The velvet protein family plays a key factor in coordinating development and secondary metabolism in many pathogenic fungi. However, no previous research has investigated the function of the velvet protein family in Fusarium oxysporum f. sp. Niveum (FON), which causes a highly destructive disease on watermelon. In this study, ∆fovel1 and ∆folae1 deletion mutants and ∆fovel1-C and ∆folae1-C corresponding complementation mutants of FON were confirmed. Meanwhile, effects of phenotype, biochemistry and virulence of the deletion mutants were protected. Compared with the wild-type strains, the ∆fovel1 and ∆folae1 mutants showed different mycelia phenotype, depressed of conidiation and reduced production of bikaverin and fusaric acid. Moreover, their virulence on watermelon plant roots was significant decreased. In addition, all of these alterations in mutants were restored in corresponding complementation strains. Importantly, yeast two hybrid results indicated an interaction relationship between FoVel1 and FoLae1. The results of this study indicated that FoVEL1 and FoLAE1 play critical roles in secondary metabolisms, conidiation, and virulence in FON. These information will deepen our understanding on the genetic and functional roles of the VEL1 and LAE1 in pathogenic fungi.

Melon (Cucumis melo) is an important economic horticulture crop cultivated worldwide. NAC (NAM, ATAC, and CUC) transcription factors play crucial roles in the transcriptional regulation of various developmental stages in plant growth and fruit development, but little about their gene function is known in melon. Here, we identified 78 CmNAC family genes containing integrated and conserved NAM (no apical meristem) domain in the melon genome by performing genome-wide identification and bioinformatics analysis. Transcriptome data analysis and qRT-PCR results showed that most CmNACs are specifically enriched in the vegetative organ or the reproductive organ in melon. Through genetic transformation, we found that overexpression of CmNAC34 in melons led to the early ripening fruits, suggesting its positive role in promoting fruit maturation. Through performing yeast two-hybrid and bimolecular fluorescence complementation assays, we verified the direct protein interaction between CmNAC34 and CmNAC-NOR. The expression pattern of CmNAC34 and CmNAC-NOR were similar in melon tissues, and subcellular localization also revealed their nuclear protein characteristic. We transformed CmNAC-NOR in melon and found that its overexpression resulted in the early ripening fruits. Then, the yeast one-hybrid and dual luciferase reporter gene assay explored that CmNAC34 protein can bind to the promoters of two Glyoxalase (GLY) genes, which were involved in the abscisic acid signal pathway and associated with the fruit regulation. These findings revealed the molecular characteristics, expression profile, and functional pattern of the NAC family transcription factors genes and provided an insight into the molecular mechanism of CmNAC34 in regulating climacteric fruit ripening.

A few Trichoderma species have been utilized as biocontrol agents in agriculture due to their ability to inhibit growth of phytopathogens. However, the antagonistic mechanism of some strains is mainly performed by direct action. The objective of our study is to explore an effective strain that has comprehensive abilities, and preliminarily clarify its practical viability and action mechanism. Trichoderma gamsii strain TC959 possessing abilities of strong antagonism and plant growth promotion was singled out. It released secondary metabolites, siderophores and chitinase/xylanase to directly inhibit the growth of plant pathogens, or released indole-3-acetic acid/gibberellin to promote plant growth. The strain also activated induced systemic resistance by increasing chlorophyll a/b ratio and jasmonic acid content of pepper seedlings through root colonization, which resulted in the improvements of defense-related gene expression levels, antioxidant enzyme activity, and indole-3-acetic acid/gibberellin production. Thereby disease resistance and plant growth were enhanced and promoted, respectively. Furthermore, TC959 had a resistance advantage to oxidation and chemical fungicides, which helped viability of the strain to be maintained, and healthy pepper seedlings were effectively ensured. In conclusion, strain TC959 has biocontrol potential and comprehensive functions against pepper damping-off disease, which is valuable for further practical applications.

Malate dehydrogenase (MDH) is a widely expressed enzyme that plays a key role in plant growth, development, and the stress response.  However, information on MDH genes in the soybean genome is limited. Seventeen members of the soybean MDH family were identified by genome-wide analysis, and the presence of conserved protein motifs was analyzed.  The genes were divided into five clusters according to their phylogenetic relationships.  The intracellular localizations of six GmMDHs were determined by confocal microscopy on Arabidopsis mesophyll protoplasts.  Transcripts of GmMDHs were significantly increased by abiotic stress (drought, salt, and alkalinity) and hormone treatments, as shown by analysis of cis-regulatory elements and quantitative real-time polymerase chain reaction (qRT-PCR).  GmMDHs displayed unique expression patterns in diverse soybean tissues.  It is noteworthy that under salt stress, the expression levels of a chloroplast isoform (GmMDH2) were unusually high, presumably indicating a critical role in soybean responses to salinity.  Expression of GmMDH2 in Escherichia coli showed that the recombinant enzyme had NADP-dependent MDH activity. The redox states of the nicotinamide adenine dinucleotide phosphate (NADPH) pool and antioxidant activities were shown to be modulated by GmMDH2 gene overexpression, which in turn reduced reactive oxygen species (ROS) formation in transgenic soybean, significantly enhancing the salt stress resistance.  Gene-based association analysis showed that variations in GmMDH2 were strongly linked to seedling salt tolerance.  A polymorphism possibly associated with salt tolerance was discovered in the promoter region of GmMDH2.  These findings not only improve our understanding of the stress response mechanism by identifying and characterizing the MDH gene family throughout the soybean genome but it also identified a potential candidate gene for the future enchancement of salt tolerance in the soybean.

In this study, we report the discovery of a novel bocaparvovirus, identified through viral metagenomic analysis of fecal samples from goats presenting with diarrhea. The complete genomic sequence of this virus shows the highest identity with the ECBOV-tdf70 strain, which was found in the wild animal Elaphodus cephalophus. Its NS1 protein shares 91.2% amino acid identity with the ECBOV-tdf70 strain. According to ICTV criteria, this strain should be classified as same species along with the ECBOV-tdf70 strain. The positive rate of diarrheal fecal samples versus non-diarrheal samples indicates the potential role of viruses in goat diarrhea. The complete VP1 genes of the five strains obtained in this study shared 74.4 - 99.2% nucleotide identity, and 63.7 - 99.1% amino acid identity. This study represents the first report of Bocaparvovirus infection in goats, providing valuable insights into the epidemiology and genetic diversity of the virus.

Root-knot nematodes (RKNs) are the most widespread soil-borne obligate endoparasites. They can infect the roots of many crops and cause significant yield losses. In tomato, the only commercially available RKN resistant gene Mi-1.2 fails at soil temperatures above 28°C. We cloned the heat stable RKN-resistant gene Mi-9 from a gene cluster composed of seven nucleotide-binding site and leucine-rich repeat (NBS-LRR) type resistant genes in Solanum arcunum accession LA2157. Screening nematode infections in individual & combinatorial knockouts of five NBS-LRR genes showed that Mi-9 Candidate 4 (MiC-4) alone is sufficient to confer heat stable RKN resistance. Our study identifies a new source of heat stable resistance to RKN in tomato for challenging environmental conditions. We also showcase a roadmap for rapid characterization of resistance genes by combining comparative genomics and genome editing, with the potential to be utilized in other crops.

