Identification and characterization of maize microRNAs involved in the very early stage of seed germination

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are a new class of endogenous small RNAs that play essential regulatory roles in plant growth, development and stress response. Extensive studies of miRNAs have been performed in model plants such as rice, <it>Arabidopsis thaliana </it>and other plants. However, the number of miRNAs discovered in maize is relatively low and little is known about miRNAs involved in the very early stage during seed germination.</p> <p>Results</p> <p>In this study, a small RNA library from maize seed 24 hours after imbibition was sequenced by the Solexa technology. A total of 11,338,273 reads were obtained. 1,047,447 total reads representing 431 unique sRNAs matched to known maize miRNAs. Further analysis confirmed the authenticity of 115 known miRNAs belonging to 24 miRNA families and the discovery of 167 novel miRNAs in maize. Both the known and the novel miRNAs were confirmed by sequencing of a second small RNA library constructed the same way as the one used in the first sequencing. We also found 10 miRNAs that had not been reported in maize, but had been reported in other plant species. All novel sequences had not been earlier described in other plant species. In addition, seven miRNA* sequences were also obtained. Putative targets for 106 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in maize imbibed seed.</p> <p>Conclusions</p> <p>This study led to the confirmation of the authenticity of 115 known miRNAs and the discovery of 167 novel miRNAs in maize. Identification of novel miRNAs resulted in significant enrichment of the repertoire of maize miRNAs and provided insights into miRNA regulation of genes expressed in imbibed seed.</p

Molecular cloning and characterization of soybean peroxidase gene families

Peroxidases are a ubiquitous class of enzymes and play major roles in many plant physiological processes. The objectives of this study were to isolate and characterize soybean peroxidase cDNA and genomic DNA, and to study in vitro folding conditions of bacterially expressed soybean peroxidase. Five full-length soybean peroxidase cDNA, GmEPa1, GmEPa2, GmEPb1, GmEPb2 and GmEPc, were isolated from a soybean seedbud cDNA library (Glycine max. Resnik). Neutral peroxidases, GmEPa1 and GmEPa2 were expressed in developing seed and root, the cationic peroxidases GmEPb1 and GmEPb2 were detected in root, stem, leaf, and seedpod, and the anionic peroxidase GmEPc was previously shown to be expressed in seed coat and root. These different peroxidases may play different roles in soybean growth, development, and/or defense system against pathogen attacks. The availability of these genes and gene-specific primers are essential for further in vivo and in vitro studies of their expressions, functions and catalytic mechanisms. Three soybean peroxidase genomic DNA fragments, GmEPA1, GmEPB1 , and GmEPC, were isolated from a soybean genomic library and correspond to cDNA GmEPa1, GmEPb1, and GmEPc, respectively. Genomic Southern blot analysis indicated that a very small number of these three genes were present in the soybean genome. The 1391 bp 5′ flanking region of GmEPA1 was capable of conferring both seed coat and root expression of GUS by transient expression assay. The 1524 bp promoter of GmEPC was capable of driving the expression of GUS in the seed coat. This promoter and regulatory sequences could be used to drive expression of proteins of industrial and human interest in soybean seed coats. The open reading frame including the 5′ leader sequence of GmEPa1 was cloned into the pET-34(+) expression vector. This peroxidase enzyme was expressed as a CBD-peroxidase fusion protein in inclusion bodies. The recovery of peroxidase activity was critically dependent on the addition of hemin and on the concentration of urea, with ∼2M being optimal at pH 8.0. The best overall in vitro folding efficiency (∼5%) was achieved when 50% ethylene glycol was included in the standard folding buffer. The folding efficiency was high enough for mutagenesis studies

Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize

In maize (Zea mays L.), unilateral cross-incompatibility (UCI) is controlled by Gametophyte factors (Ga), including Ga1, Ga2, and Tcb1; however, the molecular mechanisms underpinning this process remain unexplored. Here, we report the pollination phenotype of an inbred line, 511L, which carries a near-dominant Ga2-S allele. We performed a high-throughput RNA sequencing (RNA-Seq) analysis of the compatible and incompatible crosses between 511L and B73, to identify the transcriptomic differences associated with Ga2-mediated UCI. An in vivo kinetics analysis revealed that the growth of non-self pollen tubes was blocked at the early stages after pollination in 511L, maintaining the UCI barrier in Ga2. In total, 25,759 genes were expressed, of which, 2063 differentially expressed genes (DEGs) were induced by pollination (G_GG, G_GB, B_BB, B_BG). A gene ontology (GO) enrichment analysis revealed that these genes were specifically enriched in functions involved in cell wall strength and pectic product modification. Moreover, 1839, 4382, and 5041 genes were detected to differentially express under same pollination treatments, including B_G, BG_GG, and BB_GB, respectively. A total of 1467 DEGs were constitutively expressed between the two inbred lines following pollination treatments, which were enriched in metabolic processes, flavonoid biosynthesis, cysteine biosynthesis, and vacuole functions. Furthermore, we confirmed 14 DEGs related to cell wall modification and stress by qRT-PCR, which might be involved in Ga2-S-mediated UCI. Our results provide a comprehensive foundation for the molecular mechanisms involved in silks of UCI mediated by Ga2-S

Identification of the Potential Genes Regulating Seed Germination Speed in Maize

Seed germination is the crucial stage in plant life cycle. Rapid and uniform germination plays an essential role in plant development and grain yield improvement. However, the molecular mechanism underlying seed germination speed is largely unknown due to the complexity of the dynamic process and the difficulty in phenotyping. Here, we conducted a time-series comparative transcriptome study of two elite maize inbred lines, 72-3 and F9721, with striking difference in seed germination speed, and identified a major locus underlying maize germination speed through genome-wide association analysis (GWAS) of an F2 segregation population. Comparative transcriptome study identified 12 h after imbibition (HAI) as the critical stage responsible for the variation in germination speed. The differentially expressed genes (DEGs) between 72-3 and F9721 were mainly enriched in metabolic pathways, biosynthesis of secondary metabolites, oxidoreductase activity pathways, hormone signal transduction, and amino acid transporter activity pathways. GWAS revealed that germination speed was controlled by a major locus on chromosome 1 with the leading SNP as AX-91332814, explaining 10.63% of phenotypic variation. A total of 87 proposed protein-coding genes surrounding the locus were integrated with DEGs. Combined with evidence from the gene expression database and gene synteny with other model species, we finally anchored three genes as the likely candidates regulating germination speed in maize. This study provides clues for the further exploration of genes controlling the maize seed germination speed, thus facilitating breeding of rapid germinated elite lines through marker assistant selection

Proteomic Analysis of Seedling Roots of Two Maize Inbred Lines That Differ Significantly in the Salt Stress Response

10.1371/journal.pone.0116697PLoS ONE102e011669

Cytokinins Secreted by Agrobacterium Promote Transformation by Repressing a Plant Myb Transcription Factor

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment of bacteria to roots and decreased transformation, suggesting that AT14A may serve as an anchor point for Agrobacteria. Thus, by promoting bacterial attachment and transformation of resistant plants and increasing such processes in susceptible plants, treating roots with cytokinins may help engineer crops with improved features or yield