A Quantitative Computational Framework for Allopolyploid Single-Cell Data Integration and Core Gene Ranking in Development.

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.

TabHLH27 orchestrates root growth and drought tolerance to enhance water use efficiency in wheat.

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-A1Hap-II associated with stronger drought tolerance, larger root system, more spikelets, and higher WUE in wheat. Significantly, the excellent TabHLH27-A1Hap-II was selected during the breeding process in China, and introgression of TabHLH27-A1Hap-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.

Functional divergences of natural variations of TaNAM-A1 in controlling leaf senescence during wheat grain filling.

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.

Peroxidase gene TaPrx109-B1 enhances wheat tolerance to water deficit via modulating stomatal density.

Increasing the tolerance of crops to water deficit is crucial for the improvement of crop production in water-restricted regions. Here, a wheat peroxidase gene (TaPrx109-B1) belonging to the class III peroxidase gene family was identified and its function in water deficit tolerance was revealed. We demonstrated that overexpression of TaPrx109-B1 reduced leaf H2O2 level and stomatal density, increased leaf relative water content, water use efficiency, and tolerance to water deficit. The expression of TaEPF1 and TaEPF2, two key negative regulators of stomatal development, were significantly upregulated in TaPrx109-B1 overexpression lines. Furthermore, exogenous H2O2 downregulated the expression of TaEPF1 and TaEPF2 and increased stomatal density, while exogenous application of diphenyleneiodonium chloride, a potent NADPH oxidase inhibitor that repressed the synthesis of H2O2, upregulated the expression of TaEPF1 and TaEPF2, decreased stomatal density, and enhanced wheat tolerance to water deficit. These findings suggest that TaPrx109-B1 influences leaf stomatal density by modulation of H2O2 level and the expression of TaEPF1 and TaEPF2. The results of the field trial showed that overexpressing TaPrx109-B1 increased grain number per spike, which reduced the yield loss caused by water deficiency. Therefore, TaPrx109-B1 has great potential in breeding wheat varieties with improved water deficit tolerance.

TaLBD41 interacts with TaNAC2 to regulate nitrogen uptake and metabolism in response to nitrate availability.

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).

Application and accuracy assessment of a novel 3D-printed osteotomy guide in anterior maxillary segmental distraction osteogenesis / 上海交通大学学报（医学版）

Objective·To evaluate the effects of anterior maxillary segmental distraction osteogenesis(AMSDO)in treating sagittal maxillary hypoplasia in cleft lip and palate(CLP)patients and to report a 3D-printed surgical guide to facilitate the osteotomy.Methods·Twelve patients with CLP who underwent AMSDO were included in this study.Virtual osteotomy was performed in a 3-dimensional model and the osteotomy line were fabricated into a tooth-borne surgical guide by using 3D-printing technique.Lateral cephalograms taken before surgery(T0),at the end of consolidation(T1)and six months after consolidation(T2)were used to evaluate the effects of AMSDO.The accuracy of the osteotomy guide was measured by superimposing the postoperative CT data to virtual planning.Results·All the patients went through surgery without serious complications.SNA and overjet changed significantly both from T0 to T1 and from T0 to T2.ANB,facial convexity,and palatal length changed without significance from T0 to T1 and from T0 to T2.SNB remained stable.All the variables remained relatively stable from T1 to T2.The anteroposterior linear root-mean-square deviation(RMSD)between planning and actual results was 0.90 mm,while the angular RMSD in the sagittal plane was 5.07°.Conclusion·AMSDO is an effective treatment for maxillary hypoplasia secondary to CLP.The accuracy of this 3D-printed osteotomy guide is clinically acceptable,and this can simplify the surgery with fewer complications.

Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin: Creation of an unnatural analogue with improved anticancer properties

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin ( 1) and variecolactone ( 2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues ( 5- 7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

LHP1-mediated epigenetic buffering of subgenome diversity and defense responses confers genome plasticity and adaptability in allopolyploid wheat.

Polyploidization is a major driver of genome diversification and environmental adaptation. However, the merger of different genomes may result in genomic conflicts, raising a major question regarding how genetic diversity is interpreted and regulated to enable environmental plasticity. By analyzing the genome-wide binding of 191 trans-factors in allopolyploid wheat, we identified like heterochromatin protein 1 (LHP1) as a master regulator of subgenome-diversified genes. Transcriptomic and epigenomic analyses of LHP1 mutants reveal its role in buffering the expression of subgenome-diversified defense genes by controlling H3K27me3 homeostasis. Stripe rust infection releases latent subgenomic variations by eliminating H3K27me3-related repression. The simultaneous inactivation of LHP1 homoeologs by CRISPR-Cas9 confers robust stripe rust resistance in wheat seedlings. The conditional repression of subgenome-diversified defenses ensures developmental plasticity to external changes, while also promoting neutral-to-non-neutral selection transitions and adaptive evolution. These findings establish an LHP1-mediated buffering system at the intersection of genotypes, environments, and phenotypes in polyploid wheat. Manipulating the epigenetic buffering capacity offers a tool to harness cryptic subgenomic variations for crop improvement.

