Haiping Ding

Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing

BackgroundIn plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages.ResultsWe used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed.ConclusionsThis study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.

The Dynamics of DNA Methylation in Maize Roots under Pb Stress

Plants adapt to adverse conditions through a series of physiological, cellular, and molecular processes, culminating in stress tolerance. However, little is known about the associated regulatory mechanisms at the epigenetic level in maize under lead (Pb) stress. Therefore, in this study, we aimed to compare DNA methylation profiles during the dynamic development of maize roots following Pb treatment to identify candidate genes involved in the response to Pb stress. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) was used to investigate the genome-wide DNA methylation patterns in maize roots under normal condition (A1) and 3 mM Pb(NO3)2 stress for 12 h (K2), 24 h (K3) and 48 h (K4). The results showed that the average methylation density was the highest in CpG islands (CGIs), followed by the intergenic regions. Within the gene body, the methylation density of the introns was higher than those of the UTRs and exons. In total, 3857 methylated genes were found in 4 tested samples, including 1805 differentially methylated genes for K2 versus A1, 1508 for K3 versus A1, and 1660 for K4 versus A1. Further analysis showed that 140 genes exhibited altered DNA methylation in all three comparisons, including some well-known stress-responsive transcription factors and proteins, such as MYB, AP2/ERF, bZIP, serine-threonine/tyrosine-proteins, pentatricopeptide repeat proteins, RING zinc finger proteins, F-box proteins, leucine-rich repeat proteins and tetratricopeptide repeat proteins. This study revealed the genome-scale DNA methylation patterns of maize roots in response to Pb exposure and identified candidate genes that potentially regulate root dynamic development under Pb stress at the methylation level.

Heterosis in Early Maize Ear Inflorescence Development: A Genome-Wide Transcription Analysis for Two Maize Inbred Lines and Their Hybrid

Heterosis, or hybrid vigor, contributes to superior agronomic performance of hybrids compared to their inbred parents. Despite its importance, little is known about the genetic and molecular basis of heterosis. Early maize ear inflorescences formation affects grain yield, and are thus an excellent model for molecular mechanisms involved in heterosis. To determine the parental contributions and their regulation during maize ear-development-genesis, we analyzed genome-wide digital gene expression profiles in two maize elite inbred lines (B73 and Mo17) and their F1 hybrid using deep sequencing technology. Our analysis revealed 17,128 genes expressed in these three genotypes and 22,789 genes expressed collectively in the present study. Approximately 38% of the genes were differentially expressed in early maize ear inflorescences from heterotic cross, including many transcription factor genes and some presence/absence variations (PAVs) genes, and exhibited multiple modes of gene action. These different genes showing differential expression patterns were mainly enriched in five cellular component categories (organelle, cell, cell part, organelle part and macromolecular complex), five molecular function categories (structural molecule activity, binding, transporter activity, nucleic acid binding transcription factor activity and catalytic activity), and eight biological process categories (cellular process, metabolic process, biological regulation, regulation of biological process, establishment of localization, cellular component organization or biogenesis, response to stimulus and localization). Additionally, a significant number of genes were expressed in only one inbred line or absent in both inbred lines. Comparison of the differences of modes of gene action between previous studies and the present study revealed only a small number of different genes had the same modes of gene action in both maize seedlings and ear inflorescences. This might be an indication that in different tissues or developmental stages, different global expression patterns prevail, which might nevertheless be related to heterosis. Our results support the hypotheses that multiple molecular mechanisms (dominance and overdominance modes) contribute to heterosis.

Difference between Pb and Cd accumulation in 19 elite maize inbred lines and application prospects

In the last two decades, the accumulation of heavy metal in crop grains has become the study hotspot. In this study, 19 representative elite maize inbred lines and 3 hybrid varieties were investigated at the seedling stage, which can accumulate Pb and Cd in the stems and leaves, respectively. The results demonstrated that significant differences are among inbred lines for accumulation of heavy metals, implying that the Cd accumulation is significant correlation between the male parents and their hybrids and some inbred lines have been selected for cross-breeding with low Pb or Cd accumulation, such as S37, 9782, and ES40; Moreover, some inbred lines could be suitable for phytoremediation species for soil bioremediation with high levels of Pb and Cd accumulation, including 178, R08, 48-2, and Mo17ht.

The development dynamics of the maize root transcriptome responsive to heavy metal Pb pollution

Lead (Pb), as a heavy metal element, has become the most important metal pollutant of the environment. With allocating a relatively higher proportion of its biomass in roots, maize could be a potential important model to study the phytoremediation of Pb-contaminated soil. Here we analyzed the maize root transcriptome of inbred lines 9782 under heavy metal lead (Pb) pollution, which was identified as a non-hyperaccumulator for Pb in roots. In the present study, more than 98 millions reads were mapped to define gene structure and detect polymorphism, thereby to qualify transcript abundance along roots development under Pb treatment. A total of 17,707, 17,440, 16,998 and 16,586 genes were identified in maize roots at four developmental stages (0, 12 h, 24 h and 48 h) respectively and 2,825, 2,626, 2161 and 2260 stage-specifically expressed genes were also identified respectively. In addition, based on our RNA-Seq data, transcriptomic changes during maize root development responsive to Pb were investigated. A total of 384 differentially expressed genes (DEGs) (log2Ratio ≥ 1, FDR ≤ 0.001) were identified, of which, 36 genes with significant alteration in expression were detected in four developmental stages; 12 DEGs were randomly selected and successful validated by qRT-PCR. Additionally, many transcription factor families might act as the important regulators at different developmental stages, such as bZIP, ERF and GARP et al. These results will expand our understanding of the complex molecular and cellular events in maize root development and provide a foundation for future study on root development in maize under heavy metal pollution and other cereal crops.

A High-Through Technique to Measure DNA Methylation

MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR strategy that is used to detect and quantify DNA methylation in genomic DNA. High-throughput MethyLight allows the rapid and sensitive detection of very low frequencies of hypermethylated alleles in populations of alternated individuals. The high sensitivity and specificity of MethyLight can be applied not only to make it uniquely suited disease clinical but also quantitatively assessed of these low-frequency methylation events. Owing to its full of advantages of simple procedure, high efficiency and high sensitivity, MethyLight provides a powerful approach for clinical examination, Gene expression analysis, SNP analysis and allele analysis. Coupled with other techniques, MethyLight can be used immediately in identifying allelic alterations in genes exhibiting expressions correlating with phenotypes, Locating an allelic series of induced point mutations in genes of interest. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of samples.