Mining genic resources regulating nitrogen-use efficiency based on integrative biological analyses and their breeding applications in maize and other crops.

Nitrogen (N) is an essential factor for limiting crop yields, and cultivation of crops with low nitrogen-use efficiency (NUE) exhibits increasing environmental and ecological risks. Hence, it is crucial to mine valuable NUE improvement genes, which is very important to develop and breed new crop varieties with high NUE in sustainable agriculture system. Quantitative trait locus (QTL) and genome-wide association study (GWAS) analysis are the most common methods for dissecting genetic variations underlying complex traits. In addition, with the advancement of biotechnology, multi-omics technologies can be used to accelerate the process of exploring genetic variations. In this study, we integrate the substantial data of QTLs, quantitative trait nucleotides (QTNs) from GWAS, and multi-omics data including transcriptome, proteome, and metabolome and further analyze their interactions to predict some NUE-related candidate genes. We also provide the genic resources for NUE improvement among maize, rice, wheat, and sorghum by homologous alignment and collinearity analysis. Furthermore, we propose to utilize the knowledge gained from classical cases to provide the frameworks for improving NUE and breeding N-efficient varieties through integrated genomics, systems biology, and modern breeding technologies.

Multi-omics-driven advances in the understanding of triacylglycerol biosynthesis in oil seeds.

Vegetable oils are rich sources of polyunsaturated fatty acids and energy as well as valuable sources of human food, animal feed, and bioenergy. Triacylglycerols, which are comprised of three fatty acids attached to a glycerol backbone, are the main component of vegetable oils. Here, we review the development and application of multiple-level omics in major oilseeds and emphasize the progress in the analysis of the biological roles of key genes underlying seed oil content and quality in major oilseeds. Finally, we discuss future research directions in functional genomics research based on current omics and oil metabolic engineering strategies that aim to enhance seed oil content and quality, and specific fatty acids components according to either human health needs or industrial requirements.

Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize (Zea mays L.).

BACKGROUND: Ubiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize (Zea mays L.). RESULTS: In this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response. CONCLUSIONS: In this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Coordinated control for the auricle asymmetric development by ZmIDD14 and ZmIDD15 fine-tune the high-density planting adaption in maize.

Multiple distinct specialized regions shape the architecture of maize leaves. Among them, the fringe-like and wedge-shaped auricles alter the angle between the leaf and stalk, which is a key trait in crop plant architecture. As planting density increased, a small leaf angle (LA) was typically selected to promote crop light capture efficiency and yield. In the present study, we characterized two paralogous INDETERMINATE DOMAIN (IDD) genes, ZmIDD14 and ZmIDD15, which contain the Cys2-His2 zinc finger domain and function redundantly to regulate auricle development and LA in maize. Loss-of-function mutants showed decreased LA by reducing adaxial sclerenchyma thickness and increasing the colourless cell layers. In addition, the idd14;idd15 double mutant exhibited asymmetrically smaller auricles, which might cause by a failed maintenance of symmetric expression of the key auricle size controlling gene, LIGULELESS(LG1). The transcripts of ZmIDD14 and ZmIDD15 enriched in the ligular region, where LG1 was highly expressed, and both proteins physically interacted with ZmILI1 to promote LG1 transcription. Notably, the idd14;idd15 enhanced the grain yield of hybrids under high planting densities by shaping the plant architecture with a smaller LA. These findings demonstrate the functions of ZmIDD14 and ZmIDD15 in controlling the abaxial/adaxial development of sclerenchyma in the midrib and polar development along the medial-lateral axes of auricles and provide an available tool for high-density and high-yield breeding in maize.

ZmSPL10, ZmSPL14 and ZmSPL26 act together to promote stigmatic papilla formation in maize through regulating auxin signaling and ZmWOX3A expression.

