Seed germination is a complex, multistep developmental process that is critical for plant growth and regulated by intricate polygenic networks. Despite its agronomic significance, the precise mechanism underlying this process in rice remains incompletely understood. In this study, we determined a seed-specific gene OsDOG1L1, the ortholog of Arabidopsis DOG1, as an essential regulator that inhibits germination while promoting seed dormancy in rice. Overexpression of OsDOG1L1 led to delayed germination and enhanced dormancy, whereas loss-of-function mutants showed accelerated germination and reduced dormancy. Temporal analysis showed a progressive decline in OsDOG1L1 transcript and protein levels during germination. Furthermore, we identified two C2H2-type zinc finger transcription factors, ZFP36 (also known as Bsr-d1) and ZFP252, as direct regulators of OsDOG1L1, functioning upstream to repress OsDOG1L1 expression. Molecular and genetic analyses demonstrated that OsDOG1L1 interacts with and suppresses the phosphatase activity of clade A protein phosphatase 2Cs (PP2Cs), OsPP2C09 and OsPP2C30, thereby enhancing ABA signaling to inhibit seed germination. Together, our findings uncover a ZFP36/ZFP252-OsDOG1L1-OsPP2Cs regulatory module that governs rice seed germination, providing insights into the molecular regulation of germination control in rice.

Context or problem Increasing rice yield and nitrogen use efficiency (NUE) through improved plant architecture and canopy management is a key strategy for sustainable agriculture. Objective or research question This study investigated the role of AP2/ERF transcription factor OsRAV1 in regulating rice growth and yield under varying nitrogen (N) rates and planting densities. Methods A three-year field experiment compared lines expressing different levels of OsRAV1 with wild-type controls. OsRAV1 expression was significantly influenced by N and planting density. Results Increased expression of OsRAV1 resulted in higher grain yield, primarily through increased spikelet number per panicle. Optimized planting density, combined with a moderate N rate, further enhanced yield, largely due to a reduction in panicle number per unit area at elevated OsRAV1 expression. Furthermore, increased OsRAV1 levels promoted leaf and stem elongation, increased internode diameter, and improved lodging resistance. OsRAV1 also stimulated starch and sucrose metabolism, enhanced nitrogen uptake, increased dry matter accumulation (DMA), and delayed leaf senescence. Conversely, OsRAV1 knockout line exhibited reduced grain yield, decreased NUE, and accelerated leaf senescence. Conclusions As a crucial regulator of rice architecture and yield, OsRAV1 increases spikelet number per panicle, stimulates starch and sucrose metabolism, and delays leaf senescence, thereby enhancing DMA, enhances yield, and improves NUE. Implications or significance Modulating OsRAV1 expression in rice presents a promising strategy to optimize plant architecture, increase yield, and improve NUE - key objectives for breeding programs aimed at sustainable rice production.

Nitrogen deficiency is a common stress in agricultural soils, often coinciding with antibiotic residues that can impair crop growth and nutrient uptake. This study aimed to examine how the antibiotic roxithromycin (ROX) influences nitrate uptake, growth, and stress responses in wheat under nitrogen-deficient vs. nitrogen-sufficient conditions. Wheat seedlings were grown hydroponically at normal (5 mM) or low (2 mM) nitrate supply with ROX treatments ranging from 0 to 10 mg·L⁻1. Growth parameters (biomass, root morphology), net nitrate fluxes, and stress indicators (reactive oxygen species, malondialdehyde, antioxidant enzyme activity, nitrate reductase activity) were assessed. Under N deficiency, seedlings increased nitrate influx but showed reduced growth and lower nitrate assimilation enzyme activities compared to N-sufficient seedlings. ROX at 0.1 mg·L⁻1 slightly enhanced root growth, whereas ≥ 1 mg·L⁻1 progressively inhibited seedling growth and root development, with stronger inhibition under low N. At 10 mg·L⁻1, root length and biomass declined significantly, more so in low-N plants. Increasing ROX also triggered oxidative stress, activated antioxidant enzymes, and suppressed nitrate reductase. Adequate N supply mitigated ROX toxicity, whereas N deficiency exacerbated growth inhibition and oxidative damage. Roxithromycin disrupts wheat nitrate uptake and growth, especially under N-deficient conditions. Adequate N availability partly alleviates ROX-induced stress by supporting plant antioxidant defenses, whereas N-starved plants are more vulnerable to ROX toxicity. Soil nitrogen status thus strongly modulates the impact of antibiotic contaminants on crop physiology.

