Bile acids inhibit ferroptosis sensitivity through activating farnesoid X receptor in gastric cancer cells

BACKGROUND Gastric cancer(GC)is associated with high mortality rates.Bile acids(BAs)reflux is a well-known risk factor for GC,but the specific mechanism remains unclear.During GC development in both humans and animals,BAs serve as signaling molecules that induce metabolic reprogramming.This confers additional cancer phenotypes,including ferroptosis sensitivity.Ferroptosis is a novel mode of cell death characterized by lipid peroxidation that contributes universally to malignant progression.However,it is not fully defined if BAs can influence GC progression by modulating ferroptosis.AIM To reveal the mechanism of BAs regulation in ferroptosis of GC cells.METHODS In this study,we treated GC cells with various stimuli and evaluated the effect of BAs on the sensitivity to ferroptosis.We used gain and loss of function assays to examine the impacts of farnesoid X receptor(FXR)and BTB and CNC homology 1(BACH1)overexpression and knockdown to obtain further insights into the molecular mechanism involved.RESULTS Our data suggested that BAs could reverse erastin-induced ferroptosis in GC cells.This effect correlated with increased glutathione(GSH)concentrations,a reduced GSH to oxidized GSH ratio,and higher GSH peroxidase 4(GPX4)expression levels.Subsequently,we confirmed that BAs exerted these effects by activating FXR,which markedly increased the expression of GSH synthetase and GPX4.Notably,BACH1 was detected as an essential intermediate molecule in the promotion of GSH synthesis by BAs and FXR.Finally,our results suggested that FXR could significantly promote GC cell proliferation,which may be closely related to its anti-ferroptosis effect.CONCLUSION This study revealed for the first time that BAs could inhibit ferroptosis sensitivity through the FXR-BACH1-GSHGPX4 axis in GC cells.This work provided new insights into the mechanism associated with BA-mediated promotion of GC and may help identify potential therapeutic targets for GC patients with BAs reflux.

Natural variation in SlSOS2 promoter hinders salt resistance during tomato domestication

Increasing soil salinization seriously impairs plant growth and development,resulting in crop loss.The Salt-Overly-Sensitive(SOS)pathway is indispensable to the mitigation of Na+toxicity in plants under high salinity.However,whether natural variations of SOS2 contribute to salt tolerance has not been reported.Here a natural variation in the SlSOS2 promoter region was identified to be associated with root Na+/K+ratio and the loss of salt resistance during tomato domestication.This natural variation contains an ABI4-binding cis-element and plays an important role in the repression of SlSOS2 expression.Genetic evidence revealed that SlSOS2 mutations increase root Na+/K+ratio under salt stress conditions and thus attenuate salt resistance in tomato.Together,our findings uncovered a critical but previously unknown natural variation of SOS2 in salt resistance,which provides valuable natural resources for genetic breeding for salt resistance in cultivated tomatoes and other crops.

Sleeve gastrectomy ameliorates endothelial function and prevents lung cancer by normalizing endothelin-1 axis in obese and diabetic rats

BACKGROUND Previous evidence has implied that obesity is an independent risk factor for developing cancer.Being closely related to obesity,type 2 diabetes mellitus provides a suitable environment for the formation and metastasis of tumors through multiple pathways.Although bariatric surgeries are effective in preventing and lowering the risk of various types of cancer,the underlying mechanisms of this effect are not clearly elucidated.AIM To uncover the role and effect of sleeve gastrectomy(SG)in preventing lung cancer in obese and diabetic rats.METHODS SG was performed on obese and diabetic Wistar rats,and the postoperative transcriptional and translational alterations of the endothelin-1(ET-1)axis in the lungs were compared to sham-operated obese and diabetic rats and age-matched healthy controls to assess the improvements in endothelial function and risk of developing lung cancer at the postoperative 4 th,8 th,and 12 th weeks.The risk wasalso evaluated using nuclear phosphorylation of H2 A histone family member X as a marker of DNA damage(double-strand break).RESULTS Compared to obese and diabetic sham-operated rats,SG brought a significant reduction to body weight,food intake,and fasting blood glucose while improving oral glucose tolerance and insulin sensitivity.In addition,ameliorated levels of gene and protein expression in the ET-1 axis as well as reduced DNA damage indicated improved endothelial function and a lower risk of developing lung cancer after the surgery.CONCLUSION Apart from eliminating metabolic disorders,SG improves endothelial function and plays a protective role in preventing lung cancer via normalized ET-1 axis and reduced DNA damage.

