Single-cell RNA-seq data analysis reveals functionally relevant biomarkers of early brain development and their regulatory footprints in human embryonic stem cells (hESCs).

The complicated process of neuronal development is initiated early in life, with the genetic mechanisms governing this process yet to be fully elucidated. Single-cell RNA sequencing (scRNA-seq) is a potent instrument for pinpointing biomarkers that exhibit differential expression across various cell types and developmental stages. By employing scRNA-seq on human embryonic stem cells, we aim to identify differentially expressed genes (DEGs) crucial for early-stage neuronal development. Our focus extends beyond simply identifying DEGs. We strive to investigate the functional roles of these genes through enrichment analysis and construct gene regulatory networks to understand their interactions. Ultimately, this comprehensive approach aspires to illuminate the molecular mechanisms and transcriptional dynamics governing early human brain development. By uncovering potential links between these DEGs and intelligence, mental disorders, and neurodevelopmental disorders, we hope to shed light on human neurological health and disease. In this study, we have used scRNA-seq to identify DEGs involved in early-stage neuronal development in hESCs. The scRNA-seq data, collected on days 26 (D26) and 54 (D54), of the in vitro differentiation of hESCs to neurons were analyzed. Our analysis identified 539 DEGs between D26 and D54. Functional enrichment of those DEG biomarkers indicated that the up-regulated DEGs participated in neurogenesis, while the down-regulated DEGs were linked to synapse regulation. The Reactome pathway analysis revealed that down-regulated DEGs were involved in the interactions between proteins located in synapse pathways. We also discovered interactions between DEGs and miRNA, transcriptional factors (TFs) and DEGs, and between TF and miRNA. Our study identified 20 significant transcription factors, shedding light on early brain development genetics. The identified DEGs and gene regulatory networks are valuable resources for future research into human brain development and neurodevelopmental disorders.

Equisetin inhibits adiposity through AMPK-dependent regulation of brown adipocyte differentiation.

Obesity has a significant impact on endocrine function, which leads to metabolic diseases including diabetes, insulin resistance, and other complications associated with obesity. Development of effective and safe anti-obesity drugs is imperative and necessary. Equisetin (EQST), a tetramate-containing marine fungal product, was reported to inhibit bacterial fatty acid synthesis and affect mitochondrial metabolism. It is tempting to speculate that EQST might have anti-obesity effects. This study was designed to explore anti-obesity effects and underlying mechanism of EQST on 3T3-L1 adipocytes differentiated from 3T3-L1 cells. Oil Red O staining showed that EQST reduced lipid accumulation in 3T3-L1 adipocytes. Quantitative real-time polymerase chain reaction and Western blot analysis revealed that EQST significantly inhibited expression of adipogenesis/lipogenesis-related genes C/ebp-α, Ppar-γ, Srebp1c, Fas, and reduced protein levels. There was also increased expression of key genes and protein levels involved in lipolysis (Perilipin, Atgl, Hsl), brown adipocyte differentiation (Prdm16, Ucp1), mitochondrial biogenesis (Pgc1α, Tfam) and ß-oxidation Acsl1, Cpt1. Moreover, mitochondrial content, their membrane potential ΔΨM, and respiratory chain genes Mt-Co1, Cox7a1, Cox8b, and Cox4 (and protein) exhibited marked increase in expression upon EQST treatment, along with increased protein levels. Importantly, EQST induced expression and activation of AMPK, which was compromised by the AMPK inhibitor dorsomorphin, leading to rescue of EQST-downregulated Fas expression and a reduction of the EQST-increased expression of Pgc1α, Ucp1, and Cox4. Together, EQST robustly promotes fat clearance through the AMPK pathway, these results supporting EQST as a strong candidate for the development into an anti-obesity therapeutic agent.

Identification of reliable QTLs and designed QTL breeding for grain shape and milling quality in the reciprocal introgression lines in rice.

