MicroRNA408 negatively regulates salt tolerance by affecting secondary cell wall development in maize.

Although microRNA408 (miR408) is a highly conserved miRNA, the miR408 response to salt stress differs among plant species. Here, we show that miR408 transcripts are strongly repressed by salt stress and methyl viologen treatment in maize (Zea mays). Application of N, N1-dimethylthiourea partly relieved the NaCl-induced down-regulation of miR408. Transgenic maize overexpressing MIR408b is hypersensitive to salt stress. Overexpression of MIR408b enhanced the rate of net Na+ efflux, caused Na+ to locate in the inter-cellular space, reduced lignin accumulation, and reduced the number of cells in vascular bundles under salt stress. We further demonstrated that miR408 targets ZmLACCASE9 (ZmLAC9). Knockout of MIR408a or MIR408b or overexpression of ZmLAC9 increased the accumulation of lignin, thickened the walls of pavement cells, and improved salt tolerance of maize. Transcriptome profiles of the wild-type and MIR408b-overexpressing transgenic maize with or without salt stress indicated that miR408 negatively regulates the expression of cell wall biogenesis genes under salt conditions. These results indicate that miR408 negatively regulates salt tolerance by regulating secondary cell wall development in maize.

The auxin signaling pathway contributes to phosphorus-mediated zinc homeostasis in maize.

Although the interaction between P and Zn has long been recognized in plants, the physiological and molecular mechanisms underlying P and Zn interactions are poorly understood. We show here that P supply decreases the Zn concentration in maize shoots and roots. Compared to +P + Zn (addition of both P and Zn), +P-Zn reduced and -P-Zn increased the total length of 1° lateral roots (LRs). Under +P + Zn, both P and Zn concentrations were lower in the sl1 mutant roots than in wild-type (WT) maize roots, and P accumulation did not reduce the Zn concentration in ll1 mutant roots. Transcriptome profiling showed that the auxin signaling pathway contributed to P-mediated Zn homeostasis in maize. Auxin production and distribution were altered by changes in P and Zn supply. Cytosolic Zn co-localized with auxin accumulation under +P + Zn. Exogenous application of 1-NAA and L-Kyn altered the P-mediated root system architecture (RSA) under Zn deficiency. -P-Zn repressed the expression of miR167. Overexpression of ZmMIR167b increased the lengths of 1° LRs and the concentrations of P and Zn in maize. These results indicate that auxin-dependent RSA is important for P-mediated Zn homeostasis in maize.HighlightAuxin-dependent RSA is important for P-mediated Zn homeostasis in maize.

MIR164b represses iron uptake by regulating the NAC domain transcription factor5-Nuclear Factor Y, Subunit A8 module in Arabidopsis.

Recent findings have revealed the important roles of microRNAs (miRNAs) in the secondary responses to oxidative damage caused by iron (Fe) excess. However, the functional importance of miRNAs in plant responses to Fe deficiency remains to be explored. Here, we show that the expression level of miR164 in Arabidopsis (Arabidopsis thaliana) roots was repressed by Fe deficiency. Primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2) were higher in the mir164b mutant than in the wild-type (WT) under Fe-deficient conditions. Analysis of the Fe concentrations and ferric reductase activities in the roots of miR164 knockdown transgenic plants showed that members of the miR164 family had different functions in Fe-deficiency responses. Promoter::GUS analysis showed that NAM/ATAF/CUC (NAC) domain transcription factor5 (NAC5) is regulated at both transcriptional and posttranscriptional levels under Fe-deficient conditions. Transgenic Arabidopsis plants overexpressing NAC5 were more tolerant of Fe deficiency than the WT. NAC5 has transactivation activity and directly transactivates the expression of Nuclear Factor Y, Subunit A8 (NFYA8), as demonstrated by chromatin immunoprecipitation followed by quantitative polymerase chain reaction, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay. Like overexpression of NAC5, overexpression of NFYA8 increases primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRT1 and FRO2 under Fe-deficient conditions. Thus, MIR164b is important for Fe-deficiency responses by its regulation of the NAC5-NFYA8 module.

