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Background: Maize is sensitive to salt stress, especially during the germination and seedling stages., Methods: We conducted germination experiments on 60 maize materials under salt stress, and screened out the most salt-tolerant and salt-sensitive varieties based on germination indicators. Afterwards, transcriptome analysis was performed to screen for key regulators in the plumule and flag leaf at the germination and seedling stages, respectively. Following that, transgenic tobacco was developed to expose the roles and mechanisms of the candidate genes, enabling a deeper investigation of their functions., Results: Out of the 60 inbred lines of maize, "975-12" exhibits the highest level of salt tolerance, while "GEMS64" displays the lowest. The application of salt stress resulted in a significant increase in the levels of antioxidant enzymes in both "975-12" and "GEMS64". ABA signal transduction and jasmonic acid pathways were the pathways that mainly affected by salt stress. In addition, a significant finding has been made indicating that when exposed to high levels of salt stress, the expression of ZmHSP90 in '975-12' increased while in 'GEMS64' decreased. Furthermore, in tobacco plants overexpressing ZmHSP90 , there was an increase in antioxidant enzyme activity associated with salt tolerance. ZmHSP90 enhanced the expression levels of NtSOS1 , NtHKT1 , and NtNHX1 as compared to those in the salt treatment, causing the maintenance of Na + and K + homeostasis, suggesting that high expression of ZmHSP90 was conducive to regulate Na + transporters to maintain K + /Na + balanced in tobacco., Competing Interests: Xinyan Yu and Zhiqing Zou are employed by Win-all high-tech seed company limited. The authors declare that this work does not involve competing interests., (©2023 Yu et al.)

Moderate stimuli in mitochondria improve wide-ranging stress adaptability in animals, but whether mitochondria play similar roles in plants is largely unknown. Here, we report the enhanced stress adaptability of S-type cytoplasmic male sterility (CMS-S) maize and its association with mild expression of sterilizing gene ORF355. A CMS-S maize line exhibited superior growth potential and higher yield than those of the near-isogenic N-type line in saline fields. Moderate expression of ORF355 induced the accumulation of reactive oxygen species and activated the cellular antioxidative defense system. This adaptive response was mediated by elevation of the nicotinamide adenine dinucleotide concentration and associated metabolic homeostasis. Metabolome analysis revealed broad metabolic changes in CMS-S lines, even in the absence of salinity stress. Metabolic products associated with amino acid metabolism and galactose metabolism were substantially changed, which underpinned the alteration of the antioxidative defense system in CMS-S plants. The results reveal the ORF355-mediated superior stress adaptability in CMS-S maize and might provide an important route to developing salt-tolerant maize varieties., (© 2022 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Key Message: The novel ZmR1 CQ01 allele for maize anthocyanin synthesis was identified, and the biological function and regulatory molecular mechanisms of three ZmR1 alleles were unveiled. Anthocyanins in maize are valuable to human health. The R1 gene family is one of the important regulatory genes for the tissue-specific distribution of anthocyanins. R1 gene allelic variations are abundant and its biological function and regulatory molecular mechanisms are not fully understood. By exploiting genetic mapping and transgenic verification, we found that anthocyanin pigmentation in maize leaf midrib was controlled by ZmR1 on chromosome 10. Allelism test of maize zmr1 EMS mutants confirmed that anthocyanin pigmentation in leaf sheath was also controlled by ZmR1. ZmR1 CQ01 was a novel ZmR1 allelic variation obtained from purple maize. Its overexpression caused the whole maize plant to turn purple. ZmR1 B73 allele confers anthocyanin accumulation in near ground leaf sheath rather than in leaf midribs. The mRNA expression level of ZmR1 B73 was low in leaf midribs, resulting in no anthocyanin accumulation. ZmR1 B73 overexpression promoted anthocyanin accumulation in leaf midribs. Loss of exon 5 resulted in ZmR1 ZN3 allele function destruction and no anthocyanin accumulation in leaf midrib and leaf sheath. DNA affinity purification sequencing revealed 1010 genes targeted by ZmR1 CQ01 , including the bz2 in anthocyanin synthesis pathway. RNA-seq analysis showed 55 genes targeted by ZmR1 CQ01 changed the expression level significantly, and the expression of genes encoding key enzymes in flavonoid and phenylpropanoid biosynthesis pathways were significantly up-regulated. ZmR1 functional molecular marker was developed. These results revealed the effects of transcriptional regulation and sequence variation on ZmR1 function and identified the genes targeted by ZmR1 CQ01 at the genome-wide level., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Salt stress is a major devastating abiotic factor that affects the yield and quality of maize. However, knowledge of the molecular mechanisms of the responses to salt stress in maize is limited. To elucidate the genetic basis of salt tolerance traits, a genome-wide association study was performed on 348 maize inbred lines under normal and salt stress conditions using 557 894 single nucleotide polymorphisms (SNPs). The phenotypic data for 27 traits revealed coefficients of variation of >25%. In total, 149 significant SNPs explaining 6.6%-11.2% of the phenotypic variation for each SNP were identified. Of the 104 identified quantitative trait loci (QTLs), 83 were related to salt tolerance and 21 to normal traits. Additionally, 13 QTLs were associated with two to five traits. Eleven and six QTLs controlling salt tolerance traits and normal root growth, respectively, co-localized with QTL intervals reported previously. Based on functional annotations, 13 candidate genes were predicted. Expression levels analysis of 12 candidate genes revealed that they were all responsive to salt stress. The CRISPR/Cas9 technology targeting three sites was applied in maize, and its editing efficiency reached 70%. By comparing the biomass of three CRISPR/Cas9 mutants of ZmCLCg and one zmpmp3 EMS mutant with their wild-type plants under salt stress, the salt tolerance function of candidate genes ZmCLCg and ZmPMP3 were confirmed. Chloride content analysis revealed that ZmCLCg regulated chloride transport under sodium chloride stress. These results help to explain genetic variations in salt tolerance and provide novel loci for generating salt-tolerant maize lines., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