INTRODUCTION:Grain size and grain filling are critical determinants of rice yield and grain quality. Although transaldolase (TAL) is a key enzyme of the pentose phosphate pathway and is known to be essential for vegetative growth, whether it contributes to the regulation of grain size and filling has yet to be determined. OBJECTIVE:This study aims to systematically characterize the function of TAL, with particular emphasis on its effects on grain size and grain filling in rice. METHODS:We applied CRISPR/Cas9-mediated knockout and RNA interference (RNAi)-mediated knockdown to assess loss-of-function phenotypes. Concurrently, we established stable TAL overexpression lines to evaluate gain-of-function phenotypes. To identify transcriptional regulators upstream of TAL, we integrated multiple molecular approaches: yeast one-hybrid (Y1H) assays for protein-DNA interaction detection, electrophoretic mobility shift assays (EMSAs) for in vitro binding validation, and dual-luciferase (dual-LUC) transient expression assays for in vivo transcriptional regulatory activity quantification. RESULTS:CRISPR/Cas9-mediated knockout of the TAL gene and RNAi-based silencing of TAL affect multiple aspects of rice growth and development, including decreased grain weight and poor grain filling, accompanied by additional pleiotropic effects such as delayed flowering and a marked yield penalty. In contrast, TAL overexpression not only promoted earlier flowering but, more importantly, substantially enhanced grain size and promoted grain filling. Furthermore, TAL was associated with increased net photosynthetic performance and elevated starch accumulation in leaves and mature grains. Biochemical analysis revealed that TAL possesses transaldolase activity and modulates S7P and E4P homeostasis within the pentose phosphate pathway. We confirmed that TAL is localized to chloroplasts in rice protoplasts, and is preferentially expressed in the pericarp and aleurone layer, as well as in the vascular tissues, suggesting a role in assimilate transport during grain filling. Mechanistically, we identified that OsRA2 functions as a potential transcriptional repressor of TAL by binding to its promoter. CONCLUSION:This study reveals that TAL, as an important regulatory factor in the pentose phosphate pathway, not only affects multiple aspects of rice growth and development, but more importantly, plays a prominent role in regulating grain size and grain filling.

Abstract Maize (Zea mays L.) starch quality is a complex trait with significant implications for grain processing and industrial applications. However, the genetic basis underlying starch quality, particularly for gelatinization and thermodynamic properties, remains poorly understood. In this study, we evaluated 12 starch quality traits, including seven gelatinization characteristics, four thermodynamic traits, and kernel starch content (KSC) in a diverse panel of 335 maize inbred lines. Considerable phenotypic variation was observed for all traits. A total of 228 quantitative trait loci (QTLs) were significantly associated with 12 starch quality traits through genome-wide association studies (GWAS). By integrating a dynamic transcriptome analysis of two maize inbred lines with contrasting starch quality, we identified 60 candidate genes. One gene, waxy1, encoding a starch synthase, was found to be associated with enthalpy of gelatinization (ΔHgel) and pasting temperature (Ptemp). Six variants in waxy1 contributed to natural variation in ΔHgel and Ptemp, and a cost-effective InDel and two PARMS-based molecular markers were developed and validated in 144 maize inbred lines, enabling efficient marker-assisted selection. Our findings provide key genes and molecular markers for high-quality maize breeding with improved starch properties.

