Plant Genomics
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De Novo assembly of a plant transcriptome - SEQanswers

I work with Bioinformatics in a lab and our group received the task of mounting a plant transcriptome using sequences from 454. We still don't have the genome sequenced so we are now defining our strategies for this project.
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Scooped by Biswapriya Biswavas Misra
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Transcriptome variation along bud development in grapevine (Vitis vinifera L.)

Transcriptome variation along bud development in grapevine (Vitis vinifera L.) | Plant Genomics | Scoop.it

Abstract (provisional)

Background

Vegetative buds provide plants in temperate environments the possibility for growth and reproduction when environmental conditions are favorable. In grapevine, crucial developmental events take place within buds during two growing seasons in consecutive years. The first season, the shoot apical meristem within the bud differentiates all the basic elements of the shoot including flowering transition in lateral primordia and development of inflorescence primordia. These events practically end with bud dormancy. The second season, buds resume shoot growth associated to flower formation and development. Gene expression has been previously monitored at specific stages of bud development but has never been followed along the two growing seasons.

Results

Gene expression changes were analyzed along the bud annual cycle at eight different time points. Principal Components Analysis (PCA) revealed that the main factors explaining the global gene expression differences were the processes of bud dormancy and active growth as well as stress responses. Accordingly, non dormant buds showed an enrichment in functional categories typical of actively proliferating and growing cells together with the over abundance of transcripts belonging to stress response pathways. Differential expression analyses performed between consecutive time points indicated that major transcriptional changes were associated to para/endodormancy, endo/ecodormancy and ecodormancy/bud break transitions. Transcripts encoding key regulators of reproductive development were grouped in three major expression clusters corresponding to: (i) transcripts associated to flowering induction, (ii) transcripts associated to flower meristem specification and initiation and (iii) transcripts putatively involved in dormancy. Within this cluster, a MADS-box gene (VvFLC2) and other transcripts with similar expression patterns could participate in dormancy regulation.

Conclusions

This work provides a global view of major transcriptional changes taking place along bud development in grapevine, highlighting those molecular and biological functions involved in the main events of bud development. As reported in other woody species, the results suggest that genes regulating flowering could also be involved in dormancy regulatory pathways in grapevine.

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Rescooped by Biswapriya Biswavas Misra from Plant immunity and legume symbiosis
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Genome-Wide Distribution, Organisation and Functional Characterization of Disease Resistance and Defence Response Genes across Rice Species

Genome-Wide Distribution, Organisation and Functional Characterization of Disease Resistance and Defence Response Genes across Rice Species | Plant Genomics | Scoop.it

The resistance (R) genes and defense response (DR) genes have become very important resources for the development of disease resistant cultivars. In the present investigation, genome-wide identification, expression, phylogenetic and synteny analysis was done for R and DR-genes across three species of rice viz: Oryza sativa ssp indica cv 93-11, Oryza sativa ssp japonica and wild rice species, Oryza brachyantha. We used the in silico approach to identify and map 786 R -genes and 167 DR-genes, 672 R-genes and 142 DR-genes, 251 R-genes and 86 DR-genes in the japonica, indica and O. brachyanth a genomes, respectively. Our analysis showed that 60.5% and 55.6% of the R-genes are tandemly repeated within clusters and distributed over all the rice chromosomes in indica and japonica genomes, respectively. The phylogenetic analysis along with motif distribution shows high degree of conservation of R- and DR-genes in clusters. In silico expression analysis of R-genes and DR-genes showed more than 85% were expressed genes showing corresponding EST matches in the databases. This study gave special emphasis on mechanisms of gene evolution and duplication for R and DR genes across species. Analysis of paralogs across rice species indicated 17% and 4.38% R-genes, 29% and 11.63% DR-genes duplication in indica and Oryza brachyantha, as compared to 20% and 26% duplication of R-genes and DR-genes in japonica respectively. We found that during the course of duplication only 9.5% of R- and DR-genes changed their function and rest of the genes have maintained their identity. Syntenic relationship across three genomes inferred that more orthology is shared between indica and japonica genomes as compared to brachyantha genome. Genome wide identification of R-genes and DR-genes in the rice genome will help in allele mining and functional validation of these genes, and to understand molecular mechanism of disease resistance and their evolution in rice and related species.


