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Arabidopsis RIBA Proteins: Two out of Three Isoforms Have Lost Their Bifunctional Activity in Riboflavin Biosynthesis

Arabidopsis RIBA Proteins: Two out of Three Isoforms Have Lost Their Bifunctional Activity in Riboflavin Biosynthesis | PlantBioInnovation | Scoop.it
Abstract: Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution.
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Fast sampling method for mammalian cell metabolic analyses using liquid chromatography–mass spectrometry

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a −20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.
Biswapriya Biswavas Misra's insight:

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a −20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.

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Biomass conversion: Lignin up for break-down

Lignin is an abundant renewable resource, but its intrinsic recalcitrant nature has so far hampered its conversion into higher value chemicals. Now, a two-step strategy, oxidation followed by bond cleavage, has been shown to deconstruct lignin into high yields of low-molecular-weight aromatics.
Biswapriya Biswavas Misra's insight:

Lignin is an abundant renewable resource, but its intrinsic recalcitrant nature has so far hampered its conversion into higher value chemicals. Now, a two-step strategy, oxidation followed by bond cleavage, has been shown to deconstruct lignin into high yields of low-molecular-weight aromatics.

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Wheat rescued from fungal disease

Knockout of all six alleles of a gene in the large wheat genome confers resistance to powdery mildew.
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Knockout of all six alleles of a gene in the large wheat genome confers resistance to powdery mildew.

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The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway

The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway | PlantBioInnovation | Scoop.it
Alan Lloyd and colleagues identify a MYB gene in beet that acts as a key regulator of the betalain red pigmentation pathway. They further show that this gene resides at the classical Y pigmentation locus and shares homology with MYB genes that encode positive regulators of the anthocyanin pigmentation pathway in other species.
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Nearly all flowering plants produce red/violet anthocyanin pigments. Caryophyllales is the only order containing families that replace anthocyanins with unrelated red and yellow betalain pigments1, 2. Close biological correlation of pigmentation patterns suggested that betalains might be regulated by a conserved anthocyanin-regulating transcription factor complex consisting of a MYB, a bHLH and a WD repeat–containing protein (the MBW complex)3. Here we show that a previously uncharacterized anthocyanin MYB-like protein, Beta vulgaris MYB1 (BvMYB1), regulates the betalain pathway in beets. Silencing BvMYB1 downregulates betalain biosynthetic genes and pigmentation, and overexpressing BvMYB1 upregulates them. However, unlike anthocyanin MYBs, BvMYB1 will not interact with bHLH members of heterologous anthocyanin MBW complexes because of identified nonconserved residues. BvMYB1 resides at the historic beet pigment-patterning locus, Y, required for red-fleshed beets4. We show that Y and y express different levels of BvMYB1 transcripts. The co-option of a transcription factor regulating anthocyanin biosynthesis would be an important evolutionary event allowing betalains to largely functionally replace anthocyanins.

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Acetylation serves as a protective group in noscapine biosynthesis in opium poppy

Characterization of four enzymes involved in biosynthesis of the plant metabolite and anticancer agent noscapine completes this pathway and identifies an unusual acetyl protecting group strategy that defines the order of enzymatic steps.
Biswapriya Biswavas Misra's insight:

We have characterized four sequential enzymes that transform 1-hydroxy-N-methylcanadine to narcotoline hemiacetal, completing our elucidation of noscapine biosynthesis in opium poppy. Two cytochromes P450 catalyze hydroxylations at C13 and C8 on the protoberberine scaffold, the latter step inducing ring opening and the formation of an aldehyde moiety. Acetylation at C13 before C8 hydroxylation introduces a protective group subsequently hydrolyzed by a carboxylesterase, which triggers rearrangement to a cyclic hemiacetal.

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A U-system approach for predicting metabolic behaviors and responses based on an alleged metabolic reaction network

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Biswapriya Biswavas Misra's insight:
Background

Progress in systems biology offers sophisticated approaches toward a comprehensive understanding of biological systems. Yet, computational analyses are held back due to difficulties in determining suitable model parameter values from experimental data which naturally are subject to biological fluctuations. The data may also be corrupted by experimental uncertainties and sometimes do not contain all information regarding variables that cannot be measured for technical reasons.

