaminata can improve the rice drought resistance or not, a BIL population of 143 BC2F20 lines derived from the cross between the cultivar rice 9311 and O. longistaminata were assessed under stress of 20% PEG6000. RESULTS: In total, 28 QTLs related to drought resistance based on eight agronomic traits of seedlings were identified. Of which, thirteen QTLs including two QTLs for leaf drying, one QTL for leaf rolling, one QTL for leaf number, five QTLs for dry weight of root, two QTLs for dry weight of shoot, one QTL for maximum root length and two QTLs for maximum shoot length were derived from O. longistaminata. What's more, qDWR8.1 for dry weight of root was repeatedly detected and fine-mapped to an interval about 36.2 Kb. The unique allele of MH08g0242800 annotated as ATP-dependent Clp protease proteolytic subunit from O. longistaminata was suggested as the candidate gene for drought resistance. Further, six representative BIL lines were stably characterized showing significantly stronger drought resistance than 9311 based on principle component analysis, they each contained 2 ~ 5 QTLs including qDWR8.1 from O. longistaminata. CONCLUSIONS: Together, our results indicate that the QTLs from O. longistaminata can effectively enhance the drought tolerance of rice, showing great potential value in breeding of elite rice varieties, which will lay a novel insight into the genetic network for drought tolerance of rice.

Creation of Elite Rice with High-Yield, Superior-Quality and High Resistance to Brown Planthopper Based on Molecular Design.

Breeding rice (Oryza sativa L.) with high yield, superior quality, desired grain shape and high resistance is the goal of breeding to meet the needs of current consumers. It is usually hard to combine multiple complex traits based on traditional breeding methods because they are frequently antagonistic to each other. However, molecular design breeding, as a novel breeding method, is an optional alternative to this challenge. To demonstrate molecular design breeding, 15 favorable genes from five parent lines were pyramided together to develop elite rice with high-yield, superior-quality, desired grain shape and high resistance to brown planthopper (BPH). The parental lines were 9311, the recurrent parent, carrying APO1, Ghd7, Ghd8 and Gn1a for high yield, GS3 and qSW5 for grain shape, and Wx and ALK for eating and cooking quality; 1880 with Gn8.1 for large panicles; Luo-Yu-Xiang carrying GW7 for grain shape and SBE3, SSIV2 and SSIII for eating and cooking quality; Luoyang6 with Bph6 and Luoyang9 with Bph9 for BPH resistance. After careful screening for the 15 targeted genes, desired phenotype and maximum genetic background from 9311, three molecular design lines with desired phenotypes, named as MD1 (Molecular design 1), MD2 and MD3 were developed. MD3 carried all 15 targeted genes, and MD1 and MD2 had 14 of the 15 targeted genes. Only SBE3 was not introgressed into MD1 and MD2 but this had minimal impact on the gel consistency and alkali spreading value. These newly bred lines exhibited higher yield potential, better grain quality with slender grains, low amylose content, high gel consistency and alkali spreading value, and higher BPH resistance compared to the parent 9311. In this study, we successfully created three novel rice lines with high yield, superior quality and improved BPH resistance by rational molecular design. Our results demonstrate molecular design is a powerful strategy to improve multiple complex traits and will provide a reference for the future commercial rice improvement.

Quantitative Trait Locus Mapping of Salt Tolerance in Wild Rice Oryza longistaminata.

Salt stress is one of the most severe adverse environments in rice production; increasing salinization is seriously endangering rice production around the world. In this study, a rice backcross inbred line (BIL) population derived from the cross of 9311 and wild rice Oryza longistaminata was employed to identify the favorable genetic loci of O. longistaminata for salt tolerance. A total of 27 quantitative trait loci (QTLs) related to salt tolerance were identified in 140 rice BILs, and 17 QTLs formed seven QTL clusters on different chromosomes, of which 18 QTLs were derived from O. longistaminata, and a QTL for salt injury score (SIS), water content of seedlings (WCS) under salt treatment, and relative water content of seedlings (RWCS) was repeatedly detected and colocalized at the same site on chromosome 2, and a cytochrome P450 86B1 (MH02t0466900) was suggested as the potential candidate gene responsible for the salt tolerance based on sequence and expression analysis. These findings laid the foundation for further improving rice salt tolerance through molecular breeding in the future.