Breeding exceptionally fragrant soybeans for soy milk with strong aroma

Soybean(Glycine max(L.)Merr.)is a major source of vegetable protein and oil in human diet and animal nutrition.Soybean seeds have been extensively used in various food products and snacks.Taste quality,particularly the aroma,affects cooking and eating,and ultimately influences consumer preference.Soy milk is particularly popular in China and has been gaining popularity in many other countries in the world.

An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing.However,natural nucleases of this system often exhibit low efficiency,limiting their application.Here,we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease,Cas12i3.As a result,we developed Cas-SF01,a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells.Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases.Compared to natural Cas12i3,Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs.In addition,we identified an amino acid substitution,D876R,that markedly reduced the off-target effect while maintaining high on-target activity,leading to the development of CasSF01^(HiFi)(high-fidelity Cas-SF01).Finally,we show that Cas-SF01 has high gene editing activities in mice and plants.Our results suggest that CasSF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

Simultaneous mutations in ITPK4 and MRP5 genes result in a low phytic acid level without compromising salt tolerance in Arabidopsis

Generation of crops with low phytic acid(myoinositol-1,2,3,4,5,6-hexakisphosphate(InsP6))is an important breeding direction,but such plants often display less desirable agronomic traits.In this study,through ethyl methanesulfonate-mediated mutagenesis,we found that inositol 1,3,4-trisphosphate5/6-kinase 4(ITPK4),which is essential for producing InsP6,is a critical regulator of salt tolerance in Arabidopsis.Loss of function of ITPK4 gene leads to reduced root elongation under salt stress,which is primarily because of decreased root meristem length and reduced meristematic cell number.The itpk4 mutation also results in increased root hair density and increased accumulation of reactive oxygen species during salt exposure.RNA sequencing assay reveals that several auxin-responsive genes are down-regulated in the itpk4-1 mutant compared to the wild-type.Consistently,the itpk4-1 mutant exhibits a reduced auxin level in the root tip and displays compromised gravity response,indicating that ITPK4 is involved in the regulation of the auxin signaling pathway.Through suppressor screening,it was found that mutation of Multidrug Resistance Protein 5(MRP5)5 gene,which encodes an ATP-binding cassette(ABC)transporter required for transporting InsP6from the cytoplasm into the vacuole,fully rescues the salt hypersensitivity of the itpk4-1 mutant,but in the itpk4-1 mrp5 double mutant,InsP6remains at a very low level.These results imply that InsP6homeostasis rather than its overall amount is beneficial for stress tolerance in plants.Collectively,this study uncovers a pair of gene mutations that confer low InsP6content without impacting stress tolerance,which offers a new strategy for creating“low-phytate”crops.

Efficient and multiplex gene upregulation in plants through CRISPR-Cas-mediated knockin of enhancers

Geneupregulation through genome editing is important for plant research and breeding.Targeted insertion of short transcriptional enhancers(STEs)into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis.Here,we introduce an“in locus activation”technique in rice that leverages well-characterized STEs for refined,heritable,and multiplexed gene upregulation.To address the scarcity of potent enhancers,we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts.The in locus integration of these STEs into eight rice genes resulted in substantial tran-scriptional upregulation in the edited plants,with up to 869.1-fold increases in their transcript levels.Em-ploying a variety of STEs,we achieved delicate control of gene expression,enabling the fine-tuning of key phenotypic traits such as plant height.Our approach also enabled efficient multiplexed gene upregu-lation,with up to four genes activated simultaneously,significantly enhancing the nicotinamide mononucleotide metabolic pathway.Importantly,heritability studies from the To to T3 generations confirmed the stable and heritable nature of STE-driven gene activation.Collectively,our work demon-strates that coupled with STE mining,leveraging genome editing for in locus activation and gene upregu-lation holds great promise to be widely adopted in fundamental plant research and crop breeding.

