Maize (Zea mays) originated in southern Mexico and has spread over a wide latitudinal range. Maize expansion from tropical to temperate regions has necessitated a reduction of its photoperiod sensitivity. In this study, we cloned a quantitative trait locus (QTL) regulating flowering time in maize and show that the maize ortholog of Arabidopsis thaliana EARLY FLOWERING3, ZmELF3.1, is the causal locus. We demonstrate that ZmELF3.1 and ZmELF3.2 proteins can physically interact with ZmELF4.1/4.2 and ZmLUX1/2, to form evening complex(es; ECs) in the maize circadian clock. Loss-of-function mutants for ZmELF3.1/3.2 and ZmLUX1/2 exhibited delayed flowering under long-day and short-day conditions. We show that EC directly represses the expression of several flowering suppressor genes, such as the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) genes ZmCCT9 and ZmCCT10, ZmCONSTANS-LIKE 3, and the PSEUDORESPONSE REGULATOR (PRR) genes ZmPRR37a and ZmPRR73, thus alleviating their inhibition, allowing florigen gene expression and promoting flowering. Further, we identify two closely linked retrotransposons located in the ZmELF3.1 promoter that regulate the expression levels of ZmELF3.1 and may have been positively selected during postdomestication spread of maize from tropical to temperate regions during the pre-Columbian era. These findings provide insights into circadian clock-mediated regulation of photoperiodic flowering in maize and new targets of genetic improvement for breeding.

Increasing planting density has been adopted as an effective means to increase maize (Zea mays) yield. Competition for light from neighbors can trigger plant shade avoidance syndrome, which includes accelerated flowering. However, the regulatory networks of maize inflorescence development in response to high-density planting remain poorly understood. In this study, we showed that shade-mimicking treatments cause precocious development of the tassels and ears. Comparative transcriptome profiling analyses revealed the enrichment of phytohormone-related genes and transcriptional regulators among the genes co-regulated by developmental progression and simulated shade. Network analysis showed that three homologous Squamosa promoter binding protein (SBP)-like (SPL) transcription factors, Unbranched2 (UB2), Unbranched3 (UB3), and Tasselsheath4 (TSH4), individually exhibited connectivity to over 2,400 genes across the V3-to-V9 stages of tassel development. In addition, we showed that the ub2 ub3 double mutant and tsh4 single mutant were almost insensitive to simulated shade treatments. Moreover, we demonstrated that UB2/UB3/TSH4 could directly regulate the expression of Barren inflorescence2 (BIF2) and Zea mays teosinte branched1/cycloidea/proliferating cell factor30 (ZmTCP30). Furthermore, we functionally verified a role of ZmTCP30 in regulating tassel branching and ear development. Our results reveal a UB2/UB3/TSH4-anchored transcriptional regulatory network of maize inflorescence development and provide valuable targets for breeding shade-tolerant maize cultivars.

As transcription factors derived from transposase, FAR-RED IMPAIRED RESPONSE1 (FAR1) and its homolog FHY3 play crucial roles in the regulation of light signaling and various stress responses by coordinating the expression of downstream target genes. Despite the extensive investigation of the FAR1/FHY3 family in Arabidopsis thaliana and other species, a comprehensive examination of these genes in maize has not been conducted thus far. In this study, we employed a genomic mining approach to identify 16 ZmFAR1 genes in the maize inbred line B73, which were further classified into five subgroups based on their phylogenetic relationships. The present study characterized the predicted polypeptide sequences, molecular weights, isoelectric points, chromosomal distribution, gene structure, conserved motifs, subcellular localizations, phylogenetic relationships, and cis-regulatory elements of all members belonging to the ZmFAR1 family. Furthermore, the tissue-specific expression of the 16 ZmFAR1 genes was analyzed using RNA-seq, and their expression patterns under far-red light conditions were validated in the ear and tassel through qRT-qPCR. The observed highly temporal and spatial expression patterns of these ZmFAR1 genes were likely associated with their specific functional capabilities under different light conditions. Further analysis revealed that six ZmFAR1 genes (ZmFAR1-1, ZmFAR1-10, ZmFAR1-11, ZmFAR1-12, ZmFAR1-14, and ZmFAR1-15) exhibited a response to simulated shading treatment and actively contributed to the development of maize ears. Through the integration of expression quantitative trait loci (eQTL) analyses and population genetics, we identified the presence of potential causal variations in ZmFAR1-14 and ZmFAR1-9, which play a crucial role in regulating the kernel row number and kernel volume weight, respectively. In summary, this study represents the initial identification and characterization of ZmFAR1 family members in maize, uncovering the functional variation in candidate regulatory genes associated with the improvement of significant agronomic traits during modern maize breeding.

Long non-coding RNAs (lncRNAs) have emerged as important regulatory molecules in plants, but their potential roles during modern maize breeding remain largely unexplored. This study systematically characterized lncRNA expression dynamics using transcriptome data from 137 maize inbred lines from different breeding eras in China. We identified 18,023 lncRNAs transcripts, grouped by expression trends across historical breeding eras. Comparative analysis revealed 2228 differentially expressed lncRNAs transcripts (DElncRNAs) between modern (CN2000&10s) and early (CN1960&70s) accessions. By integrating WGCNA and cis-target analysis, we identified candidate lncRNAs and putative lncRNA-PCG associations that may be associated with maize plant architecture-related processes. Further, 771 DElncRNAs were predicted to be associated with 810 protein-coding genes, and these associated genes were significantly enriched in plant hormone signal transduction. Dual-luciferase reporter assays provided preliminary experimental support that lncrna.33063 and lncrna.33068 can repress the promoter activity of ZmPIF5.2 in a heterologous transient expression system. Furthermore, we constructed a putative ceRNA-related candidate interaction network consisting of lncRNA–miRNA–mRNA triplets that include 317 candidate miRNA-lncRNA pairs and 8325 candidate miRNA-mRNA pairs, with the associated mRNAs enriched in biological processes such as morphogenesis, stimulus response, and hormone metabolism. These findings provide a set of candidate lncRNAs and lncRNA-PCG associations for future functional validation and may offer useful clues for understanding the possible roles of lncRNAs in agronomic trait-related biological processes and maize molecular breeding. Overall, this study provides candidate genetic resources and a framework for future investigation of lncRNA-associated relationships potentially related to agronomic trait variation in maize.

Nitrogen use efficiency (NUE) is a major determinant of maize (Zea mays L.) productivity and sustainability, yet the regulatory changes associated with modern breeding remain incompletely understood. Here, we used breeding-era transcriptomic data from 137 elite Chinese maize inbred lines to identify transcriptional regulators associated with maize NUE. Breeding-era expression shifts in NUE effector genes were modest but tissue-specific, pointing to pathway-level transcriptional rewiring during modern breeding. Focusing on the first leaf above the uppermost ear at silking, we identified 69 breeding-era-responsive genes, including 10 transcription factors, and prioritized ZmSPL19 through Pearson correlation analysis with curated NUE-related genes. ZmSPL19 expression declined during modern breeding and showed a nitrate-repressed expression, with lower transcript abundance under nitrogen-sufficient conditions and rapid downregulation upon nitrate resupply. Loss of ZmSPL19 function promoted primary root elongation, biomass accumulation, leaf nitrogen content, soil–plant analysis development (SPAD), photosynthetic rate, kernel number, and grain yield under nitrogen-sufficient conditions. These results identify ZmSPL19 as a breeding-associated negative regulator of growth and yield formation under nitrogen-sufficient conditions and support the value of a breeding-informed strategy for discovering regulators with potential relevance to maize NUE improvement.