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 with 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.

The mycorrhiza-specific ammonium transporter ZmAMT3;1 mediates mycorrhiza-dependent nitrogen uptake in maize roots.

Most plant species can form symbioses with arbuscular mycorrhizal fungi (AMFs), which may enhance the host plant's acquisition of soil nutrients. In contrast to phosphorus nutrition, the molecular mechanism of mycorrhizal nitrogen (N) uptake remains largely unknown, and its physiological relevance is unclear. Here, we identified a gene encoding an AMF-inducible ammonium transporter, ZmAMT3;1, in maize (Zea mays) roots. ZmAMT3;1 was specifically expressed in arbuscule-containing cortical cells and the encoded protein was localized at the peri-arbuscular membrane. Functional analysis in yeast and Xenopus oocytes indicated that ZmAMT3;1 mediated high-affinity ammonium transport, with the substrate NH4+ being accessed, but likely translocating uncharged NH3. Phosphorylation of ZmAMT3;1 at the C-terminus suppressed transport activity. Using ZmAMT3;1-RNAi transgenic maize lines grown in compartmented pot experiments, we demonstrated that substantial quantities of N were transferred from AMF to plants, and 68%-74% of this capacity was conferred by ZmAMT3;1. Under field conditions, the ZmAMT3;1-dependent mycorrhizal N pathway contributed >30% of postsilking N uptake. Furthermore, AMFs downregulated ZmAMT1;1a and ZmAMT1;3 protein abundance and transport activities expressed in the root epidermis, suggesting a trade-off between mycorrhizal and direct root N-uptake pathways. Taken together, our results provide a comprehensive understanding of mycorrhiza-dependent N uptake in maize and present a promising approach to improve N-acquisition efficiency via plant-microbe interactions.

A genome-wide association study uncovers a ZmRap2.7-ZCN9/ZCN10 module to regulate ABA signalling and seed vigour in maize.

Seed vigour, including rapid, uniform germination and robust seedling establishment under various field conditions, is becoming an increasingly essential agronomic trait for achieving high yield in crops. However, little is known about this important seed quality trait. In this study, we performed a genome-wide association study to identify a key transcription factor ZmRap2.7, which regulates seed vigour through transcriptionally repressing expressions of three ABA signalling genes ZmPYL3, ZmPP2C and ZmABI5 and two phosphatidylethanolamine-binding genes ZCN9 and ZCN10. In addition, ZCN9 and ZCN10 proteins could interact with ZmPYL3, ZmPP2C and ZmABI5 proteins, and loss-of-function of ZmRap2.7 and overexpression of ZCN9 and ZCN10 reduced ABA sensitivity and seed vigour, suggesting a complex regulatory network for regulation of ABA signalling mediated seed vigour. Finally, we showed that four SNPs in ZmRap2.7 coding region influenced its transcriptionally binding activity to the downstream gene promoters. Together with previously identified functional variants within and surrounding ZmRap2.7, we concluded that the distinct allelic variations of ZmRap2.7 were obtained independently during maize domestication and improvement, and responded separately for the diversities of seed vigour, flowering time and brace root development. These results provide novel genes, a new regulatory network and an evolutional mechanism for understanding the molecular mechanism of seed vigour.

Genomic basis determining root system architecture in maize.

KEY MESSAGE: A total of 389 and 344 QTLs were identified by GWAS and QTL mapping explaining accumulatively 32.2-65.0% and 23.7-63.4% of phenotypic variation for 14 shoot-borne root traits using more than 1300 individuals across multiple field trails. Efficient nutrient and water acquisition from soils depends on the root system architecture (RSA). However, the genetic determinants underlying RSA in maize remain largely unexplored. In this study, we conducted a comprehensive genetic analysis for 14 shoot-borne root traits using 513 inbred lines and 800 individuals from four recombinant inbred line (RIL) populations at the mature stage across multiple field trails. Our analysis revealed substantial phenotypic variation for these 14 root traits, with a total of 389 and 344 QTLs identified through genome-wide association analysis (GWAS) and linkage analysis, respectively. These QTLs collectively explained 32.2-65.0% and 23.7-63.4% of the trait variation within each population. Several a priori candidate genes involved in auxin and cytokinin signaling pathways, such as IAA26, ARF2, LBD37 and CKX3, were found to co-localize with these loci. In addition, a total of 69 transcription factors (TFs) from 27 TF families (MYB, NAC, bZIP, bHLH and WRKY) were found for shoot-borne root traits. A total of 19 genes including PIN3, LBD15, IAA32, IAA38 and ARR12 and 19 GWAS signals were overlapped with selective sweeps. Further, significant additive effects were found for root traits, and pyramiding the favorable alleles could enhance maize root development. These findings could contribute to understand the genetic basis of root development and evolution, and provided an important genetic resource for the genetic improvement of root traits in maize.

