Advances of Apetala2/Ethylene Response Factors in Regulating Development and Stress Response in Maize.

Apetala2/ethylene response factor (AP2/ERF) is one of the largest families of transcription factors, regulating growth, development, and stress response in plants. Several studies have been conducted to clarify their roles in Arabidopsis and rice. However, less research has been carried out on maize. In this review, we systematically identified the AP2/ERFs in the maize genome and summarized the research progress related to AP2/ERF genes. The potential roles were predicted from rice homologs based on phylogenetic and collinear analysis. The putative regulatory interactions mediated by maize AP2/ERFs were discovered according to integrated data sources, implying that they involved complex networks in biological activities. This will facilitate the functional assignment of AP2/ERFs and their applications in breeding strategy.

Identification of a candidate gene underlying qHKW3, a QTL for hundred-kernel weight in maize.

KEY MESSAGE: qHKW3, a quantitative trait locus for hundred-kernel weight, harbors the proposed causal gene Zm00001d044081, encoding a homeobox-leucine zipper protein (ATHB-4) that might affect kernel size and weight. Kernel size and weight are key traits that contribute greatly to grain yield per year in maize (Zea mays). Here, we developed the chromosome segment substitution line (CSSL), H15-6-2, with smaller kernel size and lower kernel weight across environments compared to the background line Ye478. Histological analysis suggested that a slower kernel filling rate of H15-6-2 contributes to its small-kernel size and reduced hundred-kernel weight. We identified a quantitative trait locus (QTL) explaining 23% of the phenotypic variation in hundred-kernel weight. This QTL, qHKW3, was fine mapped to an interval of approximately 40.66-kb harboring the gene Zm00001d044081. The upstream sequence and its expression level of Zm00001d044081 in kernels at 6 days after pollination (DAP) showed obvious differences between the near-isogenic lines HKW3Ye478 and HKW3H15-6-2. We further confirmed the effects of the Zm00001d044081 promoter on maize kernel size and weight in an independent association mapping panel with 513 lines by candidate regional association analysis. We propose that Zm00001d044081, which encodes the homeobox-leucine zipper protein ATHB-4, is the causal gene of qHKW3, representing an attractive target for the genetic improvement of maize yield.

Starch phosphorylase 2 is essential for cellular carbohydrate partitioning in maize.

Carbohydrate partitioning is essential for plant growth and development, and its hindrance will result in excess accumulation of carbohydrates in source tissues. Most of the related mutants in maize (Zea mays L.) display impaired whole-plant sucrose transport, but other mechanisms affecting carbohydrate partitioning have seldom been reported. Here, we characterized chlorotic leaf3 (chl3), a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts, suggesting that chl3 is defective in carbohydrate partitioning. Positional cloning revealed that the chl3 phenotype results from a frameshift mutation in ZmPHOH, which encodes starch phosphorylase 2. Two mutants in ZmPHOH exhibited the same phenotype as chl3, and both alleles failed to complement the chl3 mutant phenotype in an allelism test. Inactivation of ZmPHOH in chl3 leaves reduced the efficiency of transitory starch conversion, resulting in increased leaf starch contents and altered carbohydrate metabolism patterns. RNA-seq revealed the transcriptional downregulation of genes related to photosynthesis and carbohydrate metabolism in chl3 leaves compared to the wild type. Our results demonstrate that transitory starch remobilization is very important for cellular carbohydrate partitioning in maize, in which ZmPHOH plays an indispensable role.

Loss of Function of an RNA Polymerase III Subunit Leads to Impaired Maize Kernel Development.

Kernel size is an important factor determining grain yield. Although a number of genes affecting kernel development in maize (Zea mays) have been identified by analyzing kernel mutants, most of the corresponding mutants cannot be used in maize breeding programs due to low germination or incomplete seed development. Here, we characterized small kernel7, a recessive small-kernel mutant with a mutation in the gene encoding the second-largest subunit of RNA polymerase III (RNAPΙΙΙ; NRPC2). A frame shift in ZmNRPC2 leads to a premature stop codon, resulting in significantly reduced levels of transfer RNAs and 5S ribosomal RNA, which are transcribed by RNAPΙΙΙ. Loss-of-function nrpc2 mutants created by CRISPR/CAS9 showed significantly reduced kernel size due to altered endosperm cell size and number. ZmNRPC2 affects RNAPIII activity and the expression of genes involved in cell proliferation and endoreduplication to control kernel development via physically interacting with RNAPIII subunits RPC53 and AC40, transcription factor class C1 and Floury3. Notably, unlike the semidominant negative mutant floury3, which has defects in starchy endosperm, small kernel7 only affects kernel size but not the composition of kernel storage proteins. Our findings provide novel insights into the molecular network underlying maize kernel size, which could facilitate the genetic improvement of maize in the future.