Engineering Escherichia coli to produce Bordetella pertussis oligosaccharide with multiple trisaccharide units.

The immunogenicity of the pertussis vaccine can be significantly improved by adding Bordetella pertussis oligosaccharide with multiple trisaccharide units. The more trisaccharide units there are, the better the efficiency of the immune response induction. However, natural B. pertussis oligosaccharides usually contain only a single terminal trisaccharide unit. In addition, B. pertussis is pathogenic, and there are potential safety hazards when preparing oligosaccharides from B. pertussis. In this study, Escherichia coli MG1655 was engineered to produce B. pertussis oligosaccharides containing multiple trisaccharide units. Fifty-nine genes relevant to the biosynthesis of the O-antigen and core oligosaccharide of lipopolysaccharide, enterobacterial common antigen, and colanic acid were deleted in MG1655, resulting in strain MDCO020. Then, 25 genes relevant to the biosynthesis of the oligosaccharide antigen in B. pertussis and 3 genes relevant to the repeating trisaccharide unit in Pseudomonas aeruginosa PAO1 were overexpressed in MDCO020, resulting in the recombinant E. coli MDCO020/pWpBpD5. The production of B. pertussis oligosaccharide with multiple trisaccharide units by MDCO020/pWpBpD5 was confirmed by SDS-PAGE and 1H NMR analyses, and its immune response-stimulating activity was confirmed by using rabbit anti-pertussis serum.

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 Inhibition of B7H3 by 2-HG Accumulation Is Associated With Downregulation of VEGFA in IDH Mutated Gliomas.

B7H3 (also known as CD276) is a co-stimulator checkpoint protein of the cell surface B7 superfamily. Recently, the function beyond immune regulation of B7H3 has been widely studied. However, the expression preference and the regulation mechanism underlying B7H3 in different subtypes of gliomas is rarely understood. We show here that B7H3 expression is significantly decreased in IDH-mutated gliomas and in cultured IDH1-R132H glioma cells. Accumulation of 2-HG leads to a remarkable downregulation of B7H3 protein and the activity of IDH1-R132H mutant is responsible for B7H3 reduction in glioma cells. Inhibition of autophagy by inhibitors like leupeptin, chloroquine (CQ), and Bafilomycin A1 (Baf-A1) blocks the degradation of B7H3 in glioma cells. In the meantime, the autophagy flux is more active with higher LC3B-II and lower p62 in IDH1-R132H glioma cells than in IDH1-WT cells. Furthermore, sequence alignment analysis reveals potential LC3-interacting region (LIR) motifs "F-V-S/N-I/V" in B7H3. Moreover, B7H3 interacts with p62 and CQ treatment significantly enhances this interaction. Additionally, we find that B7H3 is positively correlated with VEGFA and MMP2 by bioinformatics analysis in gliomas. B7H3 and VEGFA are decreased in IDH-mutated gliomas and further reduced in 2-HGhigh gliomas compared to 2-HGlow glioma sections by IHC staining. Our study demonstrates that B7H3 is preferentially overexpressed in IDH wild-type gliomas and could serve as a potential theranostic target for the precise treatment of glioma patients with wild-type IDH.

Quantitative Trait Loci Mapping of Mineral Element Contents in Brown Rice Using Backcross Inbred Lines Derived From Oryza longistaminata.

Mineral elements play an extremely important role in human health, and are worthy of study in rice grain. Wild rice is an important gene pool for rice improvement including grain yield, disease, and pest resistance as well as mineral elements. In this study, we identified 33 quantitative trait loci (QTL) for Fe, Zn, Se, Cd, Hg, and As contents in wild rice Oryza longistaminata. Of which, 29 QTLs were the first report, and 12 QTLs were overlapped to form five clusters as qSe1/qCd1 on chromosome 1, qCd4.2/qHg4 on chromosome 4, qFe5.2/qZn5.2 on chromosome 5, qFe9/qHg9.2/qAs9.2 on chromosome 9, and qCd10/qHg10 on chromosome 10. Importantly, qSe1/qCd1, can significantly improve the Se content while reduce the Cd content, and qFe5.2/qZn5.2 can significantly improve both the Fe and Zn contents, they were delimited to an interval about 53.8 Kb and 26.2 Kb, respectively. These QTLs detected from Oryza longistaminata not only establish the basis for subsequent gene cloning to decipher the genetic mechanism of mineral element accumulation, but also provide new genetic resource for rice quality improvement.

