Wan Teng

Engineering karyotypes could expand the genetic and breeding potential of crops, but restructuring gigabase-scale polyploid genomes remains challenging. Here we develop centromere editing-mediated chromosomal variation, a strategy using CRISPR–Cas9 to target abundant centromeric retrotransposons in hexaploid wheat. Centromere editing-mediated chromosomal variation efficiently induces simultaneous chromosome cleavage across the genome, generating diverse karyotypic variants and reconfiguring up to 11 chromosomes in one generation. Notably, it produces widespread reciprocal translocations, with one line carrying six events involving seven chromosomes. Newly formed translocation chromosomes undergo centromere remodelling, enabling their relatively stable maintenance and heritable transmission. Moreover, balanced translocations impose minimal transcriptional perturbation and maintaining normal growth. These results establish centromeres as effective sites for chromosome engineering breeding and provide a scalable framework for karyotype redesign in polyploid crops.

Precise manipulation of genome structural variations holds great potential for plant trait improvement and biological research. Here we present a genome-editing approach, dual prime editing (DualPE), that efficiently facilitates precise deletion, replacement and inversion of large DNA fragments in plants. In our experiments, DualPE enabled the production of specific genomic deletions ranging from ~500 bp to 2 Mb in wheat protoplasts and plants. DualPE was effective in directly replacing wheat genomic fragments of up to 258 kb with desired sequences in the absence of donor DNA. Additionally, DualPE allowed precise DNA inversions of up to 205.4 kb in wheat plants with efficiencies of up to 51.5%. DualPE also successfully edited large DNA fragments in the dicots Nicotiana benthamiana and tomato, with editing efficiencies of up to 72.7%. DualPE thus provides a precise and efficient approach for large DNA sequence and chromosomal engineering, expanding the availability of precision genome-editing tools for crop improvement.

Plant growth is determined by the production of cells and initiation of new organs. Exploring genes that control cell number and cell size is of great significance for understanding plant growth regulation. In this study, we characterized two wheat mutants, ah and dl, with abnormal growth. The ah mutant is a naturally occurring variant characterized by severe dwarfism, increased tiller number, and reduced grain length, while the dl mutant is derived from an ethyl methane sulfonate (EMS)‐mutagenized population and exhibits smaller grain size and slightly reduced plant height. Cytological analyses revealed abnormal cell number, cell morphology and arrangement in the stems and leaves of the ah mutant, along with reduced cell length in the grains of the dl mutant. Map‐based cloning identified that both mutants carry mutations in the same gene TavWA1‐7D, which encodes a protein with a von Willebrand factor A (vWA) domain. The ah mutant harbors a 174‐bp insertion in the 1,402‐bp coding sequence (CDS) of TavWA1‐7D, causing premature termination of protein translation, while the dl mutant contains a Glu420Lys substitution. Mimicking the TavWA1‐7D^ah through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9‐mediated genome editing leads to a severe dwarfism phenotype. The C‐terminus of the protein is crucial for its correct subcellular localization and interaction, supporting its critical role for TavWA1‐7D function. Proteomic analysis showed that the dwarf phenotype of the ah mutant is associated with impaired photosynthesis, ribosome function, and nucleosome formation. Additionally, TavWA1‐7D interacts with an E3 ligase, TaVIP1‐3B, the expression levels of which are elevated in both mutants. Overexpression and knockout studies of TaVIP1‐3B demonstrated its negative regulatory role in cell length and grain size. Together, our findings suggest that TavWA1‐7D plays a vital role in regulating wheat growth and yield‐related traits, with the dl mutant's short grain phenotype being associated with TaVIP1‐3B expression levels.