High-throughput phenotyping is increasingly becoming an important tool for rapid advancement of genetic gain in breeding programmes. Manual phenotyping of vascular bundles is tedious and time-consuming, which lags behind the rapid development of functional genomics in maize. More robust and automated techniques of phenotyping vascular bundles traits at high-throughput are urgently needed for large crop populations. In this study, we developed a standard process for stem micro-CT data acquisition and an automatic CT image process pipeline to obtain vascular bundle traits of stems including geometry-related, morphology-related and distribution-related traits. Next, we analysed the phenotypic variation of stem vascular bundles between natural population subgroup (480 inbred lines) based on 48 comprehensively phenotypic information. Also, the first database for stem micro-phenotypes, MaizeSPD, was established, storing 554 pieces of basic information of maize inbred lines, 523 pieces of experimental information, 1008 pieces of CT scanning images and processed images, and 24 192 pieces of phenotypic data. Combined with genome-wide association studies (GWASs), a total of 1562 significant single nucleotide polymorphism (SNPs) were identified for 30 stem micro-phenotypic traits, and 84 unique genes of 20 traits such as VBNum, VBAvArea and PZVBDensity were detected. Candidate genes identified by GWAS mainly encode enzymes involved in cell wall metabolism, transcription factors, protein kinase and protein related to plant signal transduction and stress response. The results presented here will advance our knowledge about phenotypic trait components of stem vascular bundles and provide useful information for understanding the genetic controls of vascular bundle formation and development., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Stalk fracture caused by strong wind can severely reduce yields in maize. Stalks with higher stiffness and flexibility will exhibit stronger lodging resistance. However, stalk flexibility is rarely studied in maize. Stalk fracture of the internode above the ear before tasseling will result in the lack of tassel and pollen, which is devastating for pollination in seed production. In this study, we focused on stalk lodging before tasseling in two maize inbred lines, JING724 and its improved line JING724A1 and their F 2:3 population., Results: JING724A1 showed a larger stalk fracture angle than JING724, indicating higher flexibility. In addition, compared to JING724, JING724A1 also had longer and thicker stalks, with a conical, frustum-shaped internode above the ear. Microscopy and X-ray microcomputed tomography of the internal stalk architecture revealed that JING724A1 had more vascular bundles and thicker sclerenchyma tissue. Furthermore, total soluble sugar content of JING724A1, especially the glucose component, was substantially higher than in JING724. Using an F 2:3 population derived from a JING724 and JING724A1 cross, we performed bulk segregant analysis for stalk fracture angle and detected one QTL located on Chr3: 14.00-19.28 Mb. Through transcriptome data analysis and ∆ (SNP-index), we identified two candidate genes significantly associated with high stalk fracture angle, which encode a RING/U-box superfamily protein (Zm00001d039769) and a MADS-box transcription factor 54 (Zm00001d039913), respectively. Two KASP markers designed from these two candidate genes also showed significant correlations with stalk fracture angle., Conclusions: The internode shape and glucose content are possibly correlated with stalk flexibility in maize. Two genes in the detected QTL are potentially associated with stalk fracture angle. These novel phenotypes and associated loci will provide a theoretical foundation for understanding the genetic mechanisms of lodging, and facilitate the selection of maize varieties with improved flexibility and robust lodging resistance.