The root system is a crucial determinant of maize yield and stress resilience, particularly under drought stress. However, the complex genetic basis governing root system architecture remains largely elusive. To dissect the genetic architecture of the maize root, a transcriptome-wide association study (TWAS) was performed for 16 root traits in a panel of 357 diverse maize inbred lines. TWAS identified 2,978 significantly associated genes, of which 530 showed root-preferential expression patterns, representing high-confidence candidates for root development. Among these candidates, ZmSAUR21, a member of the Small Auxin-Up RNA gene family, was functionally characterized. Both CRISPR-Cas9-mediated knockout and overexpression analyses demonstrated that ZmSAUR21 acts as a key positive regulator of root growth by promoting cell elongation. Furthermore, the transcription factor ZmbZIP89 was identified as a direct upstream activator that binds to the ZmSAUR21 promoter to enhance its transcription, establishing a novel ZmbZIP89-ZmSAUR21 regulatory module. Crucially, ZmSAUR21-overexpressing plants showed substantially enhanced survival rates, improved water use efficiency, and a more vigorous root system under drought conditions. Collectively, this study uncovered a key regulatory pathway controlling maize root development and demonstrates that ZmSAUR21 is a valuable target gene for improving root systems and enhancing drought tolerance in maize breeding programs.

Plant height (PH) and aboveground biomass (AGB) are critical agronomic traits that determine the yield potential of maize (Zea mays L.). However, the application of genomic selection (GS) and genome-wide association studies (GWAS) in maize breeding is often hindered by the limitations of phenotypic data collection, which is typically characterized by low throughput and inadequate accuracy. To address this challenge, we employed an unmanned aerial vehicle (UAV) equipped with LiDAR and RGB cameras for high-throughput assessment of pH and AGB in a panel of 817 maize hybrids derived from 364 inbred lines over two growing seasons. Our results demonstrated that the integration of UAV-derived LiDAR point clouds with crop surface models (CSMs) enabled robust estimation of pH across multiple years (R2 > 0.90). Furthermore, a three-dimensional AGB estimation model was developed using UAV-derived PH and canopy coverage (CC), achieving high estimation accuracy (R2 > 0.83). Subsequently, the UAV-derived PH and AGB were utilized for GS and GWAS analyses. Replicated 10-fold cross-validation showed that the mean predictability was 0.504 for PH and 0.402 for AGB across eight commonly used GS models. Moreover, of the 66,066 potential crosses derived from the 364 inbred lines, the top 200 crosses selected for AGB showed up to twice the AGB of the bottom 200 crosses. Field validation demonstrated that the mean ear weight (EW) in the AGB top group was 39.0% higher than that in the bottom group. A total of 16 and 11 significant SNPs were identified by at least two GWAS methods for PH and AGB, respectively. Based on these SNPs, 81 candidate genes were functionally annotated, six of which were simultaneously associated with both traits. The candidate gene association analysis suggested that variations in the promoter region of ZmFLA9 may affect both traits. Overall, our study highlights the potential of UAV-based high-throughput phenotyping to accelerate maize genomic breeding by enabling rapid, precise, and large-scale trait assessment.

Genomic selection (GS) holds great promise for accelerating breeding progress in plants, and the advancement of across-population GS is essential to realize its full potential. However, conventional across-population GS heavily relies on precisely aligned markers across dense genotypes, whereas the feasibility of using flexible low-density markers remains underexplored. This study developed GS-Impute, a residual convolutional denoising autoencoder-based neural network framework that enables accurate genotype imputation for low-density across-population GS. A key breakthrough of GS-Impute is an automatic matching algorithm that resolves the persistent challenge of targeted training in the presence of both sporadic and systematic missing data. Additionally, GS-Impute incorporates a data augmentation strategy and several advanced techniques to enhance imputation accuracy, including residual blocks, dynamic learning-rate optimization, and layer normalization. Comprehensive evaluations across rice and maize breeding populations demonstrated that GS-Impute outperforms the latest versions of established benchmark tools, including Beagle5.4, Minimac4, and STICI. Importantly, the results indicate that GS-Impute makes across-population GS feasible with low-density markers, establishing a resource-efficient strategy with the potential to transform genomic breeding programs.