Polyploidization drives regulatory and phenotypic innovation. How the merger of different genomes contributes to polyploid development is a fundamental issue in evolutionary developmental biology and breeding research. Clarifying this issue is challenging because of genome complexity and the difficulty in tracking stochastic subgenome divergence during development. Recent single-cell sequencing techniques enabled probing subgenome-divergent regulation in the context of cellular differentiation. However, analyzing single-cell data suffers from high error rates due to high dimensionality, noise, and sparsity, and the errors stack up in polyploid analysis due to the increased dimensionality of comparisons between subgenomes of each cell, hindering deeper mechanistic understandings. In this study, we develop a quantitative computational framework, called “pseudo-genome divergence quantification” (pgDQ), for quantifying and tracking subgenome divergence directly at the cellular level. Further comparing with cellular differentiation trajectories derived from single-cell RNA sequencing data allows for an examination of the relationship between subgenome divergence and the progression of development. pgDQ produces robust results and is insensitive to data dropout and noise, avoiding high error rates due to multiple comparisons of genes, cells, and subgenomes. A statistical diagnostic approach is proposed to identify genes that are central to subgenome divergence during development, which facilitates the integration of different data modalities, enabling the identification of factors and pathways that mediate subgenome-divergent activity during development. Case studies have demonstrated that applying pgDQ to single-cell and bulk tissue transcriptomic data promotes a systematic and deeper understanding of how dynamic subgenome divergence contributes to developmental trajectories in polyploid evolution.

Cultivating high‐yield wheat under limited water resources is crucial for sustainable agriculture in semiarid regions. Amid water scarcity, plants activate drought response signaling, yet the delicate balance between drought tolerance and development remains unclear. Through genome‐wide association studies and transcriptome profiling, we identified a wheat atypical basic helix‐loop‐helix (bHLH) transcription factor (TF), TabHLH27‐A1, as a promising quantitative trait locus candidate for both relative root dry weight and spikelet number per spike in wheat. TabHLH27‐A1/B1/D1 knock‐out reduced wheat drought tolerance, yield, and water use efficiency (WUE). TabHLH27‐A1 exhibited rapid induction with polyethylene glycol (PEG) treatment, gradually declining over days. It activated stress response genes such as TaCBL8‐B1 and TaCPI2‐A1 while inhibiting root growth genes like TaSH15‐B1 and TaWRKY70‐B1 under short‐term PEG stimulus. The distinct transcriptional regulation of TabHLH27‐A1 involved diverse interacting factors such as TaABI3‐D1 and TabZIP62‐D1. Natural variations of TabHLH27‐A1 influence its transcriptional responses to drought stress, with TabHLH27‐A1^Hap‐II associated with stronger drought tolerance, larger root system, more spikelets, and higher WUE in wheat. Significantly, the excellent TabHLH27‐A1^Hap‐II was selected during the breeding process in China, and introgression of TabHLH27‐A1^Hap‐II allele improved drought tolerance and grain yield, especially under water‐limited conditions. Our study highlights TabHLH27‐A1's role in balancing root growth and drought tolerance, providing a genetic manipulation locus for enhancing WUE in wheat.

Leaf senescence is an essential physiological process related to grain yield potential and nutritional quality. Green leaf duration (GLD) after anthesis directly reflects the leaf senescence process and exhibits large genotypic differences in common wheat; however, the underlying gene regulatory mechanism is still lacking. Here, we identified TaNAM‐A1 as the causal gene of the major loci qGLD‐6A for GLD during grain filling by map‐based cloning. Transgenic assays and TILLING mutant analyses demonstrated that TaNAM‐A1 played a critical role in regulating leaf senescence, and also affected spike length and grain size. Furthermore, the functional divergences among the three haplotypes of TaNAM‐A1 were systematically evaluated. Wheat varieties with TaNAM‐A1d (containing two mutations in the coding DNA sequence of TaNAM‐A1) exhibited a longer GLD and superior yield‐related traits compared to those with the wild type TaNAM‐A1a. All three haplotypes were functional in activating the expression of genes involved in macromolecule degradation and mineral nutrient remobilization, with TaNAM‐A1a showing the strongest activity and TaNAM‐A1d the weakest. TaNAM‐A1 also modulated the expression of the senescence‐related transcription factors TaNAC‐S‐7A and TaNAC016‐3A. TaNAC016‐3A enhanced the transcriptional activation ability of TaNAM‐A1a by protein–protein interaction, thereby promoting the senescence process. Our study offers new insights into the fine‐tuning of the leaf functional period and grain yield formation for wheat breeding under various geographical climatic conditions.