Milling quality (MQ) and grain shape (GS) of rice (Oryza sativa L.) are correlated traits, both determine farmers' final profit. More than one population under multiple environments may provide valuable information for breeding selection on these MQ-GS correlations. However, suitable analytical methods for reciprocal introgression lines with linkage map for this kind of correlation remains unclear. In this study, our major tasks were (1) to provide a set of reciprocal introgression lines (composed of two BC2RIL populations) suitable for mapping by linkage mapping using markers/bins with physical positions; (2) to test the mapping effects of different methods by using MQ-GS correlation dissection as sample case; (3) to perform genetic and breeding simulation on pyramiding favorite alleles of QTLs for representative MQ-GS traits. Finally, with four analysis methods and data collected under five environments, we identified about 28.4 loci on average for MQ-GS traits. Notably, 52.3% of these loci were commonly detected by different methods and eight loci were novel. There were also nine regions harboring loci for different MQ-GS traits which may be underlying the MQ-GS correlations. Background independent (BI) loci were also found for each MQ and GS trait. All these information may provide useful resources for rice molecular breeding.

Reduction of SARS-CoV-2 by biological nutrient removal and disinfection processes in full-scale wastewater treatment plants.

Detection of SARS-CoV-2 RNA in wastewater poses people's concerns regarding the potential risk in water bodies receiving wastewater treatment effluent, despite the infectious risk of SARS-CoV-2 in wastewater being speculated to be low. Unlike well-studied nonenveloped viruses, SARS-CoV-2 in wastewater is present abundantly in both solid and liquid fractions of wastewater. Reduction of SARS-CoV-2 in past studies were likely underestimated, as SARS-CoV-2 in influent wastewater were quantified in either solid or liquid fraction only. The objectives of this study were (i) to clarify the reduction in SARS-CoV-2 RNA during biological nutrient removal and disinfection processes in full-scale WWTPs, considering the SARS-CoV-2 present in both solid and liquid fractions of wastewater, and (ii) to evaluate applicability of pepper mild mottle virus (PMMoV) as a performance indicator for reduction of SARS-CoV-2 in WWTPs. Accordingly, large amount of SARS-CoV-2 RNA were partitioned in the solid fraction of influent wastewater for composite sampling than grab sampling. When SARS-CoV-2 RNA in the both solid and liquid fractions were considered, log reduction values (LRVs) of SARS-CoV-2 during step-feed multistage biological nitrogen removal (SM-BNR) and enhanced biological phosphorus removal (EBPR) processes ranged between>2.1-4.4 log and did not differ significantly from those in conventional activated sludge (CAS). The LRVs of SARS-CoV-2 RNA in disinfection processes by ozonation and chlorination did not differ significantly. PMMoV is a promising performance indicator to secure reduction of SARS-CoV-2 in WWTPs, because of its higher persistence in wastewater treatment processes and abundance at a detectable concentration even in the final effluent after disinfection.

Passive swab versus grab sampling for detection of SARS-CoV-2 markers in wastewater.

Early detection of the COVID-19 virus, SARS-CoV-2, is key to mitigating the spread of new outbreaks. Data from individual testing is increasingly difficult to obtain as people conduct non-reported home tests, defer tests due to logistics or attitudes, or ignore testing altogether. Wastewater based epidemiology is an alternative method for surveilling a community while maintaining individual anonymity; however, a problem is that SARS-CoV-2 markers in wastewater vary throughout the day. Collecting grab samples at a single time may miss marker presence, while autosampling throughout a day is technically challenging and expensive. This study investigates a passive sampling method that would be expected to accumulate greater amounts of viral material from sewers over a period of time. Tampons were tested as passive swab sampling devices from which viral markers could be eluted with a Tween-20 surfactant wash. Six sewersheds in Detroit were sampled 16-22 times by paired swab (4 h immersion before retrieval) and grab methods over a five-month period and enumerated for N1 and N2 SARS-CoV-2 markers using ddPCR. Swabs detected SARS-CoV-2 markers significantly more frequently (P < 0.001) than grab samples, averaging two to three-fold more copies of SARS-CoV-2 markers than their paired grab samples (p < 0.0001) in the assayed volume (10 mL) of wastewater or swab eluate. No significant difference was observed in the recovery of a spiked-in control (Phi6), indicating that the improved sensitivity is not due to improvements in nucleic acid recovery or reduction of PCR inhibition. The outcomes of swab-based sampling varied significantly between sites, with swab samples providing the greatest improvements in counts for smaller sewersheds that otherwise tend to have greater variation in grab sample counts. Swab-sampling with tampons provides significant advantages in detection of SARS-CoV-2 wastewater markers and are expected to provide earlier detection of new outbreaks than grab samples, with consequent public health benefits.

