Maize is a crucial global crop, and kernel size and weight are critical yield components. Numerous quantitative trait loci (QTLs) affecting kernel size have been identified, but few have been cloned and characterized. Previous studies have identified QTLs like *qKW9* (encoding a PPR protein affecting photosynthesis and grain filling) and *ZmVPS29* (homologous to *Arabidopsis VPS29*, affecting kernel morphology). Classical mutants with defects in embryo/endosperm (e.g., *dek*, *emp*, *smk*) typically produce small or lethal kernels. Kernel development involves the coordinated growth of the embryo, endosperm, and maternal tissues (nucellus and pericarp). The nucellus, derived from the ovule, facilitates apoplastic exchange between the mother plant and developing offspring, providing an environment for the zygote and filial tissue. However, the genetic and molecular regulation of maternal tissues and their impact on maize kernel development are largely unknown. Maize kernel development proceeds through three phases: early development, filling, and maturation. Early development involves maternal nucellar tissue degeneration via programmed cell death (PCD), with the contents of dying cells being re-mobilized to nourish the developing endosperm. This research aims to understand the genetic and molecular mechanisms underlying this process.

The literature review section extensively explores previously identified QTLs related to maize kernel size and weight. It highlights the limited success in cloning and characterizing these QTLs, contrasting previous work with the current study's focus on a major QTL, HKW9. The review also summarizes knowledge regarding the three genetically distinct components of kernel development (embryo, endosperm, and maternal tissues), emphasizing the understudied role of the maternal nucellus. Existing research on programmed cell death (PCD) in seed development and the functions of various maternal tissues, like the nucellus in other cereal crops, is reviewed to provide context for the study's findings. The role of expansins in plant cell wall loosening is also discussed, setting the stage for the identification of *ZmEXPB15* as a key player in nucellus elimination.

The study employed several methods. Near-isogenic lines (NILs) were created using two elite maize inbred lines (Mc and V671) with contrasting kernel sizes. A major QTL (HKW9) was mapped, and NILs were developed through backcrossing and self-crossing. Kernel traits (length, width, hundred kernel weight, ear weight) were measured. Cytological observation of developing kernels (0-12 days after pollination, DAP) involved semi-thin sectioning, toluidine blue staining, and scanning electron microscopy. Quantitative RT-PCR was used to analyze gene expression. RNA in situ hybridization localized gene expression. Confocal microscopy visualized the subcellular localization of ZmEXPB15-GFP. A TUNEL assay detected programmed cell death (PCD). Evans blue staining assessed cell death. RNA sequencing compared gene expression in NILs. CRISPR-Cas9 technology generated knockout mutants for *ZmEXPB15*, *ZmNAC11*, and *ZmNAC29*. *ZmEXPB15* overexpression lines were also created. Electrophoretic mobility shift assays (EMSAs) examined the binding of NAC transcription factors to the *ZmEXPB15* promoter. Transient expression assays in maize protoplasts evaluated the effect of NAC proteins on *ZmEXPB15* transcription. Recombinant proteins were expressed and purified for EMSAs.

The study identified *ZmEXPB15*, a β-expansin gene, as the key gene within the HKW9 QTL. *ZmEXPB15* is specifically expressed in the nucellus during early kernel development (2-8 DAP). Knockout mutants of *ZmEXPB15* resulted in smaller kernels, while overexpression lines showed significantly larger kernels and increased hundred kernel weight (HKW). The protein localizes to the cell wall, cytoplasm, and nucleus. *ZmEXPB15* promotes both nucellus cell expansion and programmed cell death (PCD), leading to faster nucellus elimination and more space for endosperm expansion. Two nucellus-enriched NAC transcription factors, *ZmNAC11* and *ZmNAC29*, were identified as upstream regulators of *ZmEXPB15*. These NACs bind to the *ZmEXPB15* promoter and activate its expression. Knockout mutants for *ZmNAC11* and *ZmNAC29* exhibited similar kernel defects to *ZmEXPB15* knockouts. Double knockout mutants (*zmnac11;zmnac29*) showed delayed nucellus elimination. Overexpression of *ZmEXPB15* consistently increased kernel weight across different hybrid combinations, without negatively impacting other ear traits.

The findings demonstrate a novel molecular mechanism regulating maize kernel size. The *ZmNAC11/ZmNAC29-ZmEXPB15* module plays a crucial role in coordinating the antagonistic development of the nucellus and endosperm. *ZmEXPB15*'s role in promoting both nucellus cell expansion and PCD is a unique finding, adding a new dimension to the understanding of expansin function. The discovery of this module expands our knowledge of early kernel development, moving beyond the primarily endosperm-focused previous research. The study identifies *ZmEXPB15* as a valuable resource for improving maize yield through molecular breeding.

This research identifies a previously unknown molecular mechanism governing maize kernel size and weight. The *ZmNAC11/ZmNAC29-ZmEXPB15* module controls nucellus elimination, a crucial process in early endosperm development. *ZmEXPB15*’s dual role in promoting cell expansion and programmed cell death highlights the complexity of this regulatory pathway. The successful generation of larger kernel lines through *ZmEXPB15* overexpression demonstrates its significant potential for crop improvement. Future research could investigate the detailed mechanisms of ZmEXPB15-mediated PCD and explore the potential of other NAC-EXPANSIN modules in related species.

While the study provides strong evidence for the role of the *ZmNAC11/ZmNAC29-ZmEXPB15* module, further investigation is needed to fully understand the precise molecular mechanisms. The study mainly focuses on the early stages of kernel development; future studies could explore the module's role in later developmental stages. The number of genetic backgrounds tested for the overexpression lines is relatively small, and further investigation across a broader range of genetic backgrounds is warranted. The study focuses on the maternal effect; understanding the paternal contribution to this pathway could provide additional insight.