Maize plant height and stalk mechanical strength are critical traits that influence planting density, yield, and lodging resistance. Although numerous dwarf mutants have been characterized in maize, most cannot be directly utilized in breeding programs due to associated developmental and reproductive deficiencies. In a previous study, we demonstrated that ZmNAC17 regulates mesocotyl elongation by mediating auxin and reactive oxygen species (ROS) biosynthetic pathways. Here, we characterize the role of ZmNAC17 in maize stalk development using both zmnac17 mutants and ZmNAC17-overexpressing (OE) lines. Plant height, stalk diameter, and internode length were reduced in both the zmnac17-1 EMS mutant and the zmnac17-3 CRISPR mutant. Internode cell length and cell area were decreased, whereas cell number was increased in zmnac17-1. Cellulose and lignin contents were elevated in zmnac17-1. Stalk bending force was diminished in zmnac17-3 but enhanced in the OE lines. The ratio of syringyl to guaiacyl (S/G), a key lignin monomer composition, was increased in zmnac17-3 while reduced in the OE lines. ZmNAC17 functions as a transcription factor, with its downstream targets implicated in phytohormone biosynthesis, phytohormone signaling, and lignin biosynthesis. CUT&Tag binding profile, EMSA, and dual-luciferase reporter assay demonstrate that ZmNAC17 promotes the expression of caffeoyl-CoA O-methyltransferase (CCoAOMT). IP-MS, Co-IP, and GST pull-down assays reveal that ZmNAC17 interacts with Beta glucosidase aggregating factor1 (BGAF1). Collectively, our findings indicate that ZmNAC17 regulates maize stalk development through transcriptional activation and protein-protein interactions, thereby providing new genetic resources for modifying plant architecture and mechanical strength in maize.

G-quadruplex (DG4) is folded in guanine-rich DNA sequences and regulates DNA replication and transcription. Although bioinformatics analyses have predicted the presence of DG4 in maize, its biological functions remain largely unexplored. In this study, we treated maize seedlings with 0, 100, 200, and 300 μM TMPyP4, a DG4-stabilizing ligand, and observed that TMPyP4 inhibits radicle growth by increasing reactive oxygen species (ROS) levels and inducing DNA fragmentation in radicle cells. Transcriptomic RNA-seq revealed that TMPyP4 modulated the expression of 1614 genes in maize radicle cells, which were predominantly associated with redox reactions, membrane components, and secondary metabolic pathways. BG4-ChIP-seq analysis demonstrated that DG4 structures are evenly distributed across the ten chromosomes of the maize genome, occupying 22,449 loci and showing significant enrichment for specific DG4-binding motifs. Integrative analysis of RNA-seq data and BG4-ChIP-seq identified 944 differentially expressed genes, which were significantly enriched in pathways related to redox reactions and secondary metabolism. Collectively, these findings suggest that DG4-stabilizing ligands regulate maize radicle growth by modulating ROS homeostasis, providing critical insights into the functional roles of DG4s in maize.

Target of rapamycin (TOR) signaling plays a pivotal role in regulating various cellular processes, including energy metabolism and growth. In this study, we investigated the impact of TOR inhibition on maize radicle development, focusing on energy homeostasis and starch metabolism. We found that TOR inhibition significantly decreased the ATP/AMP ratio, indicating a disruption in cellular energy balance. This was accompanied by alterations in the expression of genes related to energy metabolism, such as STP1 (SUGAR TRANSPORTER PROTEIN 1) and ASN1 (Aspartate Aminotransferase 1). Transcriptomic and metabolomic analyses revealed significant changes in carbohydrate metabolism, particularly in starch degradation pathways, with key metabolites like glucose-6-phosphate (G6P) showing marked reductions. To further elucidate the role of starch metabolism in TOR-mediated regulation of radicle growth, we performed rescue experiments using exogenous soluble starch and maltose. Both treatments alleviated the inhibitory effects of TOR suppression, with radicle length increasing by 19.7% and 37.0%, respectively. These results highlight that TOR signaling regulates radicle development by modulating starch metabolism and energy homeostasis. Our findings provide new insights into the molecular mechanisms underlying TOR-mediated growth regulation and suggest that manipulating starch metabolism could be a potential strategy to enhance seedling development under stress conditions.

