Functional analysis of ZmPHR1 and ZmPHR2 under low-phosphate stress in maize.

The PHOSPHATE STARVATION RESPONSE REGULATOR (PHR) plays a crucial regulatory role in plants during the process of responding to phosphate starvation. In this study, we combined reverse genetics and biotechnology to investigate the function of ZmPHR1 and ZmPHR2, including proteins containing the Myb_DNA_banding and Myb_CC-LHEQLE structural domains, in maize seedlings. Phylogenetic analysis revealed that ZmPHR1 and ZmPHR2 have high homology with AtPHR1 and OsPHR2, and share the characteristic features of nuclear localisation and transcriptional self-activation. Real-time quantitative PCR analysis showed that low phosphate (Pi) stress significantly induced the expression of ZmPHR1 and ZmPHR2 in maize seedling stage, and candidate gene association analysis further revealed the close association of these two genes with root traits under Pi stress conditions. Transgenic plants overexpressing ZmPHR1 and ZmPHR2 in Arabidopsis show a significant increase in lateral root number, fresh weight and total phosphorus accumulation under low-Pi stress. Besides, CHIP-PCR experiments identified target genes involved in hormone regulation, metal ion transport and homeostasis, phosphatase encoding, and photosynthesis, providing new insights into the biological functions of ZmPHR1 and ZmPHR2. Furthermore, our study showed that ZmPHR1 interacts with six SPX domain-only proteins (ZmSPXs) in maize, while ZmPHR2 interacts with five of these proteins. ZmPHR1 and ZmPHR2 expression was repressed in low Pi conditions, but was up-regulated in ZmSPX1 knockout material, according to our study of transgenic seedlings overexpressing ZmSPX1 in maize. We identified downstream target genes involved in the phosphorus signaling pathway, which are mainly involved in plant-pathogen interactions, ascorbic acid and arabinose metabolism, and ABC transporter proteins, by RNA-seq analysis of transgenic seedlings grown under low Pi stress for 7 days. Collectively, these results provide important clues to elucidate the role and functional significance of ZmPHR1 and ZmPHR2 under low Pi stress and also provide insights into understand the molecular mechanism of phosphorus homeostasis in maize. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01508-2.

SPX family response to low phosphorus stress and the involvement of ZmSPX1 in phosphorus homeostasis in maize.

Phosphorus (P) is a crucial macronutrient for plant growth and development, and low-Pi stress poses a significant limitation to maize production. While the role of the SPX domain in encoding proteins involved in phosphate (Pi) homeostasis and signaling transduction has been extensively studied in other model plants, the molecular and functional characteristics of the SPX gene family members in maize remain largely unexplored. In this study, we identified six SPX members, and the phylogenetic analysis of ZmSPXs revealed a close relationship with SPX genes in rice. The promoter regions of ZmSPXs were abundant in biotic and abiotic stress-related elements, particularly associated with various hormone signaling pathways, indicating potential intersections between Pi signaling and hormone signaling pathways. Additionally, ZmSPXs displayed tissue-specific expression patterns, with significant and differential induction in anthers and roots, and were localized to the nucleus and cytoplasm. The interaction between ZmSPXs and ZmPHRs was established via yeast two-hybrid assays. Furthermore, overexpression of ZmSPX1 enhanced root sensitivity to Pi deficiency and high-Pi conditions in Arabidopsis thaliana. Phenotypic identification of the maize transgenic lines demonstrated the negative regulatory effect on the P concentration of stems and leaves as well as yield. Notably, polymorphic sites including 34 single-nucleotide polymorphisms (SNPs) and seven insertions/deletions (InDels) in ZmSPX1 were significantly associated with 16 traits of low-Pi tolerance index. Furthermore, significant sites were classified into five haplotypes, and haplotype5 can enhance biomass production by promoting root development. Taken together, our results suggested that ZmSPX family members possibly play a pivotal role in Pi stress signaling in plants by interacting with ZmPHRs. Significantly, ZmSPX1 was involved in the Pi-deficiency response verified in transgenic Arabidopsis and can affect the Pi concentration of maize tissues and yield. This work lays the groundwork for deeper exploration of the maize SPX family and could inform the development of maize varieties with improved Pi efficiency.

Mining for QTL controlling maize low-phosphorus response genes combined with deep resequencing of RIL parental genomes and in silico GWAS analysis.