Hybrid performance evaluation and genome-wide association analysis of root system architecture in a maize association population.

KEY MESSAGE: The genetic architecture of RSA traits was dissected by GWAS and coexpression networks analysis in a maize association population. Root system architecture (RSA) is a crucial determinant of water and nutrient uptake efficiency in crops. However, the maize genetic architecture of RSA is still poorly understood due to the challenges in quantifying root traits and the lack of dense molecular markers. Here, an association mapping panel including 356 inbred lines were crossed with a common tester, Zheng58, and the test crosses were phenotyped for 12 RSA traits in three locations. We observed a 1.3 ~ sixfold phenotypic variation for measured RSA in the association panel. The association panel consisted of four subpopulations, non-stiff stalk (NSS) lines, stiff stalk (SS), tropical/subtropical (TST), and mixed. Zheng58 × TST has a 2.1% higher crown root number (CRN) and 8.6% less brace root number (BRN) than Zheng58 × NSS and Zheng58 × SS, respectively. Using a genome-wide association study (GWAS) with 1.25 million SNPs and correction for population structure, 191 significant SNPs were identified for root traits. Ninety (47%) of the significant SNPs showed positive allelic effects, and 101 (53%) showed negative effects. Each locus could explain 0.39% to 11.8% of phenotypic variation. By integrating GWAS results and comparing coexpression networks, 26 high-priority candidate genes were identified. Gene GRMZM2G377215, which belongs to the COBRA-like gene family, affected root growth and development. Gene GRMZM2G468657 encodes the aspartic proteinase nepenthesin-1, related to root development and N-deficient response. Collectively, our research provides progress in the genetic dissection of root system architecture. These findings present the further possibility for the genetic improvement of root traits in maize.

Mining genes regulating root system architecture in maize based on data integration analysis.

KEY MESSAGE: A new strategy that integrated multiple public data resources was established to construct root gene co-expression network and mine genes regulating root system architecture in maize. A root gene co-expression network, containing 13,874 genes, was constructed. A total of 53 root hub genes and 16 priority root candidate genes were identified. One priority root candidate was further functionally verified using overexpression transgenic maize lines. Root system architecture (RSA) is crucial for crops productivity and stress tolerance. In maize, few RSA genes are functionally cloned, and effective discovery of RSA genes remains a great of challenge. In this work, we established a strategy to mine maize RSA genes by integrating functionally characterized root genes, root transcriptome, weighted gene co-expression network analysis (WGCNA) and genome-wide association analysis (GWAS) of RSA traits based on public data resources. A total of 589 maize root genes were collected by searching well-characterized root genes in maize or homologous genes of other species. We performed WGCNA to construct a maize root gene co-expression network containing 13874 genes based on public available root transcriptome data, and further discovered the 53 hub genes related to root traits. In addition, by the prediction function of obtained root gene co-expression network, a total of 1082 new root candidate genes were explored. By further overlapping the obtained new root candidate gene with the root-related GWAS of RSA candidate genes, 16 priority root candidate genes were identified. Finally, a priority root candidate gene, Zm00001d023379 (encodes pyruvate kinase 2), was validated to modulate root open angle and shoot-borne roots number using its overexpression transgenic lines. Our results develop an integration analysis method for effectively exploring regulatory genes of RSA in maize and open a new avenue to mine the candidate genes underlying complex traits.

Plasticity of root anatomy during domestication of a maize-teosinte derived population.