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.

Quantitative Trait Locus Mapping of the Combining Ability for Yield-Related Traits in Wild Rice Oryza longistaminata.

In decades of hybrid rice breeding, the combining ability has been successfully used to evaluate excellent parental lines and predict heterosis. However, previous studies for the combining ability mainly focused on cultivated rice and rarely involved wild rice. In this study, for the first time, we identified 20 new quantitative trait loci (QTLs) for the combining ability in wild rice using a North Carolina II mating design. Among them, qGCA1, one of the major QTLs that can significantly improve the general combining ability of the plant height, spikelet number, and yield per plant, was delimited to an interval of about 72 kb on chromosome 1. qSCA8, another major QTL, which can significantly improve the specific combining ability of the seed-setting rate and yield per plant, was located in an interval of about 90 kb on chromosome 8. These QTLs discovered from wild rice will provide new ideas to explain the genetic mechanism of the combining ability and establish the basis for breeding of high-combining-ability rice.

Development of Elite BPH-Resistant Wide-Spectrum Restorer Lines for Three and Two Line Hybrid Rice.

Hybrid rice has contributed significantly to the world food security. Breeding of elite high-yield, strong-resistant broad-spectrum restorer line is an important strategy for hybrid rice in commercial breeding programs. Here, we developed three elite brown planthopper (BPH)-resistant wide-spectrum restorer lines by pyramiding big-panicle gene Gn8.1, BPH-resistant genes Bph6 and Bph9, fertility restorer genes Rf3, Rf4, Rf5, and Rf6 through molecular marker assisted selection. Resistance analysis revealed that the newly developed restorer lines showed stronger BPH-resistance than any of the single-gene donor parent Luoyang-6 and Luoyang-9. Moreover, the three new restorer lines had broad spectrum recovery capabilities for Honglian CMS, Wild abortive CMS and two-line GMS sterile lines, and higher grain yields than that of the recurrent parent 9,311 under nature field conditions. Importantly, the hybrid crosses also showed good performance for grain yield and BPH-resistance. Thus, the development of elite BPH-resistant wide-spectrum restorer lines has a promising future for breeding of broad spectrum BPH-resistant high-yield varieties.

Serum Human Epidermal Growth Factor 2 is a Novel Biomarker for Recurrence and Metastasis in Triple Negative Breast Cancer.

BACKGROUND: The aim of this study was to evaluate the predictive value of serum human epidermal growth factor 2 (HER2) for recurrence and metastasis in triple negative breast cancer (TNBC). METHODS: A total of 200 patients with benign breast tumors and 300 patients with breast cancer treated in the Department of Breast Surgery, Women and Children's Hospital of Ningbo City (China) between December 2006 and December 2013 were enrolled. Another 500 age- and gender-matched healthy individuals served as controls. The serum level of HER2 was determined using suspension array technology. Patients with breast cancer were further divided into ER-/PR-/HER2- and ER-/PR-/HER2+ groups and followed up for 5 years to analyze the serum concentration of HER2. RESULTS: The serum HER2 concentration was significantly higher in patients with breast cancer than in healthy controls or patients with benign tumors (both p < 0.01). The serum HER2 concentration also was significantly higher in patients with TNBC than in healthy controls (p < 0.01). The serum concentration of HER2 was significantly higher in TNBC patients who experienced recurrence and metastasis than in TNBC patients who did not experience recurrence and metastasis (both p < 0.01). Notably, the serum HER2 concentration in TNBC patients who experienced recurrence and metastasis was increased to a level statistically similar to that in patients with HER2+ breast cancer (p > 0.05). CONCLUSIONS: Patients with TNBC still have an increased serum HER2 concentration, and serum HER2 may be a valuable, novel biomarker for recurrence and metastasis in TNBC.