Iron status and root cell morphology of Arabidopsis thaliana as modified by a bacterial ferri-siderophore.

We previously provided evidence for the contribution of pyoverdine to the iron nutrition of Arabidopsis. In the present article, we further analyze the mechanisms and physiology of the adaptations underlying plant iron nutrition through Fe(III)-pyoverdine (Fe(III)-pvd). An integrated approach combining microscopy and nanoscale secondary ion mass spectrometry (NanoSIMS) on plant samples was adopted to localize pyoverdine in planta and assess the impact of this siderophore on the plant iron status and root cellular morphology. The results support a possible plant uptake mechanism of the Fe(III)-pvd complex by epidermal root cells via a non-reductive process associated with the presence of more vesicles. Pyoverdine was transported to the central cylinder via the symplastic and/or trans-cellular pathway(s), suggesting a possible root-to-shoot translocation. All these processes led to enhanced plant iron nutrition, as previously shown. Overall, these findings suggest that bacterial siderophores contribute to plant iron uptake and homeostasis.

Water soluble octa-imidazolium zinc phthalocyanine for nucleus/nucleolus cell fluorescence microscopy and photodynamic therapy.

A poly-cationic theranostic macrocycle was developed to perform confocal microscopy imaging and photodynamic therapy studies on a model of melanoma cancer, one of the most aggressive cancer. Hence, an octa-imidazolium zinc phthalocyanine was conveniently synthesized in large amount in three steps in a 44% overall yield: upon double nucleophilic aromatic substitution, cyclo-tetramerization and quaternization reactions. Such an octa-cationic molecule was readily soluble in physiological media, reaching concentrations beyond 1 mM. It showed fluorescence properties in aqueous medium (ΦF = 0.31) with no noticeable aggregation, spectroscopy studies showed. In vitro confocal fluorescence microscopy studies carried out on murine melanoma model (B16F10 cells) showed that the fluorophore was mainly located in the cell nucleolus, an organelle of interest for the treatment of cancer. The anticancer photodynamic potential of the octa-cationic photosensitizer could be measured (IC50 = 5.4 µM) using the MTS viability assay. Both fluorescence microscopy studies and photodynamic studies demonstrate the octa-cationic molecule is theranostic and could be further developed for future photodynamic diagnosis (PDD) and photodynamic inactivation of micro-organisms (PDI).

Imaging plant tissues: advances and promising clearing practices.

The study of the organ structure of plants and understanding their physiological complexity requires 3D imaging with subcellular resolution. Most plant organs are highly opaque to light, and their study under optical sectioning microscopes is therefore difficult. In animals, many protocols have been developed to make organs transparent to light using clearing protocols (CPs). By contrast, clearing plant tissues is challenging because of the presence of fibers and pigments. We describe progress in the development of plant CPs over the past 20 years through a modified taxonomy of CPs based on their physical and optical parameters that affect tissue properties. We also discuss successful approaches that combine CPs with new microscopy methods and their future applications in plant science research.

Dual Cherenkov Radiation-Induced Near-Infrared Luminescence Imaging and Photodynamic Therapy toward Tumor Resection.

Cherenkov radiation (CR), the blue light seen in nuclear reactors, is emitted by some radiopharmaceuticals. This study showed that (1) a portion of CR could be transferred in the region of the optical spectrum, where biological tissues are most transparent: as a result, upon radiance amplification in the near-infrared window, the detection of light could occur twice deeper in tissues than during classical Cherenkov luminescence imaging and (2) Cherenkov-photodynamic therapy (CR-PDT) on cells could be achieved under conditions mimicking unlimited depth using the CR-embarked light source, which is unlike standard PDT, where light penetration depth is limited in biological tissues. Both results are of utmost importance for simultaneous applications in tumor resection and post-resection treatment of remaining unresected margins, thanks to a molecular construct designed to raise its light collection efficiency (i.e., CR energy transfer) by conjugation with multiple CR-absorbing (water-soluble) antenna followed by intramolecular-FRET/TBET energy transfers.

Sugar exchanges in arbuscular mycorrhiza: RiMST5 and RiMST6, two novel Rhizophagus irregularis monosaccharide transporters, are involved in both sugar uptake from the soil and from the plant partner.