Transcriptomic profiling reveals candidate allelopathic genes in rice responsible for interactions with barnyardgrass.

Echinochloa crus-galli (barnyardgrass) is one of the most damaging weeds in rice fields worldwide. Allelopathy has been considered a possible application for weed management. Thus understanding its molecular mechanisms is important for rice production. This study generated transcriptomes from rice under mono- and co-culture with barnyardgrass at two-time points to identify the candidate genes controlling allelopathic interactions between rice and barnyardgrass. A total of 5,684 differentially expressed genes (DEGs) were detected, amongst which 388 genes were transcription factors. These DEGs include genes associated with momilactone and phenolic acid biosynthesis, which play critical roles in allelopathy. Additionally, we found significantly more DEGs at 3 hours than at 3 days, suggesting a quick allelopathic response in rice. Up-regulated DEGs involve diverse biological processes, such as response to stimulus and pathways related to phenylpropanoid and secondary metabolites biosynthesis. Down-regulated DEGs were involved in developmental processes, indicating a balance between growth and stress response to allelopathy from barnyardgrass. Comparison of DEGs between rice and barnyardgrass shows few common genes, suggesting different mechanisms underlying allelopathic interaction in these two species. Our results offer an important basis for identifying of candidate genes responsible for rice and barnyardgrass interactions and contribute valuable resources for revealing its molecular mechanisms.

Developer discussion topics on the adoption and barriers of low code software development platforms.

Low-code software development (LCSD) is an emerging approach to democratize application development for software practitioners from diverse backgrounds. LCSD platforms promote rapid application development with a drag-and-drop interface and minimal programming by hand. As it is a relatively new paradigm, it is vital to study developers' difficulties when adopting LCSD platforms. Software engineers frequently use the online developer forum Stack Overflow (SO) to seek assistance with technical issues. We observe a growing body of LCSD-related posts in SO. This paper presents an empirical study of around 33K SO posts (questions + accepted answers) containing discussions of 38 popular LCSD platforms. We use Topic Modeling to determine the topics discussed in those posts. Additionally, we examine how these topics are spread across the various phases of the agile software development life cycle (SDLC) and which part of LCSD is the most popular and challenging. Our study offers several interesting findings. First, we find 40 LCSD topics that we group into five categories: Application Customization, Database and File Management, Platform Adoption, Platform Maintenance, and Third-party API Integration. Second, while the Application Customization (30%) and Data Storage (25%) topic categories are the most common, inquiries relating to several other categories (e.g., the Platform Adoption topic category) have gained considerable attention in recent years. Third, all topic categories are evolving rapidly, especially during the Covid-19 pandemic. Fourth, the How-type questions are prevalent in all topics, but the What-type and Why-type (i.e., detail information for clarification) questions are more prevalent in the Platform Adoption and Platform Maintenance category. Fifth, LCSD practitioners find topics related to Platform Query the most popular, while topics related to Message Queue and Library Dependency Management as the most difficult to get accepted answers to. Sixth, the Why-type and What-type questions and Agile Maintenance and Deployment phase are the most challenging among practitioners. The findings of this study have implications for all three LCSD stakeholders: LCSD platform vendors, LCSD developers/practitioners, Researchers, and Educators. Researchers and LCSD platform vendors can collaborate to improve different aspects of LCSD, such as better tutorial-based documentation, testing, and DevOps support.

Dissecting Complex Traits Using Omics Data: A Review on the Linear Mixed Models and Their Application in GWAS.

