Rhizoma Drynariae-derived nanovesicles reverse osteoporosis by potentiating osteogenic differentiation of human bone marrow mesenchymal stem cells via targeting ERα signaling.

Although various anti-osteoporosis drugs are available, the limitations of these therapies, including drug resistance and collateral responses, require the development of novel anti-osteoporosis agents. Rhizoma Drynariae displays a promising anti-osteoporosis effect, while the effective component and mechanism remain unclear. Here, we revealed the therapeutic potential of Rhizoma Drynariae-derived nanovesicles (RDNVs) for postmenopausal osteoporosis and demonstrated that RDNVs potentiated osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) by targeting estrogen receptor-alpha (ERα). RDNVs, a natural product isolated from fresh Rhizoma Drynariae root juice by differential ultracentrifugation, exhibited potent bone tissue-targeting activity and anti-osteoporosis efficacy in an ovariectomized mouse model. RDNVs, effectively internalized by hBMSCs, enhanced proliferation and ERα expression levels of hBMSC, and promoted osteogenic differentiation and bone formation. Mechanistically, via the ERα signaling pathway, RDNVs facilitated mRNA and protein expression of bone morphogenetic protein 2 and runt-related transcription factor 2 in hBMSCs, which are involved in regulating osteogenic differentiation. Further analysis revealed that naringin, existing in RDNVs, was the active component targeting ERα in the osteogenic effect. Taken together, our study identified that naringin in RDNVs displays exciting bone tissue-targeting activity to reverse osteoporosis by promoting hBMSCs proliferation and osteogenic differentiation through estrogen-like effects.

Octoploids Show Enhanced Salt Tolerance through Chromosome Doubling in Switchgrass (Panicum virgatum L.).

Polyploid plants often exhibit enhanced stress tolerance. Switchgrass is a perennial rhizomatous bunchgrass that is considered ideal for cultivation in marginal lands, including sites with saline soil. In this study, we investigated the physiological responses and transcriptome changes in the octoploid and tetraploid of switchgrass (Panicum virgatum L. 'Alamo') under salt stress. We found that autoploid 8× switchgrass had enhanced salt tolerance compared with the amphidiploid 4× precursor, as indicated by physiological and phenotypic traits. Octoploids had increased salt tolerance by significant changes to the osmoregulatory and antioxidant systems. The salt-treated 8× Alamo plants showed greater potassium (K+) accumulation and an increase in the K+/Na+ ratio. Root transcriptome analysis for octoploid and tetraploid plants with or without salt stress revealed that 302 upregulated and 546 downregulated differentially expressed genes were enriched in genes involved in plant hormone signal transduction pathways and were specifically associated with the auxin, cytokinin, abscisic acid, and ethylene pathways. Weighted gene co-expression network analysis (WGCNA) detected four significant salt stress-related modules. This study explored the changes in the osmoregulatory system, inorganic ions, antioxidant enzyme system, and the root transcriptome in response to salt stress in 8× and 4× Alamo switchgrass. The results enhance knowledge of the salt tolerance of artificially induced homologous polyploid plants and provide experimental and sequencing data to aid research on the short-term adaptability and breeding of salt-tolerant biofuel plants.

Polystyrene nanoplastics and cadmium co-exposure aggravated cardiomyocyte damage in mice by regulating PANoptosis pathway.

Micro/nanoplastics (M/NPs) are the novel contaminants ubiquitous in the environment. Cadmium (Cd), a kind of heavy metal pollutant widely distributed, could potentially co-exist with PS-NPs in the environment. However, their combined effects on cardiomyocyte and its molecular mechanism in mammals remained ambiguous. Here, we examined whether PANoptosis, an emerging and complicated kind of programmed cell death, was involved in PS-NPs and Cd co-exposure-elicited cardiac injury. In this study, 60 male mice were orally subjected to environmentally relevant concentrations of PS-NPs (1 mg/kg) and/or CdCl2 (1.5 mg/kg) for 35 days. As we speculated, PS-NPs and Cd co-exposure affected the expression of pyroptosis(Caspase-1, Cleaved-Caspase-1, GSDMD, N-GSDMD, AIM2, Pyrin, NLRP3, IL-18, IL-1ß)-, apoptosis(Caspase-3, Cleaved-Caspase-3, Caspase-8, Cleaved-Caspase-8, Caspase-7, BAX)- and necroptosis (t-RIPK3, p-RIPK3, t-RIPK1, p-RIPK1, t-MLKL, p-MLKL, ZBP1)-related genes and protein, resulting in growth restriction and damaged myocardial microstructure in mice. Notably, the combined effects on Cd and PS-NPs even predominantly aggravated the toxic damage. Intriguingly, we fortuitously discovered PS-NPs and/or Cd exposure facilitated linear ubiquitination of certain proteins in mice myocardium. In summation, this study shed light toward the effects of Cd and PS-NPs on cardiotoxicity, advanced the understanding of myocardial PANoptosis and provided a scientific foundation for further exploration of the combined toxicological effects of PS-NPs and heavy metals.

