The utilization of metabolomics approaches to explore the metabolic mechanisms underlying plant fitness and adaptation to dynamic environments is growing, highlighting the need for an efficient and user-friendly toolkit tailored for analyzing the extensive datasets generated by metabolomics studies. Current protocols for metabolome data analysis often struggle with handling large-scale datasets or require programming skills. To address this, we present MetMiner (https://github.com/ShawnWx2019/MetMiner), a user-friendly, full-functionality pipeline specifically designed for plant metabolomics data analysis. Built on R shiny, MetMiner can be deployed on servers to utilize additional computational resources for processing large-scale datasets. MetMiner ensures transparency, traceability, and reproducibility throughout the analytical process. Its intuitive interface provides robust data interaction and graphical capabilities, enabling users without prior programming skills to engage deeply in data analysis. Additionally, we constructed and integrated a plant-specific mass spectrometry database into the MetMiner pipeline to optimize metabolite annotation. We have also developed MDAtoolkits, which include a complete set of tools for statistical analysis, metabolite classification, and enrichment analysis, to facilitate the mining of biological meaning from the datasets. Moreover, we propose an iterative weighted gene co-expression network analysis strategy for efficient biomarker metabolite screening in large-scale metabolomics data mining. In two case studies, we validated MetMiner's efficiency in data mining and robustness in metabolite annotation. Together, the MetMiner pipeline represents a promising solution for plant metabolomics analysis, providing a valuable tool for the scientific community to use with ease.

ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F₁ families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.

Although root nodules are essential for biological nitrogen fixation in legumes, the cell types and molecular regulatory mechanisms contributing to nodule development and nitrogen fixation in determinate nodule legumes, such as soybean (Glycine max), remain incompletely understood. Here, we generated a single-nucleus resolution transcriptomic atlas of soybean roots and nodules at 14 days post inoculation (dpi) and annotated 17 major cell types, including six that are specific to nodules. We identified the specific cell types responsible for each step in the ureides synthesis pathway, which enables spatial compartmentalization of biochemical reactions during soybean nitrogen fixation. By utilizing RNA velocity analysis, we reconstructed the differentiation dynamics of soybean nodules, which differs from those of indeterminate nodules in Medicago truncatula. Moreover, we identified several putative regulators of soybean nodulation and two of these genes, GmbHLH93 and GmSCL1, were as-yet uncharacterized in soybean. Overexpression of each gene in soybean hairy root systems validated their respective roles in nodulation. Notably, enrichment for cytokinin-related genes in soybean nodules led to identification of the cytokinin receptor, GmCRE1, as a prominent component of the nodulation pathway. GmCRE1 knockout in soybean resulted in a striking nodule phenotype with decreased nitrogen fixation zone and depletion of leghemoglobins, accompanied by downregulation of nodule-specific gene expression, as well as almost complete abrogation of biological nitrogen fixation. In summary, this study provides a comprehensive perspective of the cellular landscape during soybean nodulation, shedding light on the underlying metabolic and developmental mechanisms of soybean nodule formation.

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene‐edited cotton plants through either particle bombardment or Agrobacterium‐mediated transformation. Here, we developed a shoot apical meristem (SAM) cell‐ mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2–3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP‐ and RUBY‐positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9‐mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene‐edited plants for genetic improvement of recalcitrant cotton varieties.

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime‐editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime‐ editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA‐ evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.