Maize (Zea mays L.) has undergone profound changes in root anatomy for environmental adaptation during domestication. However, the genetic mechanism of plasticity of maize root anatomy during the domestication process remains unclear. In this study, high-resolution mapping was performed for nine root anatomical traits using a maize-teosinte population (mexicana × Mo17) across three environments. Large genetic variations were detected for different root anatomical traits. The cortex, stele, aerenchyma areas, xylem vessel number, and cortical cell number had large variations across three environments, indicating high plasticity. Sixteen quantitative trait loci (QTL) were identified, including seven QTL with QTL × environment interaction (EIQTL) for high plasticity traits and nine QTL without QTL × environment interaction (SQTL). Most of the root loci were consistent with shoot QTL depicting domestication signals. Combining transcriptome and genome-wide association studies revealed that AUXIN EFFLUX CARRIER PIN-FORMED LIKE 4 (ZmPILS4) serves as a candidate gene underlying a major QTL of xylem traits. The near-isogenic lines (NILs) with lower expression of ZmPILS4 had 18-24% more auxin concentration in the root tips and 8-15% more xylem vessels. Nucleotide diversity values analysis in the promoter region suggested that ZmPILS4 was involved in maize domestication and adaptation. These results revealed the potential genetic basis of root anatomical plasticity during domestication.

Identification of Zinc Efficiency-Associated Loci (ZEALs) and Candidate Genes for Zn Deficiency Tolerance of Two Recombination Inbred Line Populations in Maize.

Zinc (Zn) deficiency is one of the most common micronutrient disorders in cereal plants, greatly impairing crop productivity and nutritional quality. Identifying the genes associated with Zn deficiency tolerance is the basis for understanding the genetic mechanism conferring tolerance. In this study, the K22×BY815 and DAN340×K22 recombination inbred line (RIL) populations, which were derived from Zn-inefficient and Zn-efficient inbred lines, were utilized to detect the quantitative trait loci (QTLs) associated with Zn deficiency tolerance and to further identify candidate genes within these loci. The BLUP (Best Linear Unbiased Prediction) values under Zn-deficient condition (-Zn) and the ratios of the BLUP values under Zn deficient condition to the BLUP values under Zn-sufficient condition (-Zn/CK) were used to perform linkage mapping. In QTL analysis, 21 QTLs and 33 QTLs controlling the Zn score, plant height, shoot and root dry weight, and root-to-shoot ratio were detected in the K22×BY815 population and the DAN340×K22 population, explaining 5.5-16.6% and 4.2-23.3% of phenotypic variation, respectively. In addition, seventeen candidate genes associated with the mechanisms underlying Zn deficiency tolerance were identified in QTL colocalizations or the single loci, including the genes involved in the uptake, transport, and redistribution of Zn (ZmIRT1, ZmHMAs, ZmNRAMP6, ZmVIT, ZmNAS3, ZmDMAS1, ZmTOM3), and the genes participating in the auxin and ethylene signal pathways (ZmAFBs, ZmIAA17, ZmETR, ZmEIN2, ZmEIN3, ZmCTR3, ZmEBF1). Our findings will broaden the understanding of the genetic structure of the tolerance to Zn deficiency in maize.

Genetic Dissection of Phosphorus Use Efficiency in a Maize Association Population under Two P Levels in the Field.

Phosphorus (P) deficiency is an important challenge the world faces while having to increase crop yields. It is therefore necessary to select maize (Zea may L.) genotypes with high phosphorus use efficiency (PUE). Here, we extensively analyzed the biomass, grain yield, and PUE-related traits of 359 maize inbred lines grown under both low-P and normal-P conditions. A significant decrease in grain yield per plant and biomass, an increase in PUE under low-P condition, as well as significant correlations between the two treatments were observed. In a genome-wide association study, 49, 53, and 48 candidate genes were identified for eleven traits under low-P, normal-P conditions, and in low-P tolerance index (phenotype under low-P divided by phenotype under normal-P condition) datasets, respectively. Several gene ontology pathways were enriched for the genes identified under low-P condition. In addition, seven key genes related to phosphate transporter or stress response were molecularly characterized. Further analyses uncovered the favorable haplotype for several core genes, which is less prevalent in modern lines but often enriched in a specific subpopulation. Collectively, our research provides progress in the genetic dissection and molecular characterization of PUE in maize.

The physiological mechanism underlying root elongation in response to nitrogen deficiency in crop plants.

MAIN CONCLUSION: In response to low nitrogen stress, multiple hormones together with nitric oxide signaling pathways work synergistically and antagonistically in crop root elongation. Changing root morphology allows plants to adapt to soil nutrient availability. Nitrogen is the most important essential nutrient for plant growth. An important adaptive strategy for crops responding to nitrogen deficiency is root elongation, thereby accessing increased soil space and nitrogen resources. Multiple signaling pathways are involved in this regulatory network, working together to fine-tune root elongation in response to soil nitrogen availability. Based on existing research, we propose a model to explain how different signaling pathways interact to regulate root elongation in response to low nitrogen stress. In response to a low shoot nitrogen status signal, auxin transport from the shoot to the root increases. High auxin levels in the root tip stimulate the production of nitric oxide, which promotes the synthesis of strigolactones to accelerate cell division. In this process, cytokinin, ethylene, and abscisic acid play an antagonistic role, while brassinosteroids and auxin play a synergistic role in regulating root elongation. Further study is required to identify the QTLs, genes, and favorable alleles which control the root elongation response to low nitrogen stress in crops.