Genome-wide association study (GWAS) is the most popular approach to dissecting complex traits in plants, humans, and animals. Numerous methods and tools have been proposed to discover the causal variants for GWAS data analysis. Among them, linear mixed models (LMMs) are widely used statistical methods for regulating confounding factors, including population structure, resulting in increased computational proficiency and statistical power in GWAS studies. Recently more attention has been paid to pleiotropy, multi-trait, gene-gene interaction, gene-environment interaction, and multi-locus methods with the growing availability of large-scale GWAS data and relevant phenotype samples. In this review, we have demonstrated all possible LMMs-based methods available in the literature for GWAS. We briefly discuss the different LMM methods, software packages, and available open-source applications in GWAS. Then, we include the advantages and weaknesses of the LMMs in GWAS. Finally, we discuss the future perspective and conclusion. The present review paper would be helpful to the researchers for selecting appropriate LMM models and methods quickly for GWAS data analysis and would benefit the scientific society.

Removal performance of SARS-CoV-2 in wastewater treatment by membrane bioreactor, anaerobic-anoxic-oxic, and conventional activated sludge processes.

The potential risk of SARS-CoV-2 in treated effluent from a wastewater treatment plant (WWTP) is concerned since SARS-CoV-2 is contained in wastewater during the COVID-19 outbreak. However, the removal of SARS-CoV-2 in WWTP has not been well investigated. The objectives of this study were (i) to clarify the removal performance of SARS-CoV-2 during wastewater treatment, (ii) to compare the removal performance of different secondary treatment processes, and (iii) to evaluate applicability of pepper mild mottle of virus (PMMoV) as a performance indicator for the reduction of SARS-CoV-2 RNA in wastewater treatment. Influent wastewater, secondary-treatment effluent (before chlorination), and final effluent (after chlorination) samples were collected from a WWTP from May 28 to September 24, 2020, during the COVID-19 outbreak in Japan. The target WWTP had three parallel treatment systems employing conventional activated sludge (CAS), anaerobic-anoxic -oxic (A2O), and membrane bioreactor (MBR) processes. SARS-CoV-2 in both the liquid and solid fractions of the influent wastewater was concentrated and quantified using RT-qPCR. SARS-CoV-2 in treated effluent was concentrated from 10 L samples to achieve a detection limit as low as 10 copies/L. The log reduction value (LRV) of SARS-CoV-2 was 2.7 ± 0.86 log10 in CAS, 1.6 ± 0.50 log10 in A2O, and 3.6 ± 0.62 log10 in MBR. The lowest LRV observed during the sampling period was 2.8 log10 in MBR, 1.2 log10 in CAS, and 1.0 log10 in A2O process, indicating that the MBR had the most stable reduction performance. PMMoV was found to be a good indicator virus to evaluate reduction performance of SARS-CoV-2 independent of the process configuration because the LRV of PMMoV was significantly lower than that of SARS-CoV-2 in the CAS, A2O and MBR processes.

CRISPR/Cas9 Mediated Knockout of the OsbHLH024 Transcription Factor Improves Salt Stress Resistance in Rice (Oryza sativa L.).

Salinity stress is one of the most prominent abiotic stresses that negatively affect crop production. Transcription factors (TFs) are involved in the absorption, transport, or compartmentation of sodium (Na+) or potassium (K+) to resist salt stress. The basic helix-loop-helix (bHLH) is a TF gene family critical for plant growth and stress responses, including salinity. Herein, we used the CRISPR/Cas9 strategy to generate the gene editing mutant to investigate the role of OsbHLH024 in rice under salt stress. The A nucleotide base deletion was identified in the osbhlh024 mutant (A91). Exposure of the A91 under salt stress resulted in a significant increase in the shoot weight, the total chlorophyll content, and the chlorophyll fluorescence. Moreover, high antioxidant activities coincided with less reactive oxygen species (ROS) and stabilized levels of MDA in the A91. This better control of oxidative stress was accompanied by fewer Na+ but more K+, and a balanced level of Ca2+, Zn2+, and Mg2+ in the shoot and root of the A91, allowing it to withstand salt stress. Furthermore, the A91 also presented a significantly up-regulated expression of the ion transporter genes (OsHKT1;3, OsHAK7, and OsSOS1) in the shoot when exposed to salt stress. These findings imply that the OsbHLH024 might play the role of a negative regulator of salt stress, which will help to understand better the molecular basis of rice production improvement under salt stress.

