Rice black-streaked dwarf virus (RBSDV) causes maize rough dwarf disease (MRDD), which is a viral disease that significantly affects maize yields worldwide. Plants tolerate stress through transcriptional reprogramming at the alternative splicing (AS), transcriptional, and fusion gene (FG) levels. However, it is unclear whether and how AS and FG interfere with transcriptional reprogramming in MRDD. In this study, we performed global profiling of AS and FG on maize response to RBSDV and compared it with transcriptional changes. There are approximately 1.43 to 2.25 AS events per gene in maize infected with RBSDV. GRMZM2G438622 was only detected in four AS modes (A3SS, A5SS, RI, and SE), whereas GRMZM2G059392 showed downregulated expression and four AS events. A total of 106 and 176 FGs were detected at two time points, respectively, including six differentially expressed genes and five differentially spliced genes. The gene GRMZM2G076798 was the only FG that occurred at two time points and was involved in two FG events. Among these, 104 GOs were enriched, indicating that nodulin-, disease resistance-, and chloroplastic-related genes respond to RBSDV stress in maize. These results provide new insights into the mechanisms underlying post-transcriptional and transcriptional regulation of maize response to RBSDV stress.

Maize (Zea mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos. We observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed. These data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.

The NAC gene family has transcription factors specific to plants, which are involved in development and stress response and adaptation. In this study, ZmNAC89, an NAC gene in maize that plays a role in saline–alkaline tolerance, was isolated and characterized. ZmNAC89 was localized in the nucleus and had transcriptional activation activity during in vitro experiments. The expression of ZmNAC89 was strongly upregulated under saline–alkaline, drought and ABA treatments. Overexpression of the ZmNAC89 gene in transgenic Arabidopsis and maize enhanced salt tolerance at the seedling stage. Differentially expressed genes (DEGs) were then confirmed via RNA-sequencing analysis with the transgenic maize line. GO analyses showed that oxidation–reduction process-regulated genes were involved in ZmNAC89-mediated salt–alkaline stress. ZmNAC89 may regulate maize saline–alkali tolerance through the REDOX pathway and ABA signal transduction pathway. From 140 inbred maize lines, 20 haplotypes and 16 SNPs were found in the coding region of the ZmNAC89 gene, including the excellent haplotype HAP20. These results contribute to a better understanding of the response mechanism of maize to salt–alkali stress and marker-assisted selection during maize breeding.

Seed deterioration during storage poses significant challenges to agriculture, forestry, and germplasm conservation, primarily through a reduced germination potential. Maintaining the seed aging tolerance is essential for high-yield crop production. While physiological and molecular processes underpinning maize seed aging remain poorly understood, they are critical for developing improved maize varieties. This study investigated the aging mechanisms in two maize inbred lines with contrasting seed vigor (Dong 156 and Dong 237) through phenotype evaluation, physiological indices, and multiomics analyses. Artificial aging induced notable differences in phenotypic, physiological, and cellular responses among the lines. Transcriptomic analysis identified 4892 DEGs consistently expressed at three germination stages, enriched in glycolysis, starch and sucrose metabolism, antioxidant responses, and plant hormone signaling. Metabolomic profiling revealed 233 metabolites with significant enrichment in the TCA cycle, plant hormone signaling, and amino sugar/nucleotide sugar metabolism. Combined analyses pinpointed 1112 DEGs and 56 DEMs in 36 metabolic pathways. WGCNA identified 11 hub genes, including ZmARF27, as key regulators of aging tolerance. Mutants deficient in ZmARF27 exhibited significantly reduced germination indicators under aging stress, confirming its positive role in aging resistance.

Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) cause maize rough dwarf disease (MRDD) and rice black-streaked dwarf disease (RBSDD) in China. RBSDV segment 8 (S8) contains the only deletion mutation in the genomes of these viruses, which are both members of the genus Fijivirus. To illuminate the molecular differences between the RBSDV and SRBSDV genomes and better understand the evolution of these viruses, and to determine which virus is specifically associated with MRDD and RBSDD in each region, S8 was analyzed in 66 virus isolates collected from 10 geographic locations in China and 14 S8 sequences obtained from the National Center for Biotechnology Information GenBank. Phylogenetic analysis showed that the pathogen associated with MRDD and RBSDD in the Yellow and Huai River valleys was RBSDV, whereas the pathogen associated with these diseases in Sanya was SRBSDV. Codon usage bias in S8 differed significantly between RBSDV and SRBSDV, as indicated by effective number of codons used by a gene (Nc) and GC values, Nc plots, and variation explained by the first axis in correspondence analysis. The nucleotide identities among these 66 RBSDV and SRBSDV isolates ranged from 66.2 to 68.2%, and were considerably lower than the nucleotide identities within RBSDV (from 94.1 to 99.9%) or SRBSDV (from 93.9 to 100%) isolates. Most S8 polymorphisms were identified in the region from 1,000 to 1,200 bp in RBSDV and in the region from 500 to 700 bp in SRBSDV. The difference in the lengths of RBSDV (1,936 bp) and SRBSDV (1,928 bp) was due to an 8-bp deletion in the 3′-untranslated region of SRBSDV. Six recombination events were detected in S8 in RBSDV and two recombination events were detected in S8 in SRBSDV. Recombination breakpoints were found within the region containing the deletion mutation in nine isolates. However, no recombination events were detected between RBSDV and SRBSDV. Both of these viruses were under negative and purifying selection, although the ratio of nonsynonymous mutations to synonymous mutations (Ka/Ks) for RBSDV S8 (0.0530) was not significantly lower than that of SRBSDV S8 (0.0823, P = 0.1550). We found that SRBSDV was more highly genetically differentiated (product of effective population size and the migration rate among populations < 1; values for the among-populations component of genetic variation or normalized variation > 0.33; and P values of the sequence statistic, the rank statistic, and the nearest-neighbor statistic < 0.01) than RBSDV. However, gene flow between RBSDV and SRBSDV was not frequent.

Heat smut is a fungal soil-borne disease caused by Sporisorium reilianum, and affects the development of male and female tassels. Our previous research found that the tassel symptoms in maize infected with Sporisorium reilianum significantly differed in inbred lines with Sipingtou blood, and exhibited stable heredity over time at multiple locations. In this study, cytological analysis demonstrated that the cellular organization structures of three typical inbred lines (Huangzao4, Jing7, and Chang7-2) showed significant discrepancies at the VT stage. QTLs that control the different symptoms of maize tassels infected with Sporisorium reilianum were located in two F₂ populations, which were constructed using three typical inbred lines. The BSA (bulked segregation analysis) method was used to construct mixed gene pools based on typical tassel symptoms. The QTLs of different symptoms of maize tassels infected with Sporisorium reilianum were detected with 869 SSR markers covering the whole maize genome. The mixed gene pools were screened with polymorphic markers between the parents. Additional SSR markers were added near the above marker to detect genotypes in partially single plants in F₂ populations. The QTL controlling tassel symptoms in the Huangzao4 and Jing7 lines was located on the bin 1.06 region, between the markers of umc1590 and bnlg1598, and explained 21.12% of the phenotypic variation with an additive effect of 0.6524. The QTL controlling the tassel symptoms of the Jing7 and Chang7-2 lines was located on the bin 2.07 region, between the markers of umc1042 and bnlg1335, and explained 11.26% phenotypic variation with an additive effect of 0.4355. Two candidate genes (ZmABP2 and Zm00001D006403) were identified by a conjoint analysis of label-free quantification proteome sequencings.