Excess salinity is a natural stress that causes crop yield losses worldwide. The genetic bases of maize salt tolerance remain largely unknown. Here we investigated the survival rates of 445 maize natural accessions after salt treatments. A skewed distribution of the salt-tolerant phenotypes was observed in this population. Genome-wide association studies (GWAS) revealed 57 loci significantly associated with salt tolerance. Forty-nine candidate genes were detected from these loci. About 10% of these genes were co-localized with loci from QTL mapping. Forty four percent of the candidate genes were involved in stress responses, ABA signaling, stomata division, DNA binding/transcription regulation and auxin signaling, suggesting that they are key genetic mechanisms of maize salt tolerance. Transgenic studies showed that two genes, the salt-tolerance-associated-gene 4 (SAG4, GRMZM2G077295) and SAG6 (GRMZM2G106056), which encode a protein transport protein and the double-strand break repair protein MRE11, respectively, had positive roles in plant salt tolerance, and their salt-tolerant haplotypes were revealed. The genes we identified in this study provide a list of candidate targets for further study of maize salt tolerance, and of genetic markers and materials that may be used for breeding salt-tolerance in maize.

As essential B vitamin for humans, folates accumulation in edible parts of crops, such as maize kernels, is of great importance for human health. But its breeding is always limited by the prohibitive cost of folate profiling. The molecular breeding is a more executable and efficient way for folate fortification, but is limited by the molecular knowledge of folate regulation. Here we report the genetic mapping of folate quantitative trait loci (QTLs) using a segregated population crossed by two maize lines, one high in folate (GEMS31) and the other low in folate (DAN3130). Two folate QTLs on chromosome 5 were obtained by the combination of F₂ whole-exome sequencing and F₃ kernel-folate profiling. These two QTLs had been confirmed by bulk segregant analysis using F₆ pooled DNA and F₇ kernel-folate profiling, and were overlapped with QTLs identified by another segregated population. These two QTLs contributed 41.6% of phenotypic variation of 5-formyltetrahydrofolate, the most abundant storage form among folate derivatives in dry maize grains, in the GEMS31×DAN3130 population. Their fine mapping and functional analysis will reveal details of folate metabolism, and provide a basis for marker-assisted breeding aimed at the enrichment of folates in maize kernels.

Potassium (K⁺) is an essential macronutrient for plant growth and development. Transporters from the KT/HAK/KUP family play crucial roles in K⁺ homeostasis and cell growth in various plant species. However, their physiological roles in maize are still unknown. In this study, we cloned ZmHAK5 and ZmHAK1 and investigated their functions in maize (Zea mays L.). In situ hybridization showed that ZmHAK5 was mainly expressed in roots, especially in the epidermis, cortex, and vascular bundle. ZmHAK5 was characterized as a high-affinity K⁺ transporter. Loss of function of ZmHAK5 led to defective K⁺ uptake in maize, under low K⁺ conditions, whereas ZmHAK5-overexpressing plants showed increased K⁺ uptake activity and improved growth. ZmHAK1 was upregulated under low K⁺ stress, as revealed by RT-qPCR. ZmHAK1 mediated K⁺ uptake when heterologously expressed in yeast, but its transport activity was weaker than that of ZmHAK5. Overexpression of ZmHAK1 in maize significantly affected K⁺ distribution in shoots, leading to chlorosis in older leaves. These findings indicate that ZmHAK5 and ZmHAK1 play distinct roles in K⁺ homeostasis in maize, functioning in K⁺ uptake and K⁺ distribution, respectively. Genetic manipulation of ZmHAK5 may represent a feasible way to improve K⁺ utilization efficiency in maize.

