Plastid-derived signals are known to coordinate expression of nuclear genes encoding plastid-localized proteins in a process termed retrograde signaling. To date, the identity of retrograde-signaling ...molecules has remained elusive. Here, we show that methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids produced by the plastidial methylerythritol phosphate (MEP) pathway, elicits the expression of selected stress-responsive nuclear-encoded plastidial proteins. Genetic and pharmacological manipulations of the individual MEP pathway metabolite levels demonstrate the high specificity of MEcPP as an inducer of these targeted stress-responsive genes. We further demonstrate that abiotic stresses elevate MEcPP levels, eliciting the expression of the aforementioned genes. We propose that the MEP pathway, in addition to producing isoprenoids, functions as a stress sensor and a coordinator of expression of targeted stress-responsive nuclear genes via modulation of the levels of MEcPP, a specific and critical retrograde-signaling metabolite.
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► The MEP pathway produces plastidial isoprenoids and serves as a plant stress sensor ► Excess light or wounding elevates the levels of isoprenoids intermediate, MEcPP ► Accumulation of MEcPP induces expression of selected stress genes in the nucleus ► MEcPP is a retrograde-signaling metabolite coordinating stress-response pathways
A metabolite produced in plastids in response to stress in turn activates stress-response genes in the nucleus, providing insight into the signaling mechanisms that link plastids and nuclei.
Much effort has been dedicated to developing circulating tumor cells (CTC) as a noninvasive cancer biopsy, but with limited success as yet. In this study, we combine a method for isolation of highly ...pure CTCs using immunomagnetic enrichment/fluorescence-activated cell sorting with advanced whole genome sequencing (WGS), based on long fragment read technology, to illustrate the utility of an accurate, comprehensive, phased, and quantitative genomic analysis platform for CTCs. Whole genomes of 34 CTCs from a patient with metastatic breast cancer were analyzed as 3,072 barcoded subgenomic compartments of long DNA. WGS resulted in a read coverage of 23× per cell and an ensemble call rate of >95%. These barcoded reads enabled accurate detection of somatic mutations present in as few as 12% of CTCs. We found in CTCs a total of 2,766 somatic single-nucleotide variants and 543 indels and multi-base substitutions, 23 of which altered amino acid sequences. Another 16,961 somatic single nucleotide variant and 8,408 indels and multi-base substitutions, 77 of which were nonsynonymous, were detected with varying degrees of prevalence across the 34 CTCs. On the basis of our whole genome data of mutations found in all CTCs, we identified driver mutations and the tissue of origin of these cells, suggesting personalized combination therapies beyond the scope of most gene panels. Taken together, our results show how advanced WGS of CTCs can lead to high-resolution analyses of cancers that can reliably guide personalized therapy.
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Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been ...shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼ 100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.
Cereals accumulate starch in the endosperm as their major energy reserve in the grain. In most cereals the embryo, scutellum, and aleurone layer are high in oil, but these tissues constitute a very ...small part of the total seed weight. However, in oat (Avena sativa L.) most of the oil in kernels is deposited in the same endosperm cells that accumulate starch. Thus oat endosperm is a desirable model system to study the metabolic switches responsible for carbon partitioning between oil and starch synthesis. A prerequisite for such investigations is the development of an experimental system for oat that allows for metabolic flux analysis using stable and radioactive isotope labelling. An in vitro liquid culture system, developed for detached oat panicles and optimized to mimic kernel composition during different developmental stages in planta, is presented here. This system was subsequently used in analyses of carbon partitioning between lipids and carbohydrates by the administration of 14C-labelled sucrose to two cultivars having different amounts of kernel oil. The data presented in this study clearly show that a higher amount of oil in the high-oil cultivar compared with the medium-oil cultivar was due to a higher proportion of carbon partitioning into oil during seed filling, predominantly at the earlier stages of kernel development.
Brachypodium distachyon (Brachypodium) has been proposed as a model temperate grass because its physical, genetic, and genome attributes (small stature, simple growth requirements, small genome size, ...availability of diploid ecotypes, annual lifecycle and self fertility) are suitable for a model plant system. Two additional requirements that are necessary before Brachypodium can be widely accepted as a model system are an efficient transformation system and homogeneous inbred reference genotypes. Here we describe the development of inbred lines from 27 accessions of Brachypodium. Determination of c-values indicated that five of the source accessions were diploid. These diploid lines exhibit variation for a variety of morphological traits. Conditions were identified that allow generation times as fast as two months in the diploids. An Agrobacterium-mediated transformation protocol was developed and used to successfully transform 10 of the 19 lines tested with efficiencies ranging from 0.4% to 15%. The diploid accession Bd21 was readily transformed. Segregation of transgenes in the T ₁ generation indicated that most of the lines contained an insertion at a single genetic locus. The new resources and methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.
Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of ...10.9% of the Brachypodium genome based on 64,696 bacterial artificial chromosome (BAC) end sequences (BES). Analysis of repeat DNA content in BES revealed that approximately 11.0% of the genome consists of known repetitive DNA. The vast majority of the Brachypodium repetitive elements are LTR retrotransposons. While Bare-1 retrotransposons are common to wheat and barley, Brachypodium repetitive element sequence-1 (BRES-1), closely related to Bare-1, is also abundant in Brachypodium. Moreover, unique Brachypodium repetitive element sequences identified constitute approximately 7.4% of its genome. Simple sequence repeats from BES were analyzed, and flanking primer sequences for SSR detection potentially useful for genetic mapping are available at http://brachypodium.pw.usda.gov. Sequence analyses of BES indicated that approximately 21.2% of the Brachypodium genome represents coding sequence. Furthermore, Brachypodium BES have more significant matches to ESTs from wheat than rice or maize, although these species have similar sizes of EST collections. A phylogenetic analysis based on 335 sequences shared among seven grass species further revealed a closer relationship between Brachypodium and Triticeae than Brachypodium and rice or maize.
Summary
To identify the underlying molecular basis of carbon partitioning between starch and oil we conducted 454 pyrosequencing, followed by custom microarrays to profile gene expression throughout ...endosperm development, of two closely related oat cultivars that differ in oil content at the expense of starch as determined by several approaches including non‐invasive magnetic resonance imaging. Comparative transcriptome analysis in conjunction with metabolic profiling displays a close coordination between energy metabolism and carbon partitioning pathways, with increased demands for energy and reducing equivalents in kernels with a higher oil content. These studies further expand the repertoire of networks regulating carbon partitioning to those involved in metabolism of cofactors, suggesting that an elevated supply of cofactors, here called cofactomes, contribute to the allocation of higher carbon pools for production of oils and storage proteins. These data highlight a close association between cofactomes and carbon partitioning, thereby providing a biotechnological target for conversion of starch to oil.
Brachypodium distachyon (Brachypodium) is a temperate grass with the physical and genomic attributes necessary for a model system (small size, rapid generation time, self-fertile, small genome size, ...diploidy in some accessions). To increase the utility of Brachypodium as a model grass, we sequenced 20,440 expressed sequence tags (ESTs) from five cDNA libraries made from leaves, stems plus leaf sheaths, roots, callus and developing seed heads. The ESTs had an average trimmed length of 650 bp. Blast nucleotide alignments against SwissProt and GenBank non-redundant databases were performed and a total of 99.9% of the ESTs were found to have some similarity to existing protein or nucleotide sequences. Tentative functional classification of 77% of the sequences was possible by association with gene ontology or clusters of orthologous group's index descriptors. To demonstrate the utility of this EST collection for studying cell wall composition, we identified homologs for the genes involved in the biosynthesis of lignin subunits. A subset of the ESTs was used for phylogenetic analysis that reinforced the close relationship of Brachypodium to wheat and barley.
This study was conducted to identify senescence-regulated signaling pathways and genes in the monocot
Anthurium andraeanum L. for future efforts to modify leaf senescence via biotechnology. The
...Arabidopsis senescence-activated promoter (Pr-
SAG12) fused to the
gfp4 gene was expressed 20-fold higher, while the Pr35S-
gfp4 was expressed 30% lower, in senescing relative to healthy leaves of
Anthurium. This indicated that a senescence-activated pathway correctly regulated the
Arabidopsis sag12 cysteine protease gene and suggested the presence of
sag12 homologues in
Anthurium. To identify
sag12 homologs, two different
Anthurium cysteine protease cDNAs,
anth16 and
anth17, were cloned and their sequences, expression, and protein products were characterized. The Anth17 and Anth16 protein sequences were 58 and 51% identical with Sag12, respectively. Of the two genes,
anth17 expression most closely resembled the
sag12 gene.
Anth16 was expressed primarily in healthy immature leaves, while
anth17 and a control gene (ubiquitin) were significantly induced during senescence of mature leaves. The levels of known internal standards, chlorophyll, mRNAs for
cab (chlorophyll-a,b-binding protein) and
psbA (D1 protein of PSII), decreased during senescence and served to verify the progression of senescence. Cytokinin treatments repressed
anth17 mRNA accumulation and sustained
psbA mRNA levels in senescing leaves. Sucrose moderately reduced
anth17 and
psbA mRNA levels. Anth16 has a unique secretory vesicle sorting motif in the C-terminus, and produces a 70
kDa in vitro translation product that is post-translationally processed to 57
kDa by microsomal membranes, a property of secretory proteins. In contrast,
anth17 was not processed and its intracellular retention is proposed to be necessary for senescence-dependent cellular disassembly and proteolysis.
Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease ...are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2−16), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.
•Dense sequencing of an ovarian cancer identifies founder non-coding mutations.•Fallopian tube SOX2 overexpression is a common premalignant feature in ovarian cancer.•The expression of SOX2 and MYC in the fallopian tube appears mutually exclusive.
A major obstacle for effective early detection of ovarian cancer is that CA125 and ultrasound can only detect established ovarian cancer. Currently proposed pre-cancer lesions are not amenable to clinical detection because they affect only a small number of cells. In this work we demonstrated that pre-cancer SOX2 overexpression in the FTE is nearly ubiquitous in HGSOCs and is also a common feature in women with BRCA1 and BRCA2 mutations prior to ovarian cancer initiation. Thus, our data has important implications for screening, although it will need additional clinical evaluation to test the feasibility of quantitative detection of SOX2 expression or the expression of its downstream targets for this purpose.
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