Waxy maize has many excellent characteristics in terms of its nutritional and economic value. In recent decades, the waxy maize germplasm has increased dramatically as a result of different selection methods. We collected 200 waxy maize inbred accessions from different origins to study their genetic diversity and phylogenetic relationships, and to identify new waxy mutations. A simple sequence repeat (SSR) analysis revealed wide genetic diversity among the 200 waxy maize accessions. The maize accessions were clustered into three groups. We sequenced the waxy gene from the first to the 14th exon. Nucleotide variation analysis of 167 waxy maize and 14 flint maize lines revealed some nucleotide differences in the waxy gene among different waxy maize groups, and much narrower nucleotide diversity in waxy maize than in flint maize. In a phylogenetic analysis, waxy maize carrying the same mutation allele clustered together, and waxy maize carrying different mutation alleles distributed in different groups; waxy maize was intermixed with flint maize in each branch, and wx-D7 waxy maize separated significantly from waxy maize lines carrying wx-D10, wx-124 and wx-hAT mutant alleles. The wx-hAT was a new waxy mutation identified in this study. It consisted of a 2286-bp transposon inserted into the middle of exon three of the waxy gene. A PCR marker specific for the wx-hAT allele was developed. These results will be useful for the utilization and preservation of the waxy maize germplasm, and the PCR marker has potential uses in waxy maize breeding programs.

Background: Identifying genotype-phenotype links and causative genes from quantitative trait loci (QTL) is challenging for complex agronomically important traits. To accelerate maize gene discovery and breeding, we present the Complete-diallel design plus Unbalanced Breeding-like Inter-Cross (CUBIC) population, consisting of 1404 individuals created by extensively inter-crossing 24 widely used Chinese maize founders., Results: Hundreds of QTL for 23 agronomic traits are uncovered with 14 million high-quality SNPs and a high-resolution identity-by-descent map, which account for an average of 75% of the heritability for each trait. We find epistasis contributes to phenotypic variance widely. Integrative cross-population analysis and cross-omics mapping allow effective and rapid discovery of underlying genes, validated here with a case study on leaf width., Conclusions: Through the integration of experimental genetics and genomics, our study provides useful resources and gene mining strategies to explore complex quantitative traits.

Pectins, the major components of cell walls in plants, are synthesized and secreted to cell walls as highly methyl-esterified polymers and then demethyl-esterified by pectin methylesterases (PMEs). The PMEs are spatially regulated by pectin methylesterase inhibitors (PMEIs). In this study, 43 and 49 putative PME and PMEI genes were identified in maize, respectively. Gene structure and motif analysis revealed that members in the same paralogous pairs or in the same subgroup generally had common motif compositions and gene structure patterns, which indicates functional similarity between the closely related ZmPME/PMEI genes. Gene ontology annotation analysis showed that most of the ZmPME/PMEI genes are involved in cell wall modification and pectin catabolic process with molecular functions of pectinesterase or pectinesterase inhibitor activities. There are 35 ZmPME/PMEI genes expressed higher in anthers than in other tissues from the NimbleGen maize microarray data, and the semiq-RT-PCR assay revealed most of these ZmPME/PMEIs specially expressed in anthers and pollens, indicating they possibly had role in anther and pollen development. In addition, these ZmPME/PMEI genes were highly expressed in the fertile anthers, while lowly or no expressed in sterile anthers. This further indicated these genes might be involved in the development of anther and pollen.

The maize stalk is an important mechanical supporting tissue. The stalk fracture resistance is closely related to lodging resistance, and thus the yield. In this study, we showed that the basal zone (BZ) was more fragile than the middle zone (MZ) of the stalk internode before tasseling. In order to clarify the relationship between the different zones and fragile resistance between the internodes, we systematically analyzed the phenotypic, metabolomic and transcriptomic differences. The results indicated that the BZ zone had lower stalk strength, which corresponded to the results of less lignin, cellulose and hemicellulose than that of the MZ. The 27 highly enriched metabolites and 4430 highly expressed genes in the BZ mainly participated in pentose phosphate, and in ribosome and sterol synthesis pathways, respectively. In addition, the BZ had higher vascular bundles density but smaller size compared with the MZ. By contrast, the 28 highly enriched known metabolites and 4438 highly expressed genes in the MZ were mainly involved in lignin synthesis, and secondary metabolites synthesis, respectively, especially the phenylpropanoid synthesis. The results provide a deeper understanding of the relationship between development and fracture differences in stalk, and may facilitate the improvement of field management practice to reduce lodging.