KEY MESSAGE: Extensive and comprehensive phenotypic data from a maize RIL population under both low- and normal-Pi treatments were used to conduct QTL mapping. Additionally, we integrated parental resequencing data from the RIL population, GWAS results, and transcriptome data to identify candidate genes associated with low-Pi stress in maize. Phosphorus (Pi) is one of the essential nutrients that greatly affect the maize yield. However, the genes underlying the QTL controlling maize low-Pi response remain largely unknown. In this study, a total of 38 traits at both seedling and maturity stages were evaluated under low- and normal-Pi conditions using a RIL population constructed from X178 (tolerant) and 9782 (sensitive), and most traits varied significantly between low- and normal-Pi treatments. Twenty-nine QTLs specific to low-Pi conditions were identified after excluding those with common intervals under both low- and normal-Pi conditions. Furthermore, 45 additional QTLs were identified based on the index value ((Trait_under_LowPi-Trait_under_NormalPi)/Trait_under_NormalPi) of each trait. These 74 QTLs collectively were classified as Pi-dependent QTLs. Additionally, 39 Pi-dependent QTLs were clustered in nine HotspotQTLs. The Pi-dependent QTL interval contained 19,613 unique genes, 6,999 of which exhibited sequence differences with non-synonymous mutation sites between X178 and 9782. Combined with in silico GWAS results, 277 consistent candidate genes were identified, with 124 genes located within the HotspotQTL intervals. The transcriptome analysis revealed that 21 genes, including the Pi transporter ZmPT7 and the strigolactones pathway-related gene ZmPDR1, exhibited consistent low-Pi stress response patterns across various maize inbred lines or tissues. It is noteworthy that ZmPDR1 in maize roots can be sharply up-regulated by low-Pi stress, suggesting its potential importance as a candidate gene for responding to low-Pi stress through the strigolactones pathway.

Exploring the phosphorus-starch content balance mechanisms in maize grains using GWAS population and transcriptome data.

Examining the connection between P and starch-related signals can help elucidate the balance between nutrients and yield. This study utilized 307 diverse maize inbred lines to conduct multi-year and multi-plot trials, aiming to explore the relationship among P content, starch content, and 100-kernel weight (HKW) of mature grains. A significant negative correlation was found between P content and both starch content and HKW, while starch content showed a positive correlation with HKW. The starch granules in grains with high-P and low-starch content (HPLS) were significantly smaller compared to grains with low-P high-starch content (LPHS). Additionally, mian04185-4 (HPLS) exhibited irregular and loosely packed starch granules. A significant decrease in ZmPHOs genes expression was detected in the HPLS line ZNC442 as compared to the LPHS line SCML0849, while no expression difference was observed in AGPase encoding genes between these two lines. The down-regulated genes in ZNC442 grains were enriched in nucleotide sugar and fatty acid anabolic pathways, while up-regulated genes were enriched in the ABC transporters pathway. An accelerated breakdown of fat as the P content increased was also observed. This implied that HPLS was resulted from elevated lipid decomposition and inadequate carbon sources. The GWAS analysis identified 514 significantly associated genes, out of which 248 were differentially expressed. Zm00001d052392 was found to be significantly associated with P content/HKW, exhibiting high expression in SCML0849 but almost no expression in ZNC442. Overall, these findings suggested new approaches for achieving a P-yield balance through the manipulation of lipid metabolic pathways in grains.

Genome-wide association studies dissect low-phosphorus stress response genes underling field and seedling traits in maize.

Phosphorus (P) is an essential element for plant growth, and its deficiency can cause decreased crop yield. This study systematically evaluated the low-phosphate (Pi) response traits in a large population at maturity and seedling stages, and explored candidate genes and their interrelationships with specific traits. The results revealed a greater sensitivity of seedling maize to low-Pi stress compared to that at maturity stage. The phenotypic response patterns to low-Pi stress at different stages were independent. Chlorophyll content was found to be a potential indicator for screening low-Pi-tolerant materials in the field. A total of 2900 and 1446 significantly associated genes at the maturity and seedling stages were identified, respectively. Among these genes, 972 were uniquely associated with maturity traits, while 330 were specifically detected at the seedling stage under low-Pi stress. Moreover, 768 and 733 genes were specifically associated with index values (low-Pi trait/normal-Pi trait) at maturity and seedling stage, respectively. Genetic network diagrams showed that the low-Pi response gene Zm00001d022226 was specifically associated with multiple primary P-related traits under low-Pi conditions. A total of 963 out of 2966 genes specifically associated with traits under low-Pi conditions or index values were found to be induced by low-Pi stress. Notably, ZmSPX4.1 and ZmSPX2 were sharply up-regulated in response to low-Pi stress across different lines or tissues. These findings advance our understanding of maize's response to low-Pi stress at different developmental stages, shedding light on the genes and pathways implicated in this response.