Fine mapping of a kernel length-related gene with potential value for maize breeding.

KEY MESSAGE: A key candidate gene for maize kernel length was fine mapped to an interval of 942 kb; the locus significantly increases kernel length (KL) and hundred-kernel weight (HKW). Kernel size is a major determinant of yield in cereals. Kernel length, one of the determining factors of kernel size, is a target trait for both domestication and artificial breeding. However, there are few reports of fine mapping and quantitative trait loci (QTLs)/cloned genes for kernel length in maize. In this project, a novel major QTL, named qKL9, controlling maize kernel length was identified. We verified the authenticity and stability of qKL9 via BC2F2 and BC3F1 populations, respectively, and ultimately mapped qKL9 to an ~ 942-kb genomic interval by testing the progenies of recombination events derived from BC3F2 and BC4F2 populations in multiple environments. Additionally, one new line (McqKL9-A) containing the ~ 942-kb segment was screened from the BC4F2 population. Combining transcriptome analysis between McqKL9-A and Mc at 6, 9 and 14 days after pollination and candidate regional association mapping, Zm00001d046723 was preliminarily identified as the key candidate gene for qKL9. Importantly, the replacement in the Mc line of the Mc's alleles by the V671's alleles in the qKL9 region improved the performances of single-cross hybrids obtained with elite lines, illustrating the potential value of this QTL for the genetic improvement in maize kernel-related traits. These findings facilitate molecular breeding for kernel size and cloning of the gene underlying qKL9, shedding light on the genetic basis of kernel size in maize.

Knockdown NRPC2, 3, 8, NRPABC1 and NRPABC2 Affects RNAPIII Activity and Disrupts Seed Development in Arabidopsis.

RNA polymerase III (RNAPIII) contains 17 subunits forming 4 functional domains that control the different stages of RNAPIII transcription and are dedicated to the synthesis of small RNAs such as 5S rRNA and tRNAs. Here, we identified 23 genes encoding these subunits in Arabidopsis (Arabidopsis thaliana) and further analyzed 5 subunits (NRPC2, NRPC3, NRPC8, NRPABC1, and NRPABC2) encoded by 6 genes with different expression patterns and belonging to different sub-complexes. The knockdown of these genes repressed the expression of 5S rRNA and tRNAs, causing seed developmental arrest at different stages. Among these knockdown mutants, RNA-seq analysis revealed 821 common differentially expressed genes (DEGs), significantly enriched in response to stress, abscisic acid, cytokinins, and the jasmonic acid signaling pathway. Weighted gene co-expression network analysis (WGCNA) revealed several hub genes involved in embryo development, carbohydrate metabolic and lipid metabolic processes. We identified numerous unique DEGs between the mutants belonging to pathways, including cell proliferation, ribosome biogenesis, cell death, and tRNA metabolic processes. Thus, NRPC2, NRPC3, NRPC8, NRPABC1, and NRPABC2 control seed development in Arabidopsis by influencing RNAPIII activity and, thus, hormone signaling. Reduced expression of these subunit genes causes an insufficient accumulation of the total RNAPIII, leading to the phenotypes observed following the genetic knockdown of these subunits.

Fine mapping of qKRN8, a QTL for maize kernel row number, and prediction of the candidate gene.

KEY MESSAGE: qKRN8, a major QTL for kernel row number in maize, was fine mapped to an interval of ~ 520 kb on chromosome 8 and the key candidate gene was identified via expression analysis. Kernel row number (KRN) is one of the most important yield-influencing traits and is closely associated with female inflorescence development in maize (Zea mays L.). In this study, an F2:3 population derived from a cross between V54 (low KRN line) and Lian87 (high KRN line) was used to map quantitative trait loci (QTLs) conferring KRN in maize. We identified 12 QTLs for KRN in four environments, each explaining 1.40-14.95% of phenotypic variance. Among these, one novel major QTL (named qKRN8) was mapped to bin 8.03 in all four environments, explaining 8.79-14.95% of phenotypic variation. By combining map-based cloning with progeny testing of recombinants, we ultimately mapped qKRN8 to an ~ 520 kb genomic interval, harboring six putative candidate genes. Among them, one candidate gene showed contrasted expression level in immature ears of the near-isogenic lines qKRN8Lian87 and qKRN8V54. These findings should facilitate molecular breeding for KRN and the further identification of the polymorphism underlying this QTL.