Advances in plant phenotyping technologies are dramatically reducing the marginal costs of collecting multiple phenotypic measurements across several time points. Yet, most current approaches and best statistical practices implemented to link genetic and phenotypic variation in plants have been developed in an era of single-time-point data. Here, we used time-series phenotypic data collected with an unmanned aircraft system for a large panel of soybean (Glycine max (L.) Merr.) varieties to identify previously uncharacterized loci. Specifically, we focused on the dissection of canopy coverage (CC) variation from this rich data set. We also inferred the speed of canopy closure, an additional dimension of CC, from the time-series data, as it may represent an important trait for weed control. Genome-wide association studies (GWASs) identified 35 loci exhibiting dynamic associations with CC across developmental stages. The time-series data enabled the identification of 10 known flowering time and plant height quantitative trait loci (QTLs) detected in previous studies of adult plants and the identification of novel QTLs influencing CC. These novel QTLs were disproportionately likely to act earlier in development, which may explain why they were missed in previous single-time-point studies. Moreover, this time-series data set contributed to the high accuracy of the GWASs, which we evaluated by permutation tests, as evidenced by the repeated identification of loci across multiple time points. Two novel loci showed evidence of adaptive selection during domestication, with different genotypes/haplotypes favored in different geographic regions. In summary, the time-series data, with soybean CC as an example, improved the accuracy and statistical power to dissect the genetic basis of traits and offered a promising opportunity for crop breeding with quantitative growth curves.

Knowledge of the transcription factor binding landscape (TFBL) is necessary to analyze gene regulatory networks for important agronomic traits. However, a low-cost and high-throughput in vivo chromatin profiling method is still lacking in plants. Here, we developed a transient and simplified cleavage under targets and tagmentation (tsCUT&Tag) that combines transient expression of transcription factor proteins in protoplasts with a simplified CUT&Tag without nucleus extraction. Our tsCUT&Tag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP-seq. Furthermore, we developed a strategy combining tsCUT&Tag with machine learning, which has great potential for profiling the TFBL across plant development.

Rice eating and cooking quality (ECQ) is a major concern of breeders and consumers, determining market competitiveness worldwide. Rice grain protein content (GPC) is negatively related to ECQ, making it possible to improve ECQ by manipulating GPC. However, GPC is genetically complex and sensitive to environmental conditions; therefore, little progress has been made in traditional breeding for ECQ. Here, we report that CRISPR/Cas9-mediated knockout of genes encoding the grain storage protein glutelin rapidly produced lines with downregulated GPC and improved ECQ. Our finding provides a new strategy for improving rice ECQ.

A CRISPR/LbCas12a-based nucleic acid detection method that uses crude leaf extracts as samples and is rapid (≤40 min for a full run) and highly sensitive (0.01%) can be used to monitor genetically modified organisms in the field.

Doubled haploid (DH) technology is used to obtain homozygous lines in a single generation, a technique that significantly accelerates the crop breeding trajectory. Traditionally, in vitro culture is used to generate DHs, but this technique is limited by species and genotype recalcitrance. In vivo haploid induction (HI) through seed is widely and efficiently used in maize and was recently extended to several other crops. Here we show that in vivo HI can be triggered by mutation of DMP maternal haploid inducer genes in allopolyploid (allotetraploid) Brassica napus and Nicotiana tabacum. We developed a pipeline for selection of DMP orthologs for clustered regularly interspaced palindromic repeats mutagenesis and demonstrated average amphihaploid induction rates of 2.4% and 1.2% in multiple B. napus and N. tabacum genotypes, respectively. These results further confirmed the HI ability of DMP gene in polyploid dicot crops. The DMP-HI system offers a novel DH technology to facilitate breeding in these crops. The success of this approach and the conservation of DMP genes in dicots suggest the broad applicability of this technique in other dicot crops.

Current gene delivery methods for maize are limited to specific genotypes and depend on time-consuming and labor-intensive tissue culture techniques. Here, we report a new method to transfect maize that is culture-free and genotype independent. To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%, transfection was performed at cool temperature using pollen pretreated to open the germination aperture (40%–55%). Magnetic nanoparticles (MNPs) coated with DNA encoding either red fluorescent protein (RFP), β-glucuronidase gene (GUS), enhanced green fluorescent protein (EGFP) or bialaphos resistance (bar) was delivered into pollen grains, and female florets of maize inbred lines were pollinated. Red fluorescence was detected in 22% transfected pollen grains, and GUS stained 55% embryos at 18 d after pollination. Green fluorescence was detected in both silk filaments and immature kernels. The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts (29%–74%) quantitated by polymerase chain reaction, and 5%–16% of the T1 seedlings showed immunologically active EGFP or GUS protein. Moreover, 1.41% of the bar transfected T1 plants were glufosinate resistant, and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization. These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Sorghum, the fifth largest cereal crop, has high value as a staple food and raw material for liquor and vinegar brewing. Due to its high biomass and quality, it is also used as the second most planted silage resource. No fragrant sorghums are currently on the market. Through CRISPR/Cas9-mediated knockout of SbBADH2, we obtained sorghum lines with extraordinary aromatic smell in both seeds and leaves. Animal feeding experiments showed that fragrant sorghum leaves were attractable. We believe this advantage will produce great value in the sorghum market for both grain and whole biomass forage.