Chromatin remodeling analysis reveals the RdDM pathway responds to low-phosphorus stress in maize.

Chromatin in eukaryotes folds into a complex three-dimensional (3D) structure that is essential for controlling gene expression and cellular function and is dynamically regulated in biological processes. Studies on plant phosphorus signaling have concentrated on single genes and gene interactions. It is critical to expand the existing signaling pathway in terms of its 3D structure. In this study, low-Pi treatment led to greater chromatin volume. Furthermore, low-Pi stress increased the insulation score and the number of TAD-like domains, but the effects on the A/B compartment were not obvious. The methylation levels of target sites (hereafter as RdDM levels) peaked at specific TAD-like boundaries, whereas RdDM peak levels at conserved TAD-like boundaries shifted and decreased sharply. The distribution pattern of RdDM sites originating from the Helitron transposons matched that of genome-wide RdDM sites near TAD-like boundaries. RdDM pathway genes were upregulated in the middle or early stages and downregulated in the later stages under low-Pi conditions. The RdDM pathway mutant ddm1a showed increased tolerance to low-Pi stress, with shortened and thickened roots contributing to higher Pi uptake from the shallow soil layer. ChIP-seq results revealed that ZmDDM1A could bind to Pi- and root development-related genes. Strong associations were found between interacting genes in significantly different chromatin-interaction regions and root traits. These findings not only expand the mechanisms by which plants respond to low-Pi stress through the RdDM pathway but also offer a crucial framework for the analysis of biological issues using 3D genomics.

Functional analysis of ZmG6PE reveals its role in responses to low-phosphorus stress and regulation of grain yield in maize.

A previous metabolomic and genome-wide association analysis of maize screened a glucose-6-phosphate 1-epimerase (ZmG6PE) gene, which responds to low-phosphorus (LP) stress and regulates yield in maize's recombinant inbred lines (RILs). However, the relationship of ZmG6PE with phosphorus and yield remained elusive. This study aimed to elucidate the underlying response mechanism of the ZmG6PE gene to LP stress and its consequential impact on maize yield. The analysis indicated that ZmG6PE required the Aldose_epim conserved domain to maintain enzyme activity and localized in the nucleus and cell membrane. The zmg6pe mutants showed decreased biomass and sugar contents but had increased starch content in leaves under LP stress conditions. Combined transcriptome and metabolome analysis showed that LP stress activated plant immune regulation in response to the LP stress through carbon metabolism, amino acid metabolism, and fatty acid metabolism. Notably, LP stress significantly reduced the synthesis of glucose-1-phosphate, mannose-6-phosphate, and ß-alanine-related metabolites and changed the expression of related genes. ZmG6PE regulates LP stress by mediating the expression of ZmSPX6 and ZmPHT1.13. Overall, this study revealed that ZmG6PE affected the number of grains per ear, ear thickness, and ear weight under LP stress, indicating that ZmG6PE participates in the phosphate signaling pathway and affects maize yield-related traits through balancing carbohydrates homeostasis.

Modelling and Analysis of Expansion Joints' Effect on Dynamic Performance of Railway Rigid Overhead System.