The radicle of maize (Zea mays L.) is the first nutritional organ to emerge during seedling growth. However, the regulatory mechanisms underlying the pentose phosphate pathway (PPP) in radicle growth remain poorly understood. In this study, we investigated the effects of PPP inhibition on maize radicle development by treating maize seeds with the PPP inhibitor (6-amino nicotinamide, 6-AN). We evaluated radicle phenotypes and key energy and redox indicators, and performed integrated transcriptomic and metabolomic analyses to identify differentially expressed genes, metabolites, and affected pathways. Our results showed that 6-AN treatment significantly reduced radicle length and altered the NADPH/NADP+ and NADH/NAD+ ratios, with a marked decrease in reactive oxygen species (ROS) levels, total antioxidant capacity (T-AOC), and activities of SnRK1 and NADPH oxidase (NOX). These findings suggest that PPP inhibition leads to a significant reduction in energy and redox status. Additionally, transcriptomic and metabolomic analyses revealed that redox-related pathways were significantly affected. Notably, ROS levels in the radicle were restored to some extent by the addition of H2O2 and NADPH, confirming that PPP regulates radicle growth by modulating ROS homeostasis through NADPH. This study highlights the critical role of the PPP in regulating radicle development via ROS balance.

The pentose phosphate pathway (PPP) is a crucial metabolic route that influences various physiological processes in plants. In this study, we examined the effects of PPP inhibition on maize radicle growth using the PPP inhibitors and mutant. Our results showed that PPP inhibition significantly reduces radicle length, accompanied by a substantial decrease of energy level. Furthermore, molecular analyses revealed that PPP inhibition downregulates the cytokinin biosynthesis gene IPT while upregulating cytokinin oxidase/dehydrogenase (CKX) protein abundance. Bioinformatic and biochemical investigations identified a flavin adenine dinucleotide (FAD)binding domain in CKX and showed that PPP blockade reduces root FAD content, impairing CKX enzymatic activity. Transcriptomic and metabolomics analysis revealed that PPP inhibition downregulates ZmRR3, a cytokinin signaling related response regulator, through DNA methylation in its promoter region. Notably, DNA methylation inhibition via 5-Azacytidine treatment restores radicle growth and ZmRR3 expression. These findings highlight the essential role of PPP in regulating energy metabolism, cytokinin content, and gene expression during maize radicle development. This study advances understanding of the metabolic-cytokinin interplay critical for radicle growth and offers potential molecular targets for maize improvement.

The accuracy of genomic annotation is crucial for subsequent functional investigations; however, computational protocols used in high-throughput annotation of open reading frames (ORFs) can introduce inconsistencies. These inconsistencies, which lead to non-uniform extension or truncation of sequence ends, pose challenges for downstream analyses. Existing strategies to rectify these inconsistencies are time-consuming and labor-intensive, lacking specific approaches. To address this gap, we developed toGC, a tool that integrates genomic annotation with RNA-seq datasets to rectify annotation inconsistencies. Using toGC, we achieved an accuracy of nearly 100% accuracy in correcting inconsistencies in published Phytophthora sojae ORFs. We applied this innovative pipeline to the GPCR-bigrams gene family, which was predicted to have 42 members in the P. sojae genome but lacked experimental validation. By employing toGC, we identified 32 GPCR-bigram ORFs with inconsistencies between previous annotations and toGC-corrected sequences. Notably, among these were 5 genes (GPCR-TKL9, GPCR-TKL15, GPCR-PDE3, GPCR-AC3, and GPCR-AC4) showed substantial inconsistencies. Experimental gene annotation confirmed the effectiveness of toGC, as sequences obtained through cloning matched those annotated by toGC. Importantly, we discovered two novel GPCRs (GPCR-AC3 and GPCR-AC4), which were previously mispredicted as a single gene. CRISPR/Cas9-mediated knockout experiments revealed the involvement of GPCR-AC4 but not GPCR-AC3 in oospore production, further confirming their status as two separate genes. In addition to P. sojae, the reliability of the toGC pipeline in Phytophthora capsici and Pythium ultimum further emphasizes the robustness of this pipeline. Our findings highlight the utility of toGC for reliable gene model correction, facilitating investigations into biological functions and offering potential applications in diverse species analyses.