Glutamine application promotes nitrogen and biomass accumulation in the shoot of seedlings of the maize hybrid ZD958.

MAIN CONCLUSION: Glutamine (Gln) is an efficient nitrogen source in promoting aboveground nitrogen and biomass accumulation in ZD958 (an elite maize hybrid with great potential for further genetic improvement) seedlings when conditioning a smaller but adequate root system. Amino acids account for a significant part of nitrogen (N) resources in the soil. However, how amino acid-N affects crop growth remains to be further investigated. Here, glutamine (Gln) application (80% NH4NO3 + 20% Gln; mixed N) enhanced shoot growth of the maize hybrid ZD958. N concentration in the shoot increased, which is associated with favorable increases in SPAD values, GS/GOGAT activities, and accumulation of glutamate, asparagine, total free amino acids and soluble proteins in the shoot under mixed N. On the other hand, root growth was reduced when exposed to Gln as indicated by the significantly lower dry weight, root/shoot ratio, and primary, seminal, crown, and total root lengths, as well as unfavorable physiological alterations. Up-regulation of expression of ZmAMT1.3, ZmNRT2.1, and ZmAAP2 in the root and that of ZmAMT1.1, ZmAMT1.3, and ZmLHT1 in the shoot preconditioned N over-accumulation in the shoot and facilitated shoot growth, presumably via enhancing N translocation to the shoot, when Gln was supplied. Together, Gln is an efficient N source in promoting aboveground N and biomass accumulation in ZD958 seedlings when conditioning a smaller but adequate root system. Notably, ZD958's parental lines Z58 and Chang7-2 displayed a wide range of variations in Gln responses, which may be partially attributed to single nucleotide polymorphisms (SNPs) in cis-elements and coding regions revealed in this study and much larger quantities of unidentified genetic variations between Z58 and Chang7-2. Extensive genetic divergence of these two elite inbred lines implied large potentials for further genetic improvement of ZD958 in relation to organic N use efficiency.

Combined physiological, transcriptome, and genetic analysis reveals a molecular network of nitrogen remobilization in maize.

In plants, nitrogen remobilization from source to sink organs is an important process regulated by complex transcriptional regulatory networks. However, the relationship between nitrogen remobilization and leaf senescence and the molecular regulatory network that controls them are unknown in maize. Here, using 15N labeling and a transcriptome approach, a dynamic analysis of the nitrogen remobilization process was conducted in two elite maize inbred lines (PH4CV and PH6WC) with contrasting leaf senescence. PH4CV showed higher nitrogen remobilization efficiency (NRE) than PH6WC, mainly in the middle and lower leaves from 15 d to 35 d after silking. The co-expression network analysis revealed that ethylene and cytokinin metabolism-related genes triggered the onset of nitrogen remobilization, while abscisic acid and jasmonic acid biosynthesis-related genes controlled the progression of nitrogen remobilization. By integrating genetic analysis, functional annotation, and gene expression, two candidate genes underlying a major quantitative trait locus of NRE were identified, namely an early senescence acting gene (ZmASR6) and an ATP-dependent Clp protease gene (GRMZM2G172230). Hormone-coupled transcription factors and downstream target genes reveal a gene regulatory network for the nitrogen remobilization process after silking in maize. These results uncovered a sophisticated regulatory mechanism for nitrogen remobilization, and further provided characterization of valuable genes for genetic improvement of nitrogen use efficiency in maize.

Involvement of a truncated MADS-box transcription factor ZmTMM1 in root nitrate foraging.

