Summary: Kernel weight is a critical factor that essentially affects maize (Zea mays) yield. In natural inbred lines, popcorn kernels exhibit overtly smaller sizes compared to dent corn kernels, and kernel weight, which is controlled by multiple genetic loci, varies widely. Here, we characterized a major quantitative trait locus on chromosome 1, responsible for controlling kernel weight (qKW1) and size. The qKW1 locus encodes a protein containing a seven in absentia domain with E3 ubiquitin ligase activity, expressed prominently from the top to the middle region of the endosperm. The presence and function of qKW1 were confirmed through ZmKW1 gene editing, where the mutations in ZmKW1 within dent corn significantly increased kernel weight, consistent with alterations in kernel size, while overexpression of ZmKW1 had the opposite effect. ZmKW1 acts as a negative regulator of kernel weight and size by reducing both the number and size of the endosperm cells and impacting endosperm filling. Notably, the popcorn allele qKW1^N and the dent corn allele qKW1^D encode identical proteins; however, the differences in promoter activity arise due to the insertion of an Indel‐1346 sequence in the qKW1^N promoter, resulting in higher expression levels compared to qKW1^D, thus contributing to the variation in kernel weight and size between popcorn and dent corn kernels. Linkage disequilibrium analysis of the 2.8 kb promoter region of ZmKW1 in a dataset comprising 111 maize association panels identified two distinct haplotypes. Our results provide insight into the mechanisms underlying kernel development and yield regulation in dent corn and popcorn, with a specific focus on the role of the ubiquitination system.

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.

The aleurone layer, located on the outermost region of the endosperm, is rich in lipids, proteins, and minerals and can substantially enhance grain nutritional value with increased thickness. ADP-glucose pyrophosphorylase is a critical rate-limiting enzyme in starch biosynthesis, with Shrunken2 (Sh2) and Brittle2 (Bt2) encoding its large and small subunits, respectively. Nevertheless, the influence of these genes on maize (Zea mays) aleurone layer development remains poorly understood. We conducted genetic analysis, map-based cloning, and allelic validation using the Huajian1F (HJ1F) paternal line containing multialeurone layers and identified that Sh2 regulates aleurone layer thickness in HJ1F. Additional studies demonstrated that the bt2 mutant (MT) displayed the phenotype of aleurone layer thickening. Both sh2 and bt2 MTs exhibited a substantial increase in the contents of grain oil, total protein, and mineral elements. RNA sequencing analysis indicated that differentially expressed genes were enriched in fatty acid and amino acid metabolic pathways during aleurone layer formation. ZmDof3 (DNA-binding one zinc finger protein 3) and NKD2 (NAKED ENDOSPERM2), which participate in aleurone layer thickening in maize, showed significant differential expression during the aleurone layer development of sh2 and bt2 MTs. Yeast 1-hybrid assays, β-galactosidase activity assays, and the luciferase reporter system confirmed that ZmDof3 and NKD2 regulate the expression of Sh2 and Bt2. This study has established that Sh2 and Bt2 control starch biosynthesis and play a pivotal role in maize aleurone layer development.

Background/Objectives: Kernel dehydration is an important agronomic trait influencing mechanical harvesting efficiency, post-harvest drying costs, and grain storage quality in maize (Zea mays L.). Although the genetic and molecular basis of whole-kernel dehydration has been increasingly investigated, tissue-specific physiological and molecular patterns, particularly those associated with the embryo, remain insufficiently characterized. In this study, four maize inbred lines with contrasting dehydration capacities were selected to characterize tissue-specific dehydration dynamics and associated molecular patterns. Methods: Moisture content and dehydration rates of kernels, endosperms, and embryos were monitored from 10 to 55 days after pollination (DAP), followed by correlation analyses with kernel- and ear-related agronomic traits. Transcriptome sequencing, weighted gene co-expression network analysis (WGCNA), and untargeted metabolomics were integrated to characterize molecular processes associated with contrasting embryo dehydration phenotypes. Results: Embryo dehydration closely paralleled whole-kernel dehydration, and embryo dehydration rate was strongly associated with kernel dehydration rate. Transcriptomic and WGCNA analyses revealed developmental- and genotype-dependent expression changes, particularly during late maturation, and identified dehydration-associated modules enriched in lipid metabolism, glycolysis, hormone signaling, and stress-responsive processes, including fatty acid metabolism, ABA- and ethylene-related signaling, and MAPK-related genes. FDR-controlled metabolomics further revealed genotype- and developmental-stage-dependent metabolite differences associated with organic acid metabolism, amino acid metabolism, transport-associated processes, and secondary metabolism. Pathway-level integration highlighted ABA- and ethylene-associated candidate processes associated with variation in embryo dehydration. Conclusions: Collectively, these findings provide an embryo-focused characterization of physiological and multi-omics variation associated with maize kernel dehydration and identify candidate molecular processes for further functional investigation. Further validation across broader germplasm panels, environmental conditions, and experimental systems will be required to determine the causal roles and potential breeding relevance of these candidate processes.

The development of maize (Zea mays) kernels is a complex physiological process, regulated by many genes in a spatio-temporal manner. However, there remain many unknown regulatory genes involved in maize kernel development. Here, we have identified a gene (ZmZFP2), which encodes a C4HC3-type RING zinc finger protein, that regulates maize kernel weight and size based on the suppression subtractive hybridization from maize endosperm in our previous studies. Through transgenic technology, over-expression of ZmZFP2 resulted in increased kernel weight and size compared to the wild type, while mutation of the gene led to decreased kernel weight and size. Microscopic observation of developing kernels revealed that ZmZFP2 affects the number of endosperm cells and their filling, as well as the starch and protein contents of the kernels. ZmZFP2 shows constitutive expression in various maize tissues. Furthermore, transcriptome analysis revealed that the zmzfp2-ems mutation significantly interfered with multiple biological processes, especially hormone pathways and ubiquitin degradation. Together, ZmZFP2 affects kernel size and weight by influencing endosperm cell number and filling, providing a valuable potential gene for increasing maize yield.