Maize silk is a specialized type of stigma, covered with numerous papillae for pollen grain capture. However, the developmental process of stigmatic papillae and the underlying regulatory mechanisms have remained largely unknown. Here, we combined the cytological, genetic and molecular studies to demonstrate that three homologous genes ZmSPL10, ZmSPL14 and ZmSPL26 play a central role in promoting stigmatic papilla formation in maize. We show that their triple knockout mutants are nearly complete lack of stigmatic papilla, resulting in a severe reduction in kernel setting. Cellular examination reveals that stigmatic papilla is developed from a precursor cell, which is the smaller daughter cell resulting from asymmetric cell division of a silk epidermal cell. In situ hybridization shows that ZmSPL10, ZmSPL14 and their target genes SPI1, ZmPIN1b, ZmARF28 and ZmWOX3A are preferentially expressed in the precursor cells of stigmatic papillae. Moreover, ZmSPL10, ZmSPL14 and ZmSPL26 directly bind to the promoters of SPI1, ZmPIN1b, ZmARF28 and ZmWOX3A and promote their expression. Further, Zmwox3a knockout mutants display severe defects in stigmatic papilla formation and reduced seed setting. Collectively, our results demonstrate that ZmSPL10, ZmSPL14 and ZmSPL26 act together to promote stigmatic papilla development through regulating auxin signaling and ZmWOX3A expression.

Root Hairs: An under-explored target for sustainable cereal crop production.

To meet the demands of a rising human population, plant breeders will need to develop improved crop varieties that maximize yield in the face of increasing pressure on crop production. Historically, the optimization of crop root architecture has represented a challenging breeding target due to the inaccessibility of the root systems. Root hairs, single cell projections from the root epidermis, are perhaps the most overlooked component of root architecture traits. Root hairs play a central role in facilitating water, nutrient uptake, and soil cohesion. Current root hair architectures may be suboptimal under future agricultural production regimes, coupled with an increasingly variable climate. Here, we review the genetic control of root hair development in the world's three most important crops: rice, maize and wheat, and highlight conservation of gene function between monocots and the model dicot species Arabidopsis. Advances in genomic techniques including Gene-Editing combined with traditional plant breeding methods have the potential to overcome many inherent issues associated with the design of improved root hair architectures. Ultimately, this will enable detailed characterization of the effects of contrasting root hair morphology strategies on crop yield and resilience, and the development of new varieties better adapted to deliver future food security.

The Mechanism of Exogenous Salicylic Acid and 6-Benzylaminopurine Regulating the Elongation of Maize Mesocotyl.

The elongation of the mesocotyl plays an important role in the emergence of maize deep-sowing seeds. This study was designed to explore the function of exogenous salicylic acid (SA) and 6-benzylaminopurine (6-BA) in the growth of the maize mesocotyl and to examine its regulatory network. The results showed that the addition of 0.25 mmol/L exogenous SA promoted the elongation of maize mesocotyls under both 3 cm and 15 cm deep-sowing conditions. Conversely, the addition of 10 mg/L exogenous 6-BA inhibited the elongation of maize mesocotyls. Interestingly, the combined treatment of exogenous SA-6-BA also inhibited the elongation of maize mesocotyls. The longitudinal elongation of mesocotyl cells was the main reason affecting the elongation of maize mesocotyls. Transcriptome analysis showed that exogenous SA and 6-BA may interact in the hormone signaling regulatory network of mesocotyl elongation. The differential expression of genes related to auxin (IAA), jasmonic acid (JA), brassinosteroid (BR), cytokinin (CTK) and SA signaling pathways may be related to the regulation of exogenous SA and 6-BA on the growth of mesocotyls. In addition, five candidate genes that may regulate the length of mesocotyls were screened by Weighted Gene Co-Expression Network Analysis (WGCNA). These genes may be involved in the growth of maize mesocotyls through auxin-activated signaling pathways, transmembrane transport, methylation and redox processes. The results enhance our understanding of the plant hormone regulation of mesocotyl growth, which will help to further explore and identify the key genes affecting mesocotyl growth in plant hormone signaling regulatory networks.

Joint-GWAS, Linkage Mapping, and Transcriptome Analysis to Reveal the Genetic Basis of Plant Architecture-Related Traits in Maize.