This study focuses on developing a comprehensive model of a rigid overhead system, which includes essential components such as the suspension structure, positioning clamp, and expansion joint. The modelling approach utilizes finite element theory and beam elements to accurately represent the displacement, stiffness, and mass characteristics of the system. The models also incorporate the suspension structure and positioning line clamp, which play crucial roles in suspending and positioning the busbar. Various suspension structures and positioning line clamps are evaluated based on their dynamic characteristics. The expansion joint, responsible for connecting different anchor sections of the rigid overhead system, undergoes a detailed analysis. Different assembly scenarios, including ideal and deflected assembly conditions, are considered. To simulate the dynamic behaviour of the expansion joint, additional beams are introduced into the system model. The primary finding of the analysis is that the maximum stresses observed in the constructed expansion joint model, under different temperature conditions and normal/deflected assembly conditions, remain within the permissible stress limits of the material. This indicates a high level of safety. However, certain areas exhibit stress concentration, particularly at the sliding block B and sliding rod A positions. This stress concentration is primarily attributed to the unique assembly form of the expansion joint. To improve stress distribution and enhance service reliability, the analysis suggests optimizing the installation deflection angle and geometric design of the expansion joint. Furthermore, the concentrated mass at the expansion joint significantly impacts the current collection quality of the pantograph-overhead system. Mitigating this negative impact can be achieved by reducing the mass of the expansion joint.

Comprehensive analysis of transcriptional data on seed germination of two maize inbred lines under low-temperature conditions.

Seed germination directly affect maize yield and grain quality. Low-temperature reduces maize yield by affecting seed germination and seedling growth. However, the molecular mechanism of maize seed germination under low-temperature remains unclear. In this study, the transcriptome data of two maize inbred lines SCL127 (chilling-sensitive) and SCL326 (chilling-tolerant) were analyzed at five time points (0 H, 4 H, 12 H, 24 H, and 48 H) under low-temperature conditions. Through the comparison of SCL127-0 H-vs-SCL326-0 H (Group I), SCL127-4 H-vs-SCL326-4 H (Group Ⅱ), SCL127-12 H-vs-SCL326-12 H (Group Ⅲ), SCL127-24 H-vs-SCL326-24 H (Group Ⅳ), and SCL127-48 H-vs SCL326-48 H (Group Ⅴ), a total of 8,526 differentially expressed genes (DEGs) were obtained. Weighted correlation network analysis revealed that Zm00001d010445 was the hub gene involved in seed germination under low-temperature conditions. Zm00001d010445-based association analysis showed that Hap Ⅱ (G) was the excellent haplotype for seed germination under low-temperature conditions. These findings provide a new perspective for the study of the genetic architecture of maize tolerance to low-temperature and contribute to the cultivation of maize varieties with low-temperature tolerance.

GWAS across multiple environments and WGCNA suggest the involvement of ZmARF23 in embryonic callus induction from immature maize embryos.

KEY MESSAGE: Combined GWAS, WGCNA, and gene-based association studies identified the co-expression network and hub genes for maize EC induction. ZmARF23 bound to ZmSAUR15 promoter and regulated its expression, affecting EC induction. Embryonic callus (EC) induction in immature maize embryos shows high genotype dependence, which limits the application of genetic transformation in transgenic breeding and gene function elucidation in maize. Herein, we conducted a genome-wide association mapping (GWAS) for four EC induction-related traits, namely rate of embryonic callus induction (REC), increased callus diameter (ICD), ratio of shoot formation (RSF), and length of shoot (LS) across different environments. A total of 77 SNPs were significantly associated these traits under three environments and using the averages (across environments). Among these significant SNPs, five were simultaneously detected under multiple environments and 11 had respective phenotypic variation explained > 10%. A total of 257 genes were located in the linkage disequilibrium decay of these REC- and ICD-associated SNPs, of which 178 were responsive to EC induction. According to the expression values of the 178 genes, we performed a weighted gene co-expression network analysis (WGCNA) and revealed an EC induction-associated module and five hub genes. Hub gene-based association studies uncovered that the intragenic variations in GRMZM2G105473 and ZmARF23 influenced EC induction efficiency among different maize lines. Dual-luciferase reporter assay indicated that ZmARF23 bound to the promoter of a known causal gene (ZmSAUR15) for EC induction and positively regulated its expression on the transcription level. Our study will deepen the understanding of genetic and molecular mechanisms underlying EC induction and contribute to the use of genetic transformation in maize.

Genome-Wide Association Study Discovers Novel Germplasm Resources and Genetic Loci with Resistance to Gibberella Ear Rot Caused by Fusarium graminearum.