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

Plant height (PH), primary lateral branch length (PBL) and branch number (BN) are architectural components impacting peanut pod yield, biomass production and adaptivity to mechanical harvesting.  In this study, a recombinant inbred population consisting of 181 individual lines was used to determine genetic controls of PH, PBL and BN across three environments.  Phenotypic data collected from the population demonstrated continuous distributions and transgressive segregation patterns.  Broad-sense heritability of PH, PBL and BN was found to be 0.87, 0.88 and 0.92, respectively.  Unconditional individual environmental analysis revealed 35 additive QTLs with phenotypic variation explained (PVE) ranging from 4.57 to 21.68%.  A two-round meta-analysis resulted in 24 consensus and 17 unique QTLs.  Five unique QTLs exhibited pleiotropic effects and their genetic bases (pleiotropy or tight linkage) were evaluated.  Joint analysis was performed to estimate the QTL by environment interaction (QEI) effects on PH, PBL and BN, which collectively explained phenotypic variations of 10.80, 11.02, and 7.89%, respectively.  We identified 3 major and stable QTL regions (uq9-3, uq10-2 and uq16-1) on chromosomes 9, 10 and 16, spanning 1.43-1.53 Mb genomic regions.  Candidate genes involved in phytohormones biosynthesis, signaling and cell wall development were proposed to regulate these morphological traits.  These results provide valuable information for further genetic studies and development of molecular markers applicable for peanut architecture improvement.

The impact of drought stress on crop yield and quality is substantial.  Drought priming during the early growth stage of plants has been shown to improve tolerance to drought stress during the reproductive stage, although its effects on grain quality remains elusive.  This study aimed to investigate the influence of drought priming on starch and protein levels in grains under drought stress during grain filling.  Our results reveal that drought stress results in a reduction in starch content and its constituents, while simultaneously increasing glutenin macropolymers and protein fractons.  Notably,, drought primed plants under drought stress (PD) exhibit mitigated declines in starch content and its components, leading to improvements in starch swelling power and pasting properties.  Additionally, PD results in a slight increase in protein fractions, limiting the overall rise in total protein content compared to drought stress alone.  Collectively, our study underscores the efficacyof drought priming as a strategy to counteract the negative effects of drought stress on grain quality, particularly by minimizing starch losses and restraining protein content elevation.

Planting density is a major limiting factor for maize yield, and breeding for density tolerance breeding has become an urgent issue.  The leaf structure of the maize ear leaf is the main factor that restricts planting density and yield composition.  In this study, a natural population of 201 maize inbred lines was used for genome-wide association analysis, which identified nine SNPs on chromosomes 2, 5, 8, 9, and 10 that were significantly associated with ear leaf type structure.  Further verification through qRT-PCR confirmed the association of five candidate genes with these SNPs, with the Zm00001d008651 gene showing significant differential expression in compact and flat maize inbred lines.  Enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) suggested that this gene is involved in the glycolysis process.  The analysis of the basic properties of this gene revealed that it encodes a stable, basic protein consisting of 593 amino acids with some hydrophobic ability.  The promoter region contains stress and hormone (ABA) related elements.  The mutant of this gene increased the uppermost ear leaf angle (eLA) and the first leaf below the uppermost ear (bLA) by 4.96° and 0.97° compared with normal inbred lines.  Overall, this research sheds light on the regulatory mechanism of ear and leaf structure that influence density tolerance and provides solid foundational work for the development of new varieties.

Maize is a cornerstone of global food security, but it faces increasing challenges from corn aphids, particularly with the widespread adoption of genetically modified Bt maize.  This trend suggests a growing need for sustainable pest control strategies. Methyl salicylate has been proposed as a volatile compound with the potential for managing aphids.  In this study, Y-tube olfactometer and Petri dish dispersal assays showed that methyl salicylate can repel wingless and winged aphids at 0.1 to 1,000 ng μL^–1.  Moreover, at concentrations of 100 and 1,000 ng μL^–1, it was found to attract beneficial insects such as adults and larvae of Harmonia axyridis.  Exposing maize plants to methyl salicylate resulted in a prominent reduction in the number of aphids compared to the control.  In addition, clip cage experiment assays showed that the nymphal development duration was increased, while the adult duration and generation time were reduced, and the reproductive duration and total number of aphid offspring in plants treated with methyl salicylate were dramatically lower than in the control.  Over two years of field trials, methyl salicylate-impregnated alginate beads provided significant reductions in the populations of key aphid species, including Rhopalosiphum padi, Rhopalosiphum maidis, and Aphis gossypii.  Concurrently, there were marked increases in the presence of natural predators such as H. axyridis, Propylaea japonica, Syrphus corollae, and Chrysoperla sinica.  These compelling results underscore the potential of methyl salicylate as a key component in integrated pest management strategies for maize, offering a green alternative to traditional chemical control.

Soluble solids content (SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC has been shown to be transcriptionally regulated via sugar metabolism. Previous studies have been predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanism of C2H2 in the accumulation of soluble solids in tomato fruits are yet to be fully understood. This study involved the selection of eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits. The study found that the levels of SlC2H2-71 expressions were significantly reduced in high-SSC accessions compared to low-SSC accessions. The Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, leading to elevated levels of soluble solids, fructose, glucose, malic, and citric acids in mature red ripe fruits. However, sucrose content in the edited Slc2h2-71 mutant lines generally decreased. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to sugar and acid metabolic pathways, which was further confirmed by quantitative real time PCR. Specifically, there was an observed increase in the expression of SlLIN5 encoding the cell wall invertase (CWIN). The yeast one-hybrid (Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay (EMSA) demonstrated that SlC2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of SlLIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase in the SlC2H2-71 knock-out lines exhibited a significantly higher level compared to that observed in the control lines. In summary, the regulation of tomato fruit SSC by SlC2H2-71 involves the inhibition of SlLIN5 expression.

Cattle carcass traits are economically important in the beef industry. In the present study, we identified 184 significant genes and 822 alternative genes for 7 carcass traits using genome-wide association studies (GWASs) in 1566 Huaxi beef cattle. We then identified 5,860 unique cis-genes and 734 trans-genes in 227 longissimus dorsi muscle (LDM) samples to better understand the genetic regulation of gene expression. Our integration study of the GWAS and cis-eQTL analysis detected 13 variants regulating 12 identical genes, in which one variant was also detected in fine-mapping analysis. Moreover, using a transcriptome-wide association study (TWAS), we identified 4 genes (TTC30B, HMGA1, PRKD3 and FXN) that were significantly related to carcass chest depth (CCD), carcass length (CL), carcass weight (CW) and dressing percentage (DP). This study identified variants and genes that may be useful for understanding the molecular mechanism of carcass traits in beef cattle.

Leaves and glumes act as lateral organs and have essential effects on photosynthesis and seed morphology, thus affecting yield.  However, the molecular mechanisms controlling their polarity development in rice still need further study.  Here, we isolated a polarity defect of lateral organs1 (pdl1) mutant in rice, which exhibits twisted/filamentous-shaped leaves and cracked/filamentous-shaped lemmas caused by defects in polarity development.  PDL1 encodes a SUPPRESSOR OF GENE SILENCING 3 protein localized in the cytoplasmic granules.  PDL1 is expressed in the shoot apical meristem, inflorescence meristem, floral meristem, and lateral organs including leaves and floral organs.  PDL1 is involved in the synthesis of tasiR-ARF, which may subsequently modulate the expression of OsARFs.  Meanwhile, the expression levels of abaxial miR165/166 and the adaxial identity genes OSHBs were respectively increased and reduced significantly.  The results of this study clarify the molecular mechanism by which PDL1-mediated tasiR-ARF synthesis regulates the lateral organ polarity development in rice.

Deep learning (DL) methods like multilayer perceptrons (MLPs) and convolutional neural networks (CNNs) have been applied to predict the complex traits in animal and plant breeding.  However, improving the genomic prediction accuracy still presents significant challenges.  In this study, we applied CNNs to predict swine traits using previously published data.  Specifically, we extensively evaluated the CNN model’s performance by employing various sets of single nucleotide polymorphisms (SNPs) and concluded that the CNN model achieved optimal performance when utilizing SNP sets comprising 1,000 SNPs.  Furthermore, we adopted a novel approach using the one-hot encoding method that transforms the 16 different genotypes into sets of eight binary variables.  This innovative encoding method significantly enhanced the CNN’s prediction accuracy for swine traits, outperforming the traditional one-hot encoding techniques.  Our findings suggest that the expanded one-hot encoding method can improve the accuracy of DL methods in the genomic prediction of swine agricultural economic traits.  This discovery has significant implications for swine breeding programs, where genomic prediction is pivotal in improving breeding strategies.  Furthermore, future research endeavors can explore additional enhancements to DL methods by incorporating advanced data pre-processing techniques. 