ZmSRL5 is involved in drought tolerance by maintaining cuticular wax structure in maize.

Cuticular wax is a natural barrier on terrestrial plant organs, which protects plants from damages caused by a variety of stresses. Here, we report the identification and functional characterization of a cuticular-wax-related gene, Zea mays L. SEMI-ROLLED LEAF 5 (ZmSRL5). The loss-of-function mutant srl5, which was created by a 3,745 bp insertion in the first intron that led to the premature transcript, exhibited abnormal wax crystal morphology and distribution, which, in turn, caused the pleiotropic phenotypes including increased chlorophyll leaching and water loss rate, decreased leaf temperature, sensitivity to drought, as well as semi-rolled mature leaves. However, total wax amounts showed no significant difference between wild type and semi-rolled leaf5 (srl5) mutant. The phenotype of srl5 was confirmed through the generation of two allelic mutants using CRISPR/Cas9. ZmSRL5 encodes a CASPARIAN-STRIP-MEMBRANE-DOMAIN-LIKE (CASPL) protein located in plasma membrane, and highly expressed in developing leaves. Further analysis showed that the expressions of the most wax related genes were not affected or slightly altered in srl5. This study, thus, primarily uncovers that ZmSRL5 is required for the structure formation of the cuticular wax and could increase the drought tolerance by maintaining the proper cuticular wax structure in maize.

A group VII ethylene response factor gene, ZmEREB180, coordinates waterlogging tolerance in maize seedlings.

Group VII ethylene response factors (ERFVIIs) play important roles in ethylene signalling and plant responses to flooding. However, natural ERFVII variations in maize (ZmERFVIIs) that are directly associated with waterlogging tolerance have not been reported. Here, a candidate gene association analysis of the ZmERFVII gene family showed that a waterlogging-responsive gene, ZmEREB180, was tightly associated with waterlogging tolerance. ZmEREB180 expression specifically responded to waterlogging and was up-regulated by ethylene; in addition, its gene product localized to the nucleus. Variations in the 5'-untranslated region (5'-UTR) and mRNA abundance of this gene under waterlogging conditions were significantly associated with survival rate (SR). Ectopic expression of ZmEREB180 in Arabidopsis increased the SR after submergence stress, and overexpression of ZmEREB180 in maize also enhanced the SR after long-term waterlogging stress, apparently through enhanced formation of adventitious roots (ARs) and regulation of antioxidant levels. Transcriptomic assays of the transgenic maize line under normal and waterlogged conditions further provided evidence that ZmEREB180 regulated AR development and reactive oxygen species homeostasis. Our study provides direct evidence that a ZmERFVII gene is involved in waterlogging tolerance. These findings could be applied directly to breed waterlogging-tolerant maize cultivars and improve our understanding of waterlogging stress.

Gene duplication confers enhanced expression of 27-kDa γ-zein for endosperm modification in quality protein maize.

The maize opaque2 (o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection for o2 modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from a 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure of qγ27 appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa γ-zein is critical for endosperm modification in QPM, qγ27 is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.

Emp10 encodes a mitochondrial PPR protein that affects the cis-splicing of nad2 intron 1 and seed development in maize.

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

Dissecting the genetic architecture of waterlogging stress-related traits uncovers a key waterlogging tolerance gene in maize.

KEY MESSAGE: A key candidate gene, GRMZM2G110141, which could be used in marker-assisted selection in maize breeding programs, was detected among the 16 genetic loci associated with waterlogging tolerance identified through genome-wide association study. Waterlogging stress seriously affects the growth and development of upland crops such as maize (Zea mays L.). However, the genetic basis of waterlogging tolerance in crop plants is largely unknown. Here, we identified genetic loci for waterlogging tolerance-related traits by conducting a genome-wide association study using maize phenotypes evaluated in the greenhouse under waterlogging stress and normal conditions. A total of 110 trait-single nucleotide polymorphism associations spanning 16 genomic regions were identified; single associations explained 2.88-10.67% of the phenotypic variance. Among the genomic regions identified, 14 co-localized with previously detected waterlogging tolerance-related quantitative trail loci. Furthermore, 33 candidate genes involved in a wide range of stress-response pathways were predicted. We resequenced a key candidate gene (GRMZM2G110141) in 138 randomly selected inbred lines and found that variations in the 5'-UTR and in the mRNA abundance of this gene under waterlogging conditions were significantly associated with leaf injury. Furthermore, we detected favorable alleles of this gene and validated the favorable alleles in two different recombinant inbred line populations. These alleles enhanced waterlogging tolerance in segregating populations, strongly suggesting that GRMZM2G110141 is a key waterlogging tolerance gene. The set of waterlogging tolerance-related genomic regions and associated markers identified here could be valuable for isolating waterlogging tolerance genes and improving this trait in maize.

