Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors.

Maize is a major crop and a model plant for studying C4 photosynthesis and leaf development. However, a genomewide regulatory network of leaf development is not yet available. This knowledge is useful for developing C3 crops to perform C4 photosynthesis for enhanced yields. Here, using 22 transcriptomes of developing maize leaves from dry seeds to 192 h post imbibition, we studied gene up- and down-regulation and functional transition during leaf development and inferred sets of strongly coexpressed genes. More significantly, we developed a method to predict transcription factor binding sites (TFBSs) and their cognate transcription factors (TFs) using genomic sequence and transcriptomic data. The method requires not only evolutionary conservation of candidate TFBSs and sets of strongly coexpressed genes but also that the genes in a gene set share the same Gene Ontology term so that they are involved in the same biological function. In addition, we developed another method to predict maize TF-TFBS pairs using known TF-TFBS pairs in Arabidopsis or rice. From these efforts, we predicted 1,340 novel TFBSs and 253 new TF-TFBS pairs in the maize genome, far exceeding the 30 TF-TFBS pairs currently known in maize. In most cases studied by both methods, the two methods gave similar predictions. In vitro tests of 12 predicted TF-TFBS interactions showed that our methods perform well. Our study has significantly expanded our knowledge on the regulatory network involved in maize leaf development.

Assembler for de novo assembly of large genomes.

Assembling a large genome using next generation sequencing reads requires large computer memory and a long execution time. To reduce these requirements, we propose an extension-based assembler, called JR-Assembler, where J and R stand for "jumping" extension and read "remapping." First, it uses the read count to select good quality reads as seeds. Second, it extends each seed by a whole-read extension process, which expedites the extension process and can jump over short repeats. Third, it uses a dynamic back trimming process to avoid extension termination due to sequencing errors. Fourth, it remaps reads to each assembled sequence, and if an assembly error occurs by the presence of a repeat, it breaks the contig at the repeat boundaries. Fifth, it applies a less stringent extension criterion to connect low-coverage regions. Finally, it merges contigs by unused reads. An extensive comparison of JR-Assembler with current assemblers using datasets from small, medium, and large genomes shows that JR-Assembler achieves a better or comparable overall assembly quality and requires lower memory use and less central processing unit time, especially for large genomes. Finally, a simulation study shows that JR-Assembler achieves a superior performance on memory use and central processing unit time than most current assemblers when the read length is 150 bp or longer, indicating that the advantages of JR-Assembler over current assemblers will increase as the read length increases with advances in next generation sequencing technology.

Anatomical and transcriptional dynamics of maize embryonic leaves during seed germination.

Our anatomical analysis revealed that a dry maize seed contains four to five embryonic leaves at different developmental stages. Rudimentary kranz structure (KS) is apparent in the first leaf with a substantial density, but its density decreases toward younger leaves. Upon imbibition, leaf expansion occurs rapidly with new KSs initiated from the palisade-like ground meristem cells in the middle of the leaf. In parallel to the anatomical analysis, we obtained the time course transcriptomes for the embryonic leaves in dry and imbibed seeds every 6 h up to hour 72. Over this time course, the embryonic leaves exhibit transcripts of 30,255 genes at a level that can be regarded as "expressed." In dry seeds, ∼25,500 genes are expressed, showing functional enrichment in transcription, RNA processing, protein synthesis, primary metabolic pathways, and calcium transport. During the 72-h time course, ∼13,900 genes, including 590 transcription factor genes, are differentially expressed. Indeed, by 30 h postimbibition, ∼2,200 genes expressed in dry seeds are already down-regulated, and ∼2,000 are up-regulated. Moreover, the top 1% expressed genes at 54 h or later are very different from those before 30 h, reflecting important developmental and physiological transitions. Interestingly, clusters of genes involved in hormone metabolism, signaling, and responses are differentially expressed at various time points and TF gene expression is also modular and stage specific. Our dataset provides an opportunity for hypothesizing the timing of regulatory actions, particularly in the context of KS development.

Construction of gene clusters resembling genetic causal mechanisms for common complex disease with an application to young-onset hypertension.