Maize Dek407 Encodes the Nitrate Transporter 1.5 and Is Required for Kernel Development.

The kernel serves as the storage organ and harvestable component of maize, and it plays a crucial role in determining crop yield and quality. Understanding the molecular and genetic mechanisms of kernel development is of considerable importance for maize production. In this study, we obtained a mutant, which we designated defective kernel 407 (dek407), through ethyl methanesulfonate mutagenesis. The dek407 mutant exhibited reduced kernel size and kernel weight, as well as delayed grain filling compared with those of the wild type. Positional cloning and an allelism test revealed that Dek407 encodes a nitrate transporter 1/peptide transporter family (NPF) protein and is the allele of miniature 2 (mn2) that was responsible for a poorly filled defective kernel phenotype. A transcriptome analysis of the developing kernels showed that the mutation of Dek407 altered the expression of phytohormone-related genes, especially those genes associated with indole-3-acetic acid synthesis and signaling. Phytohormone measurements and analysis indicated that the endogenous indole-3-acetic acid content was significantly reduced by 66% in the dek407 kernels, which may be the primary cause of the defective phenotype. We further demonstrated that natural variation in Dek407 is associated with kernel weight and kernel size. Therefore, Dek407 is a potential target gene for improvement of maize yield.

Designing high efficiency asymmetric polarization converter for blue light: a deep reinforcement learning approach.

Conventional polarization converters selectively preserve the required polarization state by absorbing, reflecting or refracting light with unwanted polarization state, leading to a theoretical transmittance limit of 0.5 for linearly polarized light with unpolarized light incidence. In the meanwhile, due to the high-dimensional structure parameters and time-consuming numerical simulations, designing a converter with satisfactory performance is extremely difficult and closely relies on human experts' experiences and manual intervention. To address these open issues, in this paper, we first propose an asymmetric polarization converter which shows both high transmittance for one linearly polarized light and high transmittance for the orthogonal linearly polarized light with 90° rotation in blue wavelength region. To maximize the performance of the proposed structure, a deep reinforcement learning approach is further proposed to search for the optimal set of structure parameters. To avoid overly long training time by using the numerical simulations as environment, a deep neural network is proposed to serve as the surrogate model, where a prediction accuracy of 96.6% and 95.5% in two orthogonal polarization directions is achieved with micro-second grade simulation time respectively. With the optimized structure, the average transmittance is larger than 0.5 for the wavelength range from 444 to 466 nm with a maximum of 0.605 at 455 nm, which is 21% higher than the theoretical limit of 0.5 of conventional polarization converters.

Numerical simulation of optical refractometric sensing of multiple disease markers based on lab-in-a-fiber.

A multi-parameter optical refractometric sensor based on lab-in-a-fiber is proposed and its sensing properties have been investigated. Based on the particular three suspended-core fiber, the sensor has three channels for liquid circulation and three suspended cores for detection. The multiple disease markers can be detected by coating the specific bio-recognition layer on the surface of three channels. The bio-recognition layer thickness, representing the concentration of the disease markers, can then be measured by the wavelength of fiber Bragg grating inscribed in each suspended core. Owing to the triple symmetry of the fiber, the sensitivity of each core is similar. The simulation results show that the grating wavelength linearly changes with the bio-recognition layer thickness variation. Through the sensitivity matrix, the sensitivity of the sensor is 0.362 nm/nm and the sensing accuracy is ± 1 nm.

ZmEHD1 Is Required for Kernel Development and Vegetative Growth through Regulating Auxin Homeostasis.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize.

Tandem solar cells efficiency prediction and optimization via deep learning.