Pluripotent stem cells (PSCs) are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual. Pigs are one of the most important domestic ungulates, commonly used for food and as bioreactors. Generating stable pluripotent porcine PSC lines remains challenging. So far, the pluripotency gene network of porcine PSCs is poorly understood. Here we found that TBX3-derived induced pluripotent stem cells (iPSCs) closely resemble porcine 4-cell embryos with the capacity of totipotent-like stem cells (TLSCs). Interestingly, our data suggest that TBX3 facilitates the activation of H3K4me3 methyltransferase, specifically MLL1. Subsequent investigations revealed that the porcine 4-cell specific gene, MCL1, is a key downstream effector of the TBX3-MLL1 axis. Together, our study of the TBX3 regulatory network is helpful in the understanding of the totipotency characteristics of pigs.

Due to their sessile nature, plants require strong adaptability to complex environments, with stress tolerance often associated with trade-offs in growth and development (Major et al. 2020).  This antagonistic relationship between defense and growth has been interpreted as a competition for limited resources that are allocated to defense at the expense of growth, or vice versa. Recent studies have demonstrated that hormone-based signaling networks trigger transcriptional changes in key genes, leading to trade-offs between growth rates and stress defense (Huot et al. 2014).  Several genes involved in biotic and abiotic stress response have been identified.  These genes contain nonsynonymous variants that show convergent changes in allele frequency across different breeding eras in both China and the United States (Wang et al. 2020), which may reflect the selection of biotic and abiotic stress response genes during modern maize breeding.

Transcription factors (TFs) play vital roles in regulation of gene expression in plant cells, with specific key TFs exhibiting multifunctionality by coordinating various regulatory pathways to promote plant growth (Hufford et al. 2021).  Jasmonates (JAs) are identified among phytohormones for their significant roles in regulating various plant processes, particularly in defense mechanisms against pests. MYC2 is a central transcription factor that orchestrates the JA signaling pathway and defense responses in plants by regulating the expression of numerous genes (Du et al. 2022).  Although MYC2 has been extensively characterized in Arabidopsis, studies in crops have revealed the complexity of MYC2’s function, with reports addressing different aspects, such as growth in wheat (Li et al. 2023) or stress defense in maize (Ma et al. 2023).  However, lack of systematic understanding of the complex regulatory network of MYC2 in crops, particularly in maize constrain the further utilization of MYC2 and its downstream genes in maize genetic modification for breeding elite varieties.  Here, we reported that ZmMYC2 had undergone selection during domestication and modern breeding; it acts as a key regulator of the trade-off between development and defense gene expression in maize, elucidating its regulatory network, which holds significant importance in balancing yield and resistance.

Given that some resistance genes have been selected during modern breeding, we analyzed the history of ZmMYC2 over the processes of maize evolution and artificial selection.  According to maize Haplotype Map v3 (HapMap3) database consisting of 1164 modern maize accessions, 25 landraces, and 21 teosintes (Zea mays. parviglumis) (Bukowski et al. 2018), nucleotide diversity strongly decreased sharply at the promoter region (2000 bp upstream of transcription start site) of ZmMYC2 during breeding, while the coding region and 3’-downstream region of ZmMYC2 showed less dramatic changes in nucleotide diversity changes (Fig. 1-A).  Thus, we hypothesize that the genetic diversity within the promoter region of ZmMYC2 has decreased during the breeding process, with favorable variations being selected.  Moreover, the frequency of three polymorphisms underwent convergent changes during modern breeding in both the United States and China (Fig. 1-B–D).  These three polymorphisms constituted three principal haplotypes: pZmMYC2^Hap1, pZmMYC2^Hap2, and pZmMYC2^Hap3, of which the frequency of pZmMYC2^Hap1 showed an increasing trend during modern maize breeding (Fig. 1-E).  The rare haplotype pZmMYC2^Hap3 (n=4) emerged only during the breeding era of China in 2000.  LUC signal activity for pZmMYC2^Hap3 was significantly lower than that of the other two haplotypes in the promoter region (pZmMYC2^Hap1, pZmMYC2^Hap2) (Fig. 1-F–H), suggesting a differential regulatory potential among the haplotypes.  These data indicate that ZmMYC2 was under-selected during maize evolution and breeding processes of maize.  Next, we investigated the expression level of genome-wide association studies of ZmMYC2 based on 368 maize inbred lines using RNA-seq and genome resequencing data (Fu et al. 2013; Li et al. 2013).  The results showed a strong peak signal containing the genomic region of ZmMYC2 on chromosome 1 (Fig. 1-I).

To mine the genes downstream of ZmMYC2, we performed protoplast transient expression-based RNA-sequencing (PER-seq) analysis to facilitate the discovery of new downstream genes utilizing a consistent protoplast system (Zhu et al. 2023).  In total， 281.6 million clean reads were generated, among which an average of approximately 87% of reads were mapped uniquely to the reference genome (Appendices A and B).  The results demonstrated a significant increase in the expression level of ZmMYC2 in each of three replicates of the pRTL2-ZmMYC2-GFP (MYC2-GFP) construct, exceeding a 500-fold increase compared to the pRTL2-GFP-empty (GFP-empty) construct (Fig. 1-J).  Furthermore, upon analyzing differentially expressed genes (DEGs) with a false discovery rate (FDR) <0.05 as the threshold, it was found that 4480 unique DEGs of MYC2-GFP, among which 2,677 were up-regulated compared to GFP-empty (Appendix C).  These up-regulated genes are enriched in circadian rhythm, cell cycles, plant growth, and in response to stress, indicating that these genes are regulated directly or indirectly by ZmMYC2 (Appendix D-A–B).

Several potential candidate genes were selected in an unbiased manner based on their log₂(fold-change) ≥2.5 (Fig. 1-J).  Gene expression profiling analysis of ZmMYC2 and its potential targets revealed essential coincidence (Appendix E).  The interaction between MYC2 and targets observed in the PER-seq system, were further confirmed through expression quantitative trait loci (eQTL) analysis, dual-luciferase reporter assay (DLR), and electrophoretic mobility shift assay (EMSA).  Among the target genes, the members of cytochrome P450 (CYP) gene family are widely distributed in plants involving in various biological processes, such as detoxification of xenobiotics, secondary metabolites production, and terpenoid synthesis (Chakraborty et al. 2023; Sun et al. 2024).  Our results identified an unreported gene of cytochrome P450 family ZmCYP709H1 as a target of ZmMYC2.  Additionally, eQTL analysis of ZmCYP709H1 revealed a strong trans-eQTL signal in the region of chromosome 1, which contains the genomic region of ZmMYC2 (Fig. 1-K).  Subsequent validation through DLR and EMSA confirmed that ZmMYC2 interacts with the promoter region of ZmCYP709H1 and stimulates its expression (Fig. 1-L; Appendices F-A and G-A).  Moreover, the transcriptional activation effect of ZmMYC2 on the promoter of ZmCYP709H1 was suppressed by ZmJAZ8 (Fig. 1-L).  A recent report showed reduced expression of ZmCYP709H1 in three maize dwarf mutants compared to the wild-type, reflecting its potential role in regulating growth, particularly plant height.  This result supports our proposed function of the ZmMYC2-ZmCYP709H1 model (Gao et al. 2024).  Additionally, two other CYP genes, ZmBX5 and ZmBX6, were identified as potential downstream genes of ZmMYC2 that participate in benzoxazinoid synthesis, which is consistent with the findings of a previous study (Ma et al. 2023).  We further confirmed that ZmMYC2 can physically bind to the promoter region of these two genes and activate their expression (Appendix H-A–F).  Besides, the result showed that ZmMYC2 can activate ZmBRD1 expression, which is a member of the CYP gene family and responsible for the final step of brassinosteroid synthesis (Tian et al. 2019) (Fig. 4-A and B; Appendix I-A–D).