Plant height (PH) is a key factor in maize (Zea mays L.) yield, biomass, and plant architecture. We investigated the PH of diverse maize inbred lines (117 temperate lines, 135 tropical lines) at four growth stages using unmanned aerial vehicle high-throughput phenotypic platforms (UAV-HTPPs). We extracted PH data using an automated pipeline based on crop surface models and orthomosaic model. The correlation between UAV and manually measured PH data reached 0.95. Under temperate field conditions, temperate maize lines grew faster than tropical maize lines at early growth stages, but tropical lines grew faster at later growth stages and ultimately became taller than temperate lines. A genome-wide association study identified 68 unique quantitative trait loci (QTLs) for seven PH-related traits, and 35% of the QTLs coincided with those previously reported to control PH. Generally, different QTLs controlled PH at different growth stages, but eight QTLs simultaneously controlled PH and growth rate at multiple growth stages. Based on gene annotations and expression profiles, we identified candidate genes controlling PH. The PH data collected by the UAV-HTPPs were credible and the genetic mapping power was high. Therefore, UAV-HTPPs have great potential for use in studies on PH.

Maize is a globally important crop that was a classic model plant for genetic studies. Here, we report a 2.2 Gb draft genome sequence of an elite maize line, HuangZaoSi (HZS). Hybrids bred from HZS-improved lines (HILs) are planted in more than 60% of maize fields in China. Proteome clustering of six completed sequenced maize genomes show that 638 proteins fall into 264 HZS-specific gene families with the majority of contributions from tandem duplication events. Resequencing and comparative analysis of 40 HZS-related lines reveals the breeding history of HILs. More than 60% of identified selective sweeps were clustered in identity-by-descent conserved regions, and yield-related genes/QTLs were enriched in HZS characteristic selected regions. Furthermore, we demonstrated that HZS-specific family genes were not uniformly distributed in the genome but enriched in improvement/function-related genomic regions. This study provides an important and novel resource for maize genome research and expands our knowledge on the breadth of genomic variation and improvement history of maize., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

Salt stress is a major abiotic factor limiting maize yield. To characterize the mechanism underlying maize salt tolerance, we compared the seedling root proteomes of salt-tolerant Jing724 and salt-sensitive D9H. The germination rate and growth parameter values (weight and length) were higher for Jing724 than for D9H under saline conditions. Using an iTRAQ-based method, we identified 513 differentially regulated proteins (DRPs), with 83 and 386 DRPs specific to Jing724 and D9H, respectively. In salt-stressed Jing724, the DRPs were primarily associated with the pentose phosphate pathway, glutathione metabolism, and nitrogen metabolism. Key DRPs, such as glucose-6-phosphate 1-dehydrogenase, NADPH-producing dehydrogenase, glutamate synthase, and glutamine synthetase, were identified based on pathway enrichment and protein-protein interaction analyses. Moreover, salt-responsive proteins in Jing724 seedlings were implicated in energy management, maintenance of redox homeostasis, detoxification of ammonia, regulation of osmotic homeostasis, stress defense and adaptation, biotic cross-tolerance, and regulation of gene expression. Quantitative analyses of superoxide dismutase activity, malondialdehyde content, relative electrolyte leakage, and proline content were consistent with the predicted changes based on DRP functions. Furthermore, changes in the abundance of eight representative DRPs were correlated with the corresponding mRNA levels. Our results may be useful for elucidating the molecular networks mediating salt tolerance.

Background: Salt stress significantly restricts plant growth and production. Maize is an important food and economic crop but is also a salt sensitive crop. Identification of the genetic architecture controlling salt tolerance facilitates breeders to select salt tolerant lines. However, the critical quantitative trait loci (QTLs) responsible for the salt tolerance of field-grown maize plants are still unknown., Results: To map the main genetic factors contributing to salt tolerance in mature maize, a double haploid population (240 individuals) and 1317 single nucleotide polymorphism (SNP) markers were employed to produce a genetic linkage map covering 1462.05 cM. Plant height of mature maize cultivated in the saline field (SPH) and plant height-based salt tolerance index (ratio of plant height between saline and control fields, PHI) were used to evaluate salt tolerance of mature maize plants. A major QTL for SPH was detected on Chromosome 1 with the LOD score of 22.4, which explained 31.2% of the phenotypic variation. In addition, the major QTL conditioning PHI was also mapped at the same position on Chromosome 1, and two candidate genes involving in ion homeostasis were identified within the confidence interval of this QTL., Conclusions: The detection of the major QTL in adult maize plant establishes the basis for the map-based cloning of genes associated with salt tolerance and provides a potential target for marker assisted selection in developing maize varieties with salt tolerance.