Targeted G-to-T base editing for generation of novel herbicide-resistance gene alleles in rice

Deaminase-based cytosine base editors(CBEs)and adenine base editors(ABEs)induce C-to-T and A-to-G transitions,respectively,enabling single-nucleotide variants(SNVs)in plants for research and crop enhancement(Li et al.,2023a).The C-to-G base editors(CGBEs)and A-to-Y base editors(AYBEs),developed by incorporating glycosylases with CBEs and ABEs,expand the repertoire of base editing products,allowing C-to-T/G and A-to-T/G transversions in plants(Li et al.,2023b,2023c;Sretenovic et a.,2021).

Rapid and dynamic detection of endogenous proteins through in locus tagging in rice

Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remains unexplored in plants. Here, we introduce the in locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, a result attributable to low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence up to 6.9 fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in situ visualization using a cross-breeding strategy, and effective immunoprecipitation for analysis of protein interactions. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture

Of the more than 370000 species of higher plants in nature,fewer than 0.1%can be geneticallymodified due to limitations of the current gene delivery systems.Even for those that can be genetically modified,the modification involves a tedious and costly tissue culture process.Here,we describe an extremely simple cut-dip-budding(CDB)delivery system,which uses Agrobacterium rhizogene to inoculate explants,generating transformed roots that produce transformed buds due to root suckering.We have successfully used CDB to achieve the heritable transformation of plant species inmultiple plant families,including two herbaceous plants(Taraxacum kok-saghyz and Coronilla varia),a tuberous root plant(sweet potato),and three woody plant species(Ailanthus altissima,Aralia elata,and Clerodendrum chinense).These plants have previously been difficult or impossible to transform,but the CDB method enabled efficient transformation or gene editing in them using a very simple explant dipping protocol,under non-sterile conditions and without the need for tissue culture.Our work suggests that large numbers of plants could be amenable to genetic modifications using the CDB method.

High-throughput genome editing in rice with a virus-based surrogate system

With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate(WDV-surrogate), to facilitate high-throughput genome editing.WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene(HygM) for surrogate selection;the other is a WDV-based construct expressing two single guide RNAs(sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T_0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency(66.8% vs. 90.1%), plantlet regeneration efficiency(2.31-fold increase), and numbers of homozygous-edited plants(36.3%vs. 70.7%). Large-scale editing using pooled sg RNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDVGate on sequence knock-in for protein tagging.By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

Orthogroup and phylotranscriptomic analyses identify transcription factors involved in the plant cold response:Acase study of Arabidopsis BBX29

C-repeat binding factors(CBFs)are well-known transcription factors(TFs)that regulate plant cold acclimation.RNA sequencing(RNA-seq)data from diverse plant species provide opportunities to identify other TFs involved in the cold response.However,this task is challenging because gene gain and loss has led to an intertwined community of co-orthologs and in-paralogs between and within species.Using orthogroup(closely related homologs)analysis,we identified 10,549 orthogroups in five representative eudicots.A phylotranscriptomic analysis of cold-treated seedlings from eudicots identified 35 high-confidence conserved cold-responsive transcription factor orthogroups(CoCoFos).These 35 CoCoFos included the well-known cold-responsive regulators CBFs,HSFC1,ZAT6/10,and CZF1 among others.We used Arabidopsis BBX29 for experimental validation.Expression and genetic analyses showed that cold-induction of BBX29 is CBF-and abscisic acid-independent,and BBX29 is a negative regulator of cold tolerance.Integrative RNA-seq and Cleavage Under Targets and Tagmentation followed by sequencing analyses revealed that BBX29 represses a set of cold-induced TFs(ZAT12,PRR9,RVE1,MYB96,etc.).Altogether,our analysis yielded a library of eudicot CoCoFos and demonstrated that BBX29 is a negative regulator of cold tolerance in Arabidopsis.