Root metabolites are essential for plant development and environmental stress adaptation. However, the genetic basis controlling root metabolome variation in crops and its role in stress resilience remain largely uncharacterized. In this study, we employed a comprehensive multiomics approach, integrating root metabolome and transcriptome profiles of 273 maize (Zea mays L.) inbred lines at the seedling stage. Our analysis annotated 407 metabolites, of which 155 exhibited significant correlations with root traits. Using a variome-transcriptome-metabolome (VTM) association network, we identified the glutamate decarboxylase (GAD) gene ZmGAD as a crucial regulator that enhances root growth and stress tolerance by modulating gamma-aminobutyric acid (GABA) biosynthesis. ZmGAD-derived GABA confers stress tolerance by regulating stomatal aperture and scavenging reactive oxygen species. A transcription factor, ZmZIM2, acts as a negative regulator of ZmGAD expression and GABA accumulation. Moreover, a 2-bp insertion in ZmGAD causes a premature translation termination, resulting in reduced GABA content, shorter roots, and decreased stress tolerance in maize. The reduced frequency of a 2-bp deletion suggests it may have been inadvertently lost during maize domestication and modern breeding. This study elucidates the genetic and molecular framework underlying root metabolite regulation in maize and provides a valuable resource for enhancing root traits and stress tolerance in maize breeding.

Uniform and rapid germination of rice seeds is essential for successful direct seeding cultivation. In this study, we show that the tonoplast aquaporin OsTIP1;2 must be maintained within a narrow expression window to ensure optimal germination. Both loss- and gain-of-function lines exhibit reduced water uptake and delayed germination, accompanied by disturbed (abscisic acid) ABA/ (gibberellins) GA homeostasis and decreased α-amylase activity. Transcriptome profiling indicates associations with hormone signaling and starch catabolism. Moreover, altered expression of OsTIP1;2 results in differing sensitivities of germinating seeds to salt stress, impacting germination rates under saline conditions. Haplotype analysis revealed that rice varieties carrying Haplotype 1 (Hap1) allele of OsTIP1;2 are associated with improved germination under salt stress.

Lateral roots (LRs) branching is crucial for water and nutrient acquisition in plants, ultimately determining the overall plant performance and productivity. However, the transcriptional regulation of LR development in crops and its role in stress resilience remain largely unexplored. Leveraging integrated transcriptome-wide association study and single-cell RNA sequencing data, we identified a basic leucine zipper (bZIP) transcription factor ZmbZIP89 as an important regulator of LR elongation and mapped its spatial expression pattern in cortex/epidermis cell types. ZmbZIP89 can activate the expression of ZmPRX47 to regulate the production of root reactive oxygen species homeostasis, contributing to increased lateral root length (LRL) and enhanced drought resistance. Natural variations in the 3' untranslated region of ZmbZIP89 enhance gene expression by increasing mRNA stability, leading to increases in LRL and drought tolerance. These findings contribute to our understanding of the molecular mechanisms underlying LR development and provide potential gene targets for breeding stress-resilient crops.

Sheath blight (ShB), caused by necrotrophic fungus Rhizoctonia solani, is one of the most serious rice diseases worldwide. To the best of our knowledge, no genes with high potential for rice ShB resistance breeding have been previously characterized. Here we identify a ShB resistance receptor-like kinase 1 (SBRR1) gene via a genome-wide association study. The SBRR1-R elite allele, containing a 256-bp insertion in its promoter, is preferentially present in indica varieties in geographical regions with highly favorable conditions for ShB development. Introduction of SBRR1-R into a commercial japonica rice variety significantly reduces yield loss under severe ShB disease pressure. Transcription factor bHLH57 specifically binds to the 256-bp sequence and accounts for highly induced expression and stronger resistance of SBRR1-R. Localization of SBRR1 on plasma membrane, aided by SBRR1-interaction-protein 1, and phosphorylation of SBRR1 are required for SBRR1 to rapidly upregulate downstream chitinase genes for resistance. These findings offer mechanistical insights into ShB resistance hidden in natural rice varieties.