Global climate change and human activities are posing substantial threats to biodiversity. The genus Opisthopappus (O. taihangensis and O. longilobus), endemic to Taihang Mountains in china, possesses great ornamental and medicinal value. However, it is confronted with the compounding pressures of habitat fragmentation and escalating climate change. Here, we present the first haplotype-resolved, chromosome-scale genome assembly of O. longilobus (~2.95 Gb, ~58,000 protein-coding genes per haplotype) and re-sequence 115 individuals across its range. Comparative analyses show Opisthopappus is sister to Artemisia-Chrysanthemum, with Opisthopappus and Chrysanthemum diverging at 5.15-5.18 million years ago. A profound genetic divergence is evident between O. taihangensis and O. longilobus, resulting in two distinct lineages within each species, driven by geography and climate. Our analyses indicate restricted gene flow, low diversity, and recurrent demographic bottlenecks collectively contribute to their endangerment. By integrating population genomics and environmental variables, we identified 4,620 core adaptive loci and 4,437 core adaptive genes linked to water deprivation, hormone regulation, and metabolism. Genomic offset predicts higher maladaptation risk in populations under drastic climate change. Furthermore, metabolomic and experimental data demonstrate that diverged promoters of two O-methyltransferase genes, OMT250 and OMT310, account for the differential acacetin/linarin accumulation between C. morifolium and O. longilobus. These findings advance understanding of evolution and climate vulnerability of Opisthopappus, offering a model for genomics-guided biodiversity conservation. ### Competing Interest Statement The authors have declared no competing interest. National Key Research and Development Program of China, 2021YFD1200200

Phytophthora sojae-induced root rot poses a major threat to soybean production. While the molecular mechanisms underlying soybean-P. sojae interactions have been extensively studied, their biochemical basis remains largely unexplored. Previous research has identified key metabolic modules involved in pathogen defense, but structural diversity has largely been constrained by studies on single soybean accessions. Here, we broadened the chemical search space to a diverse soybean germplasm collection using high-throughput metabolomics as a powerful tool for comprehensive metabolic profiling. Chemical classes of lipids and phenylpropanoids again retrieved the most pronounced responses upon P. sojae infection in general. A two-layer analytical strategy further finely resolved metabolites into pathogenesis-, resistance-, and tolerance-type accumulation patterns, leading to the identification of cinnamaldehyde and coumestrol as potent defense metabolites. Bioassays validated cinnamaldehyde directly and strongly inhibited cyst germination and mycelial growth, and coumestrol, a benzofuran-type metabolite, exhibited broad-spectrum activity against spore germination as an identified phytoalexin. Multiomics analyses nailed down the candidate of coumestrol biosynthesis genes, and genetically overexpression of regulatory genes (Dir2a/4a/4b) in hairy root systems increased coumestrol accumulation thus positively correlating with improved host resistance. Interestingly, tolerance-type compounds may serve distinct ecological roles, as exemplified by daidzein, which, despite being classified as a tolerance-type metabolite, recruits more zoospores facilitating secondary infection in fact. This study highlights a systematic approach for population-level investigations and emphasizes the necessity of integrating bioinformatics with experimental validation to accurately predict metabolite or gene ecological functions.

Bacterial infections, particularly uropathogenic E. coli (UPEC), contribute substantially to male infertility through tissue damage and subsequent fibrosis in the testis and epididymis. The role of testicular macrophages (TMs), a diverse cell population integral to tissue maintenance and immune balance, in fibrosis is not fully understood. Here, we used single-cell RNA sequencing in a murine model of epididymo-orchitis to analyze TM dynamics during UPEC infection. Our study identified a marked increase in S100a4+ macrophages, originating from monocytes, strongly associated with fibrotic changes. This association was validated in human testicular and epididymal samples. We further demonstrated that S100a4+ macrophages transition to a myofibroblast-like phenotype, producing extracellular matrix proteins such as collagen I and fibronectin. S100a4, both extracellular and intracellular, activated collagen synthesis through the TGF-β/STAT3 signaling pathway, highlighting this pathway as a therapeutic target. Inhibition of S100a4 with niclosamide or macrophage-specific S100a4 KO markedly reduced immune infiltration, tissue damage, and fibrosis in infected murine models. Our findings establish the critical role of S100a4+ macrophages in fibrosis during UPEC-induced epididymo-orchitis and propose them as potential targets for antifibrotic therapy development.