The AUXIN RESPONSE FACTOR (ARF) family consists of plant-specific TFs that are key regulators of gene expression in response to the plant hormone auxin (AUX), and participated in various developmental processes such as vascular tissue differentiation, root and shoot development, and environmental stimuli responses (Hagen and Guilfoyle 2002; Salmon et al. 2008).  However, little evidence has been found to support the regulation of ARF gene expression by the core factor ZmMYC2 in the JA signal transduction pathway in maize.  Our data showed that the expression of ZmARF3 was regulated by a trans-eQTL signal involving the gene region of ZmMYC2 (Appendix F-B).  In addition, ZmMYC2 can bind to the promoter region of the ZmARF3 gene and activate its transcription (Fig. 1-M; Appendix G-B).  Besides, MYC2 can activate expressions of senescence-associated genes in rice and wheat, which could be repressed by physical interactions with TaARF15-A1 (Li et al. 2023).  These data demonstrate the key role of MYC2 in regulating the stress resistance and growth of maize through the synergistic regulation of JA and AUX hormone signaling pathways.

Tonoplast intrinsic proteins (TIPs), a subgroup of the aquaporin family, are integral membrane proteins that are crucial for transporting water and small solutes across cellular membrane to maintain water balance (Chaumont et al. 2001).  We found that ZmTIP3c was activated by ZmMYC2 (Fig. 1-N; Appendices F-C and G-C), which supports the potential role of ZmMYC2 in jointly regulating drought stress and JA signal transduction.  The CER2 gene, which is a member of the ECERIFERUM family, is critical for the synthesis of epicuticular wax (Bourdenx et al. 2011; Zhao et al. 2024).  A recent study demonstrated that wounding-induced wax accumulation was primarily regulated by the JA signaling pathway in Arabidopsis, suggesting the potential of JA signaling in wax synthesis (Huang et al. 2024).  We identified ZmCER2 as a ZmMYC2 target (Fig. 1-O; Appendices F-D and G-D).  Additionally, we confirmed the upregulation of ZmCER2 in response to drought stress in five elite inbred lines representing distinct heterotic groups of maize (Fig. 1-P), as observed by previous studies (Zhang et al. 2018, 2020; Jiang et al. 2023).  The result indicates that the drought-induced trait of ZmCER2 can be observed across different genetic backgrounds, thus supporting the potential role of ZmMYC2 in modulating JA signaling and response to drought stress in maize mediated by ZmCER2.

In summary, our findings support the selection of ZmMYC2 during domestication and breeding, highlighting its critical role in regulating genes involving plant growth and development.  Collectively, our eQTL, DLR, and EMSA data successfully validated several targets (ZmCER2, ZmARF3, ZmBRD1 ZmTIP3c, ZmCYP709H1, ZmBX5, and ZmBX6) of ZmMYC2, that encode diverse proteins and participate in various metabolic pathways (Fig. 1-Q).  Of these, ZmCER2 was confirmed to be induced by drought stress and activated by ZmMYC2, suggesting that ZmMYC2 may play a role in the drought response by regulating synthesizing epicuticular wax.  These findings underscore the diverse functions of ZmMYC2 in maintaining the balance between plant development and defense-response, primarily via the JA signaling pathway.  Our data represent a foundation for the further function and mechanism elucidation of of ZmMYC2 and its “Yin-Yang” roles in regulating plant defense and growth (Fig. 1-Q).  Given the crucial role of ZmMYC2 in balancing development and resistance, further work is needed to confirm to unlock the full potentials of ZmMYC2 in mediating yield and resistance through JA signaling pathway by exploring the function of those downstream targets, which is a significant step toward crop precision breeding. 

CRISPR-Cas9 emerged as a powerful tool for gene editing, which has been widely used in plant functional genomics research and crop genetic breeding (Chen et al. 2019). The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA (sgRNA), making it relatively quick and straightforward to create plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome. Several CRISPR-Cas9 mutant libraries have been developed for crops such as rice (Lu et al. 2017; Meng et al. 2017), soya bean (Bai et al. 2020), Brassica napus (He et al. 2023), and cotton (Sun et al. 2023). However, no CRISPR-Cas9 mutant library has yet been generated in woody crop plants. Grape (Vitis vinifera L.) is one of the oldest and most economically valuable fruit crops worldwide. The MYB family is one of the most abundant and versatile transcription factor families in plant (Wu et al. 2022). Here, we described a strategy for generating a collection of MYB mutant lines in grape using a sgRNA library.

We obtained 138 grape MYB transcription factor sequences from the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/family.php?sp=Vvi&fam=MYB) (Appendix A). Since genes with similar sequences often share similar functions, a phylogenetic tree was constructed and divided all MYBs into 30 sets based on the sequence similarity (Fig. 1-A and Appendix B-a). A total of 127 sgRNAs were designed, each targeting conserved regions shared by two or more MYB transcription factors with fewer than three base-pair differences. This approach aimed to simultaneously mutate multiple MYB members within a cluster using a single sgRNA, addressing the challenge of genetic redundancy (Appendix B-b). These shared target sites were designated as Target1 (T1). In addition, specific sgRNAs targeting individual MYB transcription factors were designed using the  CRISPR-P 2.0 online tool (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR) (Liu et al. 2017). The design criteria included selecting target sites within exons near the start of open reading frames (ORFs), a GC content of at least 40%, and an off-target efficiency below 0.4. Finally, 138 sgRNAs targeting specific sites for each MYB transcription factor were designed, referred to as Target2 (T2). In total, a comprehensive sgRNA library comprising 265 sgRNA was developed to target 138 MYBs, with an average  of 1.92 sgRNA per MYB (Fig. 1-A and Appendix C).

The sgRNA fragments from the same set were mixed as one sgRNA pool, ligated into the pKSE401 vector using Gibson ligation, and subsequently transformed into the Escherichia coli  TOP10 competence cells (Fig. 1-B and Appendix D-a). To evaluate the ligation efficiency of the sgRNA pool with the vector, 90 E. coli clones from sets #1-3 were randomly selected and sequenced. The results demonstrated that the ligation efficiency exceeded 90% and the sgRNA coverage ratio over 80%, confirming the feasibility of this method (Appendix D-b). Using this approach, approximately 1300 (~5×) positive E. coli clones were obtained across the 30 sets (Fig. 1-D). Plasmids extracted from each set were mixed in equal proportions and transformed into Agrobacterium tumefaciens GV3101 competence cells. Finally, all Agrobacterium colonies were collected and verified with next-generation sequencing (NGS). The results revealed that 95.31% of the sequences in the library were accurate, and 178 of 265 sgRNAs were represented by at least one read, targeting 125 (90.58%) MYB transcription factors. Most (83.93%) sgRNA read counts fell within the range of 2⁸-2¹⁵. These results indicated that the sgRNAs library in Agrobacterium exhibited high accuracy and gene coverage, which is usable for grape transformation (Fig.1-E).

