A NAC-EXPANSIN module enhances maize kernel size by controlling nucellus elimination

Maize is a globally important crop, and kernel size and weight are key yield components and important breeding targets. Numerous QTLs for kernel-related traits have been identified, yet relatively few causal genes have been cloned and functionally characterized. Prior cloned loci include qKW9, encoding a PPR protein affecting photosynthesis and grain filling, and ZmVPS29, a candidate QTL influencing kernel morphology via auxin-related processes. Many classical kernel mutants (dek, emp, smk) cause severe embryo/endosperm defects and have limited breeding utility. Kernel development involves coordinated growth of embryo, endosperm, and maternal tissues (nucellus, pericarp). The nucellus, derived from the ovule, mediates nutrient exchange and provides a developmental environment, but its genetic and molecular regulation and its impact on kernel development remain poorly understood. Early kernel development features programmed cell death (PCD) of the maternal nucellus, with remobilization of cellular contents to support developing filial tissues. The study aims to identify and characterize genetic factors controlling nucellus elimination and its coordination with early endosperm development, and to assess their potential for improving kernel size and weight.

• Kernel size/weight QTLs are abundant in maize, but few causal genes are known. qKW9 encodes a PPR protein linked to photosynthesis and grain filling; ZmVPS29 affects kernel morphology and yield via auxin-related pathways.
• Classical mutants affecting embryo/endosperm (dek, emp, smk) often produce small or lethal kernels, limiting breeding application.
• Maternal tissues, particularly the nucellus, play pivotal roles in nutrient exchange and developmental support, yet their regulation is understudied.
• Early cereal seed development involves nucellus degeneration via PCD, enabling resource remobilization. Known regulators in other species include MADS29 in rice; proteases (VPEs, cysteine, serine, aspartyl proteases) execute PCD in cereal maternal tissues.
• NAC transcription factors can regulate nutrient-related processes and bind CACG motifs; NAC-expansin modules have been implicated in Arabidopsis seed germination (NAC25/EXPA2) but nucellus-specific regulation during early kernel development in maize had not been defined.

• QTL mapping and NIL construction: Two elite inbreds (Mc and V671) differing in kernel size were crossed. An HKW QTL (HKW9) on chromosome 9 (bin 9.03–9.04) explaining >10% variance was mapped. Near-isogenic lines (NILs) HKW9Mc and HKW9V671 were developed by backcrossing (4 generations) and selfing to introgress HKW9 alleles into the Mc background.
• Candidate gene identification: Polymorphisms in the HKW9 interval identified Zm00001d045861 (ZmEXPB15, a β-expansin) as candidate. Promoter SNPs and InDels and a 4-bp exon InDel were cataloged; association analysis linked promoter variants to HKW and defined elite haplotypes.
• Expression analyses: qRT-PCR across tissues and 0–12 DAP kernels; in situ hybridization for spatial expression; generation of proZmEXPB15:ZmEXPB15-GFP transgenics and confocal imaging for localization (cell wall, cytoplasm, nucleus) and nucellus-specific accumulation dynamics.
• Functional genetics: CRISPR-Cas9 knockouts of ZmEXPB15 in C01 background (three KO lines); constitutive overexpression of ZmEXPB15 (three OE lines, B104 background). Phenotyping included kernel length, width, hundred kernel weight (HKW). Reciprocal crosses and F1 hybrids with elite inbreds assessed maternal effects and breeding potential.
• Histology and morphometrics: Longitudinal sections (0–12 DAP) prepared for nucellus and endosperm area measurements; semi-thin sections for nucellus thickness and cell size metrics (area, length, width). Image analysis via ImageJ.
• Cell death assays: TUNEL assays on sections (2–6 DAP) to detect PCD timing; Evan’s blue staining and quantification for cell death intensity.
• PCD gene expression: qRT-PCR for protease genes (VPE4, CCP5, SER1, AED1/2) in NILs at 4 DAP.
• TF identification and regulatory assays: Promoter motif analysis revealed CACG sites. Nucellus transcriptomes guided selection of NAC TFs (ZmNAC11, ZmNAC29). qRT-PCR and in situ confirmed nucellus-enriched expression. EMSA with recombinant His-SUMO-ZmNAC11/29 tested binding to promoter probes from both NILs. Transient dual-luciferase assays in maize leaf protoplasts tested transcriptional activation using 436-bp and 1308-bp promoters.
• NAC functional genetics: CRISPR-Cas9 knockouts of ZmNAC11 and ZmNAC29 (single and double mutants) characterized for kernel traits, nucellus/endosperm area ratios, TUNEL signal, and PCD gene expression; reciprocal crosses tested maternal effects.
• RNA-seq: 6-DAP kernels from HKW9Mc and HKW9V671 profiled (three biological replicates) using Illumina NovaSeq; reads processed with Trimmomatic, mapped with HISAT2 to B73 RefGen_v4, quantified with StringTie/HTSeq; DEGs via DESeq2.
• Statistics: One-way ANOVA and Tukey HSD for multiple comparisons; two-tailed Student’s t-tests where indicated. Sample sizes provided in figures; significance thresholds reported (*P<0.05, **P<0.01, ***P<0.001).