BACKGROUND: Lack of power and reproducibility are caveats of genetic association studies of common complex diseases. Indeed, the heterogeneity of disease etiology demands that causal models consider the simultaneous involvement of multiple genes. Rothman's sufficient-cause model, which is well known in epidemiology, provides a framework for such a concept. In the present work, we developed a three-stage algorithm to construct gene clusters resembling Rothman's causal model for a complex disease, starting from finding influential gene pairs followed by grouping homogeneous pairs. RESULTS: The algorithm was trained and tested on 2,772 hypertensives and 6,515 normotensives extracted from four large Caucasian and Taiwanese databases. The constructed clusters, each featured by a major gene interacting with many other genes and identified a distinct group of patients, reproduced in both ethnic populations and across three genotyping platforms. We present the 14 largest gene clusters which were capable of identifying 19.3% of hypertensives in all the datasets and 41.8% if one dataset was excluded for lack of phenotype information. Although a few normotensives were also identified by the gene clusters, they usually carried less risky combinatory genotypes (insufficient causes) than the hypertensive counterparts. After establishing a cut-off percentage for risky combinatory genotypes in each gene cluster, the 14 gene clusters achieved a classification accuracy of 82.8% for all datasets and 98.9% if the information-short dataset was excluded. Furthermore, not only 10 of the 14 major genes but also many other contributing genes in the clusters are associated with either hypertension or hypertension-related diseases or functions. CONCLUSIONS: We have shown with the constructed gene clusters that a multi-causal pie-multi-component approach can indeed improve the reproducibility of genetic markers for complex disease. In addition, our novel findings including a major gene in each cluster and sufficient risky genotypes in a cluster for disease onset (which coincides with Rothman's sufficient cause theory) may not only provide a new research direction for complex diseases but also help to reveal the disease etiology.

Characterizing regulatory and functional differentiation between maize mesophyll and bundle sheath cells by transcriptomic analysis.

To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C(4) metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.

Epigallocatechin-3-gallate-mediated cardioprotection by Akt/GSK-3ß/caveolin signalling in H9c2 rat cardiomyoblasts.

BACKGROUND: Epigallocatechin-3-gallate (EGCg) with its potent anti-oxidative capabilities is known for its beneficial effects ameliorating oxidative injury to cardiac cells. Although studies have provided convincing evidence to support the cardioprotective effects of EGCg, it remains unclear whether EGCg affect trans-membrane signalling in cardiac cells. Here, we have demonstrated the potential mechanism for cardioprotection of EGCg against H2O2-induced oxidative stress in H9c2 cardiomyoblasts. RESULTS: Exposing H9c2 cells to H2O2 suppressed cell viability and altered the expression of adherens and gap junction proteins with increased levels of intracellular reactive oxygen species and cytosolic Ca2+. These detrimental effects were attenuated by pre-treating cells with EGCg for 30 min. EGCg also attenuated H2O2-mediated cell cycle arrest at the G1-S phase through the glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin/cyclin D1 signalling pathway. To determine how EGCg targets H9c2 cells, enhanced green fluorescence protein (EGFP) was ectopically expressed in these cells. EGFP-emission fluorescence spectroscopy revealed that EGCg induced dose-dependent fluorescence changes in EGFP expressing cells, suggesting that EGCg signalling events might trigger proximity changes of EGFP expressed in these cells. Proteomics studies showed that EGFP formed complexes with the 67 kD laminin receptor, caveolin-1 and -3, ß-actin, myosin 9, vimentin in EGFP expressing cells. Using in vitro oxidative stress and in vivo myocardial ischemia models, we also demonstrated the involvement of caveolin in EGCg-mediated cardioprotection. In addition, EGCg-mediated caveolin-1 activation was found to be modulated by Akt/GSK-3ß signalling in H2O2-induced H9c2 cell injury. CONCLUSIONS: Our data suggest that caveolin serves as a membrane raft that may help mediate cardioprotective EGCg transmembrane signalling.

Response of rice (Oryza sativa L.) roots to nanoplastic treatment at seedling stage.

Potential adverse effects of nanoplastics (NPs) on marine organisms have received increased attention in recent years. In contrast, few data are available on terrestrial plants, especially on the mechanisms for transport of NPs in plants and phytotoxicity (at both phenotypic and molecular levels) of plants induced by NPs. To address this knowledge gap, we conducted a microcosm study in which hydroponically-cultured rice (Oryza sativa L.) seedlings were exposed to polystyrene (PS)-NPs at 0, 10, 50, and 100 mg L-1 for 16 d and examined for morphological and physiological phenotypes and transcriptomics. Laser confocal scanning micrographs confirmed PS-NPs were uptaken by rice roots, greatly benefitted from the transport activity of aquaporin in rice roots. The significant enhancement (p < 0.05) of antioxidant enzyme activities reflected the oxidative stress response of rice roots upon exposure to PS-NPs. Treatment by PS-NPs decreased root length and increased lateral root numbers. Carbon metabolism was activated (e.g., increased carbon and soluble sugar contents) whereas jasmonic acid and lignin biosynthesis were inhibited. The present study demonstrated the likelihood for transport of PS-NPs in rice roots and induced phytotoxicity by PS-NPs, which should inspire further investigations into the potential human health risks from rice consumption.