Transposable element-initiated enhancer-like elements generate the subgenome-biased spike specificity of polyploid wheat.

Transposable elements (TEs) comprise ~85% of the common wheat genome, which are highly diverse among subgenomes, possibly contribute to polyploid plasticity, but the causality is only assumed. Here, by integrating data from gene expression cap analysis and epigenome profiling via hidden Markov model in common wheat, we detect a large proportion of enhancer-like elements (ELEs) derived from TEs producing nascent noncoding transcripts, namely ELE-RNAs, which are well indicative of the regulatory activity of ELEs. Quantifying ELE-RNA transcriptome across typical developmental stages reveals that TE-initiated ELE-RNAs are mainly from RLG_famc7.3 specifically expanded in subgenome A. Acquisition of spike-specific transcription factor binding likely confers spike-specific expression of RLG_famc7.3-initiated ELE-RNAs. Knockdown of RLG_famc7.3-initiated ELE-RNAs resulted in global downregulation of spike-specific genes and abnormal spike development. These findings link TE expansion to regulatory specificity and polyploid developmental plasticity, highlighting the functional impact of TE-driven regulatory innovation on polyploid evolution.

Low-affinity SPL binding sites contribute to subgenome expression divergence in allohexaploid wheat.

Expression divergence caused by genetic variation and crosstalks among subgenomes of the allohexaploid bread wheat (Triticum aestivum. L., BBAADD) is hypothesized to increase its adaptability and/or plasticity. However, the molecular basis of expression divergence remains unclear. Squamosa promoter-binding protein-like (SPL) transcription factors are critical for a wide array of biological processes. In this study, we constructed expression regulatory networks by combining DAP-seq for 40 SPLs, ATAC-seq, and RNA-seq. Our findings indicate that a group of low-affinity SPL binding regions (SBRs) were targeted by diverse SPLs and caused different sequence preferences around the core GTAC motif. The SBRs including the low-affinity ones are evolutionarily conserved, enriched GWAS signals related to important agricultural traits. However, those SBRs are highly diversified among the cis-regulatory regions (CREs) of syntenic genes, with less than 8% SBRs coexisting in triad genes, suggesting that CRE variations are critical for subgenome differentiations. Knocking out of TaSPL7A/B/D and TaSPL15A/B/D subfamily further proved that both high- and low-affinity SBRs played critical roles in the differential expression of genes regulating tiller number and spike sizes. Our results have provided baseline data for downstream networks of SPLs and wheat improvements and revealed that CRE variations are critical sources for subgenome divergence in the allohexaploid wheat.

Transposable elements orchestrate subgenome-convergent and -divergent transcription in common wheat.

The success of common wheat as a global staple crop was largely attributed to its genomic diversity and redundancy due to the merge of different genomes, giving rise to the major question how subgenome-divergent and -convergent transcription is mediated and harmonized in a single cell. Here, we create a catalog of genome-wide transcription factor-binding sites (TFBSs) to assemble a common wheat regulatory network on an unprecedented scale. A significant proportion of subgenome-divergent TFBSs are derived from differential expansions of particular transposable elements (TEs) in diploid progenitors, which contribute to subgenome-divergent transcription. Whereas subgenome-convergent transcription is associated with balanced TF binding at loci derived from TE expansions before diploid divergence. These TFBSs have retained in parallel during evolution of each diploid, despite extensive unbalanced turnover of the flanking TEs. Thus, the differential evolutionary selection of paleo- and neo-TEs contribute to subgenome-convergent and -divergent regulation in common wheat, highlighting the influence of TE repertory plasticity on transcriptional plasticity in polyploid.

Utility of Triti-Map for bulk-segregated mapping of causal genes and regulatory elements in Triticeae.

Triticeae species, including wheat, barley, and rye, are critical for global food security. Mapping agronomically important genes is crucial for elucidating molecular mechanisms and improving crops. However, Triticeae includes many wild relatives with desirable agronomic traits, and frequent introgressions occurred during Triticeae evolution and domestication. Thus, Triticeae genomes are generally large and complex, making the localization of genes or functional elements that control agronomic traits challenging. Here, we developed Triti-Map, which contains a suite of user-friendly computational packages specifically designed and optimized to overcome the obstacles of gene mapping in Triticeae, as well as a web interface integrating multi-omics data from Triticeae for the efficient mining of genes or functional elements that control particular traits. The Triti-Map pipeline accepts both DNA and RNA bulk-segregated sequencing data as well as traditional QTL data as inputs for locating genes and elucidating their functions. We illustrate the usage of Triti-Map with a combination of bulk-segregated ChIP-seq data to detect a wheat disease-resistance gene with its promoter sequence that is absent from the reference genome and clarify its evolutionary process. We hope that Triti-Map will facilitate gene isolation and accelerate Triticeae breeding.

