Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.

Re-epithelialization of cutaneous wounds in adult mammals takes days to complete and relies on numerous signalling cues and multiple overlapping cellular processes that take place both within the epidermis and in other participating tissues. Re-epithelialization of partial- or full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing. Live imaging after treatment with transgene-encoded or chemical inhibitors reveals that re-epithelializing keratinocytes repopulate wounds by TGF-ß- and integrin-dependent lamellipodial crawling at the leading edges of the epidermal tongue. In addition, re-epithelialization requires long-range epithelial rearrangements, involving radial intercalations, flattening and directed elongation of cells - processes that are dependent on Rho kinase, JNK and, to some extent, planar cell polarity within the epidermis. These rearrangements lead to a massive recruitment of keratinocytes from the adjacent epidermis and make re-epithelialization independent of keratinocyte proliferation and the mitogenic effect of FGF signalling, which are only required after wound closure, allowing the epidermis outside the wound to re-establish its normal thickness. Together, these results demonstrate that the adult zebrafish is a valuable in vivo model for studying and visualizing the processes involved in cutaneous wound closure, facilitating the dissection of direct from indirect and motogenic from mitogenic effects of genes and molecules affecting wound re-epithelialization.

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation.

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.

SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development.

Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control.

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.

The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. By contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on other cues from metabolically active host cells.

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis.

By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane.

p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium.

TIME FOR COFFEE is an essential component in the maintenance of metabolic homeostasis in Arabidopsis thaliana.

Plants often respond to environmental changes by reprogramming metabolic and stress-associated pathways. Homeostatic integration of signaling is a central requirement for ensuring metabolic stability in living organisms. Under diurnal conditions, properly timed rhythmic metabolism provides fitness benefits to plants. TIME FOR COFFEE (TIC) is a circadian regulator known to be involved in clock resetting at dawn. Here we explored the mechanism of influence of TIC in plant growth