Fertilization can increase potential yield of maize, but little is known about the impact of different combinations of fertilizers on grain quality, especially under long-term located fertilization. To explore the effects of long-term nutrient applications on nutritional quality of maize grain, the maize variety Luyu 16 was treated with 12 different fertilization treatments based on 34 years of located fertilization under a maize–wheat rotation in this study. The results showed that long-term fertilization can increase iron (Fe) content but decrease sodium (Na) and potassium (K) contents in maize grain. The contents of calcium (Ca) and iron (Fe) in maize grain were highest under high nitrogen (N2) treatment, while the content of Ca was lowest under high organic manure (M2) treatment. Under the combined application of nitrogen fertilizer and organic fertilizer, the contents of magnesium (Mg) and phosphorus (P) increased significantly, and K content also increased, although not significantly. In addition, fertilization increased protein content and decreased the content of starch and oil. Furthermore, the contents of total amino acids and lysine were increased under fertilization. The X-ray diffraction spectrum of the starch was of the typical “A” type, and none of the fertilization treatments changed the starch crystal type. The results also indicated that pasting temperature, trough viscosity and final viscosity increased after fertilization treatment, while peak viscosity and breakdown reduced. High inorganic nitrogen (N2) fertilizer could significantly improve setback, while high organic fertilizer (M2) significantly reduced setback. These results suggested that rational use of fertilizers, especially the combined use of organic and inorganic nitrogen fertilizers, plays an important role in improving maize grain quality.

Shoot branching is a key process of plant growth and development, finely controlled by cytokinin and sugars. However, cytokinin fails to induce bud outgrowth in the absence of sugar, and so far nothing is known about its ability to antagonize auxin when sugar availability is limited. Here we demonstrate in rose that cytokinin requires sugar metabolism and signalling to promote bud outgrowth, to down-regulate the expression of RhBRC1, a transcription factor gene that inhibits axillary bud growth, and to antagonize the inhibitory effect of auxin on bud outgrowth. Cytokinin regulation of bud sink strength was tightly associated with sugar metabolism, which was evidenced by the expression of genes involved in sugar metabolism [e.g. glycolysis, the tricarboxylic acid, and the oxidative pentose phosphate pathway (OPPP)], a metabolomic approach, and the quantification of total carbon and nitrogen in buds. Cytokinin supply is associated with a significant up-regulation of the OPPP and nitrogen accumulation. Meanwhile, sugar up-regulated bud sensitivity to cytokinin, associated with a significant down-regulation of the cytokinin signalling regulator RhARR1. These findings highlight the key role of sugar metabolism and signalling in cytokinin-induced bud outgrowth and provide new insights into the importance of nutrient-hormone crosstalk in the regulation of shoot branching.

This article offers a comprehensive overview of the starch, protein, oil, and carotenoids content in maize kernels, while also outlining future directions for research in this area. Maize is one of the most important cereal crops globally. Maize kernels serve as a vital source of feed and food, and their nutritional quality directly impacts the dietary intake of both animals and humans. Maize kernels contain starch, protein, oil, carotenoids, and a variety of vitamins and minerals, all of which are important for maintaining life and promoting health. This review presents the current understanding of the content of starch, protein, amino acids, oil, and carotenoids in maize kernels, while also highlighting knowledge gaps that need to be addressed.