Genetic Control of Efficient Nitrogen Use for High Yield and Grain Protein Concentration in Wheat: A Review.

The increasing global population and the negative effects of nitrogen (N) fertilizers on the environment challenge wheat breeding to maximize yield potential and grain protein concentration (GPC) in an economically and environmentally friendly manner. Understanding the molecular mechanisms for the response of yield components to N availability and assimilates allocation to grains provides the opportunity to increase wheat yield and GPC simultaneously. This review summarized quantitative trait loci/genes which can increase spikes and grain number by enhancing N uptake and assimilation at relative early growth stage, and 1000-grain weight and GPC by increasing post-anthesis N uptake and N allocation to grains.

Evolutionary rewiring of the wheat transcriptional regulatory network by lineage-specific transposable elements.

More than 80% of the wheat genome consists of transposable elements (TEs), which act as major drivers of wheat genome evolution. However, their contributions to the regulatory evolution of wheat adaptations remain largely unclear. Here, we created genome-binding maps for 53 transcription factors (TFs) underlying environmental responses by leveraging DAP-seq in Triticum urartu, together with epigenomic profiles. Most TF binding sites (TFBSs) located distally from genes are embedded in TEs, whose functional relevance is supported by purifying selection and active epigenomic features. About 24% of the non-TE TFBSs share significantly high sequence similarity with TE-embedded TFBSs. These non-TE TFBSs have almost no homologous sequences in non-Triticeae species and are potentially derived from Triticeae-specific TEs. The expansion of TE-derived TFBS linked to wheat-specific gene responses, suggesting TEs are an important driving force for regulatory innovations. Altogether, TEs have been significantly and continuously shaping regulatory networks related to wheat genome evolution and adaptation.

An atlas of wheat epigenetic regulatory elements reveals subgenome divergence in the regulation of development and stress responses.

Wheat (Triticum aestivum) has a large allohexaploid genome. Subgenome-divergent regulation contributed to genome plasticity and the domestication of polyploid wheat. However, the specificity encoded in the wheat genome determining subgenome-divergent spatio-temporal regulation has been largely unexplored. The considerable size and complexity of the genome are major obstacles to dissecting the regulatory specificity. Here, we compared the epigenomes and transcriptomes from a large set of samples under diverse developmental and environmental conditions. Thousands of distal epigenetic regulatory elements (distal-epiREs) were specifically linked to their target promoters with coordinated epigenomic changes. We revealed that subgenome-divergent activity of homologous regulatory elements is affected by specific epigenetic signatures. Subgenome-divergent epiRE regulation of tissue specificity is associated with dynamic modulation of H3K27me3 mediated by Polycomb complex and demethylases. Furthermore, quantitative epigenomic approaches detected key stress responsive cis- and trans-acting factors validated by DNA Affinity Purification and sequencing, and demonstrated the coordinated interplay between epiRE sequence contexts, epigenetic factors, and transcription factors in regulating subgenome divergent transcriptional responses to external changes. Together, this study provides a wealth of resources for elucidating the epiRE regulomics and subgenome-divergent regulation in hexaploid wheat, and gives new clues for interpreting genetic and epigenetic interplay in regulating the benefits of polyploid wheat.

Genome-wide association study of yield and related traits in common wheat under salt-stress conditions.

BACKGROUND: Soil salinization is a major threat to wheat production. It is essential to understand the genetic basis of salt tolerance for breeding and selecting new salt-tolerant cultivars that have the potential to increase wheat yield. RESULT: In this study, a panel of 191 wheat accessions was subjected to genome wide association study (GWAS) to identify SNP markers linked with adult-stage characters. The population was genotyped by Wheat660K SNP array and eight phenotype traits were investigated under low and high salinity environments for three consecutive years. A total of 389 SNPs representing 11 QTLs were significantly associated with plant height, spike number, spike length, grain number, thousand kernels weight, yield and biological mass under different salt treatments, with the phenotypic explanation rate (R2) ranging from 9.14 to 50.45%. Of these, repetitive and pleiotropic loci on chromosomes 4A, 5A, 5B and 7A were significantly linked to yield and yield related traits such as thousand kernels weight, spike number, spike length, grain number and so on under low salinity conditions. Spike length-related loci were mainly located on chromosomes 1B, 3B, 5B and 7A under different salt treatments. Two loci on chromosome 4D and 5A were related with plant height in low and high salinity environment, respectively. Three salt-tolerant related loci were confirmed to be important in two bi-parental populations. Distribution of favorable haplotypes indicated that superior haplotypes of pleiotropic loci on group-5 chromosomes were strongly selected and had potential for increasing wheat salt tolerance. A total of 14 KASP markers were developed for nine loci associating with yield and related traits to improve the selection efficiency of wheat salt-tolerance breeding. CONCLUSION: Utilizing a Wheat660K SNPs chip, QTLs for yield and its related traits were detected under salt treatment in a natural wheat population. Important salt-tolerant related loci were validated in RIL and DH populations. This study provided reliable molecular markers that could be crucial for marker-assisted selection in wheat salt tolerance breeding programs.

