Ammonium (NH₄⁺) and nitrate (NO₃⁻) are major inorganic nitrogen (N) sources for plants. When serving as the sole or dominant N supply, NH₄⁺ often causes root inhibition and shoot chlorosis in plants, known as ammonium toxicity. NO₃⁻ usually causes no toxicity and can mitigate ammonium toxicity even at low concentrations, referred to as nitrate‐dependent alleviation of ammonium toxicity. Our previous studies indicated a NO₃⁻ efflux channel SLAH3 is involved in this process. However, whether additional components contribute to NO₃⁻‐mediated NH₄⁺ detoxification is unknown. Previously, mutations in NO₃⁻ transporter NRT1.1 were shown to cause enhanced resistance to high concentrations of NH₄⁺. Whereas, in this study, we found when the high‐NH₄⁺ medium was supplemented with low concentrations of NO₃⁻, nrt1.1 mutant plants showed hyper‐sensitive phenotype instead. Furthermore, mutation in NRT1.1 caused enhanced medium acidification under high‐NH₄⁺/low‐NO₃⁻ condition, suggesting NRT1.1 regulates ammonium toxicity by facilitating H⁺ uptake. Moreover, NRT1.1 was shown to interact with SLAH3 to form a transporter‐channel complex. Interestingly, SLAH3 appeared to affect NO₃⁻ influx while NRT1.1 influenced NO₃⁻ efflux, suggesting NRT1.1 and SLAH3 regulate each other at protein and/or gene expression levels. Our study thus revealed NRT1.1 and SLAH3 form a functional unit to regulate nitrate‐dependent alleviation of ammonium toxicity through regulating NO₃⁻ transport and balancing rhizosphere acidification.

Nitrogen (N) and potassium (K) are essential macronutrients for plants. Sufficient N and K uptake from the environment is required for successful growth and development. However, how N and K influence each other at the molecular level in plants is largely unknown. In this study, we found loss-of-function mutation in SLAH3 (SLAC1 HOMOLOGUE 3), encoding a NO₃⁻ efflux channel in Arabidopsis thaliana, enhanced tolerance to high KNO₃ concentrations. Surprisingly, slah3 mutants were less sensitive to high K⁺ but not NO₃⁻. Addition of NO₃⁻ led to reduced phenotypic difference between wild-type and slah3 plants, suggesting SLAH3 orchestrates NO₃⁻-K⁺ balance. Non-invasive Micro-test Technology analysis revealed reduced NO₃⁻ efflux and enhanced K⁺ efflux in slah3 mutants, demonstrating that SLAH3-mediated NO₃⁻ transport and SLAH3-affected K⁺ flux are critical in response to high K ⁺. Further investigation showed that two K⁺ efflux channels, GORK (GATED OUTWARDLY-RECTIFYING K⁺ CHANNEL) and SKOR (STELAR K⁺ OUTWARD RECTIFIER), interacted with SLAH3 and played key roles in high K⁺ response. The gork and skor mutants were slightly more sensitive to high K⁺ conditions. Less depolarization occurred in slah3 mutants and enhanced depolarization was observed in gork and skor mutants upon K⁺ treatment, suggesting NO₃⁻/K⁺ efflux-mediated membrane potential regulation is involved in high K⁺ response. Electrophysiological results showed that SLAH3 partially inhibited the activities of GORK and SKOR in Xenopus laevis oocytes. This study revealed that the anion channel SLAH3 interacts with the potassium channels GORK and SKOR to modulate membrane potential by coordinating N–K balance.

Maize (Zea mays) requires substantial amounts of nitrogen, posing a challenge for its cultivation. Recent work discovered that some ancient Mexican maize landraces harbored diazotrophic bacteria in mucilage secreted by their aerial roots. To see if this trait is retained in modern maize, we conducted a field study of aerial root mucilage (ARM) in 258 inbred lines. We observed that ARM secretion is common in modern maize, but the amount significantly varies, and only a few lines have retained the nitrogen‐fixing traits found in ancient landraces. The mucilage of the high‐ARM inbred line HN5‐724 had high nitrogen‐fixing enzyme activity and abundant diazotrophic bacteria. Our genome‐wide association study identified 17 candidate genes associated with ARM across three environments. Knockouts of one candidate gene, the subtilase family gene ZmSBT3, confirmed that it negatively regulates ARM secretion. Notably, the ZmSBT3 knockout lines had increased biomass and total nitrogen accumulation under nitrogen‐free culture conditions. High ARM was associated with three ZmSBT3 haplotypes that were gradually lost during maize domestication, being retained in only a few modern inbred lines such as HN5‐724. In summary, our results identify ZmSBT3 as a potential tool for enhancing ARM, and thus nitrogen fixation, in maize.