Plant architecture is one of the key factors affecting maize yield formation and can be divided into secondary traits, such as plant height (PH), ear height (EH), and leaf number (LN). It is a viable approach for exploiting genetic resources to improve plant density. In this study, one natural panel of 226 inbred lines and 150 family lines derived from the offspring of T32 crossed with Qi319 were genotyped by using the MaizeSNP50 chip and the genotyping by sequence (GBS) method and phenotyped under three different environments. Based on the results, a genome-wide association study (GWAS) and linkage mapping were analyzed by using the MLM and ICIM models, respectively. The results showed that 120 QTNs (quantitative trait nucleotides) and 32 QTL (quantitative trait loci) related to plant architecture were identified, including four QTL and 40 QTNs of PH, eight QTL and 41 QTNs of EH, and 20 QTL and 39 QTNs of LN. One dominant QTL, qLN7-2, was identified in the Zhangye environment. Six QTNs were commonly identified to be related to PH, EH, and LN in different environments. The candidate gene analysis revealed that Zm00001d021574 was involved in regulating plant architecture traits through the autophagy pathway, and Zm00001d044730 was predicted to interact with the male sterility-related gene ms26. These results provide abundant genetic resources for improving maize plant architecture traits by using approaches to biological breeding.

Integrated Metabolome and Transcriptome Analysis of Gibberellins Mediated the Circadian Rhythm of Leaf Elongation by Regulating Lignin Synthesis in Maize.

Plant growth exhibits rhythmic characteristics, and gibberellins (GAs) are involved in regulating cell growth, but it is still unclear how GAs crosstalk with circadian rhythm to regulate cell elongation. The study analyzed growth characteristics of wild-type (WT), zmga3ox and zmga3ox with GA3 seedlings. We integrated metabolomes and transcriptomes to study the interaction between GAs and circadian rhythm in mediating leaf elongation. The rates of leaf growth were higher in WT than zmga3ox, and zmga3ox cell length was shorter when proliferated in darkness than light, and GA3 restored zmga3ox leaf growth. The differentially expressed genes (DEGs) between WT and zmga3ox were mainly enriched in hormone signaling and cell wall synthesis, while DEGs in zmga3ox were restored to WT by GA3. Moreover, the number of circadian DEGs that reached the peak expression in darkness was more than light, and the upregulated circadian DEGs were mainly enriched in cell wall synthesis. The differentially accumulated metabolites (DAMs) were mainly attributed to flavonoids and phenolic acid. Twenty-two DAMs showed rhythmic accumulation, especially enriched in lignin synthesis. The circadian DEGs ZmMYBr41/87 and ZmHB34/70 were identified as regulators of ZmHCT8 and ZmBM1, which were enzymes in lignin synthesis. Furthermore, GAs regulated ZmMYBr41/87 and ZmHB34/70 to modulate lignin biosynthesis for mediating leaf rhythmic growth.

Phenotypic Investigation and RNA-seq of KN1 Involved in Leaf Angle Formation in Maize (Zea mays L.).

Leaf angle (LA) is one of the core agronomic traits of maize, which controls maize yield by affecting planting density. Previous studies have shown that the KN1 gene is closely related to the formation of maize LA, but its specific mechanism has not been fully studied. In this study, phenotype investigation and transcriptomic sequencing were combined to explore the mechanism of LA changes in wild type maize B73 and mutant kn1 under exogenous auxin (IAA) and abscisic acid (ABA) treatment. The results showed that the effect of exogenous phytohormones had a greater impact on the LA of kn1 compared to B73. Transcriptome sequencing showed that genes involved in IAA, gibberellins (GAs) and brassinosteroids (BRs) showed different differential expression patterns in kn1 and B73. This study provides new insights into the mechanism of KN1 involved in the formation of maize LA, and provides a theoretical basis for breeding maize varieties with suitable LA.

Co-expression of transcription factors ZmC1 and ZmR2 establishes an efficient and accurate haploid embryo identification system in maize.

Because of their high efficiency during chromosome doubling, immature haploid maize (Zea mays L.) embryos are useful for doubled haploid production. The R1-nj marker is commonly used in doubled haploid breeding and has improved the efficiency of haploid identification. However, its effectiveness is limited by genetic background and environmental factors. We addressed this technical challenge by developing an efficient and accurate haploid embryo identification marker through co-expression of two transcription factor genes (ZmC1 and ZmR2) driven by the embryo-aleurone-specific bidirectional promoter PZmBD1 ; these factors can activate anthocyanin biosynthesis in the embryo and aleurone layer during early seed development. We developed a new haploid inducer, Maize Anthocyanin Gene InduCer 1 (MAGIC1), by introducing the transgenes into the haploid inducer line CAU6. MAGIC1 could identify haploids at 12 days after pollination, which is nine days earlier than CAU6. Importantly, MAGIC1 increased haploid identification accuracy to 99.1%, compared with 88.3% for CAU6. In addition, MAGIC1 could effectively overcome the inhibition of anthocyanin synthesis in some germplasms. Furthermore, an upgraded anthocyanin marker was developed from ZmC1 and ZmR2 to generate MAGIC2, which could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. This haploid identification system is more efficient and accurate than the conventional R1-nj-based method, and it simplifies the haploid identification process. Therefore, this system provides technical support for large-scale doubled haploid line production.