Cultivating high-yield wheat under limited water resources is essential for sustainable agriculture in semiarid regions. Amid water scarcity, plants activate drought response signaling, yet the delicate balance between drought tolerance and development remains unclear. Through genome-wide-association study (GWAS) and transcriptome profiling, we identified a wheat atypical basic helix-loop-helix (bHLH) transcription factor (TF), TabHLH27-A1, as a promising quantitative trait locus (QTL) candidate for both relative root dry weight (DW.R%) and spikelet number per spike (SPS) in wheat. TabHLH27-A1/B1/D1 knockout reduced wheat drought tolerance, yield, and water use efficiency (WUE). TabHLH27-A1 exhibited rapid induction with PEG treatment, gradually declining over days. It activated stress response genes such as TaCBL8-B1 and TaCPI2-A1 while inhibiting root growth genes like TaSH15-B1 and TaWRKY70-B1 under short-term PEG stimulus. The distinct transcriptional regulation of TabHLH27-A1 involved diverse interacting factors such as TaABI3-D1 and TabZIP62-D1. Natural variations of TabHLH27-A1 influences its transcriptional responses to drought stress, with TabHLH27-A1 ^Hap-II associated with stronger drought tolerance, larger root system, more spikelets, and higher WUE in wheat. Significantly, the elite TabHLH27-A1 ^Hap-II was selected during the breeding process in China, and introgression of TabHLH27-A1 ^Hap-II allele improves drought tolerance and grain yield, especially under water-limited conditions. Our study highlights TabHLH27-A1’s role in balancing root growth and drought tolerance, providing a genetic manipulation locus for enhancing WUE in wheat.

Summary: Nitrate is the main source of nitrogen (N) available to plants and also is a signal that triggers complex regulation of transcriptional networks to modulate a wide variety of physiological and developmental responses in plants. How plants adapt to soil nitrate fluctuations is a complex process involving a fine‐tuned response to nitrate provision and N starvation, the molecular mechanisms of which remain largely uncharted. Here, we report that the wheat transcription factor TaLBD41 interacts with the nitrate‐inducible transcription factor TaNAC2 and is repressed by nitrate provision. Electrophoretic mobility shift assay and dual‐luciferase system show that the TaLBD41‐NAC2 interaction confers homeostatic coordination of nitrate uptake, reduction, and assimilation by competitively binding to TaNRT2.1, TaNR1.2, and TaNADH‐GOGAT. Knockdown of TaLBD41 expression enhances N uptake and assimilation, increases spike number, grain yield, and nitrogen harvest index under different N supply conditions. We also identified an elite haplotype of TaLBD41‐2B associated with increased spike number and grain yield. Our study uncovers a novel mechanism underlying the interaction between two transcription factors in mediating wheat adaptation to nitrate availability by antagonistically regulating nitrate uptake and assimilation, providing a potential target for designing varieties with efficient N use in wheat (Triticum aestivum).

Summary: Leaf senescence is an essential physiological process related to grain yield potential and nutritional quality. Green leaf duration (GLD) after anthesis directly reflects the leaf senescence process and exhibits large genotypic differences in common wheat; however, the underlying gene regulatory mechanism is still lacking up to now. Here, we report TaNAM-A1 as the causal gene of major loci qGLD-6A for GLD during grain filling by map-based cloning. The role of TaNAM-A1 in regulating leaf senescence, spike length, and grain size was proved by transgenic assay and TILLING mutants analyses. Furthermore, the functional divergences among TaNAM-A1 three haplotypes were systematically evaluated. Wheat varieties with TaNAM-A1d (containing two mutations in CDS of TaNAM-A1 ) had longer GLD and advantages in yield-related traits than those with the wild type TaNAM-A1a . All three haplotypes were functional in transactivating the expression of genes involved in macromolecular degradation and mineral nutrient remobilization, with TaNAM-A1a the strongest activity and TaNAM-A1d the weakest. TaNAM-A1 modulates the expression of TaNAC016-3A and TaNAC-S-7A to trigger senescence initiation. TaNAC016-3A enhances TaNAM-A1 transcriptional activation ability by protein-protein interaction. Our study provides new insights into fine-tuning the leaf functional period and grain yield formation for wheat breeding under different geographical climatic conditions.