NIN-LIKE PROTEIN3.2 inhibits repressor Aux/IAA14 expression and enhances root biomass in maize seedlings under low nitrogen.

Plants generally enhance their root growth in the form of greater biomass and/or root length to boost nutrient uptake in response to short-term low nitrogen (LN). However, the underlying mechanisms of short-term LN-mediated root growth remain largely elusive. Our genome-wide association study, haplotype analysis, and phenotyping of transgenic plants showed that the crucial nitrate signaling component NIN-LIKE PROTEIN3.2 (ZmNLP3.2), a positive regulator of root biomass, is associated with natural variations in root biomass of maize (Zea mays L.) seedlings under LN. The monocot-specific gene AUXIN/INDOLE-3-ACETIC ACID14 (ZmAux/IAA14) exhibited opposite expression patterns to ZmNLP3.2 in ZmNLP3.2 knockout and overexpression lines, suggesting that ZmNLP3.2 hampers ZmAux/IAA14 transcription. Importantly, ZmAux/IAA14 knockout seedlings showed a greater root dry weight (RDW), whereas ZmAux/IAA14 overexpression reduced RDW under LN compared with wild-type plants, indicating that ZmAux/IAA14 negatively regulates the RDW of LN-grown seedlings. Moreover, in vitro and vivo assays indicated that AUXIN RESPONSE FACTOR19 (ZmARF19) binds to and transcriptionally activates ZmAux/IAA14, which was weakened by the ZmNLP3.2-ZmARF19 interaction. The zmnlp3.2 ZmAux/IAA14-OE seedlings exhibited further reduced RDW compared to ZmAux/IAA14 overexpression lines when subjected to LN treatment, corroborating the ZmNLP3.2-ZmAux/IAA14 interaction. Thus, our study reveals a ZmNLP3.2-ZmARF19-ZmAux/IAA14 module regulating root biomass in response to nitrogen limitation in maize.

Identification of the coexisting virus-derived siRNA in maize and rice infected by rice black-streaked dwarf virus.

Rice black-streaked dwarf virus is transmitted by small brown planthoppers, which causes maize rough dwarf disease and rice black-streaked dwarf disease. This virus leads to slow growth or death of the host plants. During the co-evolutionary arms race between viruses and plants, virus-derived small interfering RNAs challenge the plant's defense response and inhibit host immunity through the RNA silencing system. However, it is currently unknown if rice black-streaked dwarf virus can produce the same small interfering RNAs to mediate the RNA silencing in different infected species. In this study, four small RNA libraries and four degradome libraries were constructed by extracting total RNAs from the leaves of the maize (Zea mays) inbred line B73 and japonica rice (Oryza sativa) variety Nipponbare exposed to feeding by viruliferous and non-viruliferous small brown planthoppers. We analyzed the characteristics of small RNAs and explored virus-derived small interfering RNAs in small RNA libraries through high-throughput sequencing. On analyzing the characteristics of small RNA, we noted that the size distributions of small RNAs were mainly 24-nt (19.74%-62.00%), whereas those of virus-derived small interfering RNAs were mostly 21-nt (41.06%-41.87%) and 22-nt (39.72%-42.26%). The 5'-terminal nucleotides of virus-derived small interfering RNAs tended to be adenine or uracil. Exploring the distribution of virus-derived small interfering RNAs hot spots on the viral genome segments revealed that the frequency of hot spots in B73 was higher than those in Nipponbare. Meanwhile, hotspots in the S9 and S10 virus genome segments were distributed similarly in both hosts. In addition, the target genes of small RNA were explored by degradome sequencing. Analyses of the regulatory pathway of these target genes unveiled that viral infection affected the ribosome-related target genes in maize and target genes in metabolism and biosynthesis pathways in rice. Here, 562 and 703 virus-derived small interfering RNAs were separately obtained in maize and rice, and 73 virus-derived small interfering RNAs named as co-vsiRNAs were detected in both hosts. Stem-loop PCR and RT-qPCR confirmed that co-vsiRNA 3.1 and co-vsiRNA 3.5 derived from genome segment S3 simultaneously play a role in maize and rice and inhibited host gene expression. The study revealed that rice black-streaked dwarf virus can produce the same small interfering RNAs in different species and provides a new direction for developing the new antiviral strategies.

ZmADF5, a Maize Actin-Depolymerizing Factor Conferring Enhanced Drought Tolerance in Maize.