Via Christophe Jacquet
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Rescooped by Biswapriya Biswavas Misra from Bioenergetic and Ion Signalling in Plant-Microbe Symbiosis
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Heterologous expression of the halophyte Zoysia matrella H+-pyrophosphatase gene improved salt tolerance in Arabidopsis thaliana

Heterologous expression of the halophyte Zoysia matrella H+-pyrophosphatase gene improved salt tolerance in Arabidopsis thaliana | Plant Genomics | Scoop.it

A number of vacuolar H+-pyrophosphatase (VP) family genes play important roles in plant growth under salt stress condition. Despite their biological importance in plant salt-stress regulation, there is no report about VP in the halophytic turfgrass Zoysia matrella. Here, we isolated ZmVP1, a type I VP homologues gene encoding 768 amino acids by using the degenerated PCR and RACE PCR methods from Zoysia matrella. The expression level of ZmVP1 was significantly induced by salinity, drought and cold, but not by heat. ZmVP1 can restore the salt-tolerant ability of a salt-sensitive yeast strain. Overexpression of ZmVP1in Arabidopsis thaliana resulted in more vigorous growth under salt stress. Moreover, the transgenic Arabidopsis accumulated more Na+ and K+ in the leaves compared to that of wild type plants under salt stress, had higher activities of V-ATPase and V-PPase, and showed higher relative gene expression levels of 5 stress-related genes (AtNHX1, AtLEA, AtP5CS, AtMn-SOD, AtAPX1). These results demonstrated that ZmVP1 from Z. matrella was a functional tonoplast H+-pyrophosphatase contributing to salt tolerance potentially through regulating the Na+ compartment in vacuole, K+ assimilation, osmotic regulation and antioxidant response.


Via Alessandro Coutinho Ramos
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Rescooped by Biswapriya Biswavas Misra from Plant & Evolution
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Genome-wide association of carbon and nitrogen metabolism in the maize nested association mapping population

Carbon (C) and nitrogen (N) metabolism are critical to plant growth and development and are at the basis of crop yield and adaptation. We performed high-throughput metabolite analyses on over 12,000 samples from the maize Nested Association Mapping population to identify genetic variation in C and N metabolism in maize (Zea mays subsp. mays). All samples were grown in the same field and used to identify natural variation controlling the levels of twelve key C and N metabolites, namely chlorophyll a, chlorophyll b, fructose, fumarate, glucose, glutamate, malate, nitrate, starch, sucrose, total amino acids, and total protein, along with the first two principal components derived from them. Our GWAS results frequently identified hits with single-gene resolution. In addition to expected genes such as invertases, natural variation was identified in key C4 metabolism genes including carbonic anhydrases and a malate transporter. Unlike several prior maize studies, extensive pleiotropy was found for C and N metabolites. This integration of field-derived metabolite data with powerful mapping and genomics resources allows for dissection of key metabolic pathways, providing avenues for future genetic improvement.


Via Pierre-Marc Delaux
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Poplar trees reconfigure the transcriptome and metabolome in response to drought in a genotype- and time-of-day-dependent manner

Drought has a major impact on tree growth and survival. Understanding tree responses to this stress can have important application in both conservation of forest health, and in production forestry. Trees of the genus Populus provide an excellent opportunity to explore the mechanistic underpinnings of forest tree drought responses, given the growing molecular resources that are available for this taxon. Here, foliar tissue of six water-deficit stressed P. balsamifera genotypes was analysed for variation in the metabolome in response to drought and time of day by using an untargeted metabolite profiling technique, gas chromatography/mass-spectrometry (GC/MS).
Biswapriya Biswavas Misra's insight:

Background Drought has a major impact on tree growth and survival. Understanding tree responses to this stress can have important application in both conservation of forest health, and in production forestry. Trees of the genus Populus provide an excellent opportunity to explore the mechanistic underpinnings of forest tree drought responses, given the growing molecular resources that are available for this taxon. Here, foliar tissue of six water-deficit stressed P. balsamifera genotypes was analysed for variation in the metabolome in response to drought and time of day by using an untargeted metabolite profiling technique, gas chromatography/mass-spectrometry (GC/MS). Results Significant variation in the metabolome was observed in response the imposition of water-deficit stress. Notably, organic acid intermediates such as succinic and malic acid had lower concentrations in leaves exposed to drought, whereas galactinol and raffinose were found in increased concentrations. A number of metabolites with significant difference in accumulation under water-deficit conditions exhibited intraspecific variation in metabolite accumulation. Large magnitude fold-change accumulation was observed in three of the six genotypes. In order to understand the interaction between the transcriptome and metabolome, an integrated analysis of the drought-responsive transcriptome and the metabolome was performed. One P. balsamifera genotype, AP-1006, demonstrated a lack of congruence between the magnitude of the drought transcriptome response and the magnitude of the metabolome response. More specifically, metabolite profiles in AP-1006 demonstrated the smallest changes in response to water-deficit conditions. Conclusions Pathway analysis of the transcriptome and metabolome revealed specific genotypic responses with respect to primary sugar accumulation, citric acid metabolism, and raffinose family oligosaccharide biosynthesis. The intraspecific variation in the molecular strategies that underpin the responses to drought among genotypes may have an important role in the maintenance of forest health and productivity.

 
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Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins | Plant Genomics | Scoop.it
Biswapriya Biswavas Misra's insight:

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.

 
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Rescooped by Biswapriya Biswavas Misra from Genomic Parasites: Coevolution between host and parasites
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Discovery of Novel Genes Derived from Transposable Elements Using Integrative Genomic Analysis

Discovery of Novel Genes Derived from Transposable Elements Using Integrative Genomic Analysis | Plant Genomics | Scoop.it
Complex eukaryotes contain millions of transposable elements (TEs), comprising large fractions of their nuclear genomes. TEs consist of structural, regulatory, and coding sequences that are ordinarily associated with transposition, but that occasionally confer on the organism a selective advantage and may thereby become exapted. Exapted transposable element genes (ETEs) are known to play critical roles in diverse systems, from vertebrate adaptive immunity to plant development. Yet despite their evident importance, most ETEs have been identified fortuitously and few systematic searches have been conducted, suggesting that additional ETEs may await discovery. To explore this possibility, we develop a comprehensive systematic approach to searching for ETEs. We use TE-specific conserved domains to identify with high precision genes derived from TEs and screen them for signatures of exaptation based on their similarities to reference sets of known ETEs, conventional (non-TE) genes, and TE genes across diverse genetic attributes including repetitiveness, conservation of genomic location and sequence, and levels of expression and repressive small RNAs. Applying this approach in the model plant Arabidopsis thaliana, we discover a surprisingly large number of novel high confidence ETEs. Intriguingly, unlike known plant ETEs, several of the novel ETE families form tandemly arrayed gene clusters, whereas others are relatively young. Our results not only identify novel TE-derived genes that may have practical applications but also challenge the notion that TE exaptation is merely a relic of ancient life, instead suggesting that it may continue to fundamentally drive evolution.

Via Gabriel Wallau
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Function of the Pseudomonas aeruginosa NrdR Transcription Factor: Global Transcriptomic Analysis and Its Role on Ribonucleotide Reductase Gene Expression

Function of the Pseudomonas aeruginosa NrdR Transcription Factor: Global Transcriptomic Analysis and Its Role on Ribonucleotide Reductase Gene Expression | Plant Genomics | Scoop.it
PLoS One. 2015 Apr 24;10(4):e0123571. doi: 10.1371/journal.pone.0123571.
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Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair development through salicylic acid signall...

Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair development through salicylic acid signall... | Plant Genomics | Scoop.it
BMC Genomics. 2015 Apr 24;16(1):341. [Epub ahead of print]
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AbstractBACKGROUND:

The impact of nano-scaled materials on photosynthetic organisms needs to be evaluated. Plants represent the largest interface between the environment and biosphere, so understanding how nanoparticles affect them is especially relevant for environmental assessments. Nanotoxicology studies in plants allude to quantum size effects and other properties specific of the nano-stage to explain increased toxicity respect to bulk compounds. However, gene expression profiles after exposure to nanoparticles and other sources of environmental stress have not been compared and the impact on plant defence has not been analysed.

RESULTS:

Arabidopsis plants were exposed to TiO2-nanoparticles, Ag-nanoparticles, and multi-walled carbon nanotubes as well as different sources of biotic (microbial pathogens) or abiotic (saline, drought, or wounding) stresses. Changes in gene expression profiles and plant phenotypic responses were evaluated. Transcriptome analysis shows similarity of expression patterns for all plants exposed to nanoparticles and a low impact on gene expression compared to other stress inducers. Nanoparticle exposure repressed transcriptional responses to microbial pathogens, resulting in increased bacterial colonization during an experimental infection. Inhibition of root hair development and transcriptional patterns characteristic of phosphate starvation response were also observed. The exogenous addition of salicylic acid prevented some nano-specific transcriptional and phenotypic effects, including the reduction in root hair formation and the colonization of distal leaves by bacteria.

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Response of Prochlorococcus to varying CO2:O2 ratios

The ISME Journal: Multidisciplinary Journal of Microbial Ecology is the official Journal of the International Society for Microbial Ecology, publishing high-quality, original research papers, short communications, commentary articles and reviews in the rapidly expanding and diverse discipline of microbial ecology.
Biswapriya Biswavas Misra's insight:

Carbon fixation has a central role in determining cellular redox poise, increasingly understood to be a key parameter in cyanobacterial physiology. In the cyanobacterium Prochlorococcus—the most abundant phototroph in the oligotrophic oceans—the carbon-concentrating mechanism is reduced to the bare essentials. Given the ability of Prochlorococcus populations to grow under a wide range of oxygen concentrations in the ocean, we wondered how carbon and oxygen physiology intersect in this minimal phototroph. Thus, we examined how CO2:O2 gas balance influenced growth and chlorophyll fluorescence in Prochlorococcus strain MED4. Under O2 limitation, per-cell chlorophyll fluorescence fell at all CO2 levels, but still permitted substantial growth at moderate and high CO2. Under CO2 limitation, we observed little growth at any O2 level, although per-cell chlorophyll fluorescence fell less sharply when O2 was available. We explored this pattern further by monitoring genome-wide transcription in cells shocked with acute limitation of CO2, O2 or both. O2 limitation produced much smaller transcriptional changes than the broad suppression seen under CO2 limitation and CO2/O2 co-limitation. Strikingly, both CO2 limitation conditions initially evoked a transcriptional response that resembled the pattern previously seen in high-light stress, but at later timepoints we observed O2-dependent recovery of photosynthesis-related transcripts. These results suggest that oxygen has a protective role in Prochlorococcus when carbon fixation is not a sufficient sink for light energy.

 
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Deep Sequencing of Virus-Derived Small Interfering RNAs and RNA from Viral Particles Shows Highly Similar Mutational Landscapes of a Plant Virus Population

Biswapriya Biswavas Misra's insight:

RNA viruses exist within a host as a population of mutant sequences, often referred to as quasispecies. Within a host, sequences of RNA viruses constitute several distinct but interconnected pools, such as RNA packed in viral particles, double-stranded RNA, and virus-derived small interfering RNAs. We aimed to test if the same representation of within-host viral population structure could be obtained by sequencing different viral sequence pools. Using ultradeep Illumina sequencing, the diversity of two coexisting Potato virus Y sequence pools present within a plant was investigated: RNA isolated from viral particles and virus-derived small interfering RNAs (the derivatives of a plant RNA silencing mechanism). The mutational landscape of the within-host virus population was highly similar between both pools, with no notable hotspots across the viral genome. Notably, all of the single-nucleotide polymorphisms with a frequency of higher than 1.6% were found in both pools. Some unique single-nucleotide polymorphisms (SNPs) with very low frequencies were found in each of the pools, with more of them occurring in the small RNA (sRNA) pool, possibly arising through genetic drift in localized virus populations within a plant and the errors introduced during the amplification of silencing signal. Sequencing of the viral particle pool enhanced the efficiency of consensus viral genome sequence reconstruction. Nonhomologous recombinations were commonly detected in the viral particle pool, with a hot spot in the 3′ untranslated and coat protein regions of the genome. We stress that they present an important but often overlooked aspect of virus population diversity.

 
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Transcriptomic profiling of linolenic acid-responsive genes in ROS signaling from RNA-seq data in Arabidopsis.

Front Plant Sci. 2015 Mar 17;6:122. doi: 10.3389/fpls.2015.00122. eCollection 2015.
Biswapriya Biswavas Misra's insight:

Linolenic acid (Ln) released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA). The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signaling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC) with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2-fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs) and Allene oxide cyclases (AOCs). In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding), such as WRKY, JAZ, MYC, and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signaling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1), methionine sulfoxide reductase (MSR) and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signaling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity) and especially in stresses mediated by ROS.

  
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Transcriptomic analysis of differentially expressed genes in an orange-pericarp mutant and wild type in pummelo (Citrus grandis).

BMC Plant Biol. 2015 Feb 12;15:44. doi: 10.1186/s12870-015-0435-3.
Biswapriya Biswavas Misra's insight:
AbstractBACKGROUND:

The external colour of fruit is a crucial quality feature, and the external coloration of most citrus fruits is due to the accumulation of carotenoids. The molecular regulation of carotenoid biosynthesis and accumulation in pericarp is limited due to the lack of mutant. In this work, an orange-pericarp mutant (MT) which showed altered pigmentation in the pericarp was used to identify genes potentially related to the regulation of carotenoid accumulation in the pericarp.

RESULTS:

High Performance Liquid Chromatography (HPLC) analysis revealed that the pericarp from MT fruits had a 10.5-fold increase of β-carotene content over that of the Wild Type (WT). Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of all downstream carotenogenic genes was lower in MT than in WT, suggesting that down-regulation is critical for the β-carotene increase in the MT pericarp. RNA-seq analysis of the transcriptome revealed extensive changes in the MT gene expression level, with 168 genes down-regulated and 135 genes up-regulated. Gene ontology (GO) and KEGG pathway analyses indicated seven reliable metabolic pathways are altered in the mutant, including carbon metabolism, starch and sucrose metabolism and biosynthesis of amino acids. The transcription factors and genes corresponding to effected metabolic pathways may involved in the carotenoid regulation was confirmed by the qRT-PCR analysis in the MT pericarp.

CONCLUSIONS:

This study has provided a global picture of the gene expression changes in a novel mutant with distinct color in the fruit pericarp of pummelo. Interpretation of differentially expressed genes (DEGs) revealed new insight into the molecular regulation of β-carotene accumulation in the MT pericarp.

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The AVRDC – The World Vegetable Center mungbean (Vigna radiata) core and mini core collections

Large ex situ germplasm collections generally harbor a wide range of crop diversity. AVRDC – The World Vegetable Center is holding in trust the world’s second largest mungbean (Vigna radiata) germplasm collection with more than 6,700 accessions. Screening large collections for traits of interest is laborious and expensive. To enhance the access of breeders to the diversity of the crop, mungbean core and mini core collections have been established.
Biswapriya Biswavas Misra's insight:

Background Large ex situ germplasm collections generally harbor a wide range of crop diversity. AVRDC – The World Vegetable Center is holding in trust the world’s second largest mungbean (Vigna radiata) germplasm collection with more than 6,700 accessions. Screening large collections for traits of interest is laborious and expensive. To enhance the access of breeders to the diversity of the crop, mungbean core and mini core collections have been established. Results The core collection of 1,481 entries has been built by random selection of 20% of the accessions after geographical stratification and subsequent cluster analysis of eight phenotypic descriptors in the whole collection. Summary statistics, especially the low differences of means, equal variance of the traits in both the whole and core collection and the visual inspection of quantile-quantile plots comparing the variation of phenotypic traits present in both collections indicated that the core collection well represented the pattern of diversity of the whole collection. The core collection was genotyped with 20 simple sequence repeat markers and a mini core set of 289 accessions was selected, which depicted the allele and genotype diversity of the core collection. Conclusions The mungbean core and mini core collections plus their phenotypic and genotypic data are available for distribution to breeders. It is expected that these collections will enhance the access to biodiverse mungbean germplasm for breeding.

 
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Combined genetic and transcriptomic analysis reveals three major signalling pathways activated by Myc-LCOs in Medicago truncatula

Combined genetic and transcriptomic analysis reveals three major signalling pathways activated by Myc-LCOs in Medicago truncatula | Plant Genomics | Scoop.it
Myc-LCOs are newly identified symbiotic signals produced by arbuscular mycorrhizal (AM) fungi. Like rhizobial Nod factors, they are lipo-chitooligosaccharides that activate the common symbiotic signalling pathway (CSSP) in plants. To increase our limited understanding of the roles of Myc-LCOs we aimed to analyse Myc-LCO-induced transcriptional changes and their genetic control.
Whole genome RNA sequencing (RNA-seq) was performed on roots of Medicago truncatula wild-type plants, and dmi3 and nsp1 symbiotic mutants affected in nodulation and mycorrhizal signalling. Plants were treated separately with the two major types of Myc-LCOs, sulphated and nonsulphated.
Generalized linear model analysis identified 2201 differentially expressed genes and classified them according to genotype and/or treatment effects. Three genetic pathways for Myc-LCO-regulation of transcriptomic reprogramming were highlighted: DMI3- and NSP1-dependent; DMI3-dependent and NSP1-independent; and DMI3- and NSP1-independent. Comprehensive analysis revealed overlaps with previous AM studies, and highlighted certain functions, especially signalling components and transcription factors.
These data provide new insights into mycorrhizal signalling mechanisms, supporting a role for NSP1, and specialisation for NSP1-dependent and -independent pathways downstream of DMI3. Our data also indicate significant Myc-LCO-activated signalling upstream of DMI3 and/or parallel to the CSSP and some constitutive activity of the CSSP.

Via Jean-Michel Ané
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G&T-seq: parallel sequencing of single-cell genomes and transcriptomes : Nature Methods : Nature Publishing Group

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes : Nature Methods : Nature Publishing Group | Plant Genomics | Scoop.it
G&T-seq offers robust full-length transcript and whole-genome sequencing simultaneously from a single cell.
Biswapriya Biswavas Misra's insight:

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

  
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Proteomic Profiling of 16 Cereal Grains and the Application of Targeted Proteomics To Detect Wheat Contamination

Proteomic Profiling of 16 Cereal Grains and the Application of Targeted Proteomics To Detect Wheat Contamination | Plant Genomics | Scoop.it
Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC–MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.
Biswapriya Biswavas Misra's insight:

Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC–MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R2 > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.

 
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Comparative proteomic analysis of melon phloem exudates in response to viral infection

Phloem vasculature is the route that most plant viruses use to spread widely around the plant. In addition, phloem sap transports signals that trigger systemic defense responses to infection. We investigated the proteome-level changes that occur in phloem sap during virus infection using the 2D-DIGE technique. Total proteins were extracted from phloem exudates of healthy and Melon necrotic spot virus infected melon plants and analyzed by 2D-DIGE. A total of 1046 spots were detected but only 25 had significant changes in abundance. After mass spectrometry, 19 different proteins corresponding to 22 spots were further identified (13 of them up-accumulated and 9 down-accumulated). Most of them were involved in controlling redox balance and cell death. Only two of the differentially altered proteins had never been described to be present in the phloem before: a carboxylesterase and the fumarylacetoacetate hydrolase 1, both considered negative regulators of cell death. RT-PCR analysis of phloem sap RNAs revealed that the transcripts corresponding to some of the identified protein could be also loaded into the sieve elements. The impact of these proteins in the host response against viral infections and the potential involvement in regulating development, growth and stress response in melon plants is discussed.
Biswapriya Biswavas Misra's insight:

Phloem vasculature is the route that most plant viruses use to spread widely around the plant. In addition, phloem sap transports signals that trigger systemic defense responses to infection. We investigated the proteome-level changes that occur in phloem sap during virus infection using the 2D-DIGE technique. Total proteins were extracted from phloem exudates of healthy and Melon necrotic spot virus infected melon plants and analyzed by 2D-DIGE. A total of 1046 spots were detected but only 25 had significant changes in abundance. After mass spectrometry, 19 different proteins corresponding to 22 spots were further identified (13 of them up-accumulated and 9 down-accumulated). Most of them were involved in controlling redox balance and cell death. Only two of the differentially altered proteins had never been described to be present in the phloem before: a carboxylesterase and the fumarylacetoacetate hydrolase 1, both considered negative regulators of cell death. RT-PCR analysis of phloem sap RNAs revealed that the transcripts corresponding to some of the identified protein could be also loaded into the sieve elements. The impact of these proteins in the host response against viral infections and the potential involvement in regulating development, growth and stress response in melon plants is discussed.

 
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Genomic variation across landscapes: insights and applications

Genomic variation across landscapes: insights and applications | Plant Genomics | Scoop.it
The distribution of genomic variation across landscapes can provide insights into the complex interactions between the environment and the genome that influence the distribution of species, and mediate phenotypic adaptation to local conditions. High throughput sequencing technologies now offer unprecedented power to explore these interactions, allowing powerful inferences about historical processes of colonization, gene flow and divergence, as well as the identification of loci that mediate local adaptation. These ‘landscape genomic’ approaches have been validated in model species and are now being applied to nonmodel organisms, including foundation species that have substantial effects on ecosystem processes. Here we review the growing field of landscape genomics from a very broad perspective. In particular, we describe the inferential power that is gained by taking a genome-wide view of genetic variation, strategies for study design to best capture adaptive variation, and how to apply this information to practical challenges, such as restoration.
Biswapriya Biswavas Misra's insight:

The distribution of genomic variation across landscapes can provide insights into the complex interactions between the environment and the genome that influence the distribution of species, and mediate phenotypic adaptation to local conditions. High throughput sequencing technologies now offer unprecedented power to explore these interactions, allowing powerful inferences about historical processes of colonization, gene flow and divergence, as well as the identification of loci that mediate local adaptation. These ‘landscape genomic’ approaches have been validated in model species and are now being applied to nonmodel organisms, including foundation species that have substantial effects on ecosystem processes. Here we review the growing field of landscape genomics from a very broad perspective. In particular, we describe the inferential power that is gained by taking a genome-wide view of genetic variation, strategies for study design to best capture adaptive variation, and how to apply this information to practical challenges, such as restoration.

 
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Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches.

Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches. | Plant Genomics | Scoop.it
BMC Plant Biol. 2015 Apr 19;15:106. doi: 10.1186/s12870-015-0485-6.
Biswapriya Biswavas Misra's insight:

The major proteins in lupin seeds are conglutins that have primary roles in supplying carbon, sulphur and nitrogen and energy for the germinating seedling. They fall into four families; α, β, γ and δ. Interest in these conglutins is growing as family members have been shown to have beneficial nutritional and pharmaceutical properties.

RESULTS:

An in-depth transcriptome and draft genome from the narrow-leafed lupin (NLL; Lupinus angustifolius) variety, Tanjil, were examined and 16 conglutin genes were identified. Using RNAseq data sets, the structure and expression of these 16 conglutin genes were analysed across eight lupin varieties from five lupin species. Phylogenic analysis suggest that the α and γ conglutins diverged prior to lupin speciation while β and δ members diverged both prior and after speciation. A comparison of the expression of the 16 conglutin genes was performed, and in general the conglutin genes showed similar levels of RNA expression among varieties within species, but quite distinct expression patterns between lupin species. Antibodies were generated against the specific conglutin families and immunoblot analyses were used to compare the levels of conglutin proteins in various tissues and during different stages of seed development in NLL, Tanjil, confirming the expression in the seed. This analysis showed that the conglutins were expressed highly at the mature seed stage, in all lupin species, and a range of polypeptide sizes were observed for each conglutin family.

CONCLUSIONS:

This study has provided substantial information on the complexity of the four conglutin families in a range of lupin species in terms of their gene structure, phylogenetic relationships as well as their relative RNA and protein abundance during seed development. The results demonstrate that the majority of the heterogeneity of conglutin polypeptides is likely to arise from post-translational modification from a limited number of precursor polypeptides rather than a large number of different genes. Overall, the results demonstrate a high degree of plasticity for conglutin expression during seed development in different lupin species.

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Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea

Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea | Plant Genomics | Scoop.it
Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37-C40) ketones (alkenones) with 2 to 4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as “alkenone body (AB)”. Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and tandem mass spectrometry for confident protein identification and annotated using BLAST tools at NCBI. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft–associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.
Biswapriya Biswavas Misra's insight:

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37-C40) ketones (alkenones) with 2 to 4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as “alkenone body (AB)”. Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and tandem mass spectrometry for confident protein identification and annotated using BLAST tools at NCBI. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft–associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.

  
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The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop (2015)

The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop (2015) | Plant Genomics | Scoop.it

Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.


Via Kamoun Lab @ TSL, Francis Martin
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Genome of octoploid plant maca (Lepidium meyenii) illuminates genomic basis for high altitude adaptation in the central Andes

Maca (Lepidium meyenii Walp, 2n = 8x = 64) of Brassicaceae family is an Andean economic plant cultivated on the 4000-4500 meters central sierra in Peru. Considering the rapid uplift of central Andes occurred 5 to 10 million years ago (Mya), an evolutionary question arises on how plants like maca acquire high altitude adaptation within short geological period. Here, we report the high-quality genome assembly of maca, in which two close-spaced maca-specific whole genome duplications (WGDs, ~ 6.7 Mya) were identified. Comparative genomics between maca and close-related Brassicaceae species revealed expansions of maca genes and gene families involved in abiotic stress response, hormone signaling pathway and secondary metabolite biosynthesis via WGDs. Retention and subsequent evolution of many duplicated genes may account for the morphological and physiological changes (i.e. small leaf shape and loss of vernalization) in maca for high altitude environment. Additionally, some duplicated maca genes under positive selection were identified with functions in morphological adaptation (i.e. MYB59) and development (i.e. GDPD5 and HDA9). Collectively, the octoploid maca genome sheds light on the important roles of WGDs in plant high altitude adaptation in the Andes.
Biswapriya Biswavas Misra's insight:

Maca (Lepidium meyenii Walp, 2n = 8x = 64) of Brassicaceae family is an Andean economic plant cultivated on the 4000-4500 meters central sierra in Peru. Considering the rapid uplift of central Andes occurred 5 to 10 million years ago (Mya), an evolutionary question arises on how plants like maca acquire high altitude adaptation within short geological period. Here, we report the high-quality genome assembly of maca, in which two close-spaced maca-specific whole genome duplications (WGDs, ~ 6.7 Mya) were identified. Comparative genomics between maca and close-related Brassicaceae species revealed expansions of maca genes and gene families involved in abiotic stress response, hormone signaling pathway and secondary metabolite biosynthesis via WGDs. Retention and subsequent evolution of many duplicated genes may account for the morphological and physiological changes (i.e. small leaf shape and loss of vernalization) in maca for high altitude environment. Additionally, some duplicated maca genes under positive selection were identified with functions in morphological adaptation (i.e. MYB59) and development (i.e. GDPD5 and HDA9). Collectively, the octoploid maca genome sheds light on the important roles of WGDs in plant high altitude adaptation in the Andes.

 
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De novo characterization of the alligator weed (Alternanthera philoxeroides) transcriptome illuminates gene expression under potassium deprivation.

