Multiplex-genome-editing based rapid directional improvement of complex traits in rice.

Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.

Prognostic implications of systemic immune-inflammation index in myocardial infarction patients with and without diabetes: insights from the NOAFCAMI-SH registry.

BACKGROUND: It is well-known that systemic inflammation plays a crucial role in the pathogenesis and prognosis of acute myocardial infarction (AMI). The systemic immune-inflammation index (SII, platelet × neutrophil/lymphocyte ratio) is a novel index that is used for the characterization of the severity of systemic inflammation. Recent studies have identified the high SII level as an independent predictor of poor outcomes in patients with AMI. We aimed to investigate the prognostic implications of SII in AMI patients with and without diabetes mellitus (DM). METHODS: We included 2111 patients with AMI from February 2014 to March 2018. Multivariable Cox regression analyses were performed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause death and cardiovascular (CV) death. Multiple imputation was used for missing covariates. RESULTS: Of 2111 patients (mean age: 65.2 ± 12.2 years, 77.5% were males) analyzed, 789 (37.4%) had DM. Generalized additive model analyses showed that as the SII increased, the C-reactive protein and peak TnT elevated while the LVEF declined, and these associations were similar in patients with and without DM. During a median of 2.5 years of follow-up, 210 all-cause deaths and 154 CV deaths occurred. When treating the SII as a continuous variable, a higher log-transformed SII was significantly associated with increased all-cause mortality (HR: 1.57, 95%CI: 1.02-2.43) and CV mortality (HR: 1.85, 95%CI 1.12-3.05), and such an association was also significant in the diabetics (HRs and 95%CIs for all-cause death and CV death were 2.90 [1.40-6.01] and 3.28 [1.43-7.57], respectively) while not significant in the nondiabetics (Pinteraction for all-cause death and CV death were 0.019 and 0.049, respectively). Additionally, compared to patients with the lowest tertiles of SII, those with the highest tertiles of SII possessed significantly higher all-cause mortality (HR: 1.82, 95%CI 1.19-2.79) and CV mortality (HR: 1.82, 95%CI 1.19-2.79) after multivariable adjustment, and this relationship remained pronounced in the diabetics (HRs and 95%CIs for all-cause death and CV death were 2.00 [1.13-3.55] and 2.09 [1.10-3.98], respectively) but was not observed in the nondiabetics (HRs and 95%CIs for all-cause death and CV death were 1.21 [0.75-1.97] and 1.60 [0.89-2.90], respectively). Our restricted cubic splines analyses indicated a pronounced linear association between SII and mortality only in diabetics. CONCLUSIONS: In AMI patients with DM, high SII is an independent predictor of poor survival and may be helpful for patient's risk stratification.

Association of stress hyperglycemia ratio with in-hospital new-onset atrial fibrillation and long-term outcomes in patients with acute myocardial infarction.

AIMS: To investigate the predictive value and prognostic impact of stress hyperglycemia ratio (SHR) for new-onset atrial fibrillation (NOAF) complicating acute myocardial infarction (AMI). MATERIALS AND METHODS: This retrospective study included 2145 AMI patients without AF history between February 2014 and March 2018. SHR was calculated using fasting blood glucose (mmol/L)/[1.59*HbA1c (%)-2.59]. The association between SHR and post-MI NOAF was assessed with multivariable logistic regression analyses. The primary outcome was a composite of cardiac death, heart failure hospitalisation, recurrent MI, and ischaemic stroke (MACE). Cox regression-adjusted hazard ratios with 95% confidence intervals (CI) were estimated for MACE. RESULTS: A total of 245 (11.4%) patients developed NOAF. In the multivariable logistic regression analyses, SHR (each 10% increase) was significantly associated with increased risks of NOAF in the whole population (OR: 1.05, 95% CI: 1.01-1.10), particularly in non-diabetic individuals (OR:1.08, 95% CI: 1.01-1.17). During a median follow-up of 2.7 years, 370 (18.5%) MACEs were recorded. The optimal cut-off value of SHR for MACE prediction was 1.119. Patients with both high SHR (≥1.119) and NOAF possessed the highest risk of MACE compared to those with neither high SHR nor NOAF after multivariable adjustment (HR: 2.18, 95% CI: 1.39-3.42), especially for diabetics (HR: 2.63, 95% CI: 1.41-4.91). Similar findings were observed using competing-risk models. CONCLUSIONS: SHR is an independent predictor of post-MI NOAF in non-diabetic individuals. Diabetic patients with both high SHR and NOAF had the highest risk of MACE, suggesting that therapies targeting SHR may be considered in these patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03533543.