cis-Regulatory variation affecting gene expression contributes to the improvement of maize kernel size.

cis-Regulatory variations contribute to trait evolution and adaptation during crop domestication and improvement. As the most important harvested organ in maize (Zea mays L.), kernel size has undergone intensive selection for size. However, the associations between maize kernel size and cis-regulatory variations remain unclear. We chose two independent association populations to dissect the genetic architecture of maize kernel size together with transcriptomic and genotypic data. The resulting phenotypes reflected a strong influence of population structure on kernel size. Compared with genome-wide association studies (GWASs), which accounted for population structure and relatedness, GWAS based on a naïve or simple linear model revealed additional associated single-nucleotide polymorphisms significantly involved in the conserved pathways controlling seed size in plants. Regulation analyses through expression quantitative trait locus mapping revealed that cis-regulatory variations likely control kernel size by fine-tuning the expression of proximal genes, among which ZmKL1 (GRMZM2G098305) was transgenically validated. We also proved that the pyramiding of the favorable cis-regulatory variations has contributed to the improvement of maize kernel size. Collectively, our results demonstrate that cis-regulatory variations, together with their regulatory genes, provide excellent targets for future maize improvement.

Genetic dissection of maize (Zea mays L.) chlorophyll content using multi-locus genome-wide association studies.

BACKGROUND: The chlorophyll content (CC) is a key factor affecting maize photosynthetic efficiency and the final yield. However, its genetic basis remains unclear. The development of statistical methods has enabled researchers to design and apply various GWAS models, including MLM, MLMM, SUPER, FarmCPU, BLINK and 3VmrMLM. Comparative analysis of their results can lead to more effective mining of key genes. RESULTS: The heritability of CC was 0.86. Six statistical models (MLM, BLINK, MLMM, FarmCPU, SUPER, and 3VmrMLM) and 1.25 million SNPs were used for the GWAS. A total of 140 quantitative trait nucleotides (QTNs) were detected, with 3VmrMLM and MLM detecting the most (118) and fewest (3) QTNs, respectively. The QTNs were associated with 481 genes and explained 0.29-10.28% of the phenotypic variation. Additionally, 10 co-located QTNs were detected by at least two different models or methods, three co-located QTNs were identified in at least two different environments, and six co-located QTNs were detected by different models or methods in different environments. Moreover, 69 candidate genes within or near these stable QTNs were screened based on the B73 (RefGen_v2) genome. GRMZM2G110408 (ZmCCS3) was identified by multiple models and in multiple environments. The functional characterization of this gene indicated the encoded protein likely contributes to chlorophyll biosynthesis. In addition, the CC differed significantly between the haplotypes of the significant QTN in this gene, and CC was higher for haplotype 1. CONCLUSION: This study's results broaden our understanding of the genetic basis of CC, mining key genes related to CC and may be relevant for the ideotype-based breeding of new maize varieties with high photosynthetic efficiency.

Sugar transporter ZmSWEET1b is responsible for assimilate allocation and salt stress response in maize.