EMPTY PERICARP11 serves as a factor for splicing of mitochondrial nad1 intron and is required to ensure proper seed development in maize.

Group II introns are common in the mitochondrial genome of higher plant species. The splicing of these introns is a complex process involving the synergistic action of multiple factors. However, few of these factors have been characterized in maize. In this study, we found that the Empty pericarp11 (Emp11) gene, which encodes a P-type pentatricopeptide repeat (PPR) protein, is required for the development of maize seeds. The loss of Emp11 function seriously impairs embryo and endosperm development, resulting in empty pericarp seeds in maize, and alteration in Emp11 expression leads to quantitative variation in kernel size and weight. We found that the emp11 mutants showed a failure in nad1 intron splicing, exhibited a severe reduction in complex I assembly and activity, mitochondrial structure disturbances, and an increase in alternative oxidase AOX2 and AOX3 levels. Interestingly, the emp11 phenotype was very severe in the W22 inbred line but could be partially recovered in B73 BC2F2 segregating ears. These results suggest that EMP11 serves as a factor for the splicing of mitochondrial nad1 introns and is required for mitochondrial function to ensure proper seed development in maize.

Comparative proteomic analysis revealing the complex network associated with waterlogging stress in maize (Zea mays L.) seedling root cells.

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

Genetic architecture of maize kernel row number and whole genome prediction.

KEY MESSAGE: Maize kernel row number might be dominated by a set of large additive or partially dominant loci and several small dominant loci and can be accurately predicted by fewer than 300 top KRN-associated SNPs. Kernel row number (KRN) is an important yield component in maize and directly affects grain yield. In this study, we combined linkage and association mapping to uncover the genetic architecture of maize KRN and to evaluate the phenotypic predictability using these detected loci. A genome-wide association study revealed 31 associated single nucleotide polymorphisms (SNPs) representing 17 genomic loci with an effect in at least one of five individual environments and the best linear unbiased prediction (BLUP) over all environments. Linkage mapping in three F2:3 populations identified 33 KRN quantitative trait loci (QTLs) representing 21 QTLs common to several population/environments. The majority of these common QTLs that displayed a large effect were additive or partially dominant. We found 70% KRN-associated genomic loci were mapped in KRN QTLs identified in this study, KRN-associated SNP hotspots detected in NAM population and/or previous identified KRN QTL hotspots. Furthermore, the KRN of inbred lines and hybrids could be predicted by the additive effect of the SNPs, which was estimated using inbred lines as a training set. The prediction accuracy using the top KRN-associated tag SNPs was obviously higher than that of the randomly selected SNPs, and approximately 300 top KRN-associated tag SNPs were sufficient for predicting the KRN of the inbred lines and hybrids. The results suggest that the KRN-associated loci and QTLs that were detected in this study show great potential for improving the KRN with genomic selection in maize breeding.

Genetic analysis and major QTL detection for maize kernel size and weight in multi-environments.

KEY MESSAGE: Twelve major QTL in five optimal clusters and several epistatic QTL are identified for maize kernel size and weight, some with pleiotropic will be promising for fine-mapping and yield improvement. Kernel size and weight are important target traits in maize (Zea mays L.) breeding programs. Here, we report a set of quantitative trait loci (QTL) scattered through the genome and significantly controlled the performance of four kernel traits including length, width, thickness and weight. From the cross V671 (large kernel) × Mc (small kernel), 270 derived F2:3 families were used to identify QTL of maize kernel-size traits and kernel weight in five environments, using composite interval mapping (CIM) for single-environment analysis along with mixed linear model-based CIM for joint analysis. These two mapping strategies identified 55 and 28 QTL, respectively. Among them, 6 of 23 coincident were detected as interacting with environment. Single-environment analysis showed that 8 genetic regions on chromosomes 1, 2, 4, 5 and 9 clustered more than 60 % of the identified QTL. Twelve stable major QTLs accounting for over 10 % of phenotypic variation were included in five optimal clusters on the genetic region of bins 1.02-1.03, 1.04-1.06, 2.05-2.07, 4.07-4.08 and 9.03-9.04; the addition and partial dominance effects of significant QTL play an important role in controlling the development of maize kernel. These putative QTL may have great promising for further fine-mapping with more markers, and genetic improvement of maize kernel size and weight through marker-assisted breeding.