Uncovering the genetic basis of seed development will provide useful tools for improving both crop yield and nutritional value. However, the genetic regulatory networks of maize (Zea mays) seed development remain largely unknown. The maize opaque endosperm and small germ 1 (os1) mutant has opaque endosperm and a small embryo. Here, we cloned OS1 and show that it encodes a putative transcription factor containing an RWP-RK domain. Transcriptional analysis indicated that OS1 expression is elevated in early endosperm development, especially in the basal endosperm transfer layer (BETL), conducting zone (CZ), and central starch endosperm (CSE) cells. RNA sequencing (RNA-Seq) analysis of the os1 mutant revealed sharp downregulation of certain genes in specific cell types, including ZmMRP-1 and Meg1 in BETL cells and a majority of zein- and starch-related genes in CSE cells. Using a haploid induction system, we show that wild-type endosperm could rescue the smaller size of os1 embryo, which suggests that nutrients are allocated by the wild-type endosperm. Therefore, our data imply that the network regulated by OS1 accomplishes a key step in nutrient allocation between endosperm and embryo within maize seeds. Identification of this network will help uncover the mechanisms regulating the nutritional balance between endosperm and embryo.

RNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation. However, the functions of RBPs in plants remain poorly understood. Maize kernel mutant dek42 has small defective kernels and lethal seedlings. Dek42 was cloned by Mutator tag isolation and further confirmed by an independent mutant allele and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 materials. Dek42 encodes an RRM_RBM48 type RNA-binding protein that localizes to the nucleus. Dek42 is constitutively expressed in various maize tissues. The dek42 mutation caused a significant reduction in the accumulation of DEK42 protein in mutant kernels. RNA-seq analysis showed that the dek42 mutation significantly disturbed the expression of thousands of genes during maize kernel development. Sequence analysis also showed that the dek42 mutation significantly changed alternative splicing in expressed genes, which were especially enriched for the U12-type intron-retained type. Yeast two-hybrid screening identified SF3a1 as a DEK42-interacting protein. DEK42 also interacts with the spliceosome component U1-70K. These results suggested that DEK42 participates in the regulation of pre-messenger RNA splicing through its interaction with other spliceosome components. This study showed the function of a newly identified RBP and provided insights into alternative splicing regulation during maize kernel development.

DNA methylation plays a crucial role in suppressing mobilization of transposable elements and regulation of gene expression. A number of studies have indicated that DNA methylation pathways and patterns exhibit distinct properties in different species, including Arabidopsis, rice, and maize. Here, we characterized the function of DDM1 in regulating genome-wide DNA methylation in maize. Two homologs of ZmDDM1 are abundantly expressed in the embryo and their simultaneous disruption caused embryo lethality with abnormalities in cell proliferation from the early stage of kernel development. We establish that ZmDDM1 is critical for DNA methylation, at CHG sites, and to a lesser extent at CG sites, in heterochromatic regions, and unexpectedly, it is required for the formation of ^mCHH islands. In addition, ZmDDM1 is indispensable for the presence of 24-nt siRNA, suggesting its involvement in the RdDM pathway. Our results provide novel insight into the role of ZmDDM1 in regulating the formation of ^mCHH islands, via the RdDM pathway maize, suggesting that, in comparison to Arabidopsis, maize may have adopted distinct mechanisms for regulating ^mCHH.

Agricultural practices could affect bacterial diversity and community structure by altering soil physical and chemical properties. Straw returning and tillage practices are widely used in agriculture, however, the effects of these agricultural practices on microbiomes are still unclear. In the present study, we compared the 18 bacterial communities of soil with different straw returning and tillage treatment combinations. The V3-V4 regions of the 16S ribosomal RNA were amplified and analyzed by high-throughput sequencing technology. The results showed that the bacterial communities were consistently dominated by Acidobacteria, Proteobacteria, Actinobacteria, and Chloroflexi. Short-term straw returning and tillage practices significantly altered the diversity, relative abundance and functions of the soil microbiome. Soil subjected to rotary tillage and straw returning (RTS) combination possessed the highest bacterial diversity and lowest ratio of G⁺/G^- bacteria, indicating that RTS could be an efficient integrated management system to improve microbiome in the short term. Double verifications based on relative abundance and network analysis, revealed close relationships of Mycobacterium and Methylibium with RTS, indicating they could serve as biomarkers for RTS. Investigating microbial changes under different agricultural practices will provide valuable foundations for land sustainable utilization and increase crop yields.