AtMCM10 promotes DNA replication-coupled nucleosome assembly in Arabidopsis

Minichromosome Maintenance protein 10(MCM10)is essential for DNA replication initiation and DNA elongation in yeasts and animals.Although the functions of MCM10 in DNA replication and repair have been well documented,the detailed mechanisms for MCM10 in these processes are not well known.Here,we identified AtMCM10 gene through a forward genetic screening for releasing a silenced marker gene.Although plant MCM10 possesses a similar crystal structure as animal MCM10,AtMCM10 is not essential for plant growth or development in Arabidopsis.AtMCM10 can directly bind to histone H3-H4 and promotes nucleosome assembly in vitro.The nucleosome density is decreased in Atmcm10,and most of the nucleosome density decreased regions in Atmcm10 are also regulated by newly synthesized histone chaperone Chromatin Assembly Factor-1(CAF-1).Loss of both AtMCM10 and CAF-1 is embryo lethal,indicating that AtM CM10 and CAF-1 are indispensable for replication-coupled nucleosome assembly.AtMCM10 interacts with both new and parental histones.Atmcm10 mutants have lower H3.1abundance and reduced H3K27me1/3 levels with releasing some silenced transposons.We propose that AtM CM10 deposits new and parental histones during nucleosome assembly,maintaining proper epigenetic modifications and genome stability during DNA replication.

The Sm core protein SmEb regulates salt stress responses through maintaining proper splicing of RCD1 pre-mRNA in Arabidopsis

Salt stress adversely impacts crop production.Several spliceosome components have been implicated in regulating salt stress responses in plants,however,the underlying molecular basis is still unclear.Here we report that the spliceosomal core protein SmEb is essential to salt tolerance in Arabidopsis.Transcriptome analysis showed that SmEb modulates alternative splicing of hundreds of pre-mRNAs in plant response to salt stress.Further study revealed that SmEb is crucial in maintaining proper ratio of two RCD1 splicing variants(RCD1.1/RCD1.2)important for salt stress response.In addition,RCD1.1 but not RCD1.2 is able to interact with the stress regulators and attenuates saltsensitivity by decreasing salt-induced cell death in smeb-1 mutant.Together,our findings uncovered the essential role of SmEb in the regulation of alternative pre-mRNA splicing in salt stress response.

Liquid-liquid phase separation as a major mechanism of plant abiotic stress sensing and responses

Identification of environmental stress sensors is one of the most important research topics in plant abiotic stress research.Traditional strategies to identify stress sensors or early signaling components based on the cell membrane as a primary site of sensing and calcium signal as a second messenger have had only limited successes.Therefore,the current theoretical framework underlying stress sensing in plants should be reconsidered and additional mechanisms need to be introduced.Recently,accumulating evidence has emerged to suggest that liquid-liquid phase separation(LLPS)is a major mechanism for environmental stress sensing and response in plants.In this review,we briefly introduce LLPS regarding its concept,compositions,and dynamics,and then summarize recent progress of LLPS research in plants,emphasizing the contribution of LLPS to the sensing of various environmental stresses,such as dehydration,osmotic stress,and low and high temperatures.Finally,we propose strategies to identify key proteins that sense and respond to environmental stimuli on the basis of LLPS,and discuss the research directions of LLPS in plant abiotic stress responses and its potential application in enhancing stress tolerance in crops.

Mitigating cadmium accumulation in rice without compromising growth via modifying the regulatory region of OsNRAMP5

Cadmium(Cd)intake poses a significant health risk to humans,and the contamination of rice grains with Cd is a major concern in regions where rice is a staple food.Although the knockout of OsNRAMP5,which encodes a key transporter responsible for Cd and manganese(Mn)uptake,can significantly reduce Cd accumulation in rice grains,recent studies have revealed that this knockout adversely affects plant growth,grain yield,and increases vulnerability to abiotic and biotic stresses due to reduced Mn accumulation.In this study,we employed CRISPR/Cas9 technology to modify the regulatory region of OsNRAMP5 with the aim of reducing Cd accumulation in rice grains.Our findings demonstrate that mutations in the regulatory region of OsNRAMP5 do not impact its expression pattern but result in a reduction in translation.The decreased translation of OsNRAMP5 effectively decreases grain Cd accumulation while leaving Mn accumulation and important agronomic traits,including yield,unaffected.Thus,our study presents a practical and viable strategy for reducing Cd accumulation in rice grains without compromising Mn accumulation or overall rice production.