Prognostic implications of the 4S-AF scheme to characterize new-onset atrial fibrillation after myocardial infarction.

AIMS: The 4S-AF scheme (Stroke risk [St], Symptom severity [Sy], Severity of atrial fibrillation burden [Sb], Substrate [Su]) is a novel approach for the holistic characterization of AF. We aimed to investigate the prognostic implications of the 4S-AF scheme score in acute myocardial infarction (AMI) patients with new-onset atrial fibrillation (NOAF). METHODS: We included 262 patients with post-MI NOAF who had complete data for the 4S-AF scheme evaluation between February 2014 and March 2018. The 4S-AF scheme score was calculated as a sum of each domain with a maximum of 9. The primary outcome was all-cause death. RESULTS: Of 262 patients (66.0% males, mean age 74.5 ± 10.4 years) were analyzed. The mean 4S-AF scheme score was 5.0 ± 1.6. There were 62 (27.3%) all-cause deaths within a median follow-up of 2.6 years. According to multivariable Cox regression models, each 1-point increase in the 4S-AF scheme score was significantly associated with 39% increased all-cause mortality (HR: 1.39, 95% CI: 1.16-1.67, P<0.001), which was mainly driven by the Sb (HR: 1.43, 95%CI: 1.05-1.95, P = 0.025) and Su (HR: 1.53, 95%CI: 1.17-2.02, P = 0.002) domains. Adding the 4S-AF scheme score on top of the Global Registry of Acute Coronary Events score could significantly improve its discriminative capability (C-index from 0.713 to 0.761, P = 0.039) and reclassification performance (continuous net reclassification improvement: 41.0% [95%CI: 12.5-69.6]; integrated discrimination improvement: 5.1% [95%CI: 2.2-8.1]) for all-cause mortality. CONCLUSIONS: Characterization of NOAF using the 4S-AF scheme aids in the risk stratification of AMI patients with NOAF.

Overexpression of a GIPC glycosyltransferase gene, OsGMT1, suppresses plant immunity and delays heading time in rice.

Glycosylinositol phosphorylceramides (GIPCs) are the major sphingolipids in the plant plasma membrane. In Arabidopsis, mutations of genes involved in the synthesis of GIPCs affect many physiological aspects of plants, including growth, pollen fertility, defense, and stress signaling. Loss of function of the GIPC MANNOSYL-TRANSFERASE1 (AtGMT1) results in GIPC misglycosylation and induces plant immune responses accompanied by a severely dwarfed phenotype, thus indicating that GIPCs play important roles in plant immunity. Here, we investigated the enzymatic activity and phenotypes of transgenic lines of OsGMT1, the ortholog of AtGMT1. Sphingolipidomic analysis indicated that OsGMT1 retained the enzymatic activity of GIPC hexose (Hex) glycosylation, but the knockout lines did not accumulate H2O2. In contrast, the OsGMT1 overexpression lines showed significant down-regulation of several defense-associated or cell wall synthesis-associated genes, and enhanced sensitivity to rice blast. Furthermore, we first demonstrated the sensitivity of rice cells to MoNLP1 protein through calcein AM release assays using rice protoplasts, thus legitimizing the presence of MoNLPs in rice blast fungus. In addition, yeast two-hybrid screens using OsGMT1 as bait revealed that OsGMT1 may regulate heading time through the OsHAP5C signaling pathway. Together, our findings suggested clear physiological functional differentiation of GMT1 orthologs between rice and Arabidopsis.