Genetic variations in ZmEREB179 are associated with waterlogging tolerance in maize.

Maize (Zea mays) is highly susceptible to waterlogging stress, which reduces both the yield and quality of this important crop. However, the molecular mechanism governing waterlogging tolerance is poorly understood. In this study, we identify a waterlogging- and ethylene-inducible gene ZmEREB179 that encodes an ethylene response factor (ERF) localized in the nucleus. Overexpression of ZmEREB179 in maize increases the sensitivity to waterlogging stress. Conversely, the zmereb179 knockout mutants are more tolerant to waterlogging, suggesting that ZmEREB179 functions as a negative regulator of waterlogging tolerance. A transcriptome analysis of the ZmEREB179-overexpressing plants reveals that the ERF-type transcription factor modulates the expression of various stress-related genes, including ZmEREB180. We find that ZmEREB179 directly targets the ZmEREB180 promoter and represses its expression. Notably, the analysis of a panel of 220 maize inbred lines reveals that genetic variations in the ZmEREB179 promoter (Hap2) are highly associated with waterlogging resistance. The functional association of Hap2 with waterlogging resistance is tightly co-segregated in two F2 segregating populations, highlighting its potential applications in breeding programs. Our findings shed light on the involvement of the transcriptional cascade of ERF genes in regulating plant-waterlogging tolerance.

Identification of combining ability loci for five yield-related traits in maize using a set of testcrosses with introgression lines.

Combining ability is essential for hybrid breeding in crops. However, the genetic basis of combining ability remains unclear and has been seldom investigated. Identifying molecular markers associated with this complex trait would help to understand its genetic basis and provide useful information for hybrid breeding in maize. In this study, we identified genetic loci of general combining ability (GCA) and specific combining ability (SCA) for five yield-related traits under three environments using a set of testcrosses with introgression lines (ILs). GCA or SCA of the five yield-related traits of the ILs was estimated by the performance of testcrosses with four testers from different heterotic groups. Genetic correlations between GCA of the traits and the corresponding traits per se were not significant or not strong, suggesting that the genetic basis between them is different. A total of 56 significant loci for GCA and 21 loci for SCA were commonly identified in at least two environments, and only 5 loci were simultaneously controlling GCA and SCA, indicating that the genetic basis of GCA and SCA is different. For all of the traits investigated, positive and significant correlations between the number of GCA loci in the ILs and the performance of the corresponding GCA of the ILs were detected, implying that pyramiding GCA loci would have positive effect on the performance of GCA. Results in this study would be useful for maize hybrid breeding.

Genome-wide identification and analysis of microRNA responding to long-term waterlogging in crown roots of maize seedlings.

MicroRNAs (miRNAs) are critical post-transcriptional modulators of gene expression involving in plant responses to abiotic stress. However, the regulation of miRNA in the morphological response to waterlogging is poorly understood in maize. In this study, we detected miRNAs and their targets that expressed in waterlogged crown roots of maize seedlings in two inbred lines (Hz32 and Mo17) by RNA sequencing. A total of 61 mature miRNAs were found including 36 known maize (zma) miRNAs and 25 potential novel miRNA candidates. Comparison of miRNA expression in both waterlogged and control crown roots revealed 32 waterlogging-responsive miRNAs, most were consistently downregulated under waterlogging in the two inbred lines. We identified the miRNA targets through degradome sequencing. Many known miRNA targets involving in transcription regulation and reactive oxygen species elimination were found in the degradome libraries, and 17 targets of 10 newly detected miRNAs were identified as well. Moreover, the miRNA-mediated pathways that respond to waterlogging and regulate the induction of crown roots were discussed. This study is a comprehensive survey of responsive miRNAs in waterlogged maize crown roots. The results will help to understand the miRNA expression in response to waterlogging and miRNA-mediated regulation of morphological adaptation to waterlogging in maize.