Extent of degradation in three stage co-composting of fecal sludge and solid waste.

Co-composting of fecal sludge (FS) and solid waste (SW) allows recycling of essential nutrients into agriculture thereby closing the nutrient circle. In this study, temperature variation, the mass balance of forced and passive aeration in the composting process, and the extent of degradation with different stages were investigated. The extent of degradation was determined through the different composting process in the first, second, and third stages with different mix proportion of fecal sludge. Four sets of the initial waste mixture were prepared using SW and FS. SW and FS were mixed at four different ratios for four sets namely 90:10, 85:15, 80:20, and 75:25 (SW:FS). Forced aeration and passive aeration composting tests were done using a series of reactors according to a planned experimental program. The results show that (i) the mean maximum temperature of the first and second stage were 65°C and 56°C of passive, 67°C and 60°C of forced aeration, respectively which raised within seven days. (ii) According to the mass balance, total mass, moisture content, and volatile solids always decreased at every stage for passive and forced aeration processes, the degradation of the volatile solid in the composting process using forced aeration was more than in the passive aeration process. (iii) For the passive and forced aeration processes, the total BVS degradation of ranged between 82% and 89%, and 73% and 91%, respectively after 60 days. Therefore, it can be concluded that the percentage of BVS degradation in forced and passive aeration was not significantly different.Implications: The extent of degradation was determined through different composting processes in the three stages with different mixture proportion of fecal sludge and organic solid waste. The study showed that maximum temperature in composting ranged from 57°C to 67°C within one week. Co-composting process with passively and forced aeration process of peak temperature were almost same. Reduction of volatile solids at first stage was greater than second and third stages. Biodegradable volatile solids reduction in passive and forced aeration processes were not significantly different. The final product of composting was used in agricultural land.

Selection of surrogate viruses for process control in detection of SARS-CoV-2 in wastewater.

Since SARS-CoV-2 RNA in wastewater is often present at low concentration or under detection limit, ensuring the reliability of detection processes using appropriate process controls is essential. The objective of this study was to evaluate applicability and limitations of candidate surrogate viruses as process controls under combinations of different virus concentration and RNA extraction methods. Detection efficiency of SARS-CoV-2 spiked in wastewater was compared with those of candidate surrogate viruses of bacteriophage ϕ6, pepper mild mottle virus (PMMoV), F-specific coliphage (F-phage), and murine norovirus (MNV). After inactivated SARS-CoV-2 and ϕ6 were spiked in two different wastewaters, the viruses in solid and liquid fractions of wastewater were concentrated by centrifuge and polyethylene glycol (PEG) precipitation, respectively. Viral RNA was extracted by using QIAamp Viral RNA Mini Kit and 3 other commercially available extraction kits, then quantified by reverse transcription-quantitative PCR using CDCN1 assay. Regardless of extraction kits, SARS-CoV-2 was consistently detected with good efficiency from both liquid (11-200%) and solid fractions (7.1-93%). Among the candidate process controls, PMMoV was widely detected at good efficiencies from both liquid and solid fractions regardless of selection of RNA extraction kits. F-phage and MNV also showed good detection efficiencies in most combinations of wastewater fractions and RNA extraction kits. An enveloped virus ɸ6 was found often undetected or to have very low detection efficiency (0.1-4.2%) even when SARS-CoV-2 spiked in wastewater was detected with good efficiency. Consequently, PMMoV is widely applicable as process control for detection of SARS-CoV-2 either in liquid fractions concentrated by PEG precipitation, or in solid fractions concentrated by centrifuge.

Neurobiochemical Cross-talk Between COVID-19 and Alzheimer's Disease.