Introduction Maize is one of the first crops to benefit from heterosis, significantly enhancing commercial breeding. Despite extensive research, the molecular mechanisms of heterosis remain elusive. Objectives This study integrates a novel genetic framework with transcriptomic and phenotypic analyses to identify heterosis-related genes and uncover their regulatory mechanisms. Methods Single-segment substitution lines (SSSLs) were combined with a novel double single-factor differential subtraction (DSDS) strategy to identify heterosis-related differentially expressed genes (DEGs). Unlike traditional parent-hybrid comparisons, DSDS subtracts background expression noise and identifies heterosis-related genes within a controlled genomic context. Five hybrid groups, including mini-F1 and sub-F1 hybrids, were generated and analyzed. RNA sequencing was conducted at 15 days after pollination (DAP) to profile gene expression. Motif enrichment and dual-luciferase assays were used for functional validation. Results Heterosis exhibited a nonlinear trajectory with parental genetic distance, displaying a rapid increase at low genomic heterogeneity levels before reaching a saturation point at moderate divergence. DSDS identified 359 and 80 heterosis-related DEGs in two mini-F1 hybrids, with most DEGs located outside the substitution regions, supporting a trans-regulatory mechanism. GO (gene ontology) analysis revealed enrichment in transport and cell expansion pathways, aligning with key processes in early kernel development. Notably, ZmWRKY67 was the only DEG in the substitution segment of mini-F1 CL137 × Ye478. Initial functional assays confirmed its regulation of Incw1, a key sucrose metabolism gene. The findings suggest a transcriptional “butterfly effect”, where minor genomic variations in heterozygous regions trigger genome-wide expression changes to drive heterosis. Conclusion This study demonstrates the effectiveness of SSSL-based DSDS in identifying heterosis-related genes and provides a framework for dissecting transcriptional regulation in heterosis. The observed transcriptional “butterfly effect” offers a novel perspective on how small genomic variations contribute to heterosis. Future research should explore additional substitution-region DEGs to further unravel the molecular basis of heterosis in maize.

Drought stress is a devastating natural stress that threatens crop productivity and quality. Mitigating the adverse effects of drought stress on wheat is a key object in agriculture. C-repeat binding transcription factor/DROUGHT RESPONSE ELEMENT BINDING FACTOR 1 (CBF/DREB1) transcription factors are well known for their role in cold acclimation. However, the involvement of CBF genes in drought stress and the mechanisms underlying their function remain poorly understood. In this study, 81 CBFs were identified in wheat, which were further clustered into four distinct lineages based on phylogenetic analysis. Chromosomal localization indicated that most CBF genes were dispersed across chromosome 5. We identified three homoeologous genes (TaCBF14A, TaCBF14B, and TaCBF14D) that were simultaneously upregulated under drought stress based on RNA-seq analysis. According to the high expression after drought stress, TaCBF14B was selected for further functional analysis. Subcellular localization and transcriptional activation activity analysis indicated that TaCBF14B likely functions as a transcription factor involved in drought stress tolerance. Overexpression of TaCBF14B in Arabidopsis enhanced the primary root growth by 13.49% (OE1), 12.56% (OE2), and 19.53% (OE3) under 200 mM mannitol treatment, and 21.65% (OE1), 16.63% (OE2), and 28.13% (OE3) under 250 mM mannitol treatment compared to WT. Meanwhile, the water loss rate of transgenic lines was 56% in WT leaves, but only 44%, 50%, and 40% in OE1, OE2, and OE3 lines, respectively. Compared to the wild type, POD activities of OE1, OE2, and OE3 were significantly increased by 42.94%, 29.41%, and 62.52%, respectively. And the Pro activities in OE1, OE2, and OE3 were significantly increased by 16.33%, 5.18%, and 29.09%, respectively, compared to the wild type. Additionally, the MDA content in OE1, OE2, and OE3 was significantly reduced by 40.53%, 15.81%, and 54.36%, respectively. Further analysis showed that the transgenic lines were hypersensitive to abscisic acid (ABA), and exhibited increased expression of AtABI3. We speculate that TaCBF14B plays an important role in enhancing drought tolerance. In summary, our findings provide new insights into the functional roles of CBF genes in drought stress tolerance.