Gibberella ear rot (GER) in maize caused by Fusarium graminearum is one of the most devastating maize diseases reducing grain yield and quality worldwide. The genetic bases of maize GER resistance remain largely unknown. Using artificial inoculation across multiple environments, the GER severity of an association panel consisting of 316 diverse inbred lines was observed with wide phenotypic variation. In the association panel, a genome-wide association study using a general linear model identified 69 single-nucleotide polymorphisms (SNPs) significantly associated with GER resistance at the threshold of 2.04 × 10-5, and the average phenotypic variation explained (PVE) of these SNPs was 5.09%. We also conducted a genome-wide association study analysis using a mixed linear model at a threshold of 1.0 × 10-4, and 16 significantly associated SNPs with an average PVE of 4.73% were detected. A combined general linear model and mixed linear model method obtained 10 co-localized significantly associated SNPs linked to GER resistance, including the most significant SNP (PZE-105079915) with the greatest PVE value, 9.07%, at bin 5.05 following 10 candidate genes. These findings are significant for the exploration of the complicated genetic variations in maize GER resistance. The regions and genes identified herein provide a list of candidate targets for further investigation, in addition to the elite germplasm resources that can be used for breeding GER resistance in maize.

Association studies of genes in a Pb response-associated network in maize (Zea mays L.) reveal that ZmPIP2;5 is involved in Pb tolerance.

Lead (Pb) in the soil affects the growth and development of plants and causes damages to the human body through the food chain. Here, we identified and cloned a Pb-tolerance gene ZmPIP2;5 based on a weighted gene co-expression network analysis and gene-based association studies. We showed that ZmPIP2;5 encodes a plasma membrane aquaporin and positively regulated Pb tolerance and accumulation in Arabidopsis and yeast. Overexpression of ZmPIP2;5 increased root length and fresh weight of Arabidopsis seedlings under Pb stress. Heterologous expression of ZmPIP2;5 in yeast caused the enhanced growth speed under Pb treatment and Pb accumulation in yeast cells. A (T/A) SNP in the ZmPIP2;5 promoter affected the expression abundance of ZmPIP2;5 and thereby led to the difference in Pb tolerance among different maize lines. Our study helps to understand the mechanism underlying plant tolerance to Pb stress and provides new ideas for breeding Pb-tolerance maize varieties via molecular marker-assisted selection.

Combined linkage mapping and association analysis uncovers candidate genes for 25 leaf-related traits across three environments in maize.

KEY MESSAGE: Combined linkage and association analysis revealed five co-localized genetic loci across multiple environments. The key gene Zm00001d026491 was further verified to influence leaf length by candidate gene association analysis. Leaf morphology and number determine the canopy structure and thus affect crop yield. Herein, the genetic basis and key genes for 25 leaf-related traits, including leaf lengths (LL), leaf widths (LW), and leaf areas (LA) of eight continuous leaves under the tassel, and the number of leaves above the primary ear (LAE), were dissected by using an association panel and a biparental population. Using an intermated B73 × Mo17 (IBM) Syn10 doubled haploid (DH) population, 290 quantitative trait loci (QTL) controlling these traits were detected across different locations, among which 115 QTL were individually repeatedly identified in at least two environments. Using the association panel, 165 unique significant single-nucleotide polymorphisms (SNPs) were associated with target traits (P < 2.15E-06), of which 35 were separately detected across multiple environments. In total, 42 pleiotropic QTL/SNPs (pQTL/SNPs) were responsible for at least two of the LL, LW, LA, and LAE traits across multiple environments. Combining the QTL mapping and association study, five unique SNPs were located within the confidence intervals of seven QTL, and 77 genes were identified based on the linkage disequilibrium regions of co-localized SNP loci. Gene-based association studies verified that the intragenic variants in the candidate gene Zm00001d026491 influenced LL of the third leaf counted from the top node. These findings will provide vital information to understanding the genetic basis of leaf-related traits and help to cultivate maize varieties with ideal plant architecture.

An Integration of Linkage Mapping and GWAS Reveals the Key Genes for Ear Shank Length in Maize.

Ear shank length (ESL) has significant effects on grain yield and kernel dehydration rate in maize. Herein, linkage mapping and genome-wide association study were combined to reveal the genetic architecture of maize ESL. Sixteen quantitative trait loci (QTL) were identified in the segregation population, among which five were repeatedly detected across multiple environments. Meanwhile, 23 single nucleotide polymorphisms were associated with the ESL in the association panel, of which four were located in the QTL identified by linkage mapping and were designated as the population-common loci. A total of 42 genes residing in the linkage disequilibrium regions of these common variants and 12 of them were responsive to ear shank elongation. Of the 12 genes, five encode leucine-rich repeat receptor-like protein kinases, proline-rich proteins, and cyclin11, respectively, which were previously shown to regulate cell division, expansion, and elongation. Gene-based association analyses revealed that the variant located in Cyclin11 promoter affected the ESL among different lines. Cyclin11 showed the highest expression in the ear shank 15 days after silking among diverse tissues of maize, suggesting its role in modulating ESL. Our study contributes to the understanding of the genetic mechanism underlying maize ESL and genetic modification of maize dehydration rate and kernel yield.