Leaf width (LW) is a critical determinant of maize architecture and yield. To uncover its genetic basis, we performed a genome-wide association study (GWAS) on 348 maize inbred lines and identified ZmCKX6, encoding cytokinin oxidase/dehydrogenase, as a key gene associated with LW. Natural variation in the ZmCKX6 promoter significantly influenced its expression levels, leading to differences in LW across various haplotypes. Functional validation using CRISPR/Cas9 revealed that ZmCKX6 knockout results in pleiotropic effects, including narrower leaves, reduced plant height, and decreased grain yield components. These phenotypes were accompanied by elevated levels of active cytokinins but reduced levels of auxin, gibberellins, and salicylic acid. Transcriptome analysis revealed a significant downregulation of photosynthesis-related genes, corresponding to reduced photosynthetic rates in knockout lines. Evolutionary analysis demonstrated that the allele associated with narrower leaves were preferentially selected during maize domestication and breeding. This study highlights the role of ZmCKX6 in modulating cytokinin homeostasis and its subsequent impact on multiple agronomic traits in maize, providing insights into the complex genetic control of plant architecture and yield. The identified natural variations could be valuable for marker-assisted selection aimed at optimizing plant architecture and improving yield.

Fructose-1,6-bisphosphate aldolase (FBA) stands as a pivotal enzyme involved within the Calvin cycle and glycolytic pathways in bacteria and higher plants, but the specific function of OsFBA in rice is still unclear. Here, we identified a chloroplast and mitochondria dual-localized FBA protein, OsFBA1, in rice. Experimental evidence showed that the functionally deficient osfba1 mutants featured a notable decline in chlorophyll content, photosynthetic rate, and severe growth impediment by the three-leaf stage, leading to eventual plant demise. Up-regulation of photosynthetic-pathway genes in the osfba1 mutants indicated the essential role of OsFBA1 in chloroplast development and suggested a compensatory mechanism of other genes in the process. Furthermore, the absence of OsFBA1 impaired the carbon assimilation in young rice seedlings, and supplying exogenous glucose could partially sustain the survival of osfba1 mutant for a few more days. Pathway-specific metabolomics analysis revealed a systemic change of metabolites in the glycolytic pathway, and consequential carbohydrates accumulation due to OsFBA1 disruption. Transcriptomics profiling corroborated the expression changes of photosynthesis, and carbon metabolism pathway genes. We further demonstrated that OsFBA1 serves as the primary FBA enzyme governing energy generation, photosynthesis and carbon metabolism. These results prove that OsFBA1 is an essential core gene in supporting the life cycle of rice, its expression has to be tightly regulated.

The cytokinin (CK) type B response regulator (RRB) gene is involved in the CK signaling pathway and performs a key function for mediating reactions to amounts of abiotic stresses. Nevertheless, the RRB gene family remains to be characterized in Poaceae (also known as Gramineae or grasses). Here, we performed a comprehensive analysis encompassing phylogenetic relationships, evolutionary pressures, and expression patterns of the RRB gene family in six Poaceae species, including rice, Panicum, Sorghum, Setaria, maize, and wheat. Phylogenetic tree and syntenic analyses revealed that the RRB genes were divided into seven orthologous gene clusters (OGCs), indicating that the common ancestor of these Poaceae species possessed at least seven RRB genes. Further analysis revealed that the evolution of the Poaceae RRB gene family was primarily driven by purifying selection. The expression pattern of rice OsRRB toward phytohormonal and abiotic stresses was also investigated. The findings revealed that several phytohormones, including cytokinin (CK), abscisic acid (ABA), and methyl jasmonate (MeJA), as well as abiotic factors such as drought and cold, significantly increased the expression levels of these genes. Importantly, haplotype analysis identified four crucial variation sites within the OsRRB5 genomic regions that may contribute to drought resistance in rice. Our findings lay the groundwork for further elucidating the biological function of OsRRB genes and provide a promising new target for developing stress-resistant rice varieties.