Upregulation of serine metabolism enzyme PSAT1 predicts poor prognosis and promotes proliferation, metastasis and drug resistance of clear cell renal cell carcinoma.

Serine metabolic reprogramming is known to be associated with oncogenesis and tumor development. The key metabolic enzyme PSAT1 has been identified as a potential prognostic marker for various cancers, but its role in ccRCC remains unkown. In this study, we investigated expression of PSAT1 in ccRCC using the TCGA database and clinical specimens. Our results showed that PSAT1 exhibited lower expression in tumor tissue compared to adjacent normal tissue, but its expression level increased with advancing stages and grades of ccRCC. Patients with elevated expression level of PSAT1 exhibited an unfavorable prognosis. Functional experiments have substantiated that the depletion of PSAT1 shows an effective activity in inhibiting the proliferation, migration and invasion of ccRCC cells, concurrently promoting apoptosis. RNA sequencing analysis has revealed that the attenuation of PSAT1 can diminish tumor resistance to therapeutic drugs. Furthermore, the xenograft model has indicated that the inhibition of PSAT1 can obviously impact the tumorigenic potential of ccRCC and mitigate lung metastasis. Notably, pharmacological targeting PSAT1 by Aminooxyacetic Acid (AOA) or knockdown of PSAT1 increased the susceptibility of sunitinib-resistant cells. Inhibition of PSAT1 increased the sensitivity of drug-resistant tumors to sunitinib in vivo. Collectively, our investigation identifies PSAT1 as an independent prognostic biomarker for advanced ccRCC patients and as a prospective therapeutic target.

EIF4A3-mediated biogenesis of circSTX6 promotes bladder cancer metastasis and cisplatin resistance.

BACKGROUND: Cisplatin (CDDP)-based chemotherapy is a standard first-line treatment for metastatic bladder cancer (BCa) patients, and chemoresistance remains a major challenge in clinical practice. Circular RNAs (circRNAs) have emerged as essential regulators in carcinogenesis and cancer progression. However, the role of circRNAs in mediating CDDP chemosensitivity has yet to be well elucidated in BCa. METHODS: CircSTX6 (hsa_circ_0007905) was identified by mining the public circRNA datasets and verified by Sanger sequencing, agarose gel electrophoresis, RNase R treatment and qRT-PCR assays. Then, function experiments were performed to evaluate the effects of circSTX6 on BCa metastasis. Luciferase reporter assay, RNA pull-down, RNA immunoprecipitation (RIP), RNA stability assay, Fluorescence in situ hybridization (FISH) and Immunofluorescence (IF) were conducted to evaluate the interaction among circSTX6, miR-515-3p, PABPC1 and SUZ12. Animal experiments were performed to explore the function of circSTX6 in tumor metastasis and CDDP sensitivity. RESULTS: We identified that circSTX6 was significantly upregulated in clinical samples and cells of BCa. Functionally, circSTX6 promoted cell migration and invasion both in vitro and in vivo. Mechanistically, circSTX6 could act as a miR-515-3p sponge and abolish its effect on SUZ12. Moreover, circSTX6 was confirmed to increase the stability of SUZ12 mRNA by interacting with a mRNA stabilizer PABPC1 and subsequently promote the expression of SUZ12. Importantly, silencing of circSTX6 improved the chemosensitivity of CDDP-resistant bladder cancer cells to CDDP. Furthermore, in vivo analysis supported that knockdown of circSTX6 attenuated CDDP resistance in BCa tumors. CONCLUSION: These studies demonstrate that circSTX6 plays a pivotal role in BCa metastasis and chemoresistance, and has potential to serve as a therapeutic target for treatment of BCa.