Drought stress is seriously affecting the growth and production of crops, especially when agricultural irrigation still remains quantitatively restricted in some arid and semi-arid areas. The identification of drought-tolerant genes is important for improving the adaptability of maize under stress. Here, we found that a new member of the actin-depolymerizing factor (ADF) family; the ZmADF5 gene was tightly linked with a consensus drought-tolerant quantitative trait locus, and the significantly associated signals were detected through genome wide association analysis. ZmADF5 expression could be induced by osmotic stress and the application of exogenous abscisic acid. Its overexpression in Arabidopsis and maize helped plants to keep a higher survival rate after water-deficit stress, which reduced the stomatal aperture and the water-loss rate, as well as improved clearance of reactive oxygen species. Moreover, seventeen differentially expressed genes were identified as regulated by both drought stress and ZmADF5, four of which were involved in the ABA-dependent drought stress response. ZmADF5-overexpressing plants were also identified as sensitive to ABA during the seed germination and seedling stages. These results suggested that ZmADF5 played an important role in the response to drought stress.

Gene pyramiding of ZmGLK36 and ZmGDIα-hel for rough dwarf disease resistance in maize.

Maize rough dwarf disease (MRDD) caused by pathogenic viruses in the genus Fijivirus in the family Reoviridae is one of the most destructive diseases in maize. The pyramiding of effective resistance genes into maize varieties is a potential approach to reduce the damage resulting from the disease. Two major quantitative trait loci (QTLs) (qMrdd2 and qMrdd8) have been previously identified. The resistance genes ZmGLK36 and ZmGDIα-hel have also been cloned with the functional markers Indel-26 and IDP25K, respectively. In this study, ZmGLK36 and ZmGDIα-hel were introgressed to improve MRDD resistance of maize lines (Zheng58, Chang7-2, B73, Mo17, and their derived hybrids Zhengdan958 and B73 × Mo17) via marker-assisted selection (MAS). The converted lines and their derived hybrids, carrying one or two genes, were evaluated for MRDD resistance using artificial inoculation methods. The double-gene pyramiding lines and their derived hybrids exhibited increased resistance to MRDD compared to the monogenic lines and the respective hybrids. The genetic backgrounds of the converted lines were highly similar (90.85-98.58%) to the recurrent parents. In addition, agronomic trait evaluation demonstrated that pyramiding lines with one or two genes and their derived hybrids were not significantly different from the recurrent parents and their hybrids under nonpathogenic stress, including period traits (tasseling, pollen shedding, and silking), yield traits (ear length, grain weight per ear and 100-kernel weight) and quality traits (protein and starch content). There were differences in plant architecture traits between the improved lines and their hybrids. This study illustrated the successful development of gene pyramiding for improving MRDD resistance by advancing the breeding process. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01466-9.

A novel maize F-bZIP member, ZmbZIP76, functions as a positive regulator in ABA-mediated abiotic stress tolerance by binding to ACGT-containing elements.

The group F-bZIP transcription factors (TFs) in Arabidopsis are involved in nutrient deficiency or salt stress responses. Nevertheless, our learning about the functions of group F-bZIP genes in maize remains limited. Here, we cloned a new F-bZIP gene (ZmbZIP76) from maize inbred line He344. The expression of ZmbZIP76 in maize was dramatically induced by high salt, osmotic stress and abscisic acid. Accordingly, overexpression of ZmbZIP76 increased tolerance of transgenic plants to salt and osmotic stress. In addition, ZmbZIP76 functions as a nuclear transcription factor and upregulates the expression of a range of abiotic stress-responsive genes by binding to the ACGT-containing elements, leading to enhanced reactive oxygen species (ROS) scavenging capability, increased abscisic acid level, proline content, and ratio of K+/Na+, reduced water loss rate, and membrane damage. These physiological changes caused by ZmbZIP76 ultimately enhanced tolerance of transgenic plants to salt and osmotic stress.

A transcription factor ZmGLK36 confers broad resistance to maize rough dwarf disease in cereal crops.