Arbuscular mycorrhizal (AM) fungi are associated with about 80% of land plants. AM fungi provide inorganic nutrients to plants and in return up to 20% of the plant-fixed CO2 is transferred to the fungal symbionts. Since AM fungi are obligate biotrophs, unraveling how sugars are provided to the fungus partner is a key for understanding the functioning of the symbiosis. In this study, we identified two new monosaccharide transporters from Rhizophagus irregularis (RiMST5 and RiMST6) that we characterized as functional high affinity monosaccharide transporters. RiMST6 was characterized as a glucose specific, high affinity H(+) co-transporter. We provide experimental support for a primary role of both RiMST5 and RiMST6 in sugar uptake directly from the soil. The expression patterns of RiMSTs in response to partial light deprivation and to interaction with different host plants were investigated. Expression of genes coding for RiMSTs was transiently enhanced after 48 h of shading and was unambiguously dependent on the host plant species. These results cast doubt on the 'fair trade' principle under carbon-limiting conditions. Therefore, in light of these findings, the possible mechanisms involved in the modulation between mutualism and parasitism in plant-AM fungus interactions are discussed.

GintAMT3 - a Low-Affinity Ammonium Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis.

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a V max of 240 nmol(-1) min(-1) 10(8) cells(-1), which is regulated by substrate concentration and carbon supply.

Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers.

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.

The family of ammonium transporters (AMT) in Sorghum bicolor: two AMT members are induced locally, but not systemically in roots colonized by arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi contribute to plant nitrogen (N) acquisition. Recent studies demonstrated the transport of N in the form of ammonium during AM symbiosis. Here, we hypothesize that induction of specific ammonium transporter (AMT) genes in Sorghum bicolor during AM colonization might play a key role in the functionality of the symbiosis. For the first time, combining a split-root experiment and microdissection technology, we were able to assess the precise expression pattern of two AM-inducible AMTs, SbAMT3;1 and SbAMT4. Immunolocalization was used to localize the protein of SbAMT3;1. The expression of SbAMT3;1 and SbAMT4 was greatly induced locally in root cells containing arbuscules and in adjacent cells. However, a split-root experiment revealed that this induction was not systemic. By contrast, a strictly AM-induced phosphate transporter (SbPt11) was expressed systemically in the split-root experiment. However, a gradient of expression was apparent. Immunolocalization analyses demonstrated that SbAMT3;1 was present only in cells containing developing arbuscules. Our results show that the SbAMT3;1 and SbAMT4 genes are expressed in root cortical cells, which makes them ready to accommodate arbuscules, a process of considerable importance in view of the short life span of arbuscules. Additionally, SbAMT3;1 might play an important role in N transfer during AM symbiosis.

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions.

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca(2+) in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca(2+)-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca(2+)-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca(2+) ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca(2+)-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.

Structure and composition of model cheeses influence sodium NMR mobility, kinetics of sodium release and sodium partition coefficients.

The mobility and release of sodium ions were assessed in model cheeses with three different lipid/protein ratios, with or without added NaCl. The rheological properties of the cheeses were analysed using uniaxial compression tests. Microstructure was characterised by confocal laser scanning microscopy. (23)Na nuclear magnetic resonance (NMR) spectroscopy was used to study the molecular mobility of sodium ions in model cheeses through measurements of the relaxation and creation times. Greater mobility was observed in cheeses containing a lower protein content and with added NaCl. The kinetics of sodium release from the cheese to an aqueous phase was correlated with the mobility of sodium ions. The highest rates of sodium release were observed with a lower protein content and with added NaCl. The water/cheese partition coefficients of sodium increased when NaCl was added or the protein content was higher. The study highlighted the effect of model cheese characteristics on molecular and macroscopic behaviours of sodium.

Overexpression of the epidermis-specific homeodomain-leucine zipper IV transcription factor Outer Cell Layer1 in maize identifies target genes involved in lipid metabolism and cuticle biosynthesis.

Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis.

Expression profiling of fungal genes during arbuscular mycorrhiza symbiosis establishment using direct fluorescent in situ RT-PCR.