Vitis vinifera L. cv. Cabernet Sauvignon is one of the most renowned red wine grape varieties, widely cultivated worldwide. In this study, pro-embryonic masses of ‘Cabernet Sauvignon’ were used as recipient material for Agrobacterium-mediated transformation (Fig. 1-C and Appendix E). A total of 1354 kanamycin-resistant seedlings were obtained, and which 341 were confirmed as transgenic lines (PCR positive). And, all the lines were determined to harbor a single correct sgRNA, representing 13 unique sgRNAs targeting 18 MYB transcription factors. Target site DNA was amplified and sequenced, revealing only 67 gene-edited lines with mutations in 8 MYB transcription factors (Fig. 1F and Appendix F and G). Among these, 56 lines were chimeric mutants, nine were biallelic mutants, one was a homozygous mutant and one was a heterozygous mutant (Appendix H). Five Target1 type transgenic lines were obtained, three of them did not mutate in all of the targeted genes, in which the sgRNA was targeting two or more completely conserved sites. Additionally, gene-edited lines for GSVIVT01032467001-T1 and GSVIVT01014770001-T1 were producted. However, the sgRNA harbored in these transgenic lines only caused the mutations in GSVIVT01032467001 and GSVIVT01014770001, without off-target effects on genes with similar sequences (Appendix I and J). All MYB-edited lines were subsequently transplanted into a greenhouse for observation (Appendix K). Phenotypic analysisrevealed that the GSVIVT01026481001 edited lines exhibited significantly enhanced tolerance to drought stress (Fig. 1-G).

CRISPR-Cas9 has greatly accelerated gene function research and breeding in plants. In this study, we developed a strategy for generating a collection of MYB mutant lines in grape using a CRISPR-Cas9 library. However, the relatively lower transformation efficiency in grape limited the number of mutants obtained. Factors affecting grape transformation efficiency primarily included the regeneration rate of the recipient material and the efficiency of Agrobacterium infection. Numerous studies have demonstrated that plant regeneration efficiency enhanced using developmental regulators such as BABY BOOM (BBM), WUSCHEL (WUS), GROWTH-REGULATING FACTOR (GRF), and REGENERATION FACTOR (REF) (Debernardi et al. 2020; Yang et al. 2022). Additionally, plant transformation and gene editing efficiency improved through optimized genetic transformation methods and gene editing vector designs (Debernardi et al. 2024; Yan et al. 2024). We confirmed that a large number of edited plants could be obtained simultaneously using a sgRNA mixed-pool library, provided that grape transformation efficiency is improved. This strategy holds significant potential for constructing genome-wide mutant libraries in woody crop plants in the future.

Red fruit peel is one of the most valuable economic traits in pear and is mainly determined by anthocyanins. Many pear cultivars with a red peel originated from bud sports; however, little is known about the genetic mechanisms underlying this trait. We have previously identified a mutant PpBBX24 containing a 14-nucleotide deletion in the coding region (Ppbbx24-del) as the only known variant associated with the red coloration of the mutant 'Red Zaosu' pear (Pyrus pyrifolia White Pear Group). Herein, we analyzed the role of the mutant gene in red coloration and its mechanism of action. The results showed that light promoted red peel coloration in 'Red Zaosu' pear, and Ppbbx24-del had positive effects on light-induced anthocyanin biosynthesis, while normal PpBBX24 had the opposite effects. Transient and stable transformation experiments confirmed that Ppbbx24-del could promote anthocyanin accumulation in pear fruit peels, calli, and tobacco flowers. Due to the loss of NLS and VP domains, Ppbbx24-del co-localized in the nucleus and cytoplasm, whereas PpBBX24 localized only in the nucleus. Real-time PCR and transcriptome analyses indicated that PpMYB10 and PpHY5 is highly expressed in 'Red Zaosu' pear. In yeast one-hybrid and dual luciferase assays, Ppbbx24-del and PpHY5 independently promoted the expression of PpCHS, PpCHI, and PpMYB10 by binding to their promoters; however, PpBBX24 did not affect the expression of these genes. Additionally, we found that Ppbbx24-del and PpHY5 had additive effects on the expression of PpCHS, PpCHI, and PpMYB10, as they promote the expression of anthocyanin synthesis genes separately. The co-expression of PpBBX24 and PpHY5 inhibited the activation of downstream genes by PpHY5, and this was attributed to the interaction between the two loci. In conclusion, our results clarify the molecular mechanism by which mutant Ppbbx24-del and PpBBX24 exert opposite effects in the regulation of anthocyanin accumulation in pear. These findings lay an important theoretical foundation for the use of Ppbbx24-del to create red pear cultivars.

The evaluation of plant stress tolerance and the screening of key regulatory genes under the combined stresses of high temperature and drought are important for the study of plant stress tolerance mechanisms. In this study, the drought tolerance of five grape varieties was evaluated under high-temperature conditions to screen key genes for further exploration of resistance mechanisms. By comparing and analysing the morphological characteristics and physiological indicators associated with the response of grapevines to drought stress and integrating them with the membership function to assess the strength of their drought tolerance, the order of drought tolerance was found to be as follows: 420A>110R>CS>fercal>188-08. To further analyse the mechanism of differences in drought tolerance, transcriptomic sequencing was performed on the drought-tolerant cultivar 420A, the drought-sensitive cultivar 188-08 and the control cultivar CS. The functional analysis of differential metabolic pathways indicated that the differentially expressed genes were enriched mainly in biological that 420A had higher antioxidant activity. Moreover, the transcription factors which differentially expressed were also analysed in the five grape varieties, and several genes, such as VvAGL15, VvLBD41, and VvMYB86, appeared to be closely related to drought tolerance, suggesting their potential involvement in the regulation of grapevine drought tolerance and their value in drought tolerance research.

Cold stress widely impairs the quality and yield of tea plants. The miR164 family and its target NAC transcription factor have been identified as crucial regulators in response to cold stress. However, the role of miR164 and CsNAC in cold tolerance in tea plants was little understood. In our study, the expression level of csn-miR164a was significantly reduced under cold stress, and was significantly negative correlation with that of CsNAC1. 5’ RACE and GUS histochemical assays clearly showed that CsNAC1 was specifically cleaved by csn-miR164a. The csn-miR164a-silenced tea leaves promoted expression level of CsNAC1 and CsCBFs, and exhibited greater cold tolerance, also overexpression of CsNAC1 enhanced cold tolerance in transgenic Arabidopsis plants by promoting the expression levels of AtCBFs. In contrast, the heterologous overexpression of csn-miR164a in Arabidopsis decreased the expression level of AtNACs and AtCBFs, and thus impaired cold tolerance. Additionally, silencing of CsNAC1-impaired the expression levels of CsCBFs resulted in greater cold sensitivity in tea leaves. Taken together, our present study demonstrated that the miR164a-CsNAC1 module may play a negative role in cold tolerance of tea plants via CsCBF-dependent pathway.

The host intestinal microbiota has emerged as the third element in the interactions between hosts and enteric viruses, and potentially affects the infection processes of enteric viruses. However, the interaction of porcine enteric coronavirus with intestinal microorganisms during infection remains unclear. In this study, we used 16S-rRNA-based Illumina NovaSeq high-throughput sequencing to identify the changes in the intestinal microbiota of piglets mediated by porcine epidemic diarrhea virus (PEDV) infection and the effects of the alterations in intestinal bacteria on PEDV infection and its molecular mechanisms. The intestinal microbiota of PEDV-infected piglets had significantly less diversity than the healthy group and different bacterial community characteristics. Among the altered intestinal bacteria, the relative abundance of Clostridium perfringens was significantly increased in the PEDV-infected group. A strain of C. perfringens type A, named DQ21, was successfully isolated from the intestines of healthy piglets. The metabolites of swine C. perfringens type A strain DQ21 significantly enhanced PEDV replication in porcine intestinal epithelial cell clone J2 (IPEC-J2) cells, and PEDV infection and pathogenicity in suckling piglets. Palmitic acid (PA) was identified as one of those metabolites with metabolomic technology, and significantly enhanced PEDV replication in IPEC-J2 cells and PEDV infection and pathogenicity in suckling piglets. PA also increased the neutralizing antibody titer in the immune sera of mice. Furthermore, PA mediated the palmitoylation of the PEDV S protein, which improved virion stability and membrane fusion, thereby enhancing viral infection. Overall, our study demonstrates a novel mechanism of PEDV infection, with implications for PEDV pathogenicity.