COVID-19, the global threat to humanity, shares etiological cofactors with multiple diseases including Alzheimer's disease (AD). Understanding the common links between COVID-19 and AD would harness strategizing therapeutic approaches against both. Considering the urgency of formulating COVID-19 medication, its AD association and manifestations have been reviewed here, putting emphasis on memory and learning disruption. COVID-19 and AD share common links with respect to angiotensin-converting enzyme 2 (ACE2) receptors and pro-inflammatory markers such as interleukin-1 (IL-1), IL-6, cytoskeleton-associated protein 4 (CKAP4), galectin-9 (GAL-9 or Gal-9), and APOE4 allele. Common etiological factors and common manifestations described in this review would aid in developing therapeutic strategies for both COVID-19 and AD and thus impact on eradicating the ongoing global threat. Thus, people suffering from COVID-19 or who have come round of it as well as people at risk of developing AD or already suffering from AD, would be benefitted.

Robustification of Linear Regression and Its Application in Genome-Wide Association Studies.

Regression analysis is one of the most popular statistical techniques that attempt to explore the relationships between a response (dependent) variable and one or more explanatory (independent) variables. To test the overall significance of regression, F-statistic is used if the parameters are estimated by the least-squares estimators (LSEs), while if the parameters are estimated by the maximum likelihood estimators (MLEs), the likelihood ratio test (LRT) statistic is used. However, both procedures produce misleading results and often fail to provide good fits to the reasonable space of the dataset in the presence of outlying observations. Moreover, outliers occur very frequently in any real datasets as well as in the molecular OMICS datasets. Hence, an effort is made in this study to robustify MLE based regression analysis by maximizing the ß-likelihood function. The tuning parameter ß is selected by cross-validation. For ß = 0, the proposed method reduces to the classical MLE based regression analysis. We inspect the performance of the proposed method using both synthetic and real data analysis. The results of simulations indicate that the proposed method performs better than traditional methods in both outliers and high leverage points to estimate the parameters and mean square errors. The results of relative efficiency analysis show that the proposed estimator is relatively less affected than the popular estimators, including S, MM, and fast-S for normal error distribution in case high dimension and outliers. Also, real data analysis results demonstrated that the proposed method shows robust properties with respect to data contaminations, overcome the drawback of the traditional methods. Genome-wide association studies (GWAS) by the proposed method identify the vital gene influencing hypertension and iron level in the liver and spleen of mice. Furthermore, we have identified 15 and 21 significant SNPs for chalkiness degree and chalkiness percentage, respectively, by GWAS based on the proposed method. The variant of the SNPs might be provided the new resources for grain quality traits and could be used for further molecular and physiological analysis to enhance the better quality of rice grain. These results offer an important basis for further understanding of the robust regression analysis, which might be applied in various fields, including business, genetics, and bioinformatics.

Gene Modules Co-regulated with Biosynthetic Gene Clusters for Allelopathy between Rice and Barnyardgrass.

Allelopathy is a central process in crop-weed interactions and is mediated by the release of allelochemicals that result in adverse growth effects on one or the other plant in the interaction. The genomic mechanism for the biosynthesis of many critical allelochemicals is unknown but may involve the clustering of non-homologous biosynthetic genes involved in their formation and regulatory gene modules involved in controlling the coordinated expression within these gene clusters. In this study, we used the transcriptomes from mono- or co-cultured rice and barnyardgrass to investigate the nature of the gene clusters and their regulatory gene modules involved in the allelopathic interactions of these two plants. In addition to the already known biosynthetic gene clusters in barnyardgrass we identified three potential new clusters including one for quercetin biosynthesis and potentially involved in allelopathic interaction with rice. Based on the construction of gene networks, we identified one gene regulatory module containing hub transcription factors, significantly positively co-regulated with both the momilactone A and phytocassane clusters in rice. In barnyardgrass, gene modules and hub genes co-expressed with the gene clusters responsible for 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) biosynthesis were also identified. In addition, we found three genes in barnyardgrass encoding indole-3-glycerolphosphate synthase that regulate the expression of the DIMBOA cluster. Our findings offer new insights into the regulatory mechanisms of biosynthetic gene clusters involved in allelopathic interactions between rice and barnyardgrass, and have potential implications in controlling weeds for crop protection.

Rice SDSFL1 plays a critical role in the regulation of plant structure through the control of different phytohormones and altered cell structure.