Sugar efflux transporter SWEET family is involved in multiple biological processes, from nectar secretion, pollen fertility to seed filling. Although roles of SWEETs in abiotic stress adaption have been revealed mainly in reference organism Arabidopsis, cereal crops SWEETs responses to abiotic stimulation remain largely elusive. Here, we report the characterization of maize SWEET family member ZmSWEET1b, with emphasis on its response to salinity stress. ZmSWEET1b is a canonical sugar transporter, characteristic of seven transmembrane helices and plasma membrane localization. ZmSWEET1b and its rice ortholog OsSWEET1b in phylogenetic clade I underwent convergent selection during evolution. Two independent knockout lines were created by the CRISPR/Cas9 method to functionally characterized ZmSWEET1b. Sucrose and fructose contents are significantly decreased in ZmSWEET1b knockout lines. Mature leaves of ZmSWEET1b-edited lines exhibit chlorosis, reminiscent of senescence-like phenotype. Ears and seeds of ZmSWEET1b knockout lines are small. Upon salinity treatment, ZmSWEET1b-edited lines become more wilted. Transcriptional abundance of genes for Na+ efflux from roots to the rhizosphere, including ZmSOS1, ZmH+-ATPASE 2, and ZmH+-ATPASE 8, is decreased in salt-treated ZmSWEET1b knockout lines. These findings indicate that convergently selected sugar transporter ZmSWEET1b is important for maize plant development and responses to salt stress. The manipulation of ZmSWEET1b may represent a feasible way forward in the breeding of salinity tolerant ideotypes through the optimization of assimilate allocation.

Genetic variation in ZmWAX2 confers maize resistance to Fusarium verticillioides.

Fusarium verticillioides (F. verticillioides) is a widely distributed phytopathogen that incites multiple destructive diseases in maize, posing a grave threat to corn yields and quality worldwide. However, there are few reports of resistance genes to F. verticillioides. Here, we reveal that a combination of two single nucleotide polymorphisms (SNPs) corresponding to ZmWAX2 gene associates with quantitative resistance variations to F. verticillioides in maize through a genome-wide association study. A lack of ZmWAX2 compromises maize resistance to F. verticillioides-caused seed rot, seedling blight and stalk rot by reducing cuticular wax deposition, while the transgenic plants overexpressing ZmWAX2 show significantly increased immunity to F. verticillioides. A natural occurrence of two 7-bp deletions within the promoter increases ZmWAX2 transcription, thus enhancing maize resistance to F. verticillioides. Upon Fusarium stalk rot, ZmWAX2 greatly promotes the yield and grain quality of maize. Our studies demonstrate that ZmWAX2 confers multiple disease resistances caused by F. verticillioides and can serve as an important gene target for the development of F. verticillioides-resistant maize varieties.

ZmBET5L1 inhibits primary root growth and decreases osmotic stress tolerance by mediating vesicle aggregation and tethering in maize.

Improving osmotic stress tolerance is critical to help crops to thrive and maintain high yields in adverse environments. Here, we characterized a core subunit of the transport protein particle (TRAPP) complex, ZmBET5L1, in maize using knowledge-driven data mining and genome editing. We found that ZmBET5L1 can interact with TRAPP I complex subunits and act as a tethering factor to mediate vesicle aggregation and targeting from the endoplasmic reticulum to the Golgi apparatus. ZmBET5L1 knock-out increased the primary root elongation rate under 20% polyethylene glycol-simulated osmotic stress and the survival rate under drought stress compared to wild-type seedlings. In addition, we found that ZmBET5L1 moderates PIN1 polar localization and auxin flow to maintain normal root growth. ZmBET5L1 knock-out optimized auxin flow to the lateral side of the root and promoted its growth to generate a robust root, which may be related to improved osmotic stress tolerance. Together, these findings demonstrate that ZmBET5L1 inhibits primary root growth and decreases osmotic stress tolerance by regulating vesicle transport and auxin distribution. This study has improved our understanding of the role of tethering factors in response to abiotic stresses and identified desirable variants for breeding osmotic stress tolerance in maize.

Salt Tolerant Gene 1 contributes to salt tolerance by maintaining photosystem II activity in maize.

Salt stress is a major environmental factor limiting crop growth and productivity. Here, we show that Salt-Tolerant Gene 1 (ZmSTG1) contributes to salt tolerance by maintaining photosystem activity in maize. ZmSTG1 encodes an endoplasmic reticulum localized protein and retrotransposon insertion in the promoter region causes differential expression levels in maize inbred lines. Overexpression of ZmSTG1 improved plant growth vigor, and knockout of ZmSTG1 weakened plant growth under normal and salt stress conditions. Transcriptome and metabolome analyses indicated that ZmSTG1 might regulate the expression of lipid trafficking-related genes dependent on the abscisic acid (ABA) signaling pathway, thereby increasing the galactolipids and phospholipid concentrations in the photosynthetic membrane under salt stress. Chlorophyll fluorescence parameters showed that the knockout of ZmSTG1 led to significant impairment of plant photosystem II (PSII) activity under normal and salt stress conditions, whereas overexpression of ZmSTG1 dramatically improved plant PSII activity under salt stress conditions. We also demonstrated that the application of the salt-tolerant locus could enhance salt tolerance in hybrid maize plants. Taken together, we propose that ZmSTG1 may modulate the lipid composition in the photosynthetic membrane by affecting the expression of lipid trafficking-related genes to maintain the photosynthetic activity of plants under salt stress.