HD-ZIP proteins (homeodomain–leucine zipper proteins) are a large family of plant-specific transcription factors that play crucial roles in regulating various physiological and developmental processes, including growth, differentiation, response to environmental stress, and reproductive development. Seed size is the main limiting factor affecting the yield of maize (Zea mays). However, the genome-wide identification and characterization of this family in maize and its biological functions in seed size have not been reported. Here, 61 ZmHD-ZIP genes were identified in the genome. Phylogenetic analysis of these ZmHD-ZIP genes revealed that they are clustered into four subfamilies: HD-ZIP I, HD-ZIP II, HD-ZIP III, and HD-ZIP IV. Domain analysis revealed that the distribution of these domains aligns perfectly with the subfamily classification criteria, with members of the same subfamily sharing similar domains. Cis-acting element analysis showed that the ZmHD-ZIP family genes are involved in the regulation of plant reproductive development. In addition, ZmHD-ZIP23-overexpressing Arabidopsis and maize had larger seed with increased grain length and heavier grain weight owing to bigger embryo and endosperm area. These findings could serve as a basis for future studies on the roles of ZmHD-ZIP genes in improving maize yield.

Maize is a crucial food crop and industrial raw material, significantly contributing to national food security. Aphids are one of the most prevalent and destructive pests in maize production, necessitating the exploration of pest-resistant germplasm and the development of resistant varieties as the most fundamental and effective strategy for mitigating aphid-induced damage. This study established an aphid resistance evaluation system and identified 17 elite resistant inbred lines through multi-year screening. A genome-wide association study (GWAS) revealed 22 significant single-nucleotide polymorphisms (SNPs) associated with aphid resistance, including genes involved in benzoxazinoid (Bx) biosynthesis (such as Bx2), insect resistance-related transcription factors (such as WRKY23), plant lectins, and other resistance pathways. RNA-seq analysis of the samples before and after aphid infestation detected 1037 differentially expressed genes (DEGs) in response to aphid infestation, with KEGG enrichment highlighting benzoxazinoid biosynthesis and starch/sucrose metabolism as primary response pathways. Integrating GWAS and RNA-seq results revealed the presence of several benzoxazinoid synthesis-related genes on the short arm of chromosome 4 (Chr4S). FMqRrm1, a Kompetitive Allele-Specific PCR (KASP) marker, was derived from the Chr4S region. We subsequently utilized this marker for marker-assisted selection (MAS) to introgress the Chr4S region from the aphid-resistant inbred line into two aphid-susceptible inbred lines. The results demonstrated that the Chr4S favorable allele significantly reduced aphid occurrence by 1.5 to 2.1 grades. This study provides a critical theoretical foundation and practical guidance for understanding the molecular mechanism of aphid resistance in maize and molecular breeding for aphid resistance.

Fusarium verticillioides can systematically infect maize through seeds, triggering stalk rot and ear rot at a later stage, thus resulting in yield loss and quality decline. Seeds carrying F. verticillioides are unsuitable for storage and pose a serious threat to human and animal health due to the toxins released by the fungus. Previously, the candidate gene ZmC2GnT was identified, using linkage and association analysis, as potentially implicated in maize seed resistance to F. verticillioides; however, its disease resistance mechanism remained unknown. Our current study revealed that ZmC2GnT codes an N-acetylglucosaminyltransferase, using sequence structure and evolutionary analysis. The candidate gene association analysis revealed multiple SNPs located in the UTRs and introns of ZmC2GnT. Cloning and comparing ZmC2GnT showed variations in the promoter and CDS of resistant and susceptible materials. The promoter of ZmC2GnT in the resistant parent contains one extra cis-element ABRE associated with the ABA signal, compared to the susceptible parent. Moreover, the amino acid sequence of ZmC2GnT in the resistant parent matches that of B73, but the susceptible parent contains ten amino acid alterations. The resistant material BT-1 and the susceptible material N6 were used as parents to observe the expression level of the ZmC2GnT. The results revealed that the expression of ZmC2GnT in disease-resistant maize seeds was significantly up-regulated after infection with F. verticillioides. After treatment with F. verticillioides or ABA, the expression activity of the ZmC2GnT promoter increased significantly in the resistant material, but no discernible difference was detected in the susceptible material. When ZmC2GnT from resistant and susceptible materials was overexpressed in Arabidopsis thaliana, the seeds’ resistance to F. verticillioides increased, although there was no significant difference between the two types of overexpressed plants. Our study revealed that ZmC2GnT could participate in the immune process of plants against pathogenic fungus. ZmC2GnT plays a significant role in regulating the disease-resistance process of maize seeds, laying the foundation for future research into the regulatory mechanism and the development of new disease-resistant maize varieties.