Optical design plays an important role in improving the performance of opto-electronic devices. However, conventional design processes using finite difference time domain (FDTD) or finite element methods are usually time and computing resource consuming, and often result in sub-optimal solutions due to an incomplete search of the parameter state space. In this paper, we propose a deep learning approach to predict and optimize the cell performance of perovskite/crystalline-silicon (c-Si) tandem solar cells. In particular, a deep neural network is established to predict the achievable short-circuit current for tandem solar cells with a given cell structure. After training on a FDTD numerical simulation data set, the proposed deep neural network achieves an accuracy of 98.3% and micro-second grade simulation time, which is an ultra-fast, highly accurate and computing resource-saving solution to investigate the current properties of tandem solar cells. Heuristic algorithms are further adopted to inversely optimize the device structure, where the optimal set of layer thicknesses is obtained to maximize the achievable short-circuit current. According to the calculated projected efficiency, the expected experimental short-circuit current and power conversion efficiency of tandem solar cells with the optimal selection of layer thickness can reach 15.79 mA cm-2 and 23.24%, which is improved by 14.42% and 28.4%, respectively, compared to the benchmark cells.

Pre-rRNA processing and its response to temperature stress in maize.

Ribosome biogenesis is a fundamental process in all eukaryotic cells and is coupled with the processing and maturation of pre-rRNAs. Maize is a primary staple crop across the world, but little is known about the exact pre-rRNA processing sites and pathways in this species. In this study, we present a detailed model of the pathway by identifying the critical endonucleolytic cleavage sites and determining the pre-rRNA intermediates by circular reverse-transcription PCR and northern blot analysis. We demonstrate that two pathways coexist in maize to promote the processing of 35S pre-rRNA, and that the processing of 27SA pre-rRNA can proceed via two different pathways, which are distinguished based on the order of ITS1 removal and ITS2 cleavage. Compared with yeast and mammals, this new 27SA pre-rRNA processing mechanism is unique to maize and other higher plants. In addition, we demonstrate that maize can modulate pre-rRNA processing levels in response to chilling and heat stress, as indicated by a significant reduction of the P-A3 intermediate. Our study provides information that will facilitate future research on ribosome biogenesis and pre-rRNA processing in maize.

The PILNCR1-miR399 Regulatory Module Is Important for Low Phosphate Tolerance in Maize.

The regulation of adaptive responses to phosphorus (P) deficiency by the microRNA399 (miR399)/PHOSPHATE2 (PHO2) pathway has been well studied in Arabidopsis (Arabidopsis thaliana) but not in maize (Zea mays). Here, we show that miR399 transcripts are strongly induced in maize by phosphate (Pi) deficiency. Transgenic maize plants that overexpressed MIR399b accumulated excessive amounts of P in their shoots and displayed typical Pi-toxicity phenotypes. We reannotated ZmPHO2 with an additional 1,165 bp of the 5' untranslated region. miR399-guided posttranscriptional repression of ZmPHO2 was mainly observed in the P-efficient lines. We identified Pi-deficiency-induced long-noncoding RNA1 (PILNCR1) from our strand-specific RNA libraries. Transient expression assays in Nicotiana benthamiana and maize leaf protoplasts demonstrated that PILNCR1 inhibits ZmmiR399-guided cleavage of ZmPHO2 The abundance of PILNCR1 was significantly higher in P-inefficient lines than in P-efficient lines, which is consistent with the abundance of ZmmiR399 transcripts. These results indicate that the interaction between PILNCR1 and miR399 is important for tolerance to low Pi in maize.

microRNA/microRNA* complementarity is important for the regulation pattern of NFYA5 by miR169 under dehydration shock in Arabidopsis.