Maize rough dwarf disease (MRDD), caused by maize rough dwarf virus (MRDV) or rice black-streaked dwarf virus (RBSDV), seriously threatens worldwide production of all major cereal crops, including maize, rice, wheat and barley. Here we report fine mapping and cloning of a previously reported major quantitative trait locus (QTL) (qMrdd2) for RBSDV resistance in maize. Subsequently, we show that qMrdd2 encodes a G2-like transcription factor named ZmGLK36 that promotes resistance to RBSDV by enhancing jasmonic acid (JA) biosynthesis and JA-mediated defence response. We identify a 26-bp indel located in the 5' UTR of ZmGLK36 that contributes to differential expression and resistance to RBSDV in maize inbred lines. Moreover, we show that ZmDBF2, an AP2/EREBP family transcription factor, directly binds to the 26-bp indel and represses ZmGLK36 expression. We further demonstrate that ZmGLK36 plays a conserved role in conferring resistance to RBSDV in rice and wheat using transgenic or marker-assisted breeding approaches. Our results provide insights into the molecular mechanisms of RBSDV resistance and effective strategies to breed RBSDV-resistant cereal crops.

Using the dominant mutation gene Ae1-5180 (amylose extender) to develop high-amylose maize.

Maize amylose is a type of high value-added starch used for medical, food, and chemical applications. Mutations in the starch branching enzyme (SBEIIb), with recessive ae (amylose extender) and dominant Ae1-5180 alleles, are the primary way to improve maize endosperm amylose content (AC). However, studies on Ae1-5180 mutation are scarce, and its roles in starch synthesis and breeding potential are unclear. We found that the AC of the Ae1-5180 mutant was 47.23%, and its kernels were tarnished and glassy and are easily distinguished from those of the wild type (WT), indicating that the dominant mutant has the classical characteristics of the ae mutant. Starch granules of Ae1-5180 became smaller, and higher in amount with irregular shape. The degree of amylopectin polymerisation changed to induce an increase in starch thermal stability. Compared with WT, the activity of granule-bound starch synthase and starch synthase was higher in early stages and lower in later stages, and other starch synthesis enzymes decreased during kernel development in the Ae1-5180 mutant. We successfully developed a marker (mu406) for the assisted selection of 17 Ae1-5180 near isogenic lines (NILs) according to the position of insertion of the Mu1 transposon in the SBEIIb promoter of Ae1-5180. JH214/Ae1-5180, CANS-1/Ae1-5180, CA240/Ae1-5180, and Z1698/Ae1-5180 have high breeding application potential with their higher AC (> 40%) and their 100-kernel weight decreased to < 25% compared to respective recurrent parents. Therefore, using the dominant Ae1-5180 mutant as a donor can detect the kernel phenotype and AC of Ae1-5180-NILs in advance, thereby accelerating the high-amylose breeding process. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01323-7.

Identification and Fine-Mapping of a Novel QTL, qMrdd2, That Confers Resistance to Maize Rough Dwarf Disease.

Maize rough dwarf disease (MRDD) is caused by a virus and seriously affects maize quality and yield worldwide. MRDD can be most effectively controlled with disease-resistant hybrids of corn. Here, MRDD-resistant (Qi319) and -susceptible (Ye478) parental inbred maize lines and their 314 recombinant inbred lines (RILs) that were derived from a cross between them were evaluated across three environments. A stable resistance QTL, qMrdd2, was identified and mapped using best linear unbiased prediction (BLUP) values to a 0.55-Mb region between the markers MK807 and MK811 on chromosome 2 (B73 RefGen_v3) and was found to explain 8.6 to 11.0% of the total phenotypic variance in MRDD resistance. We validated the effect of qMrdd2 using a chromosome segment substitution line (CSSL) that was derived from a cross between maize inbred Qi319 as the MRDD resistance donor and Ye478 as the recipient. Disease severity index of the CSSL haplotype II harboring qMrdd2 was significantly lower than that of the susceptible parent Ye478. Subsequently, we fine-mapped qMrdd2 to a 315-kb region flanked by the markers RD81 and RD87, thus testing recombinant-derived progeny using selfed backcrossed families. In this study, we identified a novel QTL for MRDD resistance by combining the RIL and CSSL populations, thus providing important genetic information that can be used for breeding MRDD-resistant varieties of maize.

ZmSNAC13, a maize NAC transcription factor conferring enhanced resistance to multiple abiotic stresses in transgenic Arabidopsis.