Expression profiling of fungal genes in the arbuscular mycorrhiza (AM) symbiosis has been based on studies of RNA extracted from fungal tissue or mycorrhizal roots, giving only a general picture of overall transcript levels in the targeted tissues. Information about the spatial distribution of transcripts within AM fungal structures during different developmental stages is essential to a better understanding of fungal activity in symbiotic interactions with host roots and to determine molecular events involved in establishment and functioning of the AM symbiosis. The obligate biotrophic nature of AM fungi is a challenge for developing new molecular methods to identify and localize their activity in situ. The direct fluorescent in situ (DIFIS) RT-PCR procedure described here represents a novel tool for spatial mapping of AM fungal gene expression simultaneously prior to root penetration, within fungal tissues in the host root and in the extraradical stage of fungal development.In order to enhance detection sensitivity of the in situ RT-PCR technique and enable localization of low abundance mRNA, we have adopted direct fluorescent labeling of primers for the amplification step to overcome the problem of low detection associated with digoxigenin or biotin-labeled primers and to avoid the multiplicity of steps associated with immunological detection. Signal detection has also been greatly improved by eliminating autofluorescence of AM fungal and root tissues using confocal microscopy.

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices.

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.

Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions.

To gain further insight into the role of the plant genome in arbuscular mycorrhiza (AM) establishment, we investigated whether symbiosis-related plant genes affect fungal gene expression in germinating spores and at the appressoria stage of root interactions. Glomus intraradices genes were identified in expressed sequence tag libraries of mycorrhizal Medicago truncatula roots by in silico expression analyses. Transcripts of a subset of genes, with predicted functions in transcription, protein synthesis, primary or secondary metabolism, or of unknown function, were monitored in spores and germinating spores and during interactions with roots of wild-type or mycorrhiza-defective (Myc-) mutants of M. truncatula. Not all the fungal genes were active in quiescent spores but all were expressed when G. intraradices spores germinated in wild-type M. truncatula root exudates or when appressoria or arbuscules were formed in association with wild-type M. truncatula roots. Most of the fungal genes were upregulated or induced at the stage of appressorium development. Inactivation of the M. truncatula genes DMI1, DMI2/MtSYM2, or DMI3/MtSYM13 was associated with altered fungal gene expression (nonactivation or inhibition), modified appressorium structure, and plant cell wall responses, providing first evidence that cell processes modified by symbiosis-related plant genes impact on root interactions by directly modulating AM fungal activity.

An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen.

Mechanisms of root penetration by arbuscular mycorrhizal (AM) fungi are unknown and investigations are hampered by the lack of transformation systems for these unculturable obligate biotrophs. Early steps of host infection by hemibiotrophic fungal phytopathogens, sharing common features with those of AM fungal colonization, depend on the transcription factor STE12. Using degenerated primers and rapid amplification of cDNA ends, we isolated the full-length cDNA of an STE12-like gene, GintSTE, from Glomus intraradices and profiled GintSTE expression by real-time and in situ RT-PCR. GintSTE activity and function were investigated by heterologous complementation of a yeast ste12Delta mutant and a Colletotrichum lindemuthianum clste12Delta mutant. * Sequence data indicate that GintSTE is similar to STE12 from hemibiotrophic plant pathogens, especially Colletotrichum spp. Introduction of GintSTE into a noninvasive mutant of C. lindemuthianum restored fungal infectivity of plant tissues. GintSTE expression was specifically localized in extraradicular fungal structures and was up-regulated when G. intraradices penetrated roots of wild-type Medicago truncatula as compared with an incompatible mutant. Results suggest a possible role for GintSTE in early steps of root penetration by AM fungi, and that pathogenic and symbiotic fungi may share common regulatory mechanisms for invasion of plant tissues.

Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR.

