RADA-dependent branch migration has a predominant role in plant mitochondria and its defect leads to mtDNA instability and cell cycle arrest.

Mitochondria of flowering plants have large genomes whose structure and segregation are modulated by recombination activities. The post-synaptic late steps of mitochondrial DNA (mtDNA) recombination are still poorly characterized. Here we show that RADA, a plant ortholog of bacterial RadA/Sms, is an organellar protein that drives the major branch-migration pathway of plant mitochondria. While RadA/Sms is dispensable in bacteria, RADA-deficient Arabidopsis plants are severely impacted in their development and fertility, correlating with increased mtDNA recombination across intermediate-size repeats and accumulation of recombination-generated mitochondrial subgenomes. The radA mutation is epistatic to recG1 that affects the additional branch migration activity. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. The physical interaction of RADA with RECA2 but not with RECA3 further indicated that RADA is required for the processing of recombination intermediates in the RECA2-depedent recombination pathway of plant mitochondria. Although RADA is dually targeted to mitochondria and chloroplasts we found little to no effects of the radA mutation on the stability of the plastidial genome. Finally, we found that the deficient maintenance of the mtDNA in radA apparently triggers a retrograde signal that activates nuclear genes repressing cell cycle progression.

Use of electron microscopy to study megakaryocytes.

Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology.

Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

Virus preparations from the mixed-infected P70 Pinot Noir accession exhibit GLRaV-1/GVA 'end-to-end' particles.

P70 is a Pinot Noir grapevine accession that displays strong leafroll disease symptoms. A high-throughput sequencing (HTS)-based analysis established that P70 was mixed-infected by two variants of grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus) and one of grapevine virus A (GVA, genus Vitivirus) as well as by two viroids (hop stunt viroid [HSVd] and grapevine yellow speckle viroid 1 [GYSVd1]) and four variants of grapevine rupestris stem pitting-associated virus (GRSPaV). Immunogold labelling using gold particles of two different diameters revealed the existence of 'hybrid' particles labelled at one end as GLRaV-1, with the rest labelled as GVA. In this work, we suggest that immunogold labelling can provide information about the biology of the viruses, going deeper than just genomic information provided by HTS, from which no recombinant or 'chimeric' GLRaV-1/GVA sequences had been identified in the dataset. Our observations suggest an unknown interaction between members of two different viral species that are often encountered together in a single grapevine, highlighting potential consequences in the vector biology and epidemiology of leafroll and rugose-wood diseases.

Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

Arabidopsis ERG28 tethers the sterol C4-demethylation complex to prevent accumulation of a biosynthetic intermediate that interferes with polar auxin transport.

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.

Histone H2B monoubiquitination is involved in the regulation of cutin and wax composition in Arabidopsis thaliana.

The plant cuticle is a chemically heterogeneous lipophilic layer composed of a cutin polymer matrix and waxes which covers the aerial parts of plants. This layer plays an essential role in the survival of plants by protecting them from desiccation and (a)biotic stresses. Knowledge on the gene networks and mechanisms regulating the synthesis of cuticle components during organ expansion or stress response remains limited however. Here, using five loss-of-function mutants for histone monoubiquitination, we report on the role of two RING E3 ligases, namely HISTONE MONOUBIQUITINATION 1 and 2 (HUB1 and HUB2), in the selective transcriptional activation of four cuticle biosynthesis genes in Arabidopsis thaliana. Microscopy observations showed that in hub1-6 and hub2-2 mutants irregular epidermal cells and disorganized cuticle layers were present in rosette leaves. Water loss measurements on excised rosettes demonstrated that cuticular permeability was significantly increased in the mutants. Chemical analysis of cuticle components revealed that the wax composition was changed and that cutin 16:0 dicarboxylic acid was significantly reduced in all hub mutants. Analysis of transcript levels of selected genes indicated that LACS2, ATT1 and HOTHEAD involved in cutin biosynthesis and CER1 involved in wax biosynthesis were down-regulated in the hub mutants, while the expression of LACERATA, CER3, CER6 and CER10 remained unchanged. Chromatin immunoprecipitation assays further showed that hub mutants are impaired in dynamic changes of histone H2B monoubiquitination at several loci of down-regulated genes. Taken together, these data establish that the regulation of cuticle composition involves chromatin remodeling by H2B monoubiquitination.

Sporopollenin biosynthetic enzymes interact and constitute a metabolon localized to the endoplasmic reticulum of tapetum cells.