Abiotic stress is the main factor that severely limits crop growth and yield. NAC (NAM, ATAF1/2 and CUC2) transcription factors play an important role in dealing with various abiotic stresses. Here, we discovered the ZmSNAC13 gene in drought-tolerant maize lines by RNA-seq analysis and verified its function in Arabidopsis thaliana. First, its gene structure showed that ZmSNAC13 had a typical NAC domain and a highly variable C-terminal. There were multiple cis-acting elements related to stress in its promoter region. Overexpression of ZmSNAC13 resulted in enhanced tolerances to drought and salt stresses in Arabidopsis, characterized by a reduction in the water loss rate, a sustained effective photosynthesis rate, and increased cell membrane stability in leaves under drought conditions. Transcriptome analysis showed that a large number of differentially expressed genes regulated by overexpression of ZmSNAC13 were identified, and the main drought tolerance regulatory pathways involved were the ABA pathway and MAPK cascade signaling pathway. Overexpression of ZmSNAC13 promoted the expression of genes, such as PYL9 and DREB3, thereby enhancing tolerance to adverse environments. Adaptability, while restraining genes expression such as WRKY53 and MPK3, facilitates regulation of senescence in Arabidopsis and improves plant responses to adversity. Therefore, ZmSNAC13 is promising gene of interest for use in transgenic breeding to improve abiotic stress tolerance in crops.

Genetic basis of maize kernel protein content revealed by high-density bin mapping using recombinant inbred lines.

Maize with a high kernel protein content (PC) is desirable for human food and livestock fodder. However, improvements in its PC have been hampered by a lack of desirable molecular markers. To identify quantitative trait loci (QTL) and candidate genes for kernel PC, we employed a genotyping-by-sequencing strategy to construct a high-resolution linkage map with 6,433 bin markers for 275 recombinant inbred lines (RILs) derived from a high-PC female Ji846 and low-PC male Ye3189. The total genetic distance covered by the linkage map was 2180.93 cM, and the average distance between adjacent markers was 0.32 cM, with a physical distance of approximately 0.37 Mb. Using this linkage map, 11 QTLs affecting kernel PC were identified, including qPC7 and qPC2-2, which were identified in at least two environments. For the qPC2-2 locus, a marker named IndelPC2-2 was developed with closely linked polymorphisms in both parents, and when tested in 30 high and 30 low PC inbred lines, it showed significant differences (P = 1.9E-03). To identify the candidate genes for this locus, transcriptome sequencing data and PC best linear unbiased estimates (BLUE) for 348 inbred lines were combined, and the expression levels of the four genes were correlated with PC. Among the four genes, Zm00001d002625, which encodes an S-adenosyl-L-methionine-dependent methyltransferase superfamily protein, showed significantly different expression levels between two RIL parents in the endosperm and is speculated to be a potential candidate gene for qPC2-2. This study will contribute to further research on the mechanisms underlying the regulation of maize PC, while also providing a genetic basis for marker-assisted selection in the future.

Transcriptome Reveals Allele Contribution to Heterosis in Maize.

Heterosis, which has greatly increased maize yields, is associated with gene expression patterns during key developmental stages that enhance hybrid phenotypes relative to parental phenotypes. Before heterosis can be more effectively used for crop improvement, hybrid maize developmental gene expression patterns must be better understood. Here, six maize hybrids, including the popular hybrid Zhengdan958 (ZC) from China, were studied. Maize hybrids created in-house were generated using an incomplete diallel cross (NCII)-based strategy from four elite inbred parental lines. Differential gene expression (DEG) profiles corresponding to three developmental stages revealed that hybrid partial expression patterns exhibited complementarity of expression of certain parental genes, with parental allelic expression patterns varying both qualitatively and quantitatively in hybrids. Single-parent expression (SPE) and parent-specific expression (PSE) types of qualitative variation were most prevalent, 43.73 and 41.07% of variation, respectively. Meanwhile, negative super-dominance (NSD) and positive super-dominance (PSD) types of quantitative variation were most prevalent, 31.06 and 24.30% of variation, respectively. During the early reproductive growth stage, the gene expression pattern differed markedly from other developmental stage patterns, with allelic expression patterns during seed development skewed toward low-value parental alleles in hybrid seeds exhibiting significant quantitative variation-associated superiority. Comparisons of qualitative gene expression variation rates between ZC and other hybrids revealed proportions of SPE-DEGs (41.36%) in ZC seed DEGs that significantly exceeded the average proportion of SPE-DEGs found in seeds of other hybrids (28.36%). Importantly, quantitative gene expression variation rate comparisons between ZC and hybrids, except for transgressive expression, revealed that the ZC rate exceeded the average rate for other hybrids, highlighting the importance of partial gene expression in heterosis. Moreover, enriched ZC DEGs exhibiting distinct tissue-specific expression patterns belonged to four biological pathways, including photosynthesis, plant hormone signal transduction, biology metabolism and biosynthesis. These results provide valuable technical insights for creating hybrids exhibiting strong heterosis.