A wheat transcription factor positively sets seed vigour by regulating the grain nitrate signal.

Seed vigour and early establishment are important factors determining the yield of crops. A wheat nitrate-inducible NAC transcription factor, TaNAC2, plays a critical role in promoting crop growth and nitrogen use efficiency (NUE), and now its role in seed vigour is revealed. A TaNAC2 regulated gene was identified that is a NRT2-type nitrate transporter TaNRT2.5 with a key role in seed vigour. Overexpressing TaNAC2-5A increases grain nitrate concentration and seed vigour by directly binding to the promoter of TaNRT2.5-3B and positively regulating its expression. TaNRT2.5 is expressed in developing grain, particularly the embryo and husk. In Xenopus oocyte assays TaNRT2.5 requires a partner protein TaNAR2.1 to give nitrate transport activity, and the transporter locates to the tonoplast in a tobacco leaf transient expression system. Furthermore, in the root TaNRT2.5 and TaNRT2.1 function in post-anthesis acquisition of soil nitrate. Overexpression of TaNRT2.5-3B increases seed vigour, grain nitrate concentration and yield, whereas RNA interference of TaNRT2.5 has the opposite effects. The TaNAC2-NRT2.5 module has a key role in regulating grain nitrate accumulation and seed vigour. Both genes can potentially be used to improve grain yield and NUE in wheat.

The bread wheat epigenomic map reveals distinct chromatin architectural and evolutionary features of functional genetic elements.

BACKGROUND: Bread wheat is an allohexaploid species with a 16-Gb genome that has large intergenic regions, which presents a big challenge for pinpointing regulatory elements and further revealing the transcriptional regulatory mechanisms. Chromatin profiling to characterize the combinatorial patterns of chromatin signatures is a powerful means to detect functional elements and clarify regulatory activities in human studies. RESULTS: In the present study, through comprehensive analyses of the open chromatin, DNA methylome, seven major chromatin marks, and transcriptomic data generated for seedlings of allohexaploid wheat, we detected distinct chromatin architectural features surrounding various functional elements, including genes, promoters, enhancer-like elements, and transposons. Thousands of new genic regions and cis-regulatory elements are identified based on the combinatorial pattern of chromatin features. Roughly 1.5% of the genome encodes a subset of active regulatory elements, including promoters and enhancer-like elements, which are characterized by a high degree of chromatin openness and histone acetylation, an abundance of CpG islands, and low DNA methylation levels. A comparison across sub-genomes reveals that evolutionary selection on gene regulation is targeted at the sequence and chromatin feature levels. The divergent enrichment of cis-elements between enhancer-like sequences and promoters implies these functional elements are targeted by different transcription factors. CONCLUSIONS: We herein present a systematic epigenomic map for the annotation of cis-regulatory elements in the bread wheat genome, which provides new insights into the connections between chromatin modifications and cis-regulatory activities in allohexaploid wheat.

Reducing expression of a nitrate-responsive bZIP transcription factor increases grain yield and N use in wheat.

Nitrogen (N) plays critical role in plant growth; manipulating N assimilation could be a target to increase grain yield and N use. Here, we show that ABRE-binding factor (ABF)-like leucine zipper transcription factor TabZIP60 mediates N use and growth in wheat. The expression of TabZIP60 is repressed when the N-deprived wheat plants is exposed to nitrate. Knock down of TabZIP60 through RNA interference (RNAi) increases NADH-dependent glutamate synthase (NADH-GOGAT) activity, lateral root branching, N uptake and spike number, and improves grain yield more than 25% under field conditions, while overexpression of TabZIP60-6D had the opposite effects. Further investigation shows TabZIP60 binds to ABRE-containing fragment in the promoter of TaNADH-GOGAT-3B and negatively regulates its expression. Genetic analysis reveals that TaNADH-GOGAT-3B overexpression overcomes the spike number and yield reduction caused by overexpressing TabZIP60-6D. As such, TabZIP60-mediated wheat growth and N use is associated with its negative regulation on TaNADH-GOGAT expression. These findings indicate that TabZIP60 and TaNADH-GOGAT interaction plays important roles in mediating N use and wheat growth, and provides valuable information for engineering N use efficiency and yield in wheat.