An enzyme-based system for extraction of small extracellular vesicles from plants.

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.

Prognostic value and model construction of preoperative inflammatory markers in patients with metastatic renal cell carcinoma.

BACKGROUND: Inflammation is considered to be one of the driving factors of cancer, and chronic inflammation plays a crucial role in tumor growth and metastasis. The aim of this study was to examine the predictive value of preoperative inflammatory biomarkers for overall survival (OS) in patients with metastatic renal cell carcinoma (mRCC), including preoperative neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and aspartate aminotransferase-to-lymphocyte ratio (ALR), a novel inflammatory biomarker. METHOD: This study included 198 patients with mRCC from a single center from 2006 to 2022. The optimal cut-off levels for the three biomarkers were derived using the receiver operating characteristic curve (ROC). Cox univariate and multivariate analyses were used to assess independent prognostic inflammatory biomarkers. Finally, independent prognostic inflammatory biomarkers were incorporated into the prognostic model to establish a nomogram to predict the postoperative survival of patients with mRCC. RESULT: The area under the ROC curve for NLR, LMR, and ALR, respectively, is 0.71 (CI: 0.635-0.784), 0.68 (CI: 0.604-0.755), and 0.75 (CI: 0.680-0.819). The optimal LMR, NLR, and ALR cut-off levels as evaluated by the ROC curve were 3.836, 3.106, and 68.056, respectively. Patients with NLR and ALR higher than the cut-off level and LMR lower than the cut-off level had a significant relationship with OS. Multivariate analysis revealed that tumor necrosis, lower LMR, and higher ALR were independent risk factors for OS. In addition, a nomogram that includes independent prognostic inflammatory biomarkers can accurately predict the OS in patients with mRCC. CONCLUSION: ALR and LMR are independent risk factors for the prognosis of individuals with mRCC. By monitoring ALR and LMR postoperatively, the prognosis of patients with mRCC can be better evaluated.

Co-exposure to environmentally relevant concentrations of cadmium and polystyrene nanoplastics induced oxidative stress, ferroptosis and excessive mitophagy in mice kidney.

Nanoplastics (NPs) are defined as a group of emerging pollutants. However, the adverse effect of NPs and/or heavy metals on mammals is still largely unclear. Therefore, we performed a 35-day chronic toxicity experiment with mice to observe the impacts of exposure to Cadmium (Cd) and/or polystyrene nanoplastics (PSNPs). This study revealed that combined exposure to Cd and PSNPs added to the mice's growth toxicity and kidney damage. Moreover, Cd and PSNPs co-exposure obviously increased the MDA level and expressions of 4-HNE and 8-OHDG while decreasing the activity of antioxidase in kidneys via inhibiting the Nrf2 pathway and its downstream genes and proteins expression. More importantly, the results suggested for the first time that Cd and PSNPs co-exposure synergistically increased iron concentration in kidneys, and induced ferroptosis through regulating expression levels of SLC7A11, GPX4, PTGS2, HMGB1, FTH1 and FTL. Simultaneously, Cd and PSNPs co-exposure further increased the expression levels of Pink, Parkin, ATG5, Beclin1, and LC3 while significantly reducing the P62 expression level. In brief, this study found that combined exposure to Cd and PSNPs synergistically caused oxidative stress, ferroptosis and excessive mitophagy ultimately aggravating kidney damage in mice, which provided new insight into the combined toxic effect between heavy metals and PSNPs on mammals.

SPI1 is a prognostic biomarker of immune infiltration and immunotherapy efficacy in clear cell renal cell carcinoma.