Gene expression profiling based on tissue extracts gives only limited information about genes associated with complex developmental processes such as those implicated in fungal interactions with plant roots during arbuscular mycorrhiza development and function. To overcome this drawback, a direct fluorescent in situ RT-PCR methodology was developed for spatial mapping of gene expression in different presymbiotic and symbiotic structures of an arbuscular mycorrhizal fungus. Transcript detection was optimized by targeting the LSU rRNA gene of Glomus intraradices and monitoring expression of a stearoyl-CoA-desaturase gene that is consistently expressed at high levels in spores, hyphae, arbuscules and vesicles. This method was further validated by localizing expression of fungal peptidylprolyl isomerase and superoxide dismutase genes, which are expressed to different extents in fungal structures. Direct fluorescent in situ RT-PCR offers new perspectives for the sensitive analysis of fungal developmental processes that occur during functional differentiation in symbiotic arbuscular mycorrhiza interactions.

Repetitive DNA sequences include retrotransposons in genomes of the Glomeromycota.

Twenty-five repetitive elements are first described in the genomes of the arbuscular mycorrhizal (AM) fungi Gigaspora margarita, Gig. rosea and Glomus mosseae. Nineteen repetitive DNA sequences isolated by genomic library screening and four by self-priming PCR had no homology to known DNA sequences, except for two Gig. margarita sequences and one Gig. rosea sequence which showed amino acid similarity to retrotransposons. Part of the Gig. rosea sequence was also similar to a DNA transposon. Two other retrotransposon sequences were isolated using PCR targeting of reverse transcriptase and ribonuclease H domains. Evidence is provided for three gypsy-like LTR retrotransposon and two non-LTR retrotransposon sequences in the AM fungal genomes. Four contain stop codons indicating that they cannot be active. Expression of three retrotransposons was not detected in germinating spores or intraradical hyphae of Gig. margarita. Southern blot analyses indicated that these three sequences are dispersed in the genome and that two are methylated. Sequence analysis of different GmarLTR1 copies showed they have undergone mutations by transitions, which may have been induced by cytosine methylation. Transposable elements may have played a major role in shaping genome structure and size during evolution of the Glomeromycota.

Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula.

Plant genes induced during early root colonization of Medicago truncatula Gaertn. J5 by a growth-promoting strain of Pseudomonas fluorescens (C7R12) have been identified by suppressive subtractive hybridization. Ten M. truncatula genes, coding proteins associated with a putative signal transduction pathway, showed an early and transient activation during initial interactions between M. truncatula and P. fluorescens, up to 8 d after root inoculation. Gene expression was not significantly enhanced, except for one gene, in P. fluorescens-inoculated roots of a Myc(-)Nod(-) genotype (TRV25) of M. truncatula mutated for the DMI3 (syn. MtSYM13) gene. This gene codes a Ca(2+) and calmodulin-dependent protein kinase, indicating a possible role of calcium in the cellular interactions between M. truncatula and P. fluorescens. When expression of the 10 plant genes was compared in early stages of root colonization by mycorrhizal and rhizobial microsymbionts, Glomus mosseae activated all 10 genes, whereas Sinorhizobium meliloti only activated one and inhibited four others. None of the genes responded to inoculation by either microsymbiont in roots of the TRV25 mutant. The similar response of the M. truncatula genes to P. fluorescens and G. mosseae points to common molecular pathways in the perception of the microbial signals by plant roots.

Effect of 2,4-diacetylphloroglucinol on pythium: cellular responses and variation in sensitivity among propagules and species.

ABSTRACT The antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) plays an important role in the suppression of plant pathogens by several strains of Pseudomonas spp. Based on the results of this study, there is variation within and among Pythium spp. to 2,4-DAPG. Also, various propagules of Pythium ultimum var. sporangiiferum, that are part of the asexual stage of the life cycle, differ considerably in their sensitivity to 2,4-DAPG. Mycelium was the most resistant structure, followed by zoosporangia, zoospore cysts, and zoospores. Additionally, we report for the first time that pH has a significant effect on the activity of 2,4-DAPG, with a higher activity at low pH. Furthermore, the level of acetylation of phloroglucinols is also a major determinant of their activity. Transmission electron microscopy studies revealed that 2,4-DAPG causes different stages of disorganization in hyphal tips of Pythium ultimum var. sporangiiferum, including alteration (proliferation, retraction, and disruption) of the plasma membrane, vacuolization, and cell content disintegration. The implications of these results for the efficacy and consistency of biological control of plant-pathogenic Pythium spp. by 2,4-DAPG-producing Pseudomonas spp. are discussed.