Plants can develop root systems with distinct anatomical features and morphological plasticity to forage nutrients distributed heterogeneously in soils. Lateral root proliferation is a typical nutrient-foraging response to a local supply of nitrate, which has been investigated across many plant species. However, the underlying mechanism in maize roots remains largely unknown. Here, we report on identification of a maize truncated MIKC-type MADS-box transcription factor (ZmTMM1) lacking K- and C-domains, expressed preferentially in the lateral root branching zone and induced by the localized supply of nitrate. ZmTMM1 belongs to the AGL17-like MADS-box transcription factor family that contains orthologs of ANR1, a key regulator for root nitrate foraging in Arabidopsis. Ectopic overexpression of ZmTMM1 recovers the defective growth of lateral roots in the Arabidopsis anr1 agl21 double mutant. The local activation of glucocorticoid receptor fusion proteins for ZmTMM1 and an artificially truncated form of AtANR1 without the K- and C-domains stimulates the lateral root growth of the Arabidopsis anr1 agl21 mutant, providing evidence that ZmTMM1 encodes a functional MADS-box that modulates lateral root development. However, no phenotype was observed in ZmTMM1-RNAi transgenic maize lines, suggesting a possible genetic redundancy of ZmTMM1 with other AGL17-like genes in maize. A comparative genome analysis further suggests that a nitrate-inducible transcriptional regulation is probably conserved in both truncated and non-truncated forms of ZmTMM1-like MADS-box transcription factors found in grass species.

Recombination Pattern Characterization via Simulation Using Different Maize Populations.

Efficient recombination is critical to both plant breeding and gene cloning. However, almost all traditional recombination studies and genetic improvements require the slow and labor-intensive population construction process, and little is known about the recombination characteristics of populations of different types, generations, and origins. Here, we provide a simple and efficient simulation method for population construction based on doubled haploid (DH) and intermated B73 × Mo17 maize (IBM) populations to predict the recombination pattern. We found that the chromosomes had 0, 1, 2, and 3 recombination events that occurred at rates of 0.16, 0.30, 0.23, and 0.15, respectively, in the DH and the recombination rate of each chromosome in the IBM population ranged from 0 to 12.1 cM per 125 kb. Based on the observed recombination parameters, we estimated the number of recombination events and constructed the linkage maps of the simulated DH and recombination inbred line (RIL) populations. These simulated populations exhibited similar recombination patterns compared with the real populations, suggesting the feasibility of this simulation approach. We then compared the recombination rates of the simulated populations of different types (DH induced or self-crossed), generations, and origins (using the 8, 16, and 32 multiparent advanced generation intercross (MAGIC) populations), and suggested a rapid and cost-effective population construction procedure for breeders and geneticists, while maintaining an optimal recombination rate. This study offers a convenient method for optimizing the population construction process and has broader implications for other crop species, thereby facilitating future population studies and genetic improvement strategies.

Increased biomass accumulation in maize grown in mixed nitrogen supply is mediated by auxin synthesis.

The use of mixed nitrate and ammonium as a nitrogen source can improve plant growth. Here, we used metabolomics and transcriptomics to study the underlying mechanisms. Maize plants were grown hydroponically in the presence of three forms of nitrogen (nitrate alone, 75%/25% nitrate/ammonium, and ammonium alone). Plants grown with mixed nitrogen had a higher photosynthetic rate than those supplied only with nitrate, and had the highest leaf area and shoot and root biomass among the three nitrogen treatments. In shoot and root, the concentration of nitrogenous compounds (ammonium, glutamine, and asparagine) and carbohydrates (sucrose, glucose, and fructose) in plants with a mixed nitrogen supply was higher than that with nitrate supply, but lower than that with ammonium supply. The activity of the related enzymes (glutamate synthase, asparagine synthase, phosphoenolpyruvate carboxylase, invertase, and ADP-glucose pyrophosphorylase) changed accordingly. Specifically, the mixed nitrogen source enhanced auxin synthesis via the shikimic acid pathway, as indicated by the higher levels of phosphoenolpyruvate and tryptophan compared with the other two treatments. The expression of corresponding genes involving auxin synthesis and response was up-regulated. Supply of only ammonium resulted in high levels of glutamine and asparagine, starch, and trehalose hexaphosphate. We conclude that, in addition to increased photosynthesis, mixed nitrogen supply enhances leaf growth via increasing auxin synthesis to build a large sink for carbon and nitrogen utilization, which, in turn, facilitates further carbon assimilation and nitrogen uptake.

Physiological and genetic analysis for maize root characters and yield in response to low phosphorus stress.