The sporopollenin polymer is the major constituent of exine, the outer pollen wall. Recently fatty acid derivatives have been shown to be the precursors of sporopollenin building units. ACYL-COA SYNTHETASE, POLYKETIDE SYNTHASE A (PKSA) and PKSB, TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 have been demonstrated to be involved in sporopollenin biosynthesis in Arabidopsis (Arabidopsis thaliana). Here all these sporopollenin biosynthetic enzymes but TKPR2 have been immunolocalized to endoplasmic reticulum of anther tapetal cells. Pull-down experiments demonstrated that tagged recombinant proteins interacted to form complexes whose constituents were characterized by immunoblotting. In vivo protein interactions were evidenced by yeast (Saccharomyces cerevisiae) two-hybrid analysis and by fluorescence lifetime imaging microscopy/Förster resonance energy transfer studies in transgenic Nicotiana benthamiana, which were used to test the possibility that the enzymes interact to form a biosynthetic metabolon. Various pairs of proteins fused to two distinct fluorochromes were coexpressed in N. benthamiana leaf tissues and fluorescence lifetime imaging microscopy/Förster resonance energy transfer measurements demonstrated that proteins interacted pairwise in planta. Taken together, these results suggest the existence of a sporopollenin metabolon.

DELLAs regulate chlorophyll and carotenoid biosynthesis to prevent photooxidative damage during seedling deetiolation in Arabidopsis.

In plants, light represents an important environmental signal that triggers the production of photosynthetically active chloroplasts. This developmental switch is critical for plant survival because chlorophyll precursors that accumulate in darkness can be extremely destructive when illuminated. Thus, plants have evolved mechanisms to adaptively control plastid development during the transition into light. Here, we report that the gibberellin (GA)-regulated DELLA proteins play a crucial role in the formation of functional chloroplasts during deetiolation. We show that Arabidopsis thaliana DELLAs accumulating in etiolated cotyledons derepress chlorophyll and carotenoid biosynthetic pathways in the dark by repressing the transcriptional activity of the phytochrome-interacting factor proteins. Accordingly, dark-grown GA-deficient ga1-3 mutants (that accumulate DELLAs) display a similar gene expression pattern to wild-type seedlings grown in the light. Consistent with this, ga1-3 seedlings accumulate higher amounts of protochlorophyllide (a phototoxic chlorophyll precursor) in darkness but, surprisingly, are substantially more resistant to photooxidative damage following transfer into light. This is due to the DELLA-dependent upregulation of the photoprotective enzyme protochlorophyllide oxidoreductase (POR) in the dark. Our results emphasize the role of DELLAs in regulating the levels of POR, protochlorophyllide, and carotenoids in the dark and in protecting etiolated seedlings against photooxidative damage during initial light exposure.

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis.

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.

Structure, function, and evolution of the Thiomonas spp. genome.

Bacteria of the Thiomonas genus are ubiquitous in extreme environments, such as arsenic-rich acid mine drainage (AMD). The genome of one of these strains, Thiomonas sp. 3As, was sequenced, annotated, and examined, revealing specific adaptations allowing this bacterium to survive and grow in its highly toxic environment. In order to explore genomic diversity as well as genetic evolution in Thiomonas spp., a comparative genomic hybridization (CGH) approach was used on eight different strains of the Thiomonas genus, including five strains of the same species. Our results suggest that the Thiomonas genome has evolved through the gain or loss of genomic islands and that this evolution is influenced by the specific environmental conditions in which the strains live.

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria.

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.

Expression of the Beet necrotic yellow vein virus p25 protein induces hormonal changes and a root branching phenotype in Arabidopsis thaliana.

The RNA-3-encoded p25 protein was previously characterized as one of the major symptom determinants of the Beet necrotic yellow vein virus. Previous analyses reported the influence of the p25 protein in root proliferation phenotype observed in rhizomania disease on infected sugar beets (Beta vulgaris). A transgenic approach was developed, in which the p25 protein was constitutively expressed in Arabidopsis thaliana Columbia (Col-0) ecotype in order to provide new clues as to how the p25 protein might promote alone disease development and symptom expression. Transgenic plants were characterized by Southern blot and independent lines carrying single and multiple copies of the transgene were selected. Mapping of the T-DNA insertion was performed on the monocopy homozygote lines. P25 protein was localized both in the nucleus and in the cytoplasm of epidermal and root cells of transgenic plants. Although A. thaliana was not described as a susceptible host for BNYVV infection, abnormal root branching was observed on p25 protein-expressing A. thaliana plants. Moreover, these transgenic plants were more susceptible than wild-type plants to auxin analog treatment (2,4-D) but more resistant to methyl jasmonate (MeJA), abscisic acid (ABA) and to lesser extend to salicylic acid (SA). Hormonal content assays measuring plant levels of auxin (IAA), jasmonate (JA) and ethylene precursor (ACC) revealed major hormonal changes. Global transcript profiling analyses on roots displayed differential gene expressions that could corroborate root branching phenotype and stress signaling modifications.

Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana.

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to Saccharomyces cerevisiae.

Corrigendum: The GIP Gamma-Tubulin Complex-Associated Proteins are Involved in Nuclear Architecture in Arabidopsis Thaliana.