The high yield potential and stability of hybrid japonica rice varieties are crucial for sustainable agricultural development and food security. Rice varieties must undergo rigorous testing through multi-site regional trials before being introduced to the market in China. The assessment of these regional trials is essential for guiding rice breeding. In this study, we evaluated the yield performance of 13 hybrid japonica rice genotypes (g1–g13) across six regional trial sites (e1–e6) in Jiangsu province, China. Variance analysis revealed that genotype (G), environment (E), and genotype-by-environment (G × E) interactions significantly influenced the yield of hybrid japonica rice varieties. The effects of G × E interactions on the yield potential and stability of these tested rice varieties were further analyzed using Genotype plus Genotype-by-Environment interaction (GGE) biplot and additive main effects and multiplicative interaction (AMMI) model analyses. The results reveal that Zhegengyou2035 (g4) and Changyou20-2 (g3) exhibited superior yield potential and stability, while Huazhongyou9413 (g12) exhibited broad adaptability. Additionally, the assessment of discrimination and representativeness among regional trial sites revealed that the Wujin Rice Research Institute (e6) served as an optimal testing location. Our findings identify the most suitable rice varieties for the area and assess their potential as initial material in the selection processes for breeding new varieties. Additionally, this work contributes to the strategic selection of optimal testing locations.

Integrating multiple modern breeding techniques in maize has always been challenging. This study aimed to address this issue by applying a flexible sparse partial diallel cross design composed of 945 maize hybrids derived from 266 inbred lines across different heterotic groups. The research integrated genome-wide association studies, genomic selection and genomic evaluation of parental inbred lines to accelerate the breeding process for developing single-cross hybrids. Significant associations were identified for 7-25 stable single nucleotide polymorphisms (SNPs) associated with the general combining abilities (GCAs) of nine yield-related traits. Using the maizeGDB and NCBI databases, 264 candidate genes were screened and functionally annotated based on significant SNPs detected by at least three statistical methods. The marker set developed from these GCA SNPs significantly improved the prediction accuracy of hybrids across all traits. The GCA estimates of the inbred lines involved in the top 100 and bottom 100 hybrids consistently ranked at the top and bottom, thereby confirming the accuracy of the predictions. Furthermore, the top 100 crosses selected using BayesB, GBLUP and LASSO showed a 105.4-108.6% increase in average ear weight compared to the bottom 100 crosses in field validation, demonstrating strong selection gains. Notably, amongst the top 100 hybrids, A017/A037 and A037/A169, each containing six superior genotypes were registered as Suyu 161 and Tongyu 1701, respectively, by the National Crop Variety Approval Committee in China. These results highlight the effectiveness of genomic selection and provide valuable insights for advancing genomic hybrid breeding in maize.

Nitrogen (N) availability determines higher plant productivity and yield. However, the molecular mechanisms governing N acquisition and utilization remain largely unknown in maize. In this study, ATAC-seq, RNA-seq, and Ribo-seq analyses were conducted in maize roots under different N supply conditions. A set of differentially expressed genes enriched in N and phenylpropanoid metabolisms at both the transcription and translation levels were highlighted. Interestingly, less than half of low-N responsive genes were shared between transcription and translation. The alteration of translational efficiency (TE) is also an important mechanism by which maize responds to LN. In addition, we identified low-N-induced open chromatin regions (OCRs) and observed an enrichment of transcription factor (TF) binding motifs. Furthermore, we constructed a transcriptional regulatory network for maize roots subjected to low-N. These findings extend our understanding of N availability response and provide new insights for improving N use efficiency (NUE).