MicroRNAs regulate gene expression at the mRNA and translational levels. Although our previous research showed that expression of miR169 and one of its targets, NFYA5, is down- and up-regulated by drought stress, respectively, the current study shows that expression of both miR169 and NFYA5 are induced by dehydration shock. Unlike overexpression of MIR169a/b, overexpression of MIR169i/l did not decrease NFYA5 transcripts but increased NFYA5 protein levels. NFYA5 protein abundance also increased in mir169a knock-out mutants and 35S::MIR169l mir169a double mutants. When bulge #11 and bulge #16 in the miR169/miR169* duplex were mutated, both NFYA5 transcripts and the corresponding protein levels were lower in 35S::MIR169l*-mut transgenic plants than in wild-type (WT) plants, and the 35S::MIR169l*-mut transgenic plants were as sensitive to drought stress as the 35S::MIR169a plants. The mRNA and protein levels of NFYA5 did not differ substantially between WT and 35S::MIR169a*-mut transgenic plants when the two bulges were introduced in the miR169a/miR169a* duplex. Both bulge #11 and bulge #16 in the miR169/miR169* duplex were essential for different regulation patterns of NFYA5 by miR169a and miR169l. These results increase the understanding of regulatory specialization in one MIR family, and also increase our understanding of the importance of microRNA/microRNA* secondary structure.

Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing.

Ribosome biogenesis is a fundamental process in eukaryotic cells. Although Urb2 protein has been implicated in ribosome biogenesis in yeast, the Urb2 domain is loosely conserved between plants and yeast, and the function of Urb2 protein in plants remains unknown. Here, we isolated a maize mutant, designated as urb2, with defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that urb2 encodes an Urb2 domain-containing protein. Compared with the wild-type (WT), the urb2 mutant showed decreased ratios of 60S/40S and 80S/40S and increased ratios of polyribosomes. The pre-rRNA intermediates of 35/33S rRNA, P-A3 and 18S-A3 were significantly accumulated in the urb2 mutant. Transcriptome profiling of the urb2 mutant indicated that ZmUrb2 affects the expression of a number of ribosome-related genes. We further demonstrated that natural variations in ZmUrb2 are significantly associated with maize kernel length. The overall results indicate that, by affecting pre-rRNA processing, the Urb2 protein is required for ribosome biogenesis in maize.

Nitrogen Limitation Adaptation (NLA) is involved in source-to-sink remobilization of nitrate by mediating the degradation of NRT1.7 in Arabidopsis.

Recent studies on nitrate transporters (NRTs) have greatly increased our knowledge of the mechanisms regulating nitrogen (N) homeostasis in plants. However, an understanding of how these NRTs are regulated is still lacking. The nitrogen limitation adaptation (nla) mutant is hypersensitive to N limitation. In the nla mutant, 15 N-nitrate spotted on old leaves preferentially accumulated in the youngest leaves. Analysis of leaf cross-sections indicated that NLA expression was expressed in the sieve element and companion cell system. The results of bimolecular fluorescence complementation (BiFC), split-ubiquitin yeast two-hybrid and co-immunoprecipitation (CoIP) assays demonstrated that NLA interacts with NRT1.7 in the plasma membrane. The following findings suggest that NLA directs the ubiquitination of NRT1.7: the down-regulation of NRT1.7 protein abundance in 35S::NLA/35S::Myc-NRT1.7 double transgenic plants compared with 35S::Myc-NRT1.7 transgenic plants; the up-regulation of NRT1.7 protein abundance in the nla mutant compared with wild-type plants; and the direct degradation of truncated NRT1.7 recombinant protein by NLA. Furthermore, analysis of NLA and NRT1.7 protein abundance in mirna827 knock-out plants showed that N deficiency-guided translational repression of NLA depends on miRNA827. Our findings reveal that plants regulate source-to-sink remobilization of nitrate by the ubiquitin-mediated post-translational regulatory pathway.

Strand-specific RNA-Seq transcriptome analysis of genotypes with and without low-phosphorus tolerance provides novel insights into phosphorus-use efficiency in maize.