Results

We show here a streamlined approach for the construction of a coarse model that allows us to set up dynamic models with minimal input information. The approach uses a hybrid between a pure mass action system and a generalized mass action (GMA) system in the framework of biochemical systems theory (BST) with rate constants of 1, normal kinetic orders of 1, and -0.5 and 0.5 for inhibitory and activating effects, named Unity (U)-system. The U-system model does not necessarily fit all data well but is often sufficient for predicting metabolic behavior of metabolites which cannot be simultaneously measured, identifying inconsistencies between experimental data and the assumed underlying pathway structure, as well as predicting system responses to a modification of gene or enzyme. The U-system approach was validated with small, generic systems and implemented to model a large-scale metabolic reaction network of a higher plant, Arabidopsis. The dynamic behaviors obtained by predictive simulations agreed with actually available metabolomic time-series data, identified probable errors in the experimental datasets, and estimated probable behavior of unmeasurable metabolites in a qualitative manner. The model could also predict metabolic responses of Arabidopsis with altered network structures due to genetic modification.

Conclusions

The U-system approach can effectively predict metabolic behaviors and responses based on structures of an alleged metabolic reaction network. Thus, it can be a useful first-line tool of data analysis, model diagnostics and aid the design of next-step experiments.

 
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Global Analysis of Photosynthesis Transcriptional Regulatory Networks

Global Analysis of Photosynthesis Transcriptional Regulatory Networks | PlantBioInnovation | Scoop.it
PLOS Genetics is an open-access
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Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ~34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

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Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic ...

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts.
Biswapriya Biswavas Misra's insight:
Abstract (provisional)Background

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts.

Results

Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme.

Conclusions

The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

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The genome sequence of the orchid Phalaenopsis equestris

The genome sequence of the orchid Phalaenopsis equestris | PlantBioInnovation | Scoop.it
Zhong-Jian Liu, Lai-Qiang Huang, Yi-Bo Luo, Hong-Hwa Chen and Yves Van de Peer report the first genome sequence of a crassulacean acid metabolism (CAM) plant, the orchid Phalaenopsis equestris. They identify genes encoding CAM pathway enzymes and find that gene duplication was likely a key process in the evolution of CAM photosynthesis.
Biswapriya Biswavas Misra's insight:

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers.

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Estimating Relative Changes of Metabolic Fluxes

Estimating Relative Changes of Metabolic Fluxes | PlantBioInnovation | Scoop.it
PLOS Computational Biology is an open-access
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Fluxes are the central trait of metabolism and Kinetic Flux Profiling (KFP) is an effective method of measuring them. To generalize its applicability, we present an extension of the method that estimates the relative changes of fluxes using only relative quantitation of 13C-labeled metabolites. Such features are directly tailored to the more common experiment that performs only relative quantitation and compares fluxes between two conditions. We call our extension rKFP. Moreover, we examine the effects of common missing data and common modeling assumptions on (r)KFP, and provide practical suggestions. We also investigate the selection of measuring times for (r)KFP and provide a simple recipe. We then apply rKFP to 13C-labeled glucose time series data collected from cells under normal and glucose-deprived conditions, estimating the relative flux changes of glycolysis and its branching pathways. We identify an adaptive response in which de novo serine biosynthesis is compromised to maintain the glycolytic flux backbone. Together, these results greatly expand the capabilities of KFP and are suitable for broad biological applications.

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Genomic Insights into the Fungal Pathogens of the Genus Pneumocystis: Obligate Biotrophs of Humans and Other Mammals

Genomic Insights into the Fungal Pathogens of the Genus Pneumocystis: Obligate Biotrophs of Humans and Other Mammals | PlantBioInnovation | Scoop.it
From molecules to physiology
Biswapriya Biswavas Misra's insight:

Pneumocystis organisms were first believed to be a single protozoan species able to colonize the lungs of all mammals. Subsequently, genetic analyses revealed their affiliation to the fungal Taphrinomycotina subphylum of the Ascomycota, a clade which encompasses plant pathogens and free-living yeasts. It also appeared that, despite their similar morphological appearance, these fungi constitute a family of relatively divergent species, each with a strict specificity for a unique mammalian species [1]. The species colonizing human lungs, Pneumocystis jirovecii, can turn into an opportunistic pathogen that causes Pneumocystis pneumonia (PCP) in immunocompromised individuals, a disease which may be fatal. Although the incidence of PCP diminished in the 1990s thanks to prophylaxis and antiretroviral therapy, PCP is nowadays the second-most-frequent, life-threatening, invasive fungal infection worldwide, with an estimated number of cases per year above 400,000 [2]. Despite this clinical importance, studies of P. jirovecii progressed slowly, at least in part because of the lack of a continuous culture system. Nevertheless, recent genomic findings provided insights into the lifestyle of these fungi.