In higher plants, many mitochondrial genes contain group II-type introns that are removed from RNAs by splicing to produce mature transcripts that are then translated into functional proteins. However, the factors involved in the splicing of mitochondrial introns and their biological functions are not well understood in maize. Here, we isolated an empty pericarp 10 (emp10) mutant and identified the underlying gene by map-based cloning. Emp10 encodes a P-type mitochondria-targeted pentatricopeptide repeat (PPR) protein with 10 PPR motifs. Loss of Emp10 function results in splicing defect of the first intron of nad2, a gene encoding subunit 2 of NADH dehydrogenase (also called complex I). The emp10 mutant has undetectable activity of complex I and has arrested development of embryo and endosperm, and thus defective seeds with empty pericarp. Additionally, the basal endosperm transfer layer cells were severely affected, indicating the deficiency of cell wall ingrowths in the emp10 kernels. Moreover, the alternative respiratory pathway involving alternative oxidase was significantly induced in the emp10 mutant. These results suggest that EMP10 is specifically required for the cis-splicing of mitochondrial nad2 intron 1, embryogenesis and endosperm development in maize., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Soil waterlogging is one of the major abiotic stresses affecting maize grain yields. To understand the molecular mechanisms underlying waterlogging tolerance in maize, the iTRAQ LC-MS/MS technique was employed to map the proteomes of seedling root cells of the A3237 (tolerant inbred) and A3239 (sensitive inbred) lines under control and waterlogging conditions. Among the 3318 proteins identified, 211 were differentially abundant proteins (DAPs), of which 81 were specific to A3237 and 57 were specific to A3239. These DAPs were categorized into 11 groups that were closely related to the plant stress response, including metabolism, energy, transport, and disease/defense. In the waterlogged A3237 root cells, NADP-malic enzyme, glutamate decarboxylase, coproporphyrinogen III oxidase, GSH S-transferase, GSH dehydrogenase, and xyloglucan endotransglycosylase 6 were specifically accumulated to manage energy consumption, maintain pH levels, and minimize oxidative damage. The evaluations of five specific physiological parameters (alcohol dehydrogenase activity and GSH, malondialdehyde, adenosine 5'-triphosphate, and nicotinamide adenine dinucleotide concentrations) were in agreement with the proteomic results. Moreover, based on the proteomic assay, eight representative genes encoding DAPs were selected for validation at the transcriptional level. qRT-PCR revealed that the expression levels of these genes correlated with their observed protein abundance. These findings shed light on the complex mechanisms underlying waterlogging tolerance in maize. All MS data have been deposited into the ProteomeXchange with the identifier PXD001125 http://proteomecentral.proteomexchange.org/dataset/PXD001125., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The RNA-binding glycine-rich protein (RB-GRP) family is characterized by the presence of a glycine-rich domain arranged in (Gly)n-X repeats and an RNA-recognition motif (RRM). RB-GRPs participate in varied physiological and biochemical processes especially in the stress response of plants. In this study, a total of 23 RB-GRPs distributed on 10 chromosomes were identified in maize (Zea mays L.), and they were divided into four subgroups according to their conserved domain architecture. Five pairs of paralogs were identified, while none of them was located on the same chromosomal region, suggesting that segmental duplication is predominant in the duplication events of the RB-GRPs in maize. Comparative analysis of RB-GRPs in maize, Arabidopsis (Arabidopsis thaliana L.), rice (Oryza sativa L.), and wheat (Triticum aestivum) revealed that two exclusive subgroups were only identified in maize. Expression of eight ZmRB-GRPs was significantly regulated by at least two kinds of stresses. In addition, cis-elements predicted in the promoter regions of the ZmRB-GRPs also indicated that these ZmRB-GRPs would be involved in stress response of maize. The preliminary genome-wide analysis of the RB-GRPs in maize would provide useful information for further study on the function of the ZmRB-GRPs., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize.