Opaque2 (O2) is a transcription factor that plays important roles during maize endosperm development. Mutation of the O2 gene improves the nutritional value of maize seeds but also confers pleiotropic effects that result in reduced agronomic quality. To reveal the transcriptional regulatory framework of O2, we studied the transcriptome of o2 mutants using RNA sequencing (RNA-Seq) and determined O2 DNA binding targets using chromatin immunoprecipitation coupled to highthroughput sequencing (ChIP-Seq). The RNA-Seq analysis revealed 1605 differentially expressed genes (DEGs) and 383 differentially expressed long, noncoding RNAs. The DEGs cover a wide range of functions related to nutrient reservoir activity, nitrogen metabolism, stress resistance, etc. ChIP-Seq analysis detected 1686 O2 DNA binding sites distributed over 1143 genes. Overlay of the RNA-Seq and ChIP-Seq results revealed 35 O2-modulated target genes. We identified four O2 binding motifs; among them, TGACGTGG appears to be the most conserved and strongest. We confirmed that, except for the 16-and 18-kD zeins, O2 directly regulates expression of all other zeins. O2 directly regulates two transcription factors, genes linked to carbon and amino acid metabolism and abiotic stress resistance. We built a hierarchical regulatory model for O2 that provides an understanding of its pleiotropic biological effects.

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BackgroundNitrogen (N) is an essential and often limiting nutrient to plant growth and development. Previous studies have shown that the mRNA expressions of numerous genes are regulated by nitrogen supplies; however, little is known about the expressed non-coding elements, for example long non-coding RNAs (lncRNAs) that control the response of maize (Zea mays L.) to nitrogen. LncRNAs are a class of non-coding RNAs larger than 200 bp, which have emerged as key regulators in gene expression.ResultsIn this study, we surveyed the intergenic/intronic lncRNAs in maize B73 leaves at the V7 stage under conditions of N-deficiency and N-sufficiency using ribosomal RNA depletion and ultra-deep total RNA sequencing approaches. By integration with mRNA expression profiles and physiological evaluations, 7245 lncRNAs and 637 nitrogen-responsive lncRNAs were identified that exhibited unique expression patterns. Co-expression network analysis showed that the nitrogen-responsive lncRNAs were enriched mainly in one of the three co-expressed modules. The genes in the enriched module are mainly involved in NADH dehydrogenase activity, oxidative phosphorylation and the nitrogen compounds metabolic process.ConclusionsWe identified a large number of lncRNAs in maize and illustrated their potential regulatory roles in response to N stress. The results lay the foundation for further in-depth understanding of the molecular mechanisms of lncRNAs’ role in response to nitrogen stresses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2650-1) contains supplementary material, which is available to authorized users.

Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca 2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.

In comparison with general FISH for preparing probes in terms of time and cost, synthesized oligonucleotide (oligo hereafter) probes for FISH have many advantages such as ease of design, synthesis, and labeling. Low cost and high sensitivity and resolution of oligo probes greatly simplify the FISH procedure as a simple, fast, and efficient method of chromosome identification. In this study, we developed new oligo and oligo multiplex probes to accurately and efficiently distinguish wheat (Triticum aestivum, 2n = 6x, AABBDD) and Thinopyrum bessarabicum (2n = 2x = 14, JJ) chromosomes. The oligo probes contained more nucleotides or more repeat units that produced stronger signals for more efficient chromosome painting. Four Th. bessarabicum-specific oligo probes were developed based on genomic DNA sequences of Th. bessarabicum chromosome arm 4JL, and one of them (oligo DP4J27982) was pooled with the oligo multiplex #1 to simultaneously detect wheat and Th. bessarabicum chromosomes for quick and accurate identification of Chinese Spring (CS) - Th. bessarabicum alien chromosome introgression lines. Oligo multiplex #4 revealed chromosome variations among CS and eight wheat cultivars by a single round of FISH analysis. This research demonstrated the high efficiency of using oligos and oligo multiplexes in chromosome identification and manipulation.