Innovations in genomics have enabled the development of low-cost, high-resolution, single nucleotide polymorphism (SNP) genotyping arrays that accelerate breeding progress and support basic research in crop science. Here, we developed and validated the SoySNP618K array (618,888 SNPs) for the important crop soybean. The SNPs were selected from whole-genome resequencing data containing 2,214 diverse soybean accessions; 29.34% of the SNPs mapped to genic regions representing 86.85% of the 56,044 annotated high-confidence genes. Identity-by-state analyses of 318 soybeans revealed 17 redundant accessions, highlighting the potential of the SoySNP618K array in supporting gene bank management. The patterns of population stratification and genomic regions enriched through domestication were highly consistent with previous findings based on resequencing data, suggesting that the ascertainment bias in the SoySNP618K array was largely compensated for. Genome-wide association mapping in combination with reported quantitative trait loci enabled fine-mapping of genes known to influence flowering time, E2 and GmPRR3b, and of a new candidate gene, GmVIP5. Moreover, genomic prediction of flowering and maturity time in 502 recombinant inbred lines was highly accurate (>0.65). Thus, the SoySNP618K array is a valuable genomic tool that can be used to address many questions in applied breeding, germplasm management, and basic crop research.

RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes. The discovery of natural trans-kingdom RNAi indicated that small RNAs act as signaling molecules and enable communication between organisms in different kingdoms. The phenomenon and potential mechanisms of trans-kingdom RNAi are among the most exciting research topics. To better understand trans-kingdom RNAi, we review the history of the discovery and elucidation of RNAi mechanisms. Based on canonical RNAi mechanisms, we summarize the major points of divergence around RNAi pathways in the main eukaryotes’ kingdoms, including plants, animals, and fungi. We review the representative incidents associated with the mechanisms and applications of trans-kingdom RNAi in crop protection, and discuss the critical factors that should be considered to develop successful trans-kingdom RNAi-based crop protection strategies.

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

A new deaminase, TadA8e, was recently evolved in the laboratory. TadA8e catalyzes DNA deamination over 1,000 times faster than ABE7.10. We developed a high-efficiency adenine base editor, rABE8e (rice ABE8e), combining monomeric TadA8e, bis-bpNLS and codon optimization. rABE8e had substantially increased editing efficiencies at NG-protospacer adjacent motif (PAM) and NGG-PAM target sequences compared with ABEmax. For most targets, rABE8e exhibited nearly 100% editing efficiency and high homozygous substitution rates in the specific editing window, especially at Positions A5 and A6. The ability to rapidly generate plant materials with homozygous base substitutions will benefit gene function research and precision molecular breeding.

An enhanced CDA-like (eCDAL) was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editor version 1(pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.

The ubiquitous volatile linalool is metabolized in plants to nonvolatile derivatives. We studied Nicotiana attenuata plants which naturally vary in (S)-(+)-linalool contents, and lines engineered to produce either (R)-(-)- or (S)-(+)-linalool. Only (S)-(+)-linalool production was associated with slower growth of a generalist herbivore, and a large fraction was present as nonvolatile derivatives. We found that variation in volatile linalool and its nonvolatile glycosides mapped to the same genetic locus which harbored the biosynthetic gene, NaLIS, but that free linalool varied more in environmental responses. This study reveals how (S)-(+)-linalool and conjugates differ in their regulation and possible functions in resistance.