Genetic Dissection of the General Combining Ability of Yield-Related Traits in Maize.

Maize yield components including row number, kernel number per row, kernel thickness, kernel width, kernel length, 100-kernel weight, and volume weight affect grain yield directly. Previous studies mainly focused on dissecting the genetic basis of per se performances for yield-related traits, but the genetic basis of general combining ability (GCA) for these traits is still unclear. In the present study, 328 RILs were crossed as males to two testers according to the NCII mating design, resulting in a hybrid panel composed of 656 hybrids. Both the hybrids and parental lines were evaluated in four environments in 2015 and 2016. Correlation analysis showed the performances of GCA effects were significantly correlated to the per se performances of RILs for all yield-related traits (0.17 ≤ r ≤ 0.64, P > 0.01). Only 17 of 95 QTL could be detected for both per se performances of RILs and GCA effects for eight yield-related traits. The QTL qKN7-1 and qHKW1-3, which could explain more than 10% of the variation in the GCA effects of KN and HKW, were also detected for per se performances for the traits. The pleiotropic loci qRN3-1 and qRN6, which together explained 14.92% of the observed variation in GCA effects for RN, were associated with the GCA effects of KW and HKW, but not with per se performances for these traits. In contrast, Incw1, which was related to seed weight in maize, was mapped to the region surrounding MK2567 at the qHKW5-2 locus, but no GCA effect was detected. The QTL identified in present study for per se performances and corresponding GCA effects for yield-related traits might be useful for maize hybrid breeding.

Integrated transcriptome, small RNA, and degradome analysis reveals the complex network regulating starch biosynthesis in maize.

BACKGROUND: Starch biosynthesis in endosperm is a key process influencing grain yield and quality in maize. Although a number of starch biosynthetic genes have been well characterized, the mechanisms by which the expression of these genes is regulated, especially in regard to microRNAs (miRNAs), remain largely unclear. RESULTS: Sequence data for small RNAs, degradome, and transcriptome of maize endosperm at 15 and 25 d after pollination (DAP) from inbred lines Mo17 and Ji419, which exhibit distinct starch content and starch granule structure, revealed the mediation of starch biosynthetic pathways by miRNAs. Transcriptome analysis of these two lines indicated that 33 of 40 starch biosynthetic genes were differentially expressed, of which 12 were up-regulated in Ji419 at 15 DAP, one was up-regulated in Ji419 at 25 DAP, 14 were up-regulated in Ji419 at both 15 and 25 DAP, one was down-regulated in Ji419 at 15 DAP, two were down-regulated in Ji419 at 25 DAP, and three were up-regulated in Ji419 at 15 DAP and down-regulated in Ji419 at 25 DAP, compared with Mo17. Through combined analyses of small RNA and degradome sequences, 22 differentially expressed miRNAs were identified, including 14 known and eight previously unknown miRNAs that could target 35 genes. Furthermore, a complex co-expression regulatory network was constructed, in which 19 miRNAs could modulate starch biosynthesis in endosperm by tuning the expression of 19 target genes. Moreover, the potential operation of four miRNA-mediated pathways involving transcription factors, miR169a-NF-YA1-GBSSI/SSIIIa and miR169o-GATA9-SSIIIa/SBEIIb, was validated via analyses of expression pattern, transient transformation assays, and transactivation assays. CONCLUSION: Our results suggest that miRNAs play a critical role in starch biosynthesis in endosperm, and that miRNA-mediated networks could modulate starch biosynthesis in this tissue. These results have provided important insights into the molecular mechanism of starch biosynthesis in developing maize endosperm.

Foxtail Millet [Setaria italica (L.) Beauv.] Grown under Low Nitrogen Shows a Smaller Root System, Enhanced Biomass Accumulation, and Nitrate Transporter Expression.