BackgroundQuinoa (Chenopodium quinoa Willd.) is a balanced nutritional crop, but its breeding improvement has been limited by the lack of information on its genetics and genomics. Therefore, it is necessary to obtain knowledge on genomic variation, population structure, and genetic diversity and to develop novel Insertion/Deletion (InDel) markers for quinoa by whole-genome re-sequencing.ResultsWe re-sequenced 11 quinoa accessions and obtained a coverage depth between approximately 7× to 23× the quinoa genome. Based on the 1453-megabase (Mb) assembly from the reference accession Riobamba, 8,441,022 filtered bi-allelic single nucleotide polymorphisms (SNPs) and 842,783 filtered InDels were identified, with an estimated SNP and InDel density of 5.81 and 0.58 per kilobase (kb). From the genomic InDel variations, 85 dimorphic InDel markers were newly developed and validated. Together with the 62 simple sequence repeat (SSR) markers reported, a total of 147 markers were used for genotyping the 129 quinoa accessions. Molecular grouping analysis showed classification into two major groups, the Andean highland (composed of the northern and southern highland subgroups) and Chilean coastal, based on combined STRUCTURE, phylogenetic tree and PCA (Principle Component Analysis) analyses. Further analysis of the genetic diversity exhibited a decreasing tendency from the Chilean coast group to the Andean highland group, and the gene flow between subgroups was more frequent than that between the two subgroups and the Chilean coastal group. The majority of the variations (approximately 70%) were found through an analysis of molecular variation (AMOVA) due to the diversity between the groups. This was congruent with the observation of a highly significant FST value (0.705) between the groups, demonstrating significant genetic differentiation between the Andean highland type of quinoa and the Chilean coastal type. Moreover, a core set of 16 quinoa germplasms that capture all 362 alleles was selected using a simulated annealing method.ConclusionsThe large number of SNPs and InDels identified in this study demonstrated that the quinoa genome is enriched with genomic variations. Genetic population structure, genetic core germplasms and dimorphic InDel markers are useful resources for genetic analysis and quinoa breeding.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4093-8) contains supplementary material, which is available to authorized users.

The roles and regulatory mechanisms of transcriptome changes during aging are unclear. It has been proposed that the transcriptome suffers decay during aging owing to age‐associated down‐regulation of transcription factors. In this study, we characterized the role of a transcription factor DAF‐16, which is a highly conserved lifespan regulator, in the normal aging process of Caenorhabditis elegans. We found that DAF‐16 translocates into the nucleus in aged wild‐type worms and activates the expression of hundreds of genes in response to age‐associated cellular stress. Most of the age‐dependent DAF‐16 targets are different from the canonical DAF‐16 targets downstream of insulin signaling. This and other evidence suggest that activation of DAF‐16 during aging is distinct from activation of DAF‐16 due to reduced signaling from DAF‐2. Further analysis showed that it is due in part to a loss of proteostasis during aging. We also found that without daf‐16, dramatic gene expression changes occur as early as on adult day 2, indicating that DAF‐16 acts to stabilize the transcriptome during normal aging. Our results thus reveal that normal aging is not simply a process in which the gene expression program descends into chaos due to loss of regulatory activities; rather, there is active transcriptional regulation during aging.

In the wake of recent progress of high throughput transcriptome profiling technologies, extensive housekeeping gene mining has been conducted in humans. However, very few studies have been reported in maize (Zea mays L.), an important crop plant, and none were conducted on a genome -wide level. In this study, we surveyed housekeeping genes throughout the maize transcriptome using RNA-seq and microarray techniques, and validated the housekeeping profile with quantitative polymerase chain reaction (qPCR) under a series of conditions including different genotypes and nitrogen supplies. Seven microarray datasets and two RNA-seq libraries representing 40 genotypes at more than 20 developmental stages were selected to screen for commonly expressed genes. A total of 1,661 genes showed constitutive expression in both microarray and RNA-seq datasets, serving as our starting housekeeping gene candidates. To determine for stably expressed housekeeping genes, NormFinder was used to select the top 20 % invariable genes to be the more likely candidates, which resulted in 48 and 489 entries from microarray and RNA-seq data, respectively. Among them, nine genes (2OG-Fe, CDK, DPP9, DUF, NAC, RPN, SGT1, UPF1 and a hypothetical protein coding gene) were expressed in all 40 maize diverse genotypes tested covering 16 tissues at more than 20 developmental stages under normal and stress conditions, implying these as being the most reliable reference genes. qPCR analysis confirmed the stable expression of selected reference gene candidates compared to two widely used housekeeping genes. All the reference gene candidates showed higher invariability than ACT and GAPDH. The hypothetical protein coding gene exhibited the most stable expression across 26 maize lines with different nitrogen treatments with qPCR, followed by CDK encoding the cyclin-dependent kinase. As the first study to systematically screen for housekeeping genes in maize, we identified candidates by examining the transcriptome atlas generated from RNA-seq and microarray technologies. The nine top-ranked qPCR-validated novel housekeeping genes provide a valuable resource of reference genes for maize gene expression analysis.