BACKGROUND: Spi-1 proto-oncogene (SPI1), which encodes an ETS-domain transcription factor, can activate gene expression in myeloid and lymphoid lineages. The role of SPI1 in the tumor immune microenvironment in clear cell renal cell carcinoma (ccRCC) remains unknown. In this study, we investigated the possible role of SPI1 in ccRCC using an independent cohort and a comprehensive bioinformatics analysis. MATERIALS AND METHODS: Quantitative real-time PCR, western blot and immunohistochemistry assays were used to compare the SPI1 expression levels between ccRCC tissues and normal tissues, analyze the relationships between SPI1 and CD68, CD8, CD4 expression levels, and explore the link between SPI1 and the efficacy of immunotherapy in our cohort. Tumor Immune Estimation Resource, UALCAN, cBioPortal, TISIDB database, and LinkedOmics database were used in our study. RESULTS: SPI1 expression level was higher in ccRCC bulk tissues than in normal bulk tissues. SPI1 was an independent prognostic factor for poor overall survival and progression-free survival in patients with ccRCC. SPI1 expression was strongly related to the infiltration of immune cells and immune-related molecules. SPI1 was more highly expressed in tumor-infiltrating immune cells rather than in cancer cells. Non-responders to immunotherapy against ccRCC were more likely to express higher SPI1 levels than responders. Genes co-expressed with SPI1 primarily correlated with immune-related pathways. CONCLUSIONS: SPI1 expression in tumor bulk tissues is associated with disease progression and poor prognosis, as well as high expression levels of immune markers and infiltration of immune cells. SPI1 can be used as a prognostic biomarker to monitor and evaluate immunotherapy efficacy.

FDX1 expression predicts favourable prognosis in clear cell renal cell carcinoma identified by bioinformatics and tissue microarray analysis.

Ferredoxin 1 (FDX1), an iron-sulphur protein, is responsible for electron transfer in a range of metabolic redox reactions. Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer characterised by metabolic reprogramming, and FDX1 is a critical regulator of cuproptosis. However, the expression profile and prognostic value of FDX1 associated with clinicopathological features in ccRCC remain largely unelucidated. In this study, we integrated a series of public bioinformatic analysis to explore the mRNA and protein profiles of FDX1 across human cancers and cell lines and validated its expression and prognostic value, especially in ccRCC. In this study, FDX1 mRNA and protein expression were aberrantly downregulated and associated with ccRCC grade, stage, and nodal metastasis, whereas in adjacent non-tumour kidney tissue, it was abundantly expressed and cytoplasmically localised in renal tubular epithelial cells. Multivariate analysis indicated that low FDX1 expression contributed to unfavourable overall and disease-free survival. The functional enrichment of FDX1 co-expressed genes in ccRCC involved mainly mitochondrial dysfunction in various metabolic processes and biological oxidation, besides iron-sulphur cluster biogenesis. Furthermore, FDX1 modulates immunological infiltration to affect prognosis. Thus, FDX1 downregulation is mechanistically because of ccRCC tumourigenesis and is a promising prognostic biomarker to stratify patients with ccRCC.

Papillary renal neoplasm with reverse polarity-a comparative study with CCPRCC, OPRCC, and PRCC1.

Papillary renal neoplasm with reverse polarity (PRNRP) is a newly defined entity with distinct histomorphology and recurrent KRAS mutation. In this study, we aimed to identify and analyze the clinicopathological, immunohistochemical (IHC), and molecular features of PRNRP in our center and to evaluate its differential diagnosis with other tumors with which it is easily confused: clear cell papillary renal cell carcinoma (CCPRCC), oncocytic papillary renal cell carcinoma (OPRCC), and papillary renal cell carcinoma type 1 (PRCC1). Nephrectomy specimens of PRNRP (n = 15), CCPRCC (n = 11), and OPRCC (n = 12) were retrieved from our pathology archives. We also selected typical cases of PRCC1 (n = 15) as a control group. PRNRP accounted for 3.05% (15/492) of all PRCC cases at our center. The median follow-up period was 41.3 months. All PRNRP cases were pT1N0M0, and only one involved recurrence (1 year after surgery). IHC analysis showed diffuse staining of CK7, EMA, and GATA3 but weak or negative staining of CD10, CD117, p504s, and vimentin in the PRNRP samples and distinctive IHC features in the other three tumor types. KRAS mutation was detected in 4/10 PRNRP cases. Among the 40 most commonly mutated genes identified, 5 (BCLAF1, PDE4DIP, NCOR1, PARP4, and PABPC1) have actionable alterations. Our study supports the suggestion that PRNRP is an entity distinct from CCPRCC, OPRCC, and PRCC1.

Identification and validation of TCONS_00093333 for regulating fertility conversion of thermo-sensitive cytoplasmic male-sterility wheat with Aegilops kotschyi cytoplasm.

An increasing number of studies have shown that long non-coding RNAs (lncRNAs) play an important role in regulating plant fertility, however, they are less studied in wheat. Here, we analyzed lncRNA sequencing data and showed that the fixation carbon sequestration pathway was closely associated with pollen development and fertility conversion in KTM3315A, and eight differentially expressed genes under different fertility conditions were significantly regulated by TCONS_00093333 (designed as TaHTMAR) and transcription factors TaBBX25 and TaOBF1. Among them, TaBBX25 and TaOBF1 were located in the nucleus and expressed in the early stage of fertile anther development. Gene silencing experiments of TaHTMAR showed that TaHTMAR positively regulated the expression of TaBBX25 and TaOBF1 under fertile conditions, thereby reducing male fertility of KTM3315A. This study confirms the effective roles of TaHTMAR, TaBBX25, and TaOBF1 in the regulation of male fertility in wheat and provides a valuable molecular basis for hybrid wheat breeding.

The gene TaPG encoding a polygalacturonase is critical for pollen development and male fertility in thermo-sensitive cytoplasmic male-sterility wheat.

Thermo-sensitive cytoplasmic male sterility is of great significance to heterosis and hybrid seed production in wheat. Consequently, it is worthwhile to research the genes associated with male sterility. Although polygalacturonases (PGs) have been studied to play a crucial role in male reproduction of many plants, their functions in the reproductive development of wheat remain unclear. Here, TaPG (TraesCS7A02G404900) encoding a polygalacturonase was isolated from the anthers of KTM3315A, a wheat thermo-sensitive cytoplasmic male sterile with Aegilops kotschyi cytoplasm. Expression pattern analyses showed that TaPG was strongly expressed in fertile anthers and its protein was localized in the cell wall. Further verification via barley stripe mosaic virus revealed that the silencing of TaPG exhibited abnormal anthers, premature degradation of tapetum, pollen abortion, and defective pollen wall formation, resulting in the declination of fertility. Conclusively, our research suggested that TaPG contributed to the pollen development and male fertility, which will provide a novel insight into the fertility conversion of thermo-sensitive cytoplasmic male sterility in wheat.

Genome-wide analysis of genomic imprinting in the endosperm and allelic variation in flax.

Genomic imprinting is an epigenetic phenomenon that causes biased expression of maternally and paternally inherited alleles. In flowering plants, genomic imprinting predominantly occurs in the triploid endosperm and plays a vital role in seed development. In this study, we identified 248 candidate imprinted genes including 114 maternally expressed imprinted genes (MEGs) and 134 paternally expressed imprinted genes (PEGs) in flax (Linum usitatissimum L.) endosperm using deep RNA sequencing. These imprinted genes were neither clustered in specific chromosomal regions nor well conserved among flax and other plant species. MEGs tended to be expressed specifically in the endosperm, whereas the expression of PEGs was not tissue-specific. Imprinted single nucleotide polymorphisms differentiated 200 flax cultivars into the oil flax, oil-fiber dual purpose flax and fiber flax subgroups, suggesting that genomic imprinting contributed to intraspecific variation in flax. The nucleotide diversity of imprinted genes in the oil flax subgroup was significantly higher than that in the fiber flax subgroup, indicating that some imprinted genes underwent positive selection during flax domestication from oil flax to fiber flax. Moreover, imprinted genes that underwent positive selection were related to flax functions. Thirteen imprinted genes related to flax seed size and weight were identified using a candidate gene-based association study. Therefore, our study provides information for further exploration of the function and genomic variation of imprinted genes in the flax population.

Factors Associated With Healthcare Workers' Insomnia Symptoms and Fatigue in the Fight Against COVID-19, and the Role of Organizational Support.

Background: Healthcare workers (HCWs) have been exposed to increased risks of insomnia and fatigue during the COVID-19 pandemic. In this study, we identify important risk factors associated with insomnia symptoms and fatigue among HCWs, and evaluate the effect of organizational support on insomnia and fatigue symptoms. Methods: This is an online cross-sectional survey of HCWs in China administered during the COVID-19 epidemic (from February 27, 2020 to March 12, 2020). We employed the AIS-8 scale for insomnia screening, and a self-reported ten-point scale to evaluate subjects' degrees of fatigue. We also designed a four-point scale to assess the degree of social support provided on an organizational level. Additionally, we conducted logistic regression analysis to identify risk factors. Results: This study included a total of 3,557 participants, 41% of which consisted of non-frontline HCWs and 59% of which was frontline HCWs. Of the non-frontline HCWs, 49% reported insomnia symptoms, and 53.8% reported a moderate to high degree of fatigue. Meanwhile, among the frontline HCWs, the percentages for insomnia and moderate to high fatigue were 63.4% and 72.2%, respectively. Additionally, frontline HCWs and HCWs employed at Centers for Disease Control and Prevention (CDCs) had elevated risks of insomnia and fatigue. However, with increased organizational support, insomnia symptoms decreased among frontline HCWs. Also, organizational support mitigated the positive correlation between daily working hours and degree of fatigue among HCWs. Conclusion: Frontline HCWs and staff in Chinese CDCs have been at a high risk of insomnia symptoms and fatigue during the fight against COVID-19. This study provides evidence for the positive effects of organizational support in relation to insomnia and fatigue among HCWs. This sheds light on government responses to the COVID-19 epidemic for other countries.

Identification and verification of genes related to pollen development and male sterility induced by high temperature in the thermo-sensitive genic male sterile wheat line.

MAIN CONCLUSION: Bioinformatic analysis identified the function of genes regulating wheat fertility. Barley stripe mosaic virus-induced gene silencing verified that the genes TaMut11 and TaSF3 are involved in pollen development and related to fertility conversion. Environment-sensitive genic male sterility is of vital importance to hybrid vigor in crop production and breeding. Therefore, it is meaningful to study the function of the genes related to pollen development and male sterility, which is still not fully understand currently. In this study, YanZhan 4110S, a new thermo-sensitive genic male sterility wheat line, and its near-isogenic line YanZhan 4110 were analyzed. Through comparative transcriptome basic bioinformatics and weighted gene co-expression network to further identify some hub genes, the genes TaMut11 and TaSF3 associated with pollen development and male sterility induced by high-temperature were identified in YanZhan 4110S. Further verification through barley stripe mosaic virus-induced gene silencing elucidated that the silencing of TaMut11 and TaSF3 caused pollen abortion, finally resulting in the declination of fertility. These findings provided data on the abortive mechanism in environment-sensitive genic male sterility wheat.

Identification and validation of genetic locus Rfk1 for wheat fertility restoration in the presence of Aegilops kotschyi cytoplasm.

KEY MESSAGE: Major fertility restorer locus for Aegilops kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm 1BS. Most likely candidate gene is TraesCS1B02G197400LC, which is predicted to encode a pectinesterase/pectinesterase inhibitor. Cytoplasmic male sterility (CMS) is widely used for heterosis and hybrid seed production in wheat. Genes related to male fertility restoration in the presence of Aegilops kotschyi cytoplasm have been reported, but the fertility restoration-associated gene loci have not been investigated systematically. In this study, a BC1F1 population derived from a backcross between KTP116A, its maintainer line TP116B, and its restorer line LK783 was employed to map fertility restoration by bulked segregant RNA-Seq (BSR-Seq). A major fertility allele restorer locus for Ae. kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm 1BS, and it was contributed by LK783. Morphological and cytological studies showed that male fertility restoration occurred mainly after the late uninucleate stage. Based on simple sequence repeat and single-nucleotide polymorphism genotyping, the gene locus was located between Xnwafu_6 and Xbarc137 on chromosome arm 1BS. To further isolate the specific region, six Kompetitive allele-specific polymerase chain reaction markers derived from BSR-Seq were developed to delimit Rfk1 within physical intervals of 26.0 Mb. After searching for differentially expressed genes within the candidate interval in the anthers and sequencing analysis, TraesCS1B02G197400LC was identified as a candidate gene for Rfk1 and it was predicted to encode a pectinesterase/pectinesterase inhibitor. Expression analysis also confirmed that it was specifically expressed in the anthers, and its expression level was higher in fertile lines compared with sterile lines. Thus, TraesCS1B02G197400LC was identified as the most likely candidate gene for Rfk1, thereby providing insights into the fertility restoration mechanism for K-type CMS in wheat.

Comprehensive analysis of polygalacturonase gene family highlights candidate genes related to pollen development and male fertility in wheat (Triticum aestivum L.).