Crude fat is an important nutritional component of maize kernels.  However, the genetic mechanisms underlying crude fat content in maize kernels remain elusive.  Previous studies used single-model genome-wide association studies (GWAS) with limited population sizes, which can result in false positives of loci and hinder the identification of functional genes.  Therefore, this study used a population consisting of 495 maize inbred lines, combined with 1.25 million single nucleotide polymorphisms (SNPs), and implemented GWAS using six models to identify quantitative trait nucleotides (QTNs) controlling crude fat content and to mine key genes.  The results revealed a wide variation in crude fat content (0.62–16.03%) and broad-sense heritability (96.23%).  In total, 744 significant QTNs were detected, with 147 co-located across different models, environments, and methods.  Based on the 147 co-located QTNs, candidate genes were searched at 50 kb up- and downstream intervals of each QTN.  We finally screened eight candidate genes (GRMZM2G169089, GRMZM2G117935, GRMZM2G002075, GRMZM2G368838, GRMZM2G058496, GRMZM2G090669, GRMZM2G001241, and GRMZM2G333454) related to crude fat content that exhibited high expression levels during kernel development in maize inbred line B73.  Notably, GRMZM2G169089, GRMZM2G117935, GRMZM2G002075, and GRMZM2G368838 are involved in the linoleic acid metabolic pathway, oil metabolism, kernel growth, and development in maize.  Furthermore, co-expression network analysis revealed that the eight candidate genes exhibited strong correlations with 30 known genes.  Proteins encoded by candidate genes interact with various other proteins and play an important role in oil content and oleic acid metabolism in maize kernels.  The best haplotypes of candidate genes might increase crude fat content without decreasing maize yield.  These results broaden the understanding of the genetic mechanism of crude fat content and facilitate marker-assisted selection for high-crude fat breeding programs for maize.

The application of organic fertilizers has become an increasingly popular substitution in maize production to reduce gaseous nitrogen (N) loss and soil degradation caused by inorganic fertilizers.  Organic fertilizer plays a key role in improving soil quality and stabilizing maize yields, but studies that refine different substitution rates remain poorly documented.  A field study was carried out in 2021 and 2022 based on a long-term trial initiated in 2016.  The experiment included five organic fertilizer N substitution rates with equal input of 200 kg N ha^–1: 0% organic fertilizer (T1, 100% inorganic fertilizer), 50.0% organic+50.0% inorganic fertilizer (T2), 37.5% organic+62.5% inorganic fertilizer (T3), 25.0% organic+75.0% inorganic fertilizer (T4), 12.5% organic+87.5% inorganic fertilizer (T5), and no fertilizer control (T6).  The average result of two years showed that T3 and T1 had the highest grain yield and biomass, respectively, and there was no significant difference between T1 and T3.  Compared with T1, 12.5, 25.0, 37.5, and 50.0% substitution rates (T5, T4, T3, and T2) significantly reduced total nitrogen loss (NH₃、N₂O) by 8.3, 16.1, 18.7, and 27.0%, respectively.  Nitrogen use efficiency (NUE) was higher in T5, T3, and T1, and there was no significant difference among them.  The organic fertilizer substitution directly reduced NH₃ volatilization and N₂O emission from farmland by lowering ammonium nitrogen and alkali-dissolved N content and by increasing soil moisture.  These substitution treatments reduced N₂O emissions indirectly by regulating the abundance of AOB and nirK-harboring genes by promoting soil moisture.  The 37.5% of organic fertilizer substitution reduces NH₃ volatilization and N₂O emission from farmland by decreasing ammonium nitrogen and alkali-dissolved N content and increasing moisture which negatively regulate the abundance of AOB and nirK-harboring genes to reduce N₂O emissions indirectly in rainfed maize fields on the Loess Plateau of China.

Primordial germ cells (PGCs) are the stem-cell population of adult animal gametes, which develop into sperm or eggs. It can be propagated in vitro and injected into the host chicken for genome editing to obtain germline chimeric chicken. However, it has the limitation that the host embryo contains endogenous PGCs, which raises complications, resultantly donor PGCs fail to compete, and transmission efficiency reduced. Therefore, to increase the transmission efficiency, we generated a novel sterile chicken with the inducible elimination of endogenous PGCs in the host. This is the first study that applied the herpes simplex virus thymidine kinase (HSV-TK) cell ablation system in avian. CRISPR/Cas9-mediated homology-directed repair was performed to localize the HSV-TK suicide gene to the last exon of the deleted in azoospermia-like (DAZL) gene, and ganciclovir (GCV) was added to induce the apoptosis in the germ cells of the host embryo. The sterilized host embryo introduced genome-edited PGCs to produce chimeric chicken carrying exogenous germ cells only. It was observed that the germline transmission efficiency was 100% achieved, and the obtained chicks were purely from donor breeds. The technologies established in the current study have important applications in germplasm conservation and gene editing in chicken.

Fusarium graminearum is a fungal plant pathogen which causes Fusarium head blight (FHB), a devastating disease on cereal crops. Here we report that FgPMA1 could be a new target to control FHB by the application of double-stranded RNA (dsRNA) of FgPMA1. FgPMA1 was divided into 6 segments to generated RNA interference (RNAi) constructs (FgPMA1RNAi-1, -2, -3, -4, -5, and -6), and these constructs were transformed in F. graminearum strain PH-1. The expression of FgPMA1 reduced by 18.48%, 33.48% and 56.93% in FgPMA1RNAi-1, FgPMA1RNAi-2 and FgPMA1RNAi-5, respectively. FgPMA1RNAi-1, -2, and -5 mutants inhibited fungal development, including mycelium growth, mycelial morphology, asexual and sexual development, and toxin production. The length of lesions on wheat leaves, wheat coleoptiles and wheat ears were shorter after infection with FgPMA1RNAi-1, -2, and -5 mutants than wild-type PH-1. These results showed that three segments (FgPMA1RNAi-1, -2, and -5) exhibited effective silencing effects. After treatment with 25 ng µL^-1 dsRNA of these segments in vitro, the growth rate of mycelium growth was significant decreased, mycelium became deformed with bulbous structure at the tip, and the mycelium lost the ability to produce conidia in F. graminearum strain PH-1, Fusarium asiacitum strain 2021 and phenamacril-resistant strain YP-1. After application of FgPMA1RNAi-1-dsRNA and FgPMA1RNAi-2-dsRNA to wheat ears, pathogenicity reduced 34.21-35.40%. 

Reducing nitrogen application rates can mitigate issues such as environmental degradation and resource wastage.  However, it can also exacerbate problems such as wheat floret degeneration, leading to reduced yields.  Therefore, investigating wheat floret degeneration mechanisms under low nitrogen stress and identifying mitigation measures are conducive to achieving high yields and sustainable development.  To investigate the physiological mechanism of low nitrogen stress affecting wheat floret degradation and whether exogenous brassinosteroids can alleviate this stress, three nitrogen application rates (N0, no nitrogen application; N1, 120 kg ha^-1 pure nitrogen; and N2, 240 kg ha^-1 pure nitrogen) and exogenous spraying experiments (N0CK, no nitrogen with water spraying; N0BR, no nitrogen with 24-epibrassinolide (an active brassinosteroids) spraying; and N1, 120 kg ha^-1 pure nitrogen with water spraying) were designed.  The results indicated that low nitrogen stress induced a large amount of reactive oxygen species generation.  Although wheat spikes synthesized flavonoids to combat oxidative stress, their energy metabolism (glycolysis and tricarboxylic acid cycle) and ascorbate-glutathione cycle were inhibited, keeping reactive oxygen levels elevated within the spike, inducing cell death and exacerbating floret degeneration.  Furthermore, brassinosteroids played a role in regulating wheat floret degeneration under low-nitrogen stress.  Exogenous foliar spraying of 24-epibrassinolide promoted energy metabolism and the ascorbate-glutathione cycle within the spike, enhancing energy charge and effectively mitigating a portion of reactive oxygen induced by low nitrogen stress, thereby alleviating floret degeneration caused by low nitrogen stress.  In summary, low-nitrogen stress disrupts the redox homeostasis of wheat spikes, leading to floret degeneration.  Brassinosteroids alleviate floret degeneration by improving the redox state of wheat spikes.  This research provides theoretical support for balancing the contradiction between high yields and sustainable development and is beneficial for the application of low nitrogen in production.