Semi-dwarfism is one of the most important agronomic traits for many cereal crops. In the present study, a mutant with semi-dwarf and short flag leaf 1, sdsfl1, was identified and characterized. The sdsfl1 mutant demonstrated some distinguished structural alterations, including shorter plant height and flag leaf length, increased tiller numbers and flag leaf width, and decreased panicle length compared with those of wild type (WT). Genetic analysis suggested that the mutant traits were completely controlled by a single recessive gene. The SDSFL1 gene was mapped to the long arm of chromosome 3 within a region of 44.6 kb between InDel markers A3P8.3 and A3P8.4. The DNA sequence analysis revealed that there was only a T to C substitution in the coding region of LOC_Os03g63970, resulting in the substitution of Tryptophan (Try) to Arginine (Arg) and encoding a GA 20 oxidase 1 protein of 372 amino acid residues. Photosynthesis analysis showed that the photosynthetic rate (Pn), stomatal conductance (Gs), and intercellular CO2 concentration (Ci) were significantly increased in sdsfl1. Chlorophyll a (Chl a), total Chl, and carotenoid contents were significantly increased in sdsfl1 compared with those in WT. sdsfl1 carried a reduced level of GA3 but reacted to exogenously applied gibberellins (GA). Moreover, the levels of abscisic acid (ABA), indole 3-acetic acid (IAA), and salicylic acid (SA) were notably improved in sdsfl1, whereas there was no noteworthy change in jasmonic acid (JA). The results thus offer a visible foundation for the molecular and physiological analysis of the SDSFL1 gene, which might participate in various functional pathways for controlling plant height and leaf length in rice breeding.

LTBSG1, a New Allele of BRD2, Regulates Panicle and Grain Development in Rice by Brassinosteroid Biosynthetic Pathway.

Panicle architecture and grain size are two important agronomic traits which determine grain yield directly in rice. In the present study, a mutant named ltbsg1 (longer top branch and shorter grain 1) was isolated from the cultivar “Zhenong 34” (Oryza sativa L. ssp. indica) by ethyl methane sulfonate (EMS) mutagenesis. The target gene was studied through phenotype observation, genetic analysis, map-based cloning and functional analysis. The histocytological analysis indicated that the elongated top branch and shorter grain of mutant ltbsg1 were caused from the defects of cell elongation. The ltbsg1 gene in mutant revealed a single nucleotide substitution (G-A) in the exon 2 of LOC_Os10g25780, causing an amino acid variation (Glycine-Arginine) in the FAD (Flavin-adenine dinucleotide)-binding domain of delta (24)-sterol reductase, which was involved in the brassinosteroid (BR) biosynthesis. LTBSG1 was constitutively expressed and the protein was widely localized in chloroplast, nucleus and cytomembrane. The ltbsg1 seedlings had a lower endogenous BR level and could be restored to the phenotype of wild type by exogenous BR. The LTBSG1 knock-out lines showed similar phenotype defects as mutant ltbsg1, which confirmed that LTBSG1 was responsible for top branch elongation and grain size reduction. Furthermore, LTBSG1 along with other BR-related genes were feedback-regulated due to their obvious altered expression in mutant ltbsg1. This study demonstrated that LTBSG1 could play a new role in regulating panicle and grain development by BR biosynthetic pathway.

The Brown Midrib Leaf (bml) Mutation in Rice (Oryza sativa L.) Causes Premature Leaf Senescence and the Induction of Defense Responses.

Isolating and characterizing mutants with altered senescence phenotypes is one of the ways to understand the molecular basis of leaf aging. Using ethyl methane sulfonate mutagenesis, a new rice (Oryza sativa) mutant, brown midrib leaf (bml), was isolated from the indica cultivar 'Zhenong34'. The bml mutants had brown midribs in their leaves and initiated senescence prematurely, at the onset of heading. The mutants had abnormal cells with degraded chloroplasts and contained less chlorophyll compared to the wild type (WT). The bml mutant showed excessive accumulation of reactive oxygen species (ROS), increased activities of superoxide dismutase, catalase, and malondialdehyde, upregulation of senescence-induced STAY-GREEN genes and senescence-related transcription factors, and down regulation of photosynthesis-related genes. The levels of abscisic acid (ABA) and jasmonic acid (JA) were increased in bml with the upregulation of some ABA and JA biosynthetic genes. In pathogen response, bml demonstrated higher resistance against Xanthomonas oryzae pv. oryzae and upregulation of four pathogenesis-related genes compared to the WT. A genetic study confirmed that the bml trait was caused by a single recessive nuclear gene (BML). A map-based cloning using insertion/deletion markers confirmed that BML was located in the 57.32kb interval between the L5IS7 and L5IS11 markers on the short arm of chromosome 5. A sequence analysis of the candidate region identified a 1 bp substitution (G to A) in the 5'-UTR (+98) of bml. BML is a candidate gene associated with leaf senescence, ROS regulation, and disease response, also involved in hormone signaling in rice. Therefore, this gene might be useful in marker-assisted backcrossing/gene editing to improve rice cultivars.