Defect Severity Identification for a Catenary System Based on Deep Semantic Learning.

A variety of Chinese textual operational text data has been recorded during the operation and maintenance of the high-speed railway catenary system. Such defect text records can facilitate defect detection and defect severity analysis if mined efficiently and accurately. Therefore, in this context, this paper focuses on a specific problem in defect text mining, which is to efficiently extract defect-relevant information from catenary defect text records and automatically identify catenary defect severity. The specific task is transformed into a machine learning problem for defect text classification. First, we summarize the characteristics of catenary defect texts and construct a text dataset. Second, we use BERT to learn defect texts and generate word embedding vectors with contextual features, fed into the classification model. Third, we developed a deep text categorization network (DTCN) to distinguish the catenary defect level, considering the contextualized semantic features. Finally, the effectiveness of our proposed method (BERT-DTCN) is validated using a catenary defect textual dataset collected from 2016 to 2018 in the China Railway Administration in Chengdu, Lanzhou, and Hengshui. Moreover, BERT-DTCN outperforms several competitive methods in terms of accuracy, precision, recall, and F1-score value.

Mining synergistic genes for nutrient utilization and disease resistance in maize based on co-expression network and consensus QTLs.

Nutrient restrictions and large-scale emergence of diseases are threatening the maize production. Recent findings demonstrated that there is a certain synergistic interaction between nutrition and diseases pathways in model plants, however there are few studies on the synergistic genes of nutrients and diseases in maize. Thus, the transcriptome data of nitrogen (N) and phosphorus (P) nutrients and diseases treatments in maize, rice, wheat and Arabidopsis thaliana were collected in this study, and four and 22 weighted co-expression modules were obtained by using Weighted Gene Co-expression Network Analysis (WGCNA) in leaf and root tissues, respectively. With a total of 5252 genes, MFUZZ cluster analysis screened 26 clusters with the same expression trend under nutrition and disease treatments. In the meantime, 1427 genes and 22 specific consensus quantitative trait loci (scQTLs) loci were identified by meta-QTL analysis of nitrogen and phosphorus nutrition and disease stress in maize. Combined with the results of cluster analysis and scQTLs, a total of 195 consistent genes were screened, of which six genes were shown to synergistically respond to nutrition and disease both in roots and leaves. Moreover, the six candidate genes were found in scQTLs associated with gray leaf spot (GLS) and corn leaf blight (CLB). In addition, subcellular localization and bioinformatics analysis of the six candidate genes revealed that they were primarily expressed in endoplasmic reticulum, mitochondria, nucleus and plasma membrane, and were involved in defense and stress, MeJA and abscisic acid response pathways. The fluorescence quantitative PCR confirmed their responsiveness to nitrogen and phosphorus nutrition as well as GLS treatments. Taken together, findings of this study indicated that the nutrition and disease have a significant synergistic response in maize.

Association mapping uncovers maize ZmbZIP107 regulating root system architecture and lead absorption under lead stress.

Lead (Pb) is a highly toxic contaminant to living organisms and the environment. Excessive Pb in soils affects crop yield and quality, thus threatening human health via the food chain. Herein, we investigated Pb tolerance among a maize association panel using root bushiness (BSH) under Pb treatment as an indicator. Through a genome-wide association study of relative BSH, we identified four single nucleotide polymorphisms (SNPs) and 30 candidate genes associated with Pb tolerance in maize seedlings. Transcriptome analysis showed that four of the 30 genes were differentially responsive to Pb treatment between two maize lines with contrasting Pb tolerance. Among these, the ZmbZIP107 transcription factor was confirmed as the key gene controlling maize tolerance to Pb by using gene-based association studies. Two 5' UTR_variants in ZmbZIP107 affected its expression level and Pb tolerance among different maize lines. ZmbZIP107 protein was specifically targeted to the nucleus and ZmbZIP107 mRNA showed the highest expression in maize seedling roots among different tissues. Heterologous expression of ZmbZIP107 enhanced rice tolerance to Pb stress and decreased Pb absorption in the roots. Our study provided the basis for revelation of the molecular mechanism underlying Pb tolerance and contributed to cultivation of Pb-tolerant varieties in maize.