Frequent drought events especially those occur in the reproductive stages severely restrict global crop productivity.  Moderate drought priming during the earlier growth stages is a promising strategy for plants to resist to recurrent severe drought stress.  However, the underlying mechanisms remain unclear.  Here, we subjected wheat plants to drought priming during the vegetative growth stage and to severe drought stress at 10 days after anthesis.  We then collected leaf samples at the ends of the drought priming, recovery periods, and at the ends of drought stress for transcriptome sequencing in combination with phenotypic and physiological determination.  The drought-primed wheat plant maintained a lower plant temperature, with higher stomatal openness and photosynthesis, thereby resulting in much less 1,000-grain weight and grain yield losses under the later drought stress than the non-primed plants.  Interestingly, 416 genes of which 27 transcription factors (e.g., MYB, NAC, HSF) seemed to be closely related to the improved drought tolerance as indicated by the dynamic transcriptome analysis.  Moreover, the candidate genes showed six temporal expression patterns and significantly enriched in several stress response related pathways such as plant hormone signal transduction, starch and sucrose metabolism, arginine and proline metabolism, inositol phosphate metabolism, and wax synthesis.  These findings illustrate new insights into physiological and molecular mechanisms of the long-term effects of early drought priming to effectively improve drought tolerance in wheat, which proved potential approaches to challenge the increasing abiotic stresses and secure food safety under global warming scenarios.

The "Greater Food" approach has replaced the older "taking grain production as a top priority" approach. The importance of feed and forage as the material basis for guaranteeing high-quality development of the livestock industry has gradually become prominent. However, owing to the tradition of "both human staple food and animal feed relying on grain production" in China and the decoupling of feed crop planting and livestock farming, the risk of feed grain security has increased, especially as it relates to the supply of high-quality protein feed ingredients from abroad, which is facing a bottleneck. To ensure food security, effective domestic agricultural production should be adopted. Nevertheless, guaranteeing the supply of high-quality protein feed through domestic soybean production is difficult because of limited arable land; furthermore, pressure on the staple food supply is still extreme. In this article, the historical and realistic implications for the security risks of feed grain in China are analyzed. Proposals are made to separate staple food grains for humans from the feed grain supply for animals and to develop high-quality forage to reduce feed grain use. High-quality forage can be supplied via intercropping with grain crops in arable land and reseeding perennial legumes or grasses into natural grasslands. However, “managing forage for grain” needs to be supported via technical paths and policies as the forage industry develops to effectively increase the capacity to ensure feed grain security.

The Rpd3 histone deacetylase complex is a multiple-subunit complex that mediates the regulation of chromatin accessibility and gene expression. Sin3, the largest subunit of Rpd3 complex, is conserved in a broad range of eukaryotes. Despite being a molecular scaffold for complex assembly, the functional sites and mechanism of action of Sin3 remain unexplored. In this study, we functionally characterized a glutamate residue (E810) in FgSin3, the ortholog of yeast Sin3 in Fusarium graminearum (known as wheat scab fungus). Our findings indicate that E810 was important for the functions of FgSin3 in regulating vegetative growth, sexual reproduction, wheat infection, and DON biosynthesis. Furthermore, the E810K missense mutation restored the reduced H4 acetylation caused by the deletion of FNG1, the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum. Correspondingly, the defects of the fng1 mutant were also partially rescued by the E810K mutation in FgSin3. Sequence alignment and evolutionary analysis revealed that E810 residue is well-conserved in fungi, animals, and plants. Based on Alphafold2 structure modeling, E810 localized on the FgRpd3-FgSin3 interface for the formation of a hydrogen bond with FgRpd3. Mutation of E810 disrupts the hydrogen bond and likely affects the FgRpd3-FgSin3 interaction. Taken together, E810 of FgSin3 is functionally associated with Fng1 in the regulation of H4 acetylation and related biological processes, probably by affecting the assembly of the Rpd3 complex.

Genotype imputation is essential for increasing marker density and maximizing the utility of existing SNP array data in animal breeding. Although a wide range of software is available for genotype imputation, a comprehensive benchmark in pigs is still lacking. In this study, we benchmarked 24 combinations of genotype imputation software for SNP arrays in pigs, comprising six independent pre-phasing software (fastPHASE, MaCH, BIMBAM, Eagle, SHAPEIT, Beagle) and four distinct imputation software (pbwt, Minimac, IMPUTE, Beagle), using 1,602 whole-genome sequencing (WGS) pigs from a multibreed pig genomics reference panel (PGRP) in PigGTEx. Our results indicated that the combination of Beagle for pre-phasing and Minimac for imputation achieves the highest imputation accuracy with a concordance of 0.983, especially for low-frequency SNPs (MAF<0.05). Finally, we proposed three recommended strategies: i) the combination of Beagle and Minimac is optimal for achieving the highest accuracy; ii) the combination of Beagle and Beagle is recognized for its convenience and relatively high accuracy despite it being memory-intensive; iii) the combination of Eagle and pbwt is feasible for its minimal computational cost with relatively high accuracy. This study provides valuable insights for implementing genotype imputation for pig SNP arrays toward sequence data and offers a basis for applications in livestock and poultry breeding.

种子的落粒性和品质是维持稳定产量及确保优良食用品质的关键因素。本研究成功鉴定了一个AP2/ERF转录因子—SHAT2，该基因能正向调控种子落粒性和品质。shat2突变体由于离层发育异常，导致成熟期种子难落粒。同时，与野生型相比，shat2突变体的淀粉颗粒排列松散，总淀粉以及直链淀粉含量和蛋白质含量显著降低，而可溶性糖含量显著增加。qRT-PCR 的检测结果显示，大部分与种子落粒性以及品质相关基因在shat2突变体中表达显著下调，表明了SHAT2在水稻种子落粒性和品质中起重要作用。此外，EMSA结果显示SHAT2可以结合GCC-box，暗示SHAT2可能通过调控下游基因的表达来影响种子发育。本研究不仅丰富了遗传资源，还突显了SHAT2在培育具有理想落粒特性和优良稻米品质的水稻品种中的巨大潜力。

Long-term excessive nitrogen (N) application does not increase or even decreases grain yield and N use efficiency (NUE) of maize, in which the roles of root morphological and physiological characteristics are not clear.  The goal of this study was to explain the mechanism underlying no increment in grain yield under excessive N application from the perspective of root morpho-physiological characteristics.  A 10-year long-term N fertilizer trial was conducted in Jilin Province, Northeast China, growing maize at three N fertilizer levels (zero N, N0; recommended N, N2; and high N level, N4) in 2019, 2020 and 2021.  Two widely planted maize genotypes: ‘Xianyu 335’ (XY335) and ‘Zhengdan 958’ (ZD958) were used.  Grain yield, N content, root morphology and other physiological characteristics were analyzed to further evaluate the relationships between N uptake, N utilization, plant growth, and root systems under different N treatments.  Compared with N0, root biomass, post-silking N uptake and grain yield were significantly improved with increased N input, whereas no significant differences were observed between recommended N and high N.  High N application increased root length and root surface area, but decreased root activity (measured by TTC (2,3,5-triphenyltetrazolium chloride) method), nitrate reductase activity and root activity absorbing area regardless of genotypes.  Root length and root to shoot ratio negatively contributed to N uptake (by -1.2% and -24.6%), while root surface area, root activity, nitrate reductase activity and root activity absorbing area were positively contributed to N uptake.  The interaction effect between cultivar and N application was significant on NUE.  XY335 obtained the highest NUE (11.6%) and N recovery efficiency (18.4%) through higher root surface area (23.6%), root activity (12.5%), nitrate reductase activity (8.3%) and root activity absorbing area (6.9%) compared with other treatments.  Overall, recommended N application promoted Post N uptake, NUE and grain yield by root surface area and root activity, nitrate reductase activity and root activity absorbing area, while high N application did not increase or even decreased NUE by reducing root surface area, root activity, nitrate reductase activity and root activity absorbing area.  Our case study successfully revealed that root surface area, root activity, nitrate reductase activity and root activity absorbing area were the limiting factors of NUE increase under high N application.