TRIM21 deficiency protects against atrial inflammation and remodeling post myocardial infarction by attenuating oxidative stress.

Atrial remodeling is a major contributor to the onset of atrial fibrillation (AF) after myocardial infarction (MI). Tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin protein ligase, is associated with pathological cardiac remodeling and dysfunction. However, the role of TRIM21 in postmyocardial infarction atrial remodeling and subsequent AF remains unclear. This study investigated the role of TRIM21 in post myocardial infarction atrial remodeling using TRIM21 knockout mice and explored the underlying mechanisms by overexpressing TRIM21 in HL-1 atrial myocytes using a lentiviral vector. The expression of TRIM21 in the left atrium of the mouse MI model was significantly elevated. TRIM21 deficiency alleviated MI-induced atrial oxidative damage, Cx43 downregulation, atrial fibrosis and enlargement, and abnormalities in electrocardiogram parameters (prolongation of the P-wave and PR interval). TRIM21 overexpression in atrial myocyte HL-1 cells further enhanced oxidative damage and Cx43 downregulation, whereas these effects were reversed by the reactive oxygen species scavenger N-acetylcysteine. The findings suggest that TRIM21 likely induces Nox2 expression mechanistically by activating the NF-κB pathway, which in turn leads to myocardial oxidative damage, inflammation, and atrial remodeling.

IPA1 improves drought tolerance by activating SNAC1 in rice.

Drought is a major abiotic stress to rice (Oryza sativa) during growth. Ideal Plant Architecture (IPA1), the first cloned gene controlling the ideal plant type in rice, has been reported to function in both ideal rice plant architecture and biotic resistance. Here, we report that the IPA1/OsSPL14, encoding a transcriptional factor, positively regulates drought tolerance in rice. The IPA1 is constitutively expressed and regulated by H2O2, abscisic acid, NaCl and polyethylene glycol 6000 treatments in rice. Furthermore, the IPA1-knockout plants showed much greater accumulation of H2O2 as measured by 3,3'-diaminobenzidine staining in leaves compared with WT plants. Yeast one-hybrid, dual-luciferase and electrophoretic mobility shift assays indicated that the IPA1 directly activates the promoter of SNAC1. Expression of SNAC1 is significantly down-regulated in IPA1 knockout plants. Further investigation indicated that the IPA1 plays a positive role in drought-stress tolerance by inducing reactive oxygen species scavenging in rice. Together, these findings indicated that the IPA1 played important roles in drought tolerance by regulating SNAC1, thus activating the antioxidant system in rice.

Analysis of co-expression and gene regulatory networks associated with sterile lemma development in rice.

BACKGROUND: The sterile lemma is a unique organ of the rice (Oryza sativa L.) spikelet. However, the characteristics and origin of the rice sterile lemma have not been determined unequivocally, so it is important to elucidate the molecular mechanism of the development of the sterile lemma. RESULTS: In the paper, we outline the regulatory mechanism of sterile lemma development by LONG STERILE LEMMA1 (G1), which has been identified as the gene controlling sterile lemma development. Based on the comprehensive analyses of transcriptome dynamics during sterile lemma development with G1 alleles between wild-type (WT) and mutant (MT) in rice, we obtained co-expression data and regulatory networks related to sterile lemma development. Co-transfection assays of rice protoplasts confirmed that G1 affects the expression of various phytohormone-related genes by regulating a number of critical transcription factors, such as OsLBD37 and OSH1. The hormone levels in sterile lemmas from WT and MT of rice supports the hypotheses that lower auxin, lower gibberellin, and higher cytokinin concentrations are required to maintain a normal phenotype of sterile lemmas. CONCLUSION: The regulatory networks have considerable reference value, and some of the regulatory relationships exhibiting strong correlations are worthy of further study. Taken together, these work provided a detailed guide for further studies into the molecular mechanism of sterile lemma development.

SH3P2, an SH3 domain-containing protein that interacts with both Pib and AvrPib, suppresses effector-triggered, Pib-mediated immunity in rice.