BACKGROUND: Phosphorus (P) stress is a global problem in maize production. Although macro/microarray technologies have greatly increased our general knowledge of maize responses to P stress, a greater understanding of the diversity of responses in maize genotypes is still needed. RESULTS: In this study, we first evaluated the tolerance to low P of 560 accessions under field conditions, and selected the low P-tolerant line CCM454 and the low P-sensitive line 31778 for further research. We then generated 24 strand-specific RNA libraries from shoots and roots of CCM454 and 31778 that had been subjected to P stress for 2 and 8 days. The P deficiency-responsive genes common to CCM454 and 31778 were involved in various metabolic processes, including acid phosphatase (APase) activity. Determination of root-secretory APase activities showed that the induction of APase by P stress occurred much earlier in CCM454 than that in 31778. Gene Ontology analysis of differentially expressed genes (DEGs) and CAT/POD activities between CCM454 and 31778 under P-sufficient and -deficient conditions demonstrated that CCM454 has a greater ability to eliminate reactive oxygen species (ROS) than 31778. In addition, 16 miRNAs in roots and 12 miRNAs in shoots, including miRNA399s, were identified as DEGs between CCM454 and 31778. CONCLUSIONS: The results indicate that the tolerance to low P of CCM454 is mainly due to the rapid responsiveness to P stress and efficient elimination of ROS. Our findings increase the understanding of the molecular events involved in the diversity of responses to P stress among maize accessions.

Probing the intrinsic optical Bloch-mode emission from a 3D photonic crystal.

We report experimental observation of intrinsic Bloch-mode emission from a 3D tungsten photonic crystal at low thermal excitation. After the successful removal of conventional metallic emission (normal emission), it is possible to make an accurate comparison of the Bloch-mode and the normal emission. For all biases, we found that the emission intensity of the Bloch-mode is higher than that of the normal emission. The Bloch-mode emission also exhibits a slower dependence on [Formula: see text] than that of the normal emission. The observed higher emission intensity and a different T-dependence is attributed to Bloch-mode assisted emission where emitters have been located into a medium having local density of states different than the isotropic case. Furthermore, our finite-difference time-domain (FDTD) simulation shows the presence of localized spots at metal-air boundaries and corners, having intense electric field. The enhanced plasmonic field and local non-equilibrium could induce a strong thermally stimulated emission and may be the cause of our unusual observation.

Biofortification of iron content by regulating a NAC transcription factor in maize.

Iron (Fe) deficiency remains widespread among people in developing countries. To help solve this problem, breeders have been attempting to develop maize cultivars with high yields and high Fe concentrations in the kernels. We conducted a genome-wide association study and identified a gene, ZmNAC78 (NAM/ATAF/CUC DOMAIN TRANSCRIPTION FACTOR 78), that regulates Fe concentrations in maize kernels. We cultivated maize varieties with both high yield and high Fe concentrations in their kernels by using a molecular marker developed from a 42-base pair insertion or deletion (indel) in the promoter of ZmNAC78. ZmNAC78 expression is enriched in the basal endosperm transfer layer of kernels, and the ZmNAC78 protein directly regulates messenger RNA abundance of Fe transporters. Our results thus provide an approach to develop maize varieties with Fe-enriched kernels.

The long non-coding RNA PILNCR2 increases low phosphate tolerance in maize by interfering with miRNA399-guided cleavage of ZmPHT1s.

The open reading regions of ZmPHT1s (inorganic phosphate [Pi] transporters) in maize possess target sites of microRNA399 (miR399). However, the relationship between miR399 and ZmPHT1s and its functional importance in response to Pi deficiency remain to be explored. We show here that ZmPHT1;1, ZmPHT1;3, and ZmPHT1;13 are the targets of ZmmiRNA399. We found that a long non-coding RNA, PILNCR2 (Pi-deficiency-induced lncRNA 2), is transcribed from the opposing DNA strand of ZmPHT1;1 and predominantly localized in the cytoplasm. A ribonuclease protection assay and an RNA-RNA binding assay showed that PILNCR2 and ZmPHT1s could form the RNA/RNA duplexes in vivo and in vitro. A co-expression assay in N. benthamiana revealed that the PILNCR2/ZmPHT1 RNA/RNA duplexes interfere with miR399-guided cleavage of ZmPHT1 mRNAs. Overexpression of PILNCR2 increased low-Pi tolerance in maize, whereas its knockout and knockdown decreased low-Pi tolerance in maize. Consistently, ZmPHT1;3 and ZmPHT1;13 mRNA abundance was increased in transgenic plants overexpressing PILNCR2 but reduced in its knock-out mutants, suggesting that PILNCR2 positively regulates the mRNA abundance of ZmPHT1;3 and ZmPHT1;13 in maize. Collectively, these results indicate that PILNCR2 plays an important role in maize Pi homeostasis by interfering with miRNA399-guided cleavage of ZmPHT1 mRNAs.