Root system architecture (RSA) plays an important role in the acquisition of mineral nutrients. Nevertheless, RSA has seldom been selected as an important agricultural trait in conventional breeding programs. Little is known about the response of RSA and phosphorus use efficiency (PUE) in regards to phosphorus (P) availability between parental inbred lines and their F1. In this study, 6 maize inbred lines and their 15 F1 generated by a diallel mating system, were used to analyze the genetic associations between RSA and PUE. Heterosis for PUE-related traits were comparatively greater under high P condition and reduced significantly under low P condition. Relative mid-parent heterosis for root traits were expressed more under the low P condition. Low P supply had a significant effect on heterosis, GCA and SCA of RSA- and PUE- related traits. The hybrid C3 (7922 × 8703-2), which had the highest PUE, showed an average yield with a lower P uptake under the both P conditions. Results from this study suggested breeding for a relatively high grain yield with reducing aboveground P demand and grain P concentration should be sufficient to reduce P fertilizer input and improve P efficiency.

A RNA-Seq Analysis of the Response of Photosynthetic System to Low Nitrogen Supply in Maize Leaf.

Nitrogen is a major limiting factor for crop productivity. The relationship between photosynthesis and nitrogen nutrition has been widely studied. However, the molecular response of leaf photosynthesis to low nitrogen supply in crops is less clear. In this study, RNA sequencing technology (RNA-Seq) was used to investigate the gene expressions related to photosynthesis in maize in response to low nitrogen supply. It was found that low nitrogen supply down-regulated the expression of genes involved in photosystem I (PSI) and photosystem II (PSII). Thus, low nitrogen supply down-regulated the expression of genes related to the antenna system, reduced light absorption, light transport, and electron transport. Correspondingly, the parameters related to chlorophyll fluorescence were very sensitive to nitrogen deficiency. Under low nitrogen supply, leaf chlorophyll content, actual quantum yield of PSII photochemistry, photochemical quenching, and electron transport rate, were reduced. However, the thermal diffusion and chlorophyll fluorescence were increased. RNA-Seq was used to analyze the genes involved in the response of leaf photosynthesis to low nitrogen supply in maize. These results highlight the possibility of utilizing chlorophyll fluorescence parameters, and the related genes, as indicators for plant nitrogen nutrition. This could lead to the development of new tools to make precise nitrogen fertilizer recommendations and select nitrogen-efficient genotypes.

Use of genotype-environment interactions to elucidate the pattern of maize root plasticity to nitrogen deficiency.

Maize (Zea mays L.) root morphology exhibits a high degree of phenotypic plasticity to nitrogen (N) deficiency, but the underlying genetic architecture remains to be investigated. Using an advanced BC4 F3 population, we investigated the root growth plasticity under two contrasted N levels and identified the quantitative trait loci (QTLs) with QTL-environment (Q × E) interaction effects. Principal components analysis (PCA) on changes of root traits to N deficiency (ΔLN-HN) showed that root length and biomass contributed for 45.8% in the same magnitude and direction on the first PC, while root traits scattered highly on PC2 and PC3. Hierarchical cluster analysis on traits for ΔLN-HN further assigned the BC4 F3 lines into six groups, in which the special phenotypic responses to N deficiency was presented. These results revealed the complicated root plasticity of maize in response to N deficiency that can be caused by genotype-environment (G × E) interactions. Furthermore, QTL mapping using a multi-environment analysis identified 35 QTLs for root traits. Nine of these QTLs exhibited significant Q × E interaction effects. Taken together, our findings contribute to understanding the phenotypic and genotypic pattern of root plasticity to N deficiency, which will be useful for developing maize tolerance cultivars to N deficiency.

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.

A comprehensive analysis of root morphological changes and nitrogen allocation in maize in response to low nitrogen stress.

The plasticity of root architecture is crucial for plants to acclimate to unfavourable environments including low nitrogen (LN) stress. How maize roots coordinate the growth of axile roots and lateral roots (LRs), as well as longitudinal and radial cell behaviours in response to LN stress, remains unclear. Maize plants were cultivated hydroponically under control (4 mm nitrate) and LN (40 µm) conditions. Temporal and spatial samples were taken to analyse changes in the morphology, anatomical structure and carbon/nitrogen (C/N) ratio in the axile root and LRs. LN stress increased axile root elongation, reduced the number of crown roots and decreased LR density and length. LN stress extended cell elongation zones and increased the mature cell length in the roots. LN stress reduced the cell diameter and total area of vessels and increased the amount of aerenchyma, but the number of cell layers in the crown root cortex was unchanged. The C/N ratio was higher in the axile roots than in the LRs. Maize roots acclimate to LN stress by optimizing the anatomical structure and N allocation. As a result, axile root elongation is favoured to efficiently find available N in the soil.