Plant abiotic stress response and nutrient use efficiency

Abiotic stresses and soil nutrient limitations are major environmental conditions that reduce plant growth,productivity and quality.Plants have evolved mechanisms to perceive these environmental challenges,transmit the stress signals within cells as well as between cells and tissues,and make appropriate adjustments in their growth and development in order to survive and reproduce.In recent years,significant progress has been made on many fronts of the stress signaling research,particularly in understanding the downstream signaling events that culminate at the activation of stress-and nutrient limitation-responsive genes,cellular ion homeostasis,and growth adjustment.However,the revelation of the early events of stress signaling,particularly the identification of primary stress sensors,still lags behind.In this review,we summarize recent work on the genetic and molecular mechanisms of plant abiotic stress and nutrient limitation sensing and signaling and discuss new directions for future studies.

Abscisic Acid-mediated Epigenetic Processes in Plant Development and Stress Responses

Abscisic acid （ABA） regulates diverse plant processes, growth and development under non-stress conditions and plays a pivotal role in abiotic stress tolerance. Although ABA-regulated genetic processes are well known, recent discoveries reveal that epigenetic processes are an integral part of ABA-regulated processes. Epigenetic mechanisms, namely, histone modifications and cytosine DNA methylation-induced modification of genome give rise to epigenomes, which add diversity and complexity to the genome of organisms. Histone monoubiquitination appears to regulate ABA levels in developing seeds through histone H2B monoubiquitination. ABA and H2B ubiquitination dependent chromatin remodeling regulate seed dormancy. Transcription factor networks necessary for seed maturation are repressed by histone deacetylases （HDACs）-dependent and PICKLE chromatin remodeling complexes （CRCs）, whereas ABA induces the expression of these genes directly or through repression of HDACs. Abiotic stress-induced ABA regulates stomatal response and stress- responsive gene expression through HDACs and HOS15-dependent histone deacetylation, as well as through the ATP- dependent SWITCH/SUCROSE NONFERMENTING CRC. ABA also probably regulates the abiotic stress response through DNA methylation and short interfering RNA pathways. Further studies on ABA-regulated epigenome will be of immense use to understand the plant development, stress adaptation and stress memory.

Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences

CRISPR/Cas9 genome editing relies on sgRNA-target DNA base pairing and a short downstream PAM sequence to recognize target DNA. The strict protospacer adjacent motif (PAM) requirement hinders applications of the CRISPR/Cas9 system since it restricts the targetable sites in the genomes. xCas9 and SpCas9-NG are two recently engineered SpCas9 variants that can recognize more relaxed NG PAMs, implying a great potential in addressing the issue of PAM constraint. Here we use stable transgenic lines to evaluate the efficacies of xCas9 and SpCas9-NG in performing gene editing and base editing in rice. We found that xCas9 can efficiently induce mutations at target sites with NG and GAT PAM sequences in rice. However, base editors containing xCas9 failed to edit most of the tested target sites. SpCas9-NG exhibited a robust editing activity at sites with various NG PAMs without showing any preference for the third nucleotide after NG. Moreover, we showed that xCas9 and SpCas9-NG have higher specificity than SpCas9 at the CGG PAM site. We further demonstrated that different forms of cytosine or adenine base editors containing SpCas9-NG worked efficiently in rice with broadened PAM compatibility. Taken together, our work has yielded versatile genome-engineering tools that will significantly expand the target scope in rice and other crops.

Abscisic acid dynamics, signaling, and functions in plants

Abscisic acid(ABA)is an important phytohormone regulating plant growth,development,and stress responses.It has an essential role in multiple physiological processes of plants,such as stomatal closure,cuticular wax accumulation,leaf senescence,bud dormancy,seed germination,osmotic regulation,and growth inhibition among many others.Abscisic acid controls downstream responses to abiotic and biotic environmental changes through both transcriptional and posttranscriptional mechanisms.During the past 20 years,ABA biosynthesis and many of its signaling pathways have been well characterized.Here we review the dynamics of ABA metabolic pools and signaling that affects many of its physiological functions.