A guanine insert in OsBBS1 leads to early leaf senescence and salt stress sensitivity in rice (Oryza sativa L.).

KEY MESSAGE: A rice receptor-like kinase gene OSBBS1/OsRLCK109 was identified; this gene played vital roles in leaf senescence and the salt stress response. Early leaf senescence can cause negative effects on rice yield, but the underlying molecular regulation is not fully understood. bilateral blade senescence 1 (bbs1), an early leaf senescence mutant with a premature senescence phenotype that occurs mainly performing at the leaf margins, was isolated from a rice mutant population generated by ethylmethane sulfonate (EMS) treatment. The mutant showed premature leaf senescence beginning at the tillering stage and exhibited severe symptoms at the late grain-filling stage. bbs1 showed accelerated dark-induced leaf senescence. The OsBBS1 gene was cloned by a map-based cloning strategy, and a guanine (G) insertion was found in the first exon of LOC_Os03g24930. This gene encodes a receptor-like cytoplasmic kinase and was named OsRLCK109 in a previous study. Transgenic LOC_Os03g24930 knockout plants generated by a CRISPR/Cas9 strategy exhibited similar early leaf senescence phenotypes as did the bbs1 mutant, which confirmed that LOC_Os03g24930 was the OsBBS1 gene. OsBBS1/OsRLCK109 was expressed in all detected tissues and was predominantly expressed in the main vein region of mature leaves. The expression of OsBBS1 could be greatly induced by salt stress, and the bbs1 mutant exhibited hypersensitivity to salt stress. In conclusion, this is the first identification of OsRLCKs participating in leaf senescence and playing critical roles in the salt stress response in rice (Oryza sativa L.).

Rice gene SDL/RNRS1, encoding the small subunit of ribonucleotide reductase, is required for chlorophyll synthesis and plant growth development.

A new mutant named sdl (stripe and drooping leaf) was characterized from indica cultivar Zhenong 34 by ethylmethane sulfonate (EMS) mutagenesis. The mutant sdl exhibited development defects including stripe and drooping leaf, dwarfism and deformed floral organs. The gene SDL was found allelic to RNRS1 by map-based cloning, which was homologous to Arabidopsis TSO2 encoding the small subunit of ribonucleotide reductase. The gDNA sequencing results of sdl in mutant showed that there was a repetitive sequence insertion of 138-bp at the 475th bp in the exon. The redundant sequence was conserved in SDL homologous proteins, which contained the active site (tyrosine), as well as two amino acids glutamate and histidine involved in the binding of iron. There were fewer chloroplasts and grana lamellas in sdl leaf compared with those of wild-type. Additionally, the stripe leaves of sdl seedlings were highly sensitive to temperature, since the chlorophyll content was increased with the temperature rising. The drooping leaf of sdl might be resulted from the disappearance of vascular bundles and mesophyll cells in both leaf midrib and lateral veins. Fittingly to the phenotypes of mutant sdl, the expression levels of genes associated with photosynthesis and chlorophyll synthesis were found to be down- or up-regulated at different temperatures in mutant sdl. Also, the transcriptional levels of genes related to plant height and floral organ formation showed obvious differences between wild-type and sdl. The "SDL/RNRS1" was, hence, required for the chlorophyll biosynthesis and also played pleiotropic roles in the regulation of plant development.