Crop domestication has long been known to reshape rhizosphere microbial communities, yet research has focused disproprotionately on bacteria and fungal responses to crop domestication while neglecting protist communities. Protists, as key microbial predators regulating bacterial populations and thereby their functionalities, remain understudied in this context. Here, we investigate the influence of soybean domestication on both bacterial and protist communities, with a focus on the reorganization of ecological strategies, specifically generalists and specialists, within these microbiomes. We analyzed 270 rhizosphere samples from 27 domesticated and 63 wild soybean varieties. Domestication significantly altered community compositions of bacterial communities, with wild soybeans harboring higher proprotions of Pseudomonadota (71.4 %) and Bacillota (4.8 %), while domesticated soybeans exhibited an enrichment of Bacteroidota (11.0 %). Protist communities also diverged: wild soybeans were dominated by Cercozoa (58.2 %) and Gyrista (23.5 %), while domesticated plants had more Ciliophora (7.1 %) and Evosea (5.7 %). Domesticated soybeans hosted fewer generalist and specialist bacteria but more generalist protists, suggesting divergent microbial responses to domestication. Correlation analyses revealed that bacterial and protist generalists exhibited strong positive correlations with each other. At the same time, bacterial and protist specialists also showed positive correlations in wild soybeans-patterns that were largely absent in their domesticated counterparts. Functionally, wild soybeans supported more ureolytic and methylotrophic bacteria, while domesticated soybeans favored nitrate-respiration taxa. Notably, predatory protists in wild soybeans were significantly correlated with bacteria involved in carbon and nitrogen cycling, a key ecological relationship lost with domestication. These findings suggest that domestication exerts different selection pressures on bacteria and protists, disrupting potential relationships between bacterial and protist functional groups.

Intercropping has the potential to improve plant nutrition as well as crop yield. However, the exact mechanism promoting improved nutrient acquisition and the role the rhizosphere microbiome may play in this process remains poorly understood. Here, we use a peanut/maize intercropping system to investigate the role of root-associated microbiota in iron nutrition in these crops, combining microbiome profiling, strain and substance isolation and functional validation. We find that intercropping increases iron nutrition in peanut but not in maize plants and that the microbiota composition changes and converges between the two plants tested in intercropping experiments. We identify a Pseudomonas secreted siderophore, pyoverdine, that improves iron nutrition in glasshouse and field experiments. Our results suggest that the presence of siderophore-secreting Pseudomonas in peanut and maize intercropped plays an important role in iron nutrition. These findings could be used to envision future intercropping practices aiming to improve plant nutrition.

Phosphatases are important regulators of protein phosphorylation and various cellular processes, and they serve as counterparts to kinases. In this study, our comprehensive analysis of oomycete complete proteomes unveiled the presence of approximately 3833 phosphatases, with most species estimated to have between 100 and 300 putative phosphatases. Further investigation of these phosphatases revealed a significant increase in protein serine/threonine phosphatases (PSP) within oomycetes. In particular, we extensively studied the metallo-dependent protein phosphatase (PPM) within the PSP family in the model oomycete Phytophthora sojae. Our results showed notable differences in the expression patterns of PPMs throughout 10 life stages of P. sojae, indicating their vital roles in various stages of oomycete pathogens. Moreover, we identified 29 PPMs in P. sojae, and eight of them possessed accessory domains in addition to phosphate domains. We investigated the biological function of one PPM protein with an extra PH domain (PPM1); this protein exhibited high expression levels in both asexual developmental and infectious stages. Our analysis confirmed that PPM1 is indeed an active protein phosphatase, and its accessory domain does not affect its phosphatase activity. To delve further into its function, we generated knockout mutants of PPM1 and validated its essential roles in mycelial growth, sporangia and oospore production, as well as infectious stages. To the best of our knowledge, this study provides the first comprehensive inventory of phosphatases in oomycetes and identifies an important phosphatase within the expanded serine/threonine phosphatase group in oomycetes.

Two waxy maize hybrids were selected to explore the relationship between amylopectin fine structure and gene expression of starch synthesis-related enzymes. High-performance anion-exchange chromatograph analysis showed that waxy maize had the highest content of amylopectin B1 chain (DP 13–24) and the secondary proportion of A chain (DP 6–12) while the low proportions of B2 (DP 25–36) and B3 chains (DP ≥ 37). SBE activity was positively correlated with the proportion of A chain and negatively correlated with the proportion of B3 chain and average chain length, but DBE activity showed the almost opposite relation. AGPase and SSS activities were not found to be significant with chain-length distribution. SSI preferentially generated short A chain, which would be elongated further by SSIIa to produce long B1 chain that was continued to extend by SSIIIa to produce longer B2 and B3 chains. SBEI was more likely responsible for the synthesis of B2 and B3 chains, while SBEIIb played a specific role in the formation of A chain. ISA2 regulated amylopectin structure through the removal of A and B1 chains and the formation of B2 and B3 chains.