[This corrects the article DOI: 10.3389/fpls.2013.00480/full.].

A non-canonical transferred DNA insertion at the BRI1 locus in Arabidopsis thaliana.

Agrobacterium-mediated transformation is widely used in transgenic plant engineering and has been proven to be a powerful tool for insertional mutagenesis of the plant genome. The transferred DNA (T-DNA) from Agrobacterium is integrated into the plant genome through illegitimate recombination between the T-DNA and the plant DNA. Contrasting to the canonical insertion, here we report on a locus showing a complex mutation associated with T-DNA insertion at the BRI1 gene in Arabidopsis thaliana. We obtained a mutant line, named salade for its phenotype of dwarf stature and proliferating rosette. Molecular characterization of this mutant revealed that in addition to T-DNA a non-T-DNA-localized transposon from bacteria was inserted in the Arabidopsis genome and that a region of more than 11.5 kb of the Arabidopsis genome was deleted at the insertion site. The deleted region contains the brassinosteroid receptor gene BRI1 and the transcription factor gene WRKY13. Our finding reveals non-canonical T-DNA insertion, implicating horizontal gene transfer and cautioning the use of T-DNA as mutagen in transgenic research.

Functionalized Tobacco Mosaic Virus Coat Protein Monomers and Oligomers as Nanocarriers for Anti-Cancer Peptides.

Components with self-assembly properties derived from plant viruses provide the opportunity to design biological nanoscaffolds for the ordered display of agents of diverse nature and with complementing functions. With the aim of designing a functionalized nanoscaffold to target cancer, the coat protein (CP) of Tobacco mosaic virus (TMV) was tested as nanocarrier for an insoluble, highly hydrophobic peptide that targets the transmembrane domain of the Neuropilin-1 (NRP1) receptor in cancer cells. The resulting construct CPL-K (CP-linker-"Kill") binds to NRP1 in cancer cells and disrupts NRP1 complex formation with PlexA1 as well as downstream Akt survival signaling. The application of CPL-K also inhibits angiogenesis and cell migration. CP was also fused to a peptide that targets the extracellular domain of NRP1 and this fusion protein (CPL-F, CP-Linker-"Find") is shown to bind to cultured cancer cells and to inhibit NRP1-dependent angiogenesis as well. CPL-K and CPL-F maintain their anti-angiogenic properties upon co-assembly to oligomers/nanoparticles together with CPL. The observations show that the CP of TMV can be employed to generate a functionalized nanoparticle with biological activity. Remarkably, fusion to CPL allowed us to solubilize the highly insoluble transmembrane NRP1 peptide and to retain its anti-angiogenic effect.

Formation of two-dimensional crystals of icosahedral RNA viruses.

UNLABELLED: The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.

Crosstalk between PTGS and TGS pathways in natural antiviral immunity and disease recovery.

Virus-induced diseases cause severe damage to cultivated plants, resulting in crop losses. Certain plant-virus interactions allow disease recovery at later stages of infection and have the potential to reveal important molecular targets for achieving disease control. Although recovery is known to involve antiviral RNA silencing1,2, the specific components of the many plant RNA silencing pathways 3 required for recovery are not known. We found that Arabidopsis thaliana plants infected with oilseed rape mosaic virus (ORMV) undergo symptom recovery. The recovered leaves contain infectious, replicating virus, but exhibit a loss of viral suppressor of RNA silencing (VSR) protein activity. We demonstrate that recovery depends on the 21-22 nt siRNA-mediated post-transcriptional gene silencing (PTGS) pathway and on components of a transcriptional gene silencing (TGS) pathway that is known to facilitate non-cell-autonomous silencing signalling. Collectively, our observations indicate that recovery reflects the establishment of a tolerant state in infected tissues and occurs following robust delivery of antiviral secondary siRNAs from source to sink tissues, and establishment of a dosage able to block the VSR activity involved in the formation of disease symptoms.

Sequence variation within Beet necrotic yellow vein virus p25 protein influences its oligomerization and isolate pathogenicity on Tetragonia expansa.

The p25 protein encoded by Beet necrotic yellow vein virus (BNYVV) RNA-3 is a pathogenicity determinant that has been implicated in symptom exacerbation on Chenopodiaceae hosts. Several p25 variants exist within natural isolates and p25 sequence variation may influence the degree of pathogenicity of such BNYVV isolates. Expression of p25 from natural A- and P-type isolates in the background of B-type BNYVV cDNA clones gave symptom discrepancies when compared to B-type p25 expression. Such pathogenicity fluctuation was not due to a different subcellular localization of p25 but was correlated with the nature of the tetrad motif present between amino acid residues 67-70, as well as with the capacity of p25 to self-associate and to activate transcription in a yeast one-hybrid system. Our data suggest that the complete sequence of p25 is required for its functions and the identified sequence variations may contribute to correct folding of the protein.