A Combination of a Genome-Wide Association Study and a Transcriptome Analysis Reveals circRNAs as New Regulators Involved in the Response to Salt Stress in Maize.

Salinization seriously threatens the normal growth of maize, especially at the seedling stage. Recent studies have demonstrated that circular RNAs (circRNAs) play vital roles in the regulation of plant stress resistance. Here, we performed a genome-wide association study (GWAS) on the survival rate of 300 maize accessions under a salt stress treatment. A total of 5 trait-associated SNPs and 86 candidate genes were obtained by the GWAS. We performed RNA sequencing for 28 transcriptome libraries derived from 2 maize lines with contrasting salt tolerance under normal and salt treatment conditions. A total of 1217 highly expressed circRNAs were identified, of which 371 were responsive to a salt treatment. Using PCR and Sanger sequencing, we verified the reliability of these differentially expressed circRNAs. An integration of the GWAS and RNA-Seq analyses uncovered two differentially expressed hub genes (Zm00001eb013650 and Zm00001eb198930), which were regulated by four circRNAs. Based on these results, we constructed a regulation model of circRNA/miRNA/mRNA that mediated salt stress tolerance in maize. By conducting hub gene-based association analyses, we detected a favorable haplotype in Zm00001eb198930, which was responsible for high salt tolerance. These results help to clarify the regulatory relationship between circRNAs and their target genes as well as to develop salt-tolerant lines for maize breeding.

Identification of a new mutant allele of ZmYSL2 that regulates kernel development and nutritional quality in maize.

The discovery and characterization of the opaque endosperm gene provide ideas and resources for the production and application of maize. We found an o213 mutant whose phenotype was opaque and shrunken endosperm with semi-dwarf plant height. The protein, lipid, and starch contents in the o213 endosperm were significantly decreased, while the free amino acid content in the o213 endosperm significantly increased. The aspartic acid, asparagine, and lysine contents were raised in the o213 endosperm by 6.5-, 8.5-, and 1.7-fold, respectively. Genetic analysis showed that this o213 mutant is a recessive single-gene mutation. The position mapping indicated that o213 is located in a 468-kb region that contains 11 protein-encoding genes on the long arm of chromosome 5. The coding sequence analysis of candidate genes between the WT and o213 showed that ZmYSL2 had only a single-base substitution (A-G) in the fifth exon, which caused methionine substitution to valine. Sequence analysis and the allelic test showed that o213 is a new mutant allele of ZmYSL2. The qRT-PCR results indicated that o213 is highly expressed in the stalks and anthers. Subcellular localization studies showed that o213 is a membrane transporter. In the variation analysis of o213, the amplification of 65 inbred lines in GWAS showed that this 3-bp deletion of the first exon of o213 was found only in temperate inbred lines, implying that the gene was artificially affected in the selection process. Our results suggest that o213 is an important endosperm development gene and may serve as a genetic resource. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01278-9.

Genetic variations in ZmSAUR15 contribute to the formation of immature embryo-derived embryonic calluses in maize.

The ability of immature maize (Zea mays) embryos to form embryonic calluses (ECs) is highly genotype dependent, which limits transgenic breeding development in maize. Here, we report the association map-based cloning of ZmSAUR15 using an association panel (AP) consisting of 309 inbred lines with diverse formation abilities for ECs. We demonstrated that ZmSAUR15, which encodes a small auxin-upregulated RNA, acts as a negative effector in maize EC induction. Polymorphisms in the ZmSAUR15 promoter that influence the expression of ZmSAUR15 transcripts modulate the EC induction capacity in maize. ZmSAUR15 is involved in indole-3-acetic acid biosynthesis and cell division in immature embryo-derived callus. The ability of immature embryos to induce EC formation can be improved by the knockout of ZmSAUR15, which consequently increases the callus regeneration efficiency. Our study provides new insights into overcoming the genotypic limitations associated with EC formation and improving genetic transformation in maize.