Toxoplasma gondii is an important zoonotic parasite which has over 200 genotypes worldwide. N⁶-methyladenosine (m⁶A) methylation is a common epigenetic modification in messenger RNAs (mRNAs), and has been implicated in many aspects of mRNA biology. However, little is known about the difference in m⁶A methylation among different genotypes of T. gondii. In the present study, we employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) technology to identify key genes exhibiting m⁶A methylation in the three major clonal lineages (Types I, II and III) of T. gondii tachyzoites. A total of 7650, 8359 and 7264 m⁶A peaks were identified in 5211, 5607 and 4974 genes in tachyzoites of RH strain (Type I), ME49 strain (Type II) and VEG strain (Type III), respectively. By comparing RH vs. ME49, RH vs. VEG, and ME49 vs. VEG, 735, 192 and 615 differentially methylated peaks (DMPs) were identified in 676, 168 and 553 genes, respectively. A combined MeRIP-seq and RNA-seq analysis revealed 172, 41 and 153 differentially methylated genes (DMGs) at both the m⁶A methylation and transcriptional level. Gene ontology term enrichment analysis of the DMPs identified differences related to Golgi apparatus, plasma membrane, signal transduction, RNA processing and catalytic step 2 spliceosome. KEGG pathway enrichment analysis showed that the DMGs are mainly involved in endocytosis, systemic lupus erythematosus and mTOR signaling pathway. These findings reveal genotype-specific differences in m⁶A methylation, which provide new resources for further investigations of the role of m⁶A in the pathobiology of T. gondii.

The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials. In this study, we isolated four Escherichia albertii (E. albertii) strains from broilers in Eastern China. The antimicrobial susceptibility and genomic characterization of these E. albertii isolates were determined. Our results revealed that these four E. albertii isolates exhibited resistance to tetracyclines, chloramphenicol, β-lactams, aminoglycosides, polymyxin B, sulfonamides, quinolones, and other antimicrobials. Among them, EA04 isolate was multidrug resistant and harbored extended-spectrum β-lactamases (ESBL) genes bla_CTX-M and bla_TEM. Whole genome sequencing and core-genome Multilocus sequence typing (cgMLST) based on all ST4638 E. albertii for EA04 inferred highly probable epidemiological links between selected human isolates. Additionally, the ESBL genes bla_TEM-141 and bla_CTX-M-55 were coexistent in an approximately 75 kb IncFII plasmid pEA04.2 in EA04. Comparative analysis indicated that genes bla_TEM-141 and bla_CTX-M-55 were located in IS15-bla_CTX-M-55-wbuC-bla_TEM-141-IS26 region, which similar structures were identified in various bacteria. Furthermore, the plasmid pEA04.2 could be transferable to Escherichia coli EC600 and lead to the resistance to third-generation cephalosporins. These results suggested that chicken potentially serve as a reservoir for multidrug resistant E. albertii, which increases the risk of horizontal transfer of antimicrobial resistance between humans, animals and environment.

Comprehending the genetic basis of economically important traits is of significant importance to enhance livestock breeding. In domestic ruminants, hornlessness is becoming a more desirable trait since horns could lead to accidental injuries to animals and producers. Recent studies have identified the relaxin family peptide receptor 2 (RXFP2) as a primary candidate gene associated with the presence and absence of horns in sheep. However, no sufficient molecular biology-based analyses were performed to validate the association and function of RXFP2 in sheep. Noticeably, previous studies in both humans and mice have provided evidence supporting the involvement of RXFP2 in testicular descent. To validate the potential function of the RXFP2 gene in sheep, we used the CRISPR/Cas9 technology to obtain RXFP2-disrupted sheep individuals. Initially, highly efficient sgRNAs, targeting RXFP2, were screening through in vitro cleavage assays and cellular assessments. Then, two RXFP2-disrupted lambs were generated by intracytoplasmic microinjection of CRISPR/Cas9-sg1 ribonucleoprotein, with an efficiency of 81.84 and 37.17%, respectively. No potential off-target events were detected. Western blot analysis showed that RXFP2 expression was significantly reduced in the pedicle skin of edited lambs (P=0.034). Intriguingly, although the partial disruption of RXFP2 did not affect the horn phenotype in sheep, it led to an obvious unilateral cryptorchidism. These results provide evidence for a hitherto ambiguous link between both horn and testicular development. In conclusion, this study represents the first successful generation of cryptorchid sheep models via the disruption of RXFP2 using CRISPR/Cas9. These findings provide new insights into the roles of RXFP2, whose partial disruption is associated with testicular descent rather than horn formation.

Aligning leaf nitrogen (N) distribution to match the light gradient is crucial for maximizing canopy dry matter production (DMP) and improving N utilization efficiency.  However, the relationship between the gradient of root-derived cytokinins and N distribution in rice leaves, along with its impact on DMP and the underlying mechanisms, remains poorly understood.  A two-year field experiment was conducted using two japonica N-efficient varieties (NEVs) and two japonica N-inefficient varieties (NIVs) under four different N rates (0, 90, 180 and 360 kg N ha⁻¹). These selected varieties exhibited similar values in the coefficient of light extinction (K_L).  Results showed that, at lower N rates (0-180 kg N ha⁻¹), the NEVs exhibited greater dry matter weight at maturity, higher grain yield and improved internal N use efficiency (IE_N), compared to the NIVs, despite possessing comparable total N uptake.  Compared with the NIVs, the NEVs exhibited a more pronounced nitrogen distribution gradient in leaves, as indicated by the coefficient of nitrogen extinction (K_N) values during the middle and early grain filling stages.  This enhanced gradient led to improved coordination between light and nitrogen, resulting in greater photosynthetic production, particularly at lower N rates. Furthermore, the NEVs demonstrated a larger gradient of zeatin (Z)+zeatin riboside (ZR) in leaves (i.e., higher ratios of Z+ZR levels between upper and lower leaves), enhanced expression levels of genes related to N export in lower leaves and Z+ZR loading in root, respectively, elevated enzymes activities related to N assimilation in upper leaves, in relative to the NIVs.  Correlation and random forest analyses demonstrated a strong positive correlation between Z+ZR gradient, K_N, and DMP, and the gradient facilitated the export of N from lower leaves and its assimilation in upper leaves, contributing significantly to both K_N and DMP.  This process was closely linked to root activity, including root oxidation activity, root Z+ZR content, and Z+ZR loading capacity, as confirmed by applying an inhibitor or a promoter of cytokinins biosynthesis to roots.  Interestingly, at the N rate of 360 kg N ha⁻¹, both NEVs and NIVs showed indistinguishable plant traits, achieving a super high-yielding level (over 10.5 t ha⁻¹) but with remarkably low IE_N.  The results suggest that increasing Z+ZR gradient can improve K_N and DMP, where it needs to maintain higher root activity, thus leading to high yield and high IE_N.  Further research is needed to explore and develop cultivation practices with reduced N to unlock the super high-yielding potential of the NEVs.