Plants usually keep resistance (R) proteins in a static state under normal conditions to avoid autoimmunity and save energy for growth, but R proteins can be rapidly activated upon perceiving pathogen invasion. Pib, the first cloned blast disease R gene in rice, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, mediates resistance to the blast fungal (Magnaporthe oryzae) isolates carrying the avirulence gene AvrPib. However, the molecular mechanisms about how Pib recognizes AvrPib and how it is inactivated and activated remain largely unclear. In this study, through map-based cloning and CRISPR-Cas9 gene editing, we proved that Pib contributes to the blast disease resistance of rice cultivar Yunyin (YY). Furthermore, an SH3 domain-containing protein, SH3P2, was found to associate with Pib mainly at clathrin-coated vesicles in rice cells, via direct binding with the coiled-coil (CC) domain of Pib. Interestingly, overexpression of SH3P2 in YY compromised Pib-mediated resistance to M. oryzae isolates carrying AvrPib and Pib-AvrPib recognition-induced cell death. SH3P2 competitively inhibits the self-association of the Pib CC domain in vitro, suggesting that binding of SH3P2 with Pib undermines its homodimerization. Moreover, SH3P2 can also interact with AvrPib and displays higher affinity to AvrPib than to Pib, which leads to dissociation of SH3P2 from Pib in the presence of AvrPib. Taken together, our results suggest that SH3P2 functions as a "protector" to keep Pib in a static state by direct interaction during normal growth but could be triggered off by the invasion of AvrPib-carrying M. oryzae isolates. Our study reveals a new mechanism about how an NLR protein is inactivated under normal conditions but is activated upon pathogen infection.

TRIM21 aggravates cardiac injury after myocardial infarction by promoting M1 macrophage polarization.

Macrophage polarization followed by myocardial infarction (MI) is essential for wound healing. Tripartite motif-containing protein 21 (TRIM21), a member of E3 ubiquitin ligases, is emerging as a mediator in cardiac injury and heart failure. However, its function in modulating post-MI macrophage polarization remains elusive. Here, we detected that the levels of TRIM21 significantly increased in macrophages of wild-type (WT) mice after MI. In contrast, MI was ameliorated in TRIM21 knockout (TRIM21-/-) mice with improved cardiac remodeling, characterized by a marked decrease in mortality, decreased infarct size, and improved cardiac function compared with WT-MI mice. Notably, TRIM21 deficiency impeded the post-MI apoptosis and DNA damage in the hearts of mice. Consistently, the accumulation of M1 phenotype macrophages in the infarcted tissues was significantly reduced with TRIM21 deletion. Mechanistically, the deletion of TRIM21 orchestrated the process of M1 macrophage polarization at least partly via a PI3K/Akt signaling pathway. Overall, we identify TRIM21 drives the inflammatory response and cardiac remodeling by stimulating M1 macrophage polarization through a PI3K/Akt signaling pathway post-MI.

Prognostic impact of stress hyperglycemia ratio in acute myocardial infarction patients with and without diabetes mellitus.

BACKGROUND AND AIMS: Stress hyperglycemia ratio (SHR) is associated with increased in-hospital morbidity and mortality in patients with acute myocardial infarction (AMI). We aimed to investigate the impact of stress "hyperglycemia" on long-term mortality after AMI in patients with and without diabetes mellitus (DM). METHODS AND RESULTS: We included 2089 patients with AMI between February 2014 and March 2018. SHR was measured with the fasting glucose divided by the estimated average glucose derived from glycosylated hemoglobin (HbA1c). The primary endpoint was all-cause death. Of 2 089 patients (mean age: 65.7 ± 12.4, 76.7% were men) analyzed, 796 (38.1%) had DM. Over a median follow-up of 2.7 years, 141 (6.7%) and 150 (7.2%) all-cause deaths occurred in the diabetic and nondiabetic cohorts, respectively. Compared with participants with low SHR (<1.24 in DM; <1.14 in non-DM), the hazard ratios and 95% confidence intervals for those with high SHR (≥1.24 in DM; ≥1.14 in non-DM) for all-cause mortality were 2.23 (1.54-3.23) and 1.79 (1.15-2.78); for cardiovascular mortality were 2.42 (1.63-3.59) and 2.10 (1.32-3.35) in DM and non-DM subjects, respectively. The mortality prediction was improved in the diabetic individuals with the incorporation of SHR into the Global Registry of Acute Coronary Events (GRACE) score, showing an increase in a continuous net reclassification index of 0.184 (95%CI: 0.003-0.365) and an absolute integrated discrimination improvement of 0.014 (95%CI: 0.002-0.025). CONCLUSION: The improvement in the prediction of long-term mortality beyond the GRACE score indicates the potential of SHR as a biomarker for post-MI risk stratification among patients with DM. REGISTRATION NUMBER FOR CLINICAL TRIALS: NCT03533543.