Overexpression of ZmEXPA5 reduces anthesis-silking interval and increases grain yield under drought and well-watered conditions in maize.

Drought is one of the major abiotic stresses affecting the maize production worldwide. As a cross-pollination crop, maize is sensitive to water stress at flowering stage. Drought at this stage leads to asynchronous development of male and female flower organ and increased interval between anthesis and silking, which finally causes failure of pollination and grain yield loss. In the present study, the expansin gene ZmEXPA5 was cloned and its function in drought tolerance was characterized. An indel variant in promoter of ZmEXPA5 is significantly associated with natural variation in drought-induced anthesis-silking interval. The drought susceptible haplotypes showed lower expression level of ZmEXPA5 than tolerant haplotypes and lost the cis-regulatory activity of ZmDOF29. Increasing ZmEXPA5 expression in transgenic maize decreases anthesis-silking interval and improves grain yield under both drought and well-watered environments. In addition, the expression pattern of ZmEXPA5 was analyzed. These findings provide insights into the genetic basis of drought tolerance and a promising gene for drought improvement in maize breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01432-x.

Integrated IBD Analysis, GWAS Analysis and Transcriptome Analysis to Identify the Candidate Genes for White Spot Disease in Maize.

Foundation parents (FPs) play an irreplaceable role in maize breeding practices. Maize white spot (MWS) is an important disease in Southwest China that always seriously reduces production. However, knowledge about the genetic mechanism of MWS resistance is limited. In this paper, a panel of 143 elite lines were collected and genotyped by using the MaizeSNP50 chip with approximately 60,000 single nucleotide polymorphisms (SNPs) and evaluated for resistance to MWS among 3 environments, and a genome-wide association study (GWAS) and transcriptome analysis were integrated to reveal the function of the identity-by-descent (IBD) segments for MWS. The results showed that (1) 225 IBD segments were identified only in the FP QB512, 192 were found only in the FP QR273 and 197 were found only in the FP HCL645. (2) The GWAS results showed that 15 common quantitative trait nucleotides (QTNs) were associated with MWS. Interestingly, SYN10137 and PZA00131.14 were in the IBD segments of QB512, and the SYN10137-PZA00131.14 region existed in more than 58% of QR273's descendants. (3) By integrating the GWAS and transcriptome analysis, Zm00001d031875 was found to located in the region of SYN10137-PZA00131.14. These results provide some new insights for the detection of MWS's genetic variation mechanisms.

A kelch-repeat superfamily gene, ZmNL4, controls leaf width in maize (Zea mays L.).

Leaf width (LW) is an important component of plant architecture that extensively affects both light capture during photosynthesis and grain yield, particularly under dense planting conditions. However, the genetic and molecular mechanisms regulating LW remain largely elusive in maize (Zea mays L.). In this study, qLW4a, a major quantitative trait locus controlling LW, was identified in a population constructed with maize inbred lines PH6WC, with wide leaves, and Lin387, with narrow leaves. Map-based cloning revealed that ZmNL4, a kelch-repeat superfamily gene, emerged to be the candidate for qLW4a, and a single-base deletion in the conserved SMC_prok_B domain of ZmNL4 in Lin387 caused a frame shift, leading to premature termination. Consistently, the knockout of ZmNL4 by CRISPR/Cas9 editing significantly reduced the LW, which was attributed to a reduction in the cell number instead of cell size, indicating a role of ZmNL4 in regulating cell division. Transcriptomic comparison of ZmNL4 knockout lines with the wild type B73-329 revealed that ZmNL4 might participate in cell wall biogenesis, asymmetric cell division, metabolic processes, transmembrane transport and response to external stimulus, etc. These results provide insights into the genetic and molecular mechanisms of ZmNL4 in controlling LW and could potentially contribute to optimizing plant architecture for maize breeding.