Nitrogen supply affects ion homeostasis by modifying root Casparian strip formation through the miR528-LAC3 module in maize.

Although nitrogen (N) is known to affect mineral element homeostasis in plants, the molecular mechanisms of interactions between N and other nutrients remain largely unclear. We report here that N supply affects ion homeostasis in maize. Berberine hemisulfate staining and a propidium iodide penetration assay showed that N luxury significantly delayed Casparian strip (CS) formation in maize roots. We further demonstrated that N-mediated CS formation in maize was independent of RBOHF-activated H2O2 production. N luxury induced the expression of ZmmiR528 in whole roots and root tips. Knockdown and loss-of-function of ZmmiR528 promoted CS formation under both N-luxury and N-deficient conditions. Both ZmMIR528a and ZmMIR528b contribute to early CS formation under different N conditions. RNA-seq and real-time RT-PCR analysis demonstrated that ZmLAC3, but not ZmLAC5, responded to N treatments. Consistent with results obtained with ZmmiR528 TM transgenic maize and mir528a/b loss-of-function mutants, transgenic maize overexpressing ZmLAC3 displayed early CS formation under different N conditions. Under field conditions, K, Ca, Mn, Cu, Mg, and Zn concentrations were greater in the ear leaf of ZmLAC3-overexpressing transgenic maize than in the wild type. These results indicate that ZmmiR528 affects CS formation in maize by regulating the expression of ZmLAC3, and modification of CS formation has the potential to improve maize quality.

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.

Flowering time regulation model revisited by pooled sequencing of mass selection populations.

Maize is one of the most broadly cultivated crops throughout the world, and flowering time is a major adaptive trait for its diffusion. The biggest challenge in understanding maize flowering genetic architecture is that the trait is confounded with population structure. To eliminate the effect, we revisited the flower time genetic network by using a tropical maize population Pop32, which was under mass selection for adaptation to early flowering time in China for six generations from tropical to temperate regions. The days to anthesis (DTA) of the initial (Pop32C0), intermedia (Pop32C3), and final population (Pop32C5) was 90.77, 84.63, and 79.72 days on average, respectively. To examine the genetic mechanism and identify the genetic loci underlying this rapid change in flowering time of Pop32, we bulked 30 individuals from C0, C3, and C5 to conduct the whole genome sequencing. And we finally identified 4,973,810 high-quality single nucleotide polymorphisms (SNPs) and 6,517 genes with allele frequency significantly changed during the artificial improvement process. We speculate that these genes might participate in the adaptive improvement process and control flowering time. To identify the candidate genes for flowering time from the gene set with allele frequency changed, we carried out weighted gene co-expression network analysis (WGCNA), and identified four co-expression modules that highly associated with the flowering time development, as well as constructed the co-expression network of key flowering time genes. Gene Ontology (GO) enrichment analysis revealed that the GO terms photosynthesis/light reaction, carbohydrate binding, auxin mediated signaling pathway, response to temperature stimulus that are closely connected with flowering time. Furthermore, targeted GWAS revealed the genes are significantly connected with the flowering time. qRT-PCR of four candidate genes GRMZM2G019879, GRMZM2G055905, GRMZM2G058158, and GRMZM2G171365 showed that their expression level is similar to the flowering time genes, which playing a key role in maize flowering time transition. This study revealed that the changes of flowering time in mass selection process may be strongly associated with the variations of allele frequency changes, and we identified some important candidate genes for flowering time, which will provide a new insight for the rapid improvement of maize important agronomic traits and promote the gene cloning of maize flowering time.