Transcriptome Analyses Indicate Significant Association of Increased Non-Additive and Allele-Specific Gene Expression with Hybrid Weakness in Rice (Oryza sativa L.).

The heterosis in hybrid rice is highly affected by the environment and hybrid weakness occurs frequently depending on the genotypes of the hybrid and its parents. Hybrid weakness was also observed in our field experiments on nine rice hybrids produced by 3 × 3 incomplete diallel crosses. Among the nine hybrids, five displayed mid-parent heterosis (MPH) for grain yield per plant, while four showed mid-parent hybrid weakness (MPHW). A sequencing analysis of transcriptomes in panicles at the seed-filling stage revealed a significant association between enhanced non-additive gene expression (NAE) and allele-specific gene expression (ASE) with hybrid weakness. High proportions of ASE genes, with most being of mono-allele expression, were detected in the four MPHW hybrids, ranging from 22.65% to 45.97%; whereas only 4.80% to 5.69% of ASE genes were found in the five MPH hybrids. Moreover, an independence test indicated that the enhancements of NAE and ASE in the MPHW hybrids were significantly correlated. Based on the results of our study, we speculated that an unfavorable environment might cause hybrid weakness by enhancing ASE and NAE at the transcriptome level.

ZPI prevents ox-LDL-mediated endothelial injury leading to inhibition of EndMT, inflammation, apoptosis, and oxidative stress through activating Pi3k/Akt signal pathway.

Oxidized low-density lipoprotein (ox-LDL)-mediated endothelial dysfunction exerts an essential role in the development of atherosclerosis. Protein Z-dependent protease inhibitor (ZPI), a member of the serine protease inhibitor superfamily, could inhibit the function of activated coagulation factor X (FXa) via interaction with protein Z (PZ). Studies have pointed out that ZPI was statistically related to atherosclerotic diseases, which may have a robust cardiovascular protective effect. However, the underlying mechanism of ZPI on ox-LDL-mediated endothelial injury requires further elucidation. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL (100 µg/ml) and ZPI (10 µg/ml). Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, oxidative stress, and endothelial-to-mesenchymal transition (EndMT) were analyzed by immunofluorescence (IF). Cell migration was measured using a wound-healing assay. Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine messenger RNA and protein expression. Ox-LDL (100 µg/ml, 48 h) significantly reduced cell viability and migration, increased EndMT, inflammation, apoptosis, and oxidative stress. The related protein expression of phosphatidylinositol 3 kinase/protein kinase B (Pi3k/Akt) signal pathway in HUVECs was also simultaneously decreased. We also discovered that ZPI treatment could prevent ox-LDL-mediated endothelial injury through the improvement of cell viability and alleviation of apoptosis, oxidative stress, EndMT, and inflammation. Thus, the protective effect of ZPI on HUVECs may be mediated by activation of the Pi3k/Akt signal pathway. ZPI may exert an important protective role in HUVECs dysfunction triggered by ox-LDL via activation of the Pi3k/Akt signal pathway. Therefore, ZPI may possess potential therapeutic effects on atherosclerotic endothelial injury-related diseases.

Characterization and expression analysis of the glycosyltransferase 64 family in rice (Oryza sativa).

The glycosyltransferase 64 (GT64) family is widely conserved in many species, including animals and plants. The functions of GT64 family genes in animals have been well characterized in the biosynthesis of extracellular heparan sulfate, whereas two GT64 members in Arabidopsis thaliana are involved in the glycosylation of plasma membrane glycosylinositol phosphorylceramides (GIPCs). GIPCs are the main components of plant sphingolipids and serve as important signal molecules in various developmental processes and stress responses. Rice (Oryza sativa), a model monocot plant, contains four GT64 members in its genome. Using phylogenetic analysis, 73 GT64s from 19 plant species were divided into three main groups. Each group can be represented by the three members in Arabidopsis and show a trend of monocot-eudicot divergence. A promoter and genomic variation analysis of GT64s in rice showed that various stress-related regulatory elements exist in their promoters, and many sequence variations were found between the two main rice subspecies, japonica and indica. Additionally, transmembrane domain and subcellular localization analyses revealed that these genes all encode membrane-bound glycosyltransferases and are localized to the Golgi apparatus. Finally, expression analysis of the four GT64 genes in rice, as assessed by quantitative real-time PCR, showed that they have distinct tissue-specific expression patterns and respond to different hormone treatments or abiotic stresses. Our results indicated that this family of genes may play a role in different stress responses and hormone signaling pathways in rice, which will provide fundamental information for further investigation of their functions in future.

Prognostic Significance of New-Onset Atrial Fibrillation in Heart Failure with Preserved, Mid-Range, and Reduced Ejection Fraction Following Acute Myocardial Infarction: Data from the NOAFCAMI-SH Registry.

Purpose: The prognostic impact of new-onset atrial fibrillation (NOAF) among different heart failure (HF) subtypes including HF with preserved (HFpEF, ejection fraction [EF] ≥50%), mid-range (HFmrEF, EF 40%~49%), and reduced (HFrEF, EF <40%) EF following acute myocardial infarction (AMI) remains unclear. We aimed to investigate the incidence and prognostic implication of post-MI NOAF across HF subtypes. Patients and Methods: We included 1413 patients with post-MI HF (743 with HFpEF, 342 with HFmrEF and 328 with HFrEF) between February 2014 and March 2018. NOAF was considered as patients without a preexisting AF who developed AF during the AMI hospitalization. The primary endpoint was all-cause mortality. Results: Of 1413 patients (mean age 66.8 ± 12.6 years, 72.9% men) analyzed, 200 (14.2%) developed post-MI NOAF. Patients with HFrEF were more likely to experience NOAF compared to those with HFmrEF or HFrEF (18.9%, 13.7% and 12.2% in HFrEF, HFmrEF and HFpEF, respectively; p for trend = 0.006). During a median follow-up of 28.5 months, 192 patients died (70 with HFrEF, 35 with HFmrEF and 87 with HFpEF) and 195 patients experienced HF rehospitalization (79 with HFrEF, 37 with HFmrEF and 79 with HFpEF). After multivariable adjustment, NOAF was independently associated with all-cause mortality (hazard ratio [HR]: 1.79, 95% confidence interval [CI]: 1.03-3.12) only in the HFrEF group compared to sinus rhythm (SR), whereas an increased risk of HF rehospitalization was found in all HF subtypes, particularly in HFmrEF (HR: 5.08, 95% CI: 2.29-11.25) and HFpEF (HR: 2.83 95% CI: 1.64-4.90). Conclusion: In patients with post-MI HF, NOAF carried a worse prognosis for all-cause death in the HFrEF group and for HF rehospitalization in all HF subtypes.

Ubiquitylomes and proteomes analyses provide a new interpretation of the molecular mechanisms of rice leaf senescence.

MAIN CONCLUSION: We identified a typical rice premature senescence leaf mutant 86 (psl86) and exhibited the first global ubiquitination data during rice leaf senescence. Premature leaf senescence affects the yield and quality of rice, causing irreparable agricultural economic losses. In this study, we reported a rice premature senescence leaf mutant 86 (psl86) in the population lines of rice (Oryza sativa) japonica cultivar 'Yunyin' (YY) mutagenized using ethyl methane sulfonate (EMS) treatment. Immunoblotting analysis revealed that a higher ubiquitination level in the psl86 mutant compared with YY. Thus, we performed the proteome and ubiquitylome analyses to identify the differential abundance proteins and ubiquitinated proteins (sites) related to leaf senescence. Among 885 quantified lysine ubiquitination (Kub) sites in 492 proteins, 116 sites in 94 proteins were classified as up-regulated targets and seven sites in six proteins were classified as down-regulated targets at a threshold of 1.5. Proteins with up-regulated Kub sites were mainly enriched in the carbon fixation in photosynthetic organisms, glycolysis/gluconeogenesis and the pentose phosphate pathway. Notably, 14 up-regulated Kub sites in 11 proteins were enriched in the carbon fixation in photosynthetic organism pathway, and seven proteins (rbcL, PGK, GAPA, FBA5, ALDP, CFBP1 and GGAT) were down-regulated, indicating this pathway is tightly regulated by ubiquitination during leaf senescence. To our knowledge, we present the first global data on ubiquitination during rice leaf senescence.

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein, regulates light-dependent cell death and defence responses in rice.

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross-talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map-based cloning, a short-chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7-6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light-dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity-related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000-grain weight, grain weight per plant, panicle length, and plant height. SDR7-6 is a new lesion mimic gene that encodes a short-chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7-6 led to cell death and had adverse effects on multiple agricultural characters. SDR7-6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.

RNA-seq profiling of primary calli induced by different media and photoperiods for japonica rice 'Yunyin'.

The induction of embryogenic calli plays a vital role in the genetic transformation and regeneration of rice (Oryza sativa L.). Despite progress in rice tissue culture, the molecular mechanisms of embryogenic callus induction remain unknown. In this study, gene expression profiles associated with calli were comprehensively analyzed during callus induction of japonica rice 'Yunyin'. We first confirmed that NMB medium with 24 h of light and 0 h of dark (NMB-L) was the optimal condition for 'Yunyin' callus induction, while J3 medium with 0 h of light and 24 h of dark (J3-D) was the worst condition. After transcriptome analysis, 33,597 unigenes were assembled, among which we identified 6,063 DEGs (Differentially Expressed Genes) related to media and seven DEGs related to photoperiod. Phenylpropanoid biosynthesis, plant hormone signal, and starch and sucrose metabolism were the top three pathways affected by media, while the circadian rhythm-plant pathway was associated with photoperiod. Furthermore, we identified two candidate genes, Os01g0965900 and Os12g0555200, affected by both medium and photoperiod. Statistical analysis of RNA-seq libraries showed that the expression levels of these two genes in J3-D calli were over 2.5 times higher than those in NMB-L calli, which was further proved by RT-qPCR analysis. Based on FPKM (Fragments Per Kilobase of transcript Per Million mapped reads), unigenes belonging to the NMB-L group were mainly assigned to ribosome, carbon metabolism, biosynthesis of amino acids, protein processing in endoplasmic reticulum, and plant hormone signal transduction pathways. We transformed Os12g0555200Nip and Os12g05552009311 into 'Nipponbare' calli and observed their effects on the growth and development process of rice calli using TEM (Transmission Electron Microscopy) and SEM (Scanning Electron Microscopy). Observations showed that Os12g05552009311 was more disadvantageous to rice callus growth than Os12g0555200Nip. Our results reveal that the Os12g0555200, identified from transcriptomic profiles, has a negative influence during 'Yunyin' callus induction. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-022-01283-y.

MiR-126-HMGB1-HIF-1 Axis Regulates Endothelial Cell Inflammation during Exposure to Hypoxia-Acidosis.