De novo characterization of the alligator weed (Alternanthera philoxeroides) transcriptome illuminates gene expression under potassium deprivation. | Plant Genomics | Scoop.it
J Genet. 2015 Mar;94(1):95-104.
Biswapriya Biswavas Misra's insight:

As one of the three macronutrients, potassium participates in many physiological processes in plant life cycle. Recently, potassium-dependent transcriptome analysis has been reported in Arabidopsis, rice and soybean. Alligator weed is well known, particularly for its strong ability to accumulate potassium. However, the molecular mechanism that underlies potassium starvation responses has not yet been described. In this study, we used Illumina (Solexa) sequencing technology to analyse the root transcriptome information of alligator weed under low potassium stress. Further analysis suggested that 9253 differentially expressed genes (DEGs) were upregulated, and 2138 DEGs were downregulated after seven days of potassium deficiency. These factors included 121 transcription factors, 108 kinases, 136 transporters and 178 genes that were related to stress. Twelve transcription factors were randomly selected for further analysis. The expression level of each transcription factor was confirmed by quantitative RT-PCR, and the results of this secondary analysis were consistent with the results of Solexa sequencing. Enrichment analysis indicated that 10,993 DEGs were assigned to 54 gene ontology terms and 123 KEGG pathways. Approximately 24% of DEGs belong to the metabolic, ribosome and biosynthesis of secondary metabolite KEGG pathways. Our results provide a comprehensive analysis of the gene regulatory network of alligator weed under low potassium stress, and afford a valuable resource for genetic and genomic research on plant potassium deficiency.

  
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Transcriptomic insights into antagonistic effects of gibberellin and abscisic acid on petal growth in Gerbera hybrida.

Front Plant Sci. 2015 Mar 17;6:168. doi: 10.3389/fpls.2015.00168. eCollection 2015.
Biswapriya Biswavas Misra's insight:

Petal growth is central to floral morphogenesis, but the underlying genetic basis of petal growth regulation is yet to be elucidated. In this study, we found that the basal region of the ray floret petals of Gerbera hybrida was the most sensitive to treatment with the phytohormones gibberellin (GA) and abscisic acid (ABA), which regulate cell expansion during petal growth in an antagonistic manner. To screen for differentially expressed genes (DEGs) and key regulators with potentially important roles in petal growth regulation by GA or/and ABA, the RNA-seq technique was employed. Differences in global transcription in petals were observed in response to GA and ABA and target genes antagonistically regulated by the two hormones were identified. Moreover, we also identified the pathways associated with the regulation of petal growth after application of either GA or ABA. Genes relating to the antagonistic GA and ABA regulation of petal growth showed distinct patterns, with genes encoding transcription factors (TFs) being active during the early stage (2 h) of treatment, while genes from the "apoptosis" and "cell wall organization" categories were expressed at later stages (12 h). In summary, we present the first study of global expression patterns of hormone-regulated transcripts in G. hybrida petals; this dataset will be instrumental in revealing the genetic networks that govern petal morphogenesis and provides a new theoretical basis and novel gene resources for ornamental plant breeding.

  
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DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato.

PLoS One. 2015 Apr 7;10(4):e0121864. doi: 10.1371/journal.pone.0121864. eCollection 2015.
Biswapriya Biswavas Misra's insight:

We reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L., cv. Rutgers), an ABA overproducer, is defective in blue light (B) signaling leading to B-specific resistance to abiotic and biotic stresses. Using a methylation-sensitive amplified polymorphism (MSAP) assay, a number of genes were identified, which were differentially methylated between 7B-1 and its wild type (WT) seedlings in white (W), blue (B), red (R) lights and dark (D) or in response to exogenous ABA and mannitol-induced stresses. The genomic methylation level was almost similar in different lights between 7B-1 and WT seedlings, while significant differences were observed in response to stresses in D, but not B. Using a cDNA-AFLP assay, several transcripts were identified, which were differentially regulated between 7B-1 and WT by B or D or in response to stresses. Blue light receptors cryptochrome 1 and 2 (CRY1 and CRY2) and phototropin 1 and 2 (PHOT1 and PHOT2) were not affected by the 7B-1 mutation at the transcriptional level, instead the mutation had likely affected downstream components of the light signaling pathway. 5-azacytidine (5-azaC) induced DNA hypomethylation, inhibited stem elongation and differentially regulated the expression of a number of genes in 7B-1. In addition, it was shown that mir167 and mir390 were tightly linked to auxin signaling pathway in 5-azaC-treated 7B-1 seedlings via the regulation of auxin-response factor (ARF) transcripts. Our data showed that DNA methylation remodeling is an active epigenetic response to different lights and stresses in 7B-1 and WT, and highlighted the differences in epigenetic and transcriptional regulation of light and stress responses between 7B-1 and WT. Furthermore, it shed lights on the crosstalk between DNA hypomethylation and miRNA regulation of ARFs expression. This information could also be used as a benchmark for future studies of male-sterility in other crops.

  
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