MAIN CONCLUSION: Four polygalacturonase gene family members were highlighted that contribute to elucidate the roles of polygalacturonase during the fertility conversion process in male-sterile wheat. Polygalacturonase (PG) belongs to a large family of hydrolases with important functions in cell separation during plant growth and development via the degradation of pectin. Specific expressed PGs in anthers may be significant for male sterility research and hybrid wheat breeding, but they have not been characterized in wheat (Triticum aestivum L.). In this study, we systematically studied the PG gene family using the latest published wheat reference genomic information. In total, 113 wheat PG genes were identified, which could be classified into six categories A-F according to their structure characteristics and phylogenetic comparisons with Arabidopsis and rice. Polyploidy and segmental duplications in wheat were proved to be mainly responsible for the expansion of the wheat PG gene family. RNA-seq showed that TaPGs have specific temporal and spatial expression characteristics, in which 12 TaPGs with spike-specific expression patterns were detected by qRT-PCR in different fertility anthers of KTM3315A, a thermo-sensitive cytoplasmic male-sterile wheat. Four of them specific upregulated (TaPG09, TaPG95, and TaPG93) or downregulated (TaPG87) at trinucleate stage of fertile anthers, and further aligning with the homologous in Arabidopsis revealed that they may undertake functions such as anther dehiscence, separation of pollen, pollen development, and pollen tube elongation, thereby inducing male fertility conversion in KTM3315A. These findings facilitate function investigations of the wheat PG gene family and provide new insights into the fertility conversion mechanism in male-sterile wheat.

Genome-Wide Investigation of Heat Shock Transcription Factor Family in Wheat (Triticum aestivum L.) and Possible Roles in Anther Development.

Heat shock transcription factors (HSFs) play crucial roles in resisting heat stress and regulating plant development. Recently, HSFs have been shown to play roles in anther development. Thus, investigating the HSF family members and identifying their protective roles in anthers are essential for the further development of male sterile wheat breeding. In the present study, 61 wheat HSF genes (TaHsfs) were identified in the whole wheat genome and they are unequally distributed on 21 chromosomes. According to gene structure and phylogenetic analyses, the 61 TaHsfs were classified into three categories and 12 subclasses. Genome-wide duplication was identified as the main source of the expansion of the wheat HSF gene family based on 14 pairs of homeologous triplets, whereas only a very small number of TaHsfs were derived by segmental duplication and tandem duplication. Heat shock protein 90 (HSP90), HSP70, and another class of chaperone protein called htpG were identified as proteins that interact with wheat HSFs. RNA-seq analysis indicated that TaHsfs have obvious period- and tissue-specific expression patterns, and the TaHsfs in classes A and B respond to heat shock, whereas the C class TaHsfs are involved in drought regulation. qRT-PCR identified three TaHsfA2bs with differential expression in sterile and fertile anthers, and they may be candidate genes involved in anther development. This comprehensive analysis provides novel insights into TaHsfs, and it will be useful for understanding the mechanism of plant fertility conversion.

Blocked synthesis of sporopollenin and jasmonic acid leads to pollen wall defects and anther indehiscence in genic male sterile wheat line 4110S at high temperatures.

Environment-sensitive genic male sterility is a valid tool for hybrid production and hybrid breeding, but there are no previous reports of the molecular mechanism of fertility conversion. In this study, RNA-seq, phenotypic and cytological observations, and physiological indexes were applied to analyze thermo-sensitive genic male sterility line 4110S under different temperature conditions to explore the fertility transformation mechanism. In total, 3420 differentially expressed genes (DEGs) were identified comprising 2331 upregulated genes and 1089 downregulated genes. The DEGs were apparently distributed among 54 Gene Ontology functional groups. The phenylpropanoid, long-chain fatty acid, and jasmonic acid (JA) biosynthesis pathways were related to male sterility, where their downregulation blocked the synthesis of sporopollenin and JA. Phenotypic and cytological analyses showed that pollen wall defects and anther indehiscence at high temperatures induced sterility. Moreover, enzyme-linked immunosorbent assay results indicated that the abundance of JA was lower in 4110S under restrictive conditions (high temperature) than permissive conditions (low temperature). A possible regulated network of pathways associated with male sterility was suggested. These results provided insights into the molecular mechanism of fertility conversion in the thermosensitive male sterility system.