Foxtail millet (FM) [Setaria italica (L.) Beauv.] is a grain and forage crop well adapted to nutrient-poor soils. To date little is known how FM adapts to low nitrogen (LN) at the morphological, physiological, and molecular levels. Using the FM variety Yugu1, we found that LN led to lower chlorophyll contents and N concentrations, and higher root/shoot and C/N ratios and N utilization efficiencies under hydroponic culture. Importantly, enhanced biomass accumulation in the root under LN was in contrast to a smaller root system, as indicated by significant decreases in total root length; crown root number and length; and lateral root number, length, and density. Enhanced carbon allocation toward the root was rather for significant increases in average diameter of the LN root, potentially favorable for wider xylem vessels or other anatomical alterations facilitating nutrient transport. Lower levels of IAA and CKs were consistent with a smaller root system and higher levels of GA may promote root thickening under LN. Further, up-regulation of SiNRT1.1, SiNRT2.1, and SiNAR2.1 expression and nitrate influx in the root and that of SiNRT1.11 and SiNRT1.12 expression in the shoot probably favored nitrate uptake and remobilization as a whole. Lastly, more soluble proteins accumulated in the N-deficient root likely as a result of increases of N utilization efficiencies. Such "excessive" protein-N was possibly available for shoot delivery. Thus, FM may preferentially transport carbon toward the root facilitating root thickening/nutrient transport and allocate N toward the shoot maximizing photosynthesis/carbon fixation as a primary adaptive strategy to N limitation.

Down-regulation of nitrogen/carbon metabolism coupled with coordinative hormone modulation contributes to developmental inhibition of the maize ear under nitrogen limitation.

MAIN CONCLUSION: Developmental inhibition of the maize ear by nitrogen limitation is due to overall down-regulation of nitrogen/carbon metabolism, coordinative hormonal modulation, and probable early senescence. The kernel number is primarily determined from 2 weeks pre-silking to 3 weeks post-silking, largely depending on dynamic nitrogen (N) and carbohydrate metabolism and accumulation in the maize ear. Underlying physiological and molecular mechanisms of kernel abortion caused by N limitation needs to be further investigated. Using a widely grown maize hybrid ZD958, we found that the N deficient ear was shorter, with less biomass accumulation, lower N concentrations, and overall lower concentrations of N assimilates and soluble sugars at 1- or 2-week after silking. Such negative alterations were probably due to significant decreases in activities of nitrate reductase, glutamine synthetase, sucrose phosphate synthetase, and sucrose synthetase in the N deficient maize ear especially after silking. Compensatory up-regulation of corresponding gene expression, together with co-downregulation of gene expression and enzyme activities in certain circumstances, suggested regulatory complexity and mechanistic differentiation from gene expression to functioning at physiological and molecular levels in quickly developing maize ear in counteracting N deficiency. Importantly, auxin, gibberellin, cytokinin, and abscisic acid may act in a coordinative manner to negatively modulate ear development under N limitation, as indicated by their concentration variations and substantial up-regulation of IAA14, GA2-ox1, and CKX12. Lastly, early senescence may occur in the low-N ear driven by interplay of hormone functioning and senescence-related gene regulation.

ZD958 is a low-nitrogen-efficient maize hybrid at the seedling stage among five maize and two teosinte lines.

MAIN CONCLUSION: ZD958 was the most low-N-efficient line among five maize and two teosinte lines. Zea parviglumis and Zea diploperennis were insensitive to N limitation. Maize and teosinte genetically and evolutionarily diverged in gene regulation. GDH2, ASN2, and T4 were consistently down-regulated across seven lines. Maternal asymmetric inheritance and heterosis vigor made ZD958 low-N-efficient. Nitrogen (N) deficiency remains a serious limiting factor for maize production in many developing countries. It is particularly important to better understand how hybrid maize responds to N limitation. ZD958, a dominant high-yield hybrid in North China, was comparatively analyzed with four other maize and two teosinte lines at physiological and transcriptional levels. ZD958 was the most low-N-efficient line among five maize and two teosinte lines due to its largest biomass accumulation at a lowest N concentration under N limitation; while Zea parviglumis and Zea diploperennis had large root systems and were insensitive to N limitation. In anti-parallel with down-regulation of N metabolic genes in the ZD958 root, carbon allocation towards the root was enhanced for the significant increase in the root length. Variations in expression patterns of ten genes mediating N uptake, transport, and metabolism indicated large genetic and evolutionary divergence among seven lines under N limitation. Notably, GDH2, ASN2, and VAAT5 were consistently down-regulated under N limitation across these maize and teosinte lines, suggesting essential evolutionary conservation of gene regulation in response to N limitation and providing molecular markers for N nutritional diagnosis. Asymmetric inheritance, mostly from its maternal donor Z58, and heterosis vigor made ZD958 low-N-efficient at the seedling stage. The superior traits in crown roots in ZD958 may be derived from its paternal donor Chang7-2. Thus, Z58, Chang7-2, and two wild maize lines (Z. parviglumis and Z. diploperennis) provide valuable germplasms for N-efficient and large-root maize breeding.