Crosstalk between molecular regulators miR-126, hypoxia-inducible factor 1-alpha (HIF-1-α), and high-mobility group box-1 (HMGB1) contributes to the regulation of inflammation and angiogenesis in multiple physiological and pathophysiological settings. Here, we present evidence of an overriding role for miR-126 in the regulation of HMGB1 and its downstream proinflammatory effectors in endothelial cells subjected to hypoxia with concurrent acidosis (H/A). Methods. Primary mouse endothelial cells (PMEC) were exposed to hypoxia or H/A to simulate short or chronic low-flow ischemia, respectively. RT-qPCR quantified mRNA transcripts, and proteins were measured by western blot. ROS were quantified by fluorogenic ELISA and luciferase reporter assays employed to confirm an active miR-126 target in the HMGB1 3'UTR. Results. Enhanced expression of miR-126 in PMECs cultured under neutral hypoxia was suppressed under H/A, whereas the HMGB1 expression increased sequentially under both conditions. Enhanced expression of HMGB1 and downstream inflammation markers was blocked by the premiR-126 overexpression and optimized by antagomiR. Compared with neutral hypoxia, H/A suppressed the HIF-1α expression independently of miR-126. The results show that HMGB1 and downstream effectors are optimally induced by H/A relative to neutral hypoxia via crosstalk between hypoxia signaling, miR-126, and HIF-1α, whereas B-cell lymphoma 2(Bcl2), a HIF-1α, and miR-126 regulated gene expressed optimally under neutral hypoxia. Conclusion. Inflammatory responses of ECs to H/A are dynamically regulated by the combined actions of hypoxia, miR-126, and HIF-1α on the master regulator HMGB1. The findings may be relevant to vascular diseases including atherosclerotic occlusion and interiors of plaque where coexisting hypoxia and acidosis promote inflammation as a defining etiology.

Prognostic Impact of the Symptom of New-Onset Atrial Fibrillation in Acute Myocardial Infarction: Insights From the NOAFCAMI-SH Registry.

Background: New-onset atrial fibrillation (NOAF) is a common complication during acute myocardial infarction (AMI) and sometimes can be completely asymptomatic, but the clinical implications of these asymptomatic episodes require further characterization. The objective of this study was to investigate the short- and long-term prognostic impact of post-MI NOAF based on the presence of AF-related symptoms. Methods: The New-Onset Atrial Fibrillation Complicating Acute Myocardial Infarction in ShangHai (NOAFCAMI-SH) registry was a retrospective cohort including participants with AMI without a documented history of AF. Patients with NOAF were divided into two groups according to the AF-related symptoms. The primary endpoint was all-cause mortality. Results: Of 2,399 patients included, 278 (11.6%) developed NOAF of whom 145 (6.0%) with asymptomatic episodes and 133 (5.5%) with symptomatic ones. During hospitalization, 148 patients died [106, 10, and 32 in the sinus rhythm (SR), asymptomatic, and symptomatic NOAF groups, respectively]. After multivariable adjustment, only symptomatic NOAF was associated with in-hospital mortality [odds ratio (OR): 2.32, 95% confidence interval (CI): 1.36-3.94] compared with SR. Over a median follow-up of 2.7 years, all-cause mortality was 3.2, 12.4, and 11.8% per year in the SR, asymptomatic, and symptomatic NOAF groups, respectively. After adjustment for confounders, it was the asymptomatic NOAF [hazard ratio (HR): 1.61, 95% CI: 1.09-2.37) rather than the symptomatic one (HR: 1.37, 95% CI: 0.88-2.12) that was significantly related to mortality. Similar results were also observed for cardiovascular mortality [HRs and 95% CI were 1.71 (1.10-2.67) and 1.25 (0.74-2.11) for asymptomatic and symptomatic NOAF, respectively]. Both asymptomatic and symptomatic NOAF episodes were associated with heart failure, whereas only those with symptomatic NOAF were at heightened risk of ischemic stroke. Our exploratory analysis further identified patients with asymptomatic high-burden NOAF as the highest-risk population (mortality: 19.6% per year). Conclusion: Among patients with AMI, symptomatic NOAF is related to in-hospital mortality and asymptomatic NOAF is associated with poor long-term survival. Registration: URL: https://clinicaltrials.gov/; Unique identifier: NCT03533543.