• ZmEXPB15 is the causal gene underlying a major kernel weight QTL (HKW9). It encodes a β-expansin with nucellus-specific expression during early kernel development (2–8 DAP, peak at 4 DAP), and its expression correlates with HKW across 75 inbred lines.
• Promoter polymorphisms in ZmEXPB15 associate with HKW. An elite promoter haplotype (Hap1) confers higher ZmEXPB15 expression and increased HKW relative to Hap2 (sample sizes n=40 vs n=180, respectively).
• Functional validation: CRISPR knockout of ZmEXPB15 reduces kernel length, width, and HKW; overexpression increases these traits. Maternal effects confirmed: crosses show HKW primarily influenced when the high-expression allele is in the maternal parent; most reciprocal crosses using OE as pollen did not increase HKW.
• Cellular basis: Higher ZmEXPB15 expression (HKW9Mc) enlarges nucellus thickness and nucellar cell size (area, length, width) without altering cell number, indicating promotion of cell expansion. During 0–12 DAP, nucellus-to-endosperm area ratio declines faster in HKW9Mc than in HKW9V671; at 12 DAP, the ratio is ~25% in HKW9Mc vs ~50% in HKW9V671, indicating accelerated nucellus elimination and endosperm expansion.
• PCD dynamics: TUNEL-positive nuclei appear earlier and more abundantly in nucellus of HKW9Mc (from 2 DAP) compared to HKW9V671 (nearly absent at 3 DAP); KO lines delay and OE lines accelerate TUNEL signal onset. Evan’s blue staining corroborates enhanced nucellus cell death in high ZmEXPB15 contexts. PCD-related protease gene expression (VPE4, CCP5, SER1, AED1/2) is significantly lower in HKW9V671 than HKW9Mc at 4 DAP.
• Upstream regulation: ZmNAC11 and ZmNAC29 are nucellus-enriched TFs peaking at 4 DAP, bind the ZmEXPB15 promoter CACG motifs by EMSA, with stronger binding to the intact motif from HKW9m than the disrupted motif from HKW9V671. Both TFs activate ZmEXPB15 promoter-driven luciferase in maize protoplasts, with stronger activation on the HKW9m promoter. A longer promoter with additional CACG sites further enhances activation.
• NAC mutant phenotypes: Single mutants zmnac11 and zmnac29 reduce kernel size and HKW; the double mutant is not significantly more severe, suggesting a shared pathway. The double mutant shows larger nucellus and smaller endosperm areas at 4–6 DAP, delayed TUNEL signal at 4 DAP, and downregulation of PCD genes (e.g., AED1, SER1). Reciprocal crosses support maternal control.
• Breeding relevance: Overexpression of ZmEXPB15 increases HKW and kernel size without obvious adverse effects on other ear traits in multiple hybrid combinations; most hybrids with OE as female show increased HKW, while using OE as male generally does not, consistent with maternal action.

The study identifies a nucellus-specific regulator, ZmEXPB15, as a key mediator of early kernel development by promoting nucellus cell expansion and programmed cell death, thereby coordinating maternal tissue elimination with endosperm expansion. This fills a gap in understanding how maternal tissues influence maize kernel growth, extending beyond previously characterized genes acting predominantly within endosperm after 6 DAP. The localization of ZmEXPB15 to the cell wall (and detectable in cytoplasm and nucleus) aligns with expansin-mediated wall loosening underlying nucellar cell expansion and suggests additional roles connected to PCD initiation. Upstream, the nucellus-enriched NAC TFs ZmNAC11 and ZmNAC29 bind and activate the ZmEXPB15 promoter, and their loss phenocopies ZmEXPB15 disruption, supporting a NAC–EXPANSIN regulatory module driving nucellus PCD. Genetic interactions indicate ZmNAC11/29 act in a common pathway with ZmEXPB15, though the more severe phenotypes in higher-order mutants imply additional targets beyond ZmEXPB15, consistent with observed expression of these NACs in endosperm as well. The discovery of this module reveals a previously unknown mechanism coupling maternal tissue remodeling with filial tissue growth and provides a framework for improving kernel traits through maternal tissue regulation.

This work demonstrates that a NAC–EXPANSIN regulatory module, comprising nucellus-expressed ZmNAC11/ZmNAC29 and their downstream target ZmEXPB15, enhances maize kernel size and weight by promoting nucellus cell expansion and programmed cell death to facilitate timely endosperm development. ZmEXPB15 underlies a major HKW QTL (HKW9); favorable promoter haplotypes and transgenic overexpression increase kernel weight without evident penalties in other ear traits, underscoring breeding utility. Future research should elucidate the molecular mechanism by which expansin activity interfaces with PCD signaling, define the broader regulatory network and additional targets of ZmNAC11/29 in nucellus and endosperm, and evaluate the stability and agronomic impacts of ZmEXPB15-based improvements across diverse environments and germplasm.

• The precise molecular mechanism linking expansin function to initiation and execution of PCD in nucellus remains unresolved; subcellular localization suggests potential non–cell wall roles that require further dissection.
• ZmNAC11/29 likely regulate additional targets beyond ZmEXPB15, as indicated by higher-order mutant phenotypes and their expression in endosperm; the full target network and possible pleiotropic effects are not fully characterized.
• Most quantitative effects (e.g., across environments and genetic backgrounds) are shown in selected lines and hybrids; broader field validation and assessment of potential trade-offs under diverse conditions are needed.