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A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis

A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis | PlantBioInnovation | Scoop.it
From molecules to physiology
Biswapriya Biswavas Misra's insight:

Microbe- or host damage-derived patterns mediate activation of pattern-triggered immunity (PTI) in plants. Microbial virulence factor (effector)-triggered immunity (ETI) constitutes a second layer of plant protection against microbial attack. Various necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) produced by bacterial, oomycete and fungal microbes are phytotoxic virulence factors that exert immunogenic activities through phytotoxin-induced host cell damage. We here show that multiple cytotoxic NLPs also carry a pattern of 20 amino acid residues (nlp20) that triggers immunity-associated plant defenses and immunity to microbial infection in Arabidopsis thaliana and related plant species with similar characteristics as the prototype pattern, bacterial flagellin. Characteristic differences in flagellin and nlp20 plant responses exist however, as nlp20s fail to trigger extracellular alkalinization in Arabidopsis cell suspensions and seedling growth inhibition. Immunogenic nlp20 peptide motifs are frequently found in bacterial, oomycete and fungal NLPs. Such an unusually broad taxonomic distribution within three phylogenetic kingdoms is unprecedented among microbe-derived triggers of immune responses in either metazoans or plants. Our findings suggest that cytotoxic NLPs carrying immunogenic nlp20 motifs trigger PTI in two ways as typical patterns and by inflicting host cell damage. We further propose that conserved structures within a microbial virulence factor might have driven the emergence of a plant pattern recognition system mediating PTI. As this is reminiscent of the evolution of immune receptors mediating ETI, our findings support the idea that there is a continuum between PTI and ETI.

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GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement | PlantBioInnovation | Scoop.it
From molecules to physiology
Biswapriya Biswavas Misra's insight:

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

 
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Plant genomics: High-throughput phenotyping of rice growth traits

A high-throughput phenotyping tool enables the study of rice traits during the growth period and after harvest. Yang et al. combined imaging techniques with a yield traits scorer to automatically screen for 15 agronomic traits, including two newly defined traits — plant compactness and grain-projected area — that cannot be identified by…
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A high-throughput phenotyping tool enables the study of rice traits during the growth period and after harvest. Yang et al. combined imaging techniques with a yield traits scorer to automatically screen for 15 agronomic traits, including two newly defined traits — plant compactness and grain-projected area — that cannot be identified by…

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Cell walls: Cellulose snakes along

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Cellulose polymers found in plant cell walls are synthesized by cellulose synthase complexes (CESA), but the mechanism for conversion of individual polymers into microfibrils remains unclear. COBRA is coexpressed with CESA, and mutations of COBRA are linked to changes in cellulose, but its specific function is unknown.…

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Gene duplication of type-B ARR transcription factors systematically extends transcriptional regulatory structures in Arabidopsis

Gene duplication of type-B ARR transcription factors systematically extends transcriptional regulatory structures in Arabidopsis | PlantBioInnovation | Scoop.it
Many of duplicated genes are enriched in signaling pathways. Recently, gene duplication of kinases has been shown to provide genetic buffering and functional diversification in cellular signaling. Transcription factors (TFs) are also often duplicated. However, how duplication of TFs affects their regulatory structures and functions of target genes has not been explored at the systems level. Here, we examined regulatory and functional roles of duplication of three major ARR TFs (ARR1, 10, and 12) in Arabidopsis cytokinin signaling using wild-type and single, double, and triple deletion mutants of the TFs. Comparative analysis of gene expression profiles obtained from Arabidopsis roots in wild-type and these mutants showed that duplication of ARR TFs systematically extended their transcriptional regulatory structures, leading to enhanced robustness and diversification in functions of target genes, as well as in regulation of cellular networks of target genes. Therefore, our results suggest that duplication of TFs contributes to robustness and diversification in functions of target genes by extending transcriptional regulatory structures.
Biswapriya Biswavas Misra's insight:

Many of duplicated genes are enriched in signaling pathways. Recently, gene duplication of kinases has been shown to provide genetic buffering and functional diversification in cellular signaling. Transcription factors (TFs) are also often duplicated. However, how duplication of TFs affects their regulatory structures and functions of target genes has not been explored at the systems level. Here, we examined regulatory and functional roles of duplication of three major ARR TFs (ARR1, 10, and 12) in Arabidopsis cytokinin signaling using wild-type and single, double, and triple deletion mutants of the TFs. Comparative analysis of gene expression profiles obtained from Arabidopsis roots in wild-type and these mutants showed that duplication of ARR TFs systematically extended their transcriptional regulatory structures, leading to enhanced robustness and diversification in functions of target genes, as well as in regulation of cellular networks of target genes. Therefore, our results suggest that duplication of TFs contributes to robustness and diversification in functions of target genes by extending transcriptional regulatory structures.

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A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice

The brown planthopper (BPH) is the most destructive pest of rice (Oryza sativa) and a substantial threat to rice production, causing losses of billions of dollars annually1, 2. Breeding of resistant cultivars is currently hampered by the rapid breakdown of BPH resistance2. Thus, there is an urgent need to identify…
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The brown planthopper (BPH) is the most destructive pest of rice (Oryza sativa) and a substantial threat to rice production, causing losses of billions of dollars annually1, 2. Breeding of resistant cultivars is currently hampered by the rapid breakdown of BPH resistance2. Thus, there is an urgent need to identify…

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The phytoplasmal virulence factor TENGU causes plant sterility by downregulating of the jasmonic acid and auxin pathways

The phytoplasmal virulence factor TENGU causes plant sterility by downregulating of the jasmonic acid and auxin pathways | PlantBioInnovation | Scoop.it
Despite plants infected by pathogens are often unable to produce offspring, it remains unclear how sterility is induced in host plants. In this study, we demonstrate that TENGU, a phytoplasmal virulence peptide known as a dwarfism inducer, acts as an inducer of sterility. Transgenic expression of TENGU induced both male and female sterility in Arabidopsis thaliana flowers similar to those observed in double knockout mutants of auxin response factor 6 (ARF6) and ARF8, which are known to regulate floral development in a jasmonic acid (JA)-dependent manner. Transcripts of ARF6 and ARF8 were significantly decreased in both tengu-transgenic and phytoplasma-infected plants. Furthermore, JA and auxin levels were actually decreased in tengu-transgenic buds, suggesting that TENGU reduces the endogenous levels of phytohormones by repressing ARF6 and ARF8, resulting in impaired flower maturation. TENGU is the first virulence factor with the effects on plant reproduction by perturbation of phytohormone signaling.
Biswapriya Biswavas Misra's insight:

Despite plants infected by pathogens are often unable to produce offspring, it remains unclear how sterility is induced in host plants. In this study, we demonstrate that TENGU, a phytoplasmal virulence peptide known as a dwarfism inducer, acts as an inducer of sterility. Transgenic expression of TENGU induced both male and female sterility in Arabidopsis thaliana flowers similar to those observed in double knockout mutants of auxin response factor 6 (ARF6) and ARF8, which are known to regulate floral development in a jasmonic acid (JA)-dependent manner. Transcripts of ARF6 and ARF8 were significantly decreased in both tengu-transgenic and phytoplasma-infected plants. Furthermore, JA and auxin levels were actually decreased in tengu-transgenic buds, suggesting that TENGU reduces the endogenous levels of phytohormones by repressing ARF6 and ARF8, resulting in impaired flower maturation. TENGU is the first virulence factor with the effects on plant reproduction by perturbation of phytohormone signaling.

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Analysis of a Plant Complex Resistance Gene Locus Underlying Immune-Related Hybrid Incompatibility and Its Occurrence in Nature

Analysis of a Plant Complex Resistance Gene Locus Underlying Immune-Related Hybrid Incompatibility and Its Occurrence in Nature | PlantBioInnovation | Scoop.it
PLOS Genetics is an open-access
Biswapriya Biswavas Misra's insight:

Mechanisms underlying speciation in plants include detrimental (incompatible) genetic interactions between parental alleles that incur a fitness cost in hybrids. We reported on recessive hybrid incompatibility between an Arabidopsis thaliana strain from Poland, Landsberg erecta (Ler), and many Central Asian A. thaliana strains. The incompatible interaction is determined by a polymorphic cluster of Toll/interleukin-1 receptor-nucleotide binding-leucine rich repeat (TNL) RPP1 (Recognition of Peronospora parasitica1)-like genes in Ler and alleles of the receptor-like kinase Strubbelig Receptor Family 3 (SRF3) in Central Asian strains Kas-2 or Kond, causing temperature-dependent autoimmunity and loss of growth and reproductive fitness. Here, we genetically dissected the RPP1-like Ler locus to determine contributions of individual RPP1-like Ler (R1–R8) genes to the incompatibility. In a neutral background, expression of most RPP1-like Ler genes, except R3, has no effect on growth or pathogen resistance. Incompatibility involves increased R3 expression and engineered R3 overexpression in a neutral background induces dwarfism and sterility. However, no individual RPP1-like Ler gene is sufficient for incompatibility between Ler and Kas-2 or Kond, suggesting that co-action of at least two RPP1-like members underlies this epistatic interaction. We find that the RPP1-like Ler haplotype is frequent and occurs with other Ler RPP1-like alleles in a local population in Gorzów Wielkopolski (Poland). Only Gorzów individuals carrying the RPP1-like Ler haplotype are incompatible with Kas-2 and Kond, whereas other RPP1-like alleles in the population are compatible. Therefore, the RPP1-like Ler haplotype has been maintained in genetically different individuals at a single site, allowing exploration of forces shaping the evolution of RPP1-like genes at local and regional population scales.

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Metabolic and co-expression network-based analyses associated with nitrate response in rice

Understanding gene expression and metabolic re-programming that occur in response to limiting nitrogen (N) conditions in crop plants is crucial for the ongoing progress towards the development of varieties with improved nitrogen use efficiency (NUE). To unravel new details on the molecular and metabolic responses to N availability in a major food crop, we conducted analyses on a weighted gene co-expression network and metabolic profile data obtained from leaves and roots of rice plants adapted to sufficient and limiting N as well as after shifting them to limiting (reduction) and sufficient (induction) N conditions.
Biswapriya Biswavas Misra's insight:
Abstract (provisional)Background

Understanding gene expression and metabolic re-programming that occur in response to limiting nitrogen (N) conditions in crop plants is crucial for the ongoing progress towards the development of varieties with improved nitrogen use efficiency (NUE). To unravel new details on the molecular and metabolic responses to N availability in a major food crop, we conducted analyses on a weighted gene co-expression network and metabolic profile data obtained from leaves and roots of rice plants adapted to sufficient and limiting N as well as after shifting them to limiting (reduction) and sufficient (induction) N conditions.

Results

A gene co-expression network representing clusters of rice genes with similar expression patterns across four nitrogen conditions and two tissue types was generated. The resulting 18 clusters were analyzed for enrichment of significant gene ontology (GO) terms. Four clusters exhibited significant correlation with limiting and reducing nitrate treatments. Among the identified enriched GO terms, those related to nucleoside/nucleotide, purine and ATP binding, defense response, sugar/carbohydrate binding, protein kinase activities, cell-death and cell wall enzymatic activity are enriched. Although a subset of functional categories are more broadly associated with the response of rice organs to limiting N and N reduction, our analyses suggest that N reduction elicits a response distinguishable from that to adaptation to limiting N, particularly in leaves. This observation is further supported by metabolic profiling which shows that several compounds in leaves change proportionally to the nitrate level (i.e. higher in sufficient N vs. imitimg N) and respond with even higher levels when the nitrate level is reduced. Notably, these compounds are directly involved in N assimilation, transport, and storage (glutamine, asparagine, glutamate and allantoin) and extend to most amino acids. Based on these data, we hypothesize that plants respond by rapidly mobilizing stored vacuolar nitrate when N deficit is perceived, and that the response likely involves phosphorylation signal cascades and transcriptional regulation.

Conclusions

The co-expression network analysis and metabolic profiling performed in rice pinpoint the relevance of signal transduction components and regulation of N mobilization in response to limiting N conditions and deepen our understanding of N responses and N use in crops.

 
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The Evolution of Fungal Metabolic Pathways

The Evolution of Fungal Metabolic Pathways | PlantBioInnovation | Scoop.it
PLOS Genetics is an open-access
Biswapriya Biswavas Misra's insight:
Abstract

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.

 
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Multi-level Modeling of Light-Induced Stomatal Opening Offers New Insights into Its Regulation by Drought

Multi-level Modeling of Light-Induced Stomatal Opening Offers New Insights into Its Regulation by Drought | PlantBioInnovation | Scoop.it
Plant guard cells gate CO2 uptake and transpirational water loss through stomatal pores. As a result of decades of experimental investigation, there is an abundance of information on the involvement of specific proteins and secondary messengers in the regulation of stomatal movements and on the pairwise relationships between guard cell components. We constructed a multi-level dynamic model of guard cell signal transduction during light-induced stomatal opening and of the effect of the plant hormone abscisic acid (ABA) on this process. The model integrates into a coherent network the direct and indirect biological evidence regarding the regulation of seventy components implicated in stomatal opening. Analysis of this signal transduction network identified robust cross-talk between blue light and ABA, in which [Ca2+]c plays a key role, and indicated an absence of cross-talk between red light and ABA. The dynamic model captured more than 1031 distinct states for the system and yielded outcomes that were in qualitative agreement with a wide variety of previous experimental results. We obtained novel model predictions by simulating single component knockout phenotypes. We found that under white light or blue light, over 60%, and under red light, over 90% of all simulated knockouts had similar opening responses as wild type, showing that the system is robust against single node loss. The model revealed an open question concerning the effect of ABA on red light-induced stomatal opening. We experimentally showed that ABA is able to inhibit red light-induced stomatal opening, and our model offers possible hypotheses for the underlying mechanism, which point to potential future experiments. Our modelling methodology combines simplicity and flexibility with dynamic richness, making it well suited for a wide class of biological regulatory systems.Plant guard cells gate CO2 uptake and transpirational water loss through stomatal pores. As a result of decades of experimental investigation, there is an abundance of information on the involvement of specific proteins and secondary messengers in the regulation of stomatal movements and on the pairwise relationships between guard cell components. We constructed a multi-level dynamic model of guard cell signal transduction during light-induced stomatal opening and of the effect of the plant hormone abscisic acid (ABA) on this process. The model integrates into a coherent network the direct and indirect biological evidence regarding the regulation of seventy components implicated in stomatal opening. Analysis of this signal transduction network identified robust cross-talk between blue light and ABA, in which [Ca2+]c plays a key role, and indicated an absence of cross-talk between red light and ABA. The dynamic model captured more than 1031 distinct states for the system and yielded outcomes that were in qualitative agreement with a wide variety of previous experimental results. We obtained novel model predictions by simulating single component knockout phenotypes. We found that under white light or blue light, over 60%, and under red light, over 90% of all simulated knockouts had similar opening responses as wild type, showing that the system is robust against single node loss. The model revealed an open question concerning the effect of ABA on red light-induced stomatal opening. We experimentally showed that ABA is able to inhibit red light-induced stomatal opening, and our model offers possible hypotheses for the underlying mechanism, which point to potential future experiments. Our modelling methodology combines simplicity and flexibility with dynamic richness, making it well suited for a wide class of biological regulatory systems.
Biswapriya Biswavas Misra's insight:

Plant guard cells gate CO2 uptake and transpirational water loss through stomatal pores. As a result of decades of experimental investigation, there is an abundance of information on the involvement of specific proteins and secondary messengers in the regulation of stomatal movements and on the pairwise relationships between guard cell components. We constructed a multi-level dynamic model of guard cell signal transduction during light-induced stomatal opening and of the effect of the plant hormone abscisic acid (ABA) on this process. The model integrates into a coherent network the direct and indirect biological evidence regarding the regulation of seventy components implicated in stomatal opening. Analysis of this signal transduction network identified robust cross-talk between blue light and ABA, in which [Ca2+]c plays a key role, and indicated an absence of cross-talk between red light and ABA. The dynamic model captured more than 1031 distinct states for the system and yielded outcomes that were in qualitative agreement with a wide variety of previous experimental results. We obtained novel model predictions by simulating single component knockout phenotypes. We found that under white light or blue light, over 60%, and under red light, over 90% of all simulated knockouts had similar opening responses as wild type, showing that the system is robust against single node loss. The model revealed an open question concerning the effect of ABA on red light-induced stomatal opening. We experimentally showed that ABA is able to inhibit red light-induced stomatal opening, and our model offers possible hypotheses for the underlying mechanism, which point to potential future experiments. Our modelling methodology combines simplicity and flexibility with dynamic richness, making it well suited for a wide class of biological regulatory systems.

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Genomic Insights into the Fungal Pathogens of the Genus Pneumocystis: Obligate Biotrophs of Humans and Other Mammals

Genomic Insights into the Fungal Pathogens of the Genus Pneumocystis: Obligate Biotrophs of Humans and Other Mammals | PlantBioInnovation | Scoop.it
From molecules to physiology
Biswapriya Biswavas Misra's insight:

Pneumocystis organisms were first believed to be a single protozoan species able to colonize the lungs of all mammals. Subsequently, genetic analyses revealed their affiliation to the fungal Taphrinomycotina subphylum of the Ascomycota, a clade which encompasses plant pathogens and free-living yeasts. It also appeared that, despite their similar morphological appearance, these fungi constitute a family of relatively divergent species, each with a strict specificity for a unique mammalian species [1]. The species colonizing human lungs, Pneumocystis jirovecii, can turn into an opportunistic pathogen that causes Pneumocystis pneumonia (PCP) in immunocompromised individuals, a disease which may be fatal. Although the incidence of PCP diminished in the 1990s thanks to prophylaxis and antiretroviral therapy, PCP is nowadays the second-most-frequent, life-threatening, invasive fungal infection worldwide, with an estimated number of cases per year above 400,000 [2]. Despite this clinical importance, studies of P. jirovecii progressed slowly, at least in part because of the lack of a continuous culture system. Nevertheless, recent genomic findings provided insights into the lifestyle of these fungi.

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The Relationship between Host Lifespan and Pathogen Reservoir Potential: An Analysis in the System Arabidopsis thaliana-Cucumber mosaic virus

The Relationship between Host Lifespan and Pathogen Reservoir Potential: An Analysis in the System Arabidopsis thaliana-Cucumber mosaic virus | PlantBioInnovation | Scoop.it
From molecules to physiology
Biswapriya Biswavas Misra's insight:

Identification of the determinants of pathogen reservoir potential is central to understand disease emergence. It has been proposed that host lifespan is one such determinant: short-lived hosts will invest less in costly defenses against pathogens, so that they will be more susceptible to infection, more competent as sources of infection and/or will sustain larger vector populations, thus being effective reservoirs for the infection of long-lived hosts. This hypothesis is sustained by analyses of different hosts of multihost pathogens, but not of different genotypes of the same host species. Here we examined this hypothesis by comparing two genotypes of the plant Arabidopsis thaliana that differ largely both in life-span and in tolerance to its natural pathogen Cucumber mosaic virus (CMV). Experiments with the aphid vector Myzus persicae showed that both genotypes were similarly competent as sources for virus transmission, but the short-lived genotype was more susceptible to infection and was able to sustain larger vector populations. To explore how differences in defense against CMV and its vector relate to reservoir potential, we developed a model that was run for a set of experimentally-determined parameters, and for a realistic range of host plant and vector population densities. Model simulations showed that the less efficient defenses of the short-lived genotype resulted in higher reservoir potential, which in heterogeneous host populations may be balanced by the longer infectious period of the long-lived genotype. This balance was modulated by the demography of both host and vector populations, and by the genetic composition of the host population. Thus, within-species genetic diversity for lifespan and defenses against pathogens will result in polymorphisms for pathogen reservoir potential, which will condition within-population infection dynamics. These results are relevant for a better understanding of host-pathogen co-evolution, and of the dynamics of pathogen emergence.

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GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement | PlantBioInnovation | Scoop.it
From molecules to physiology
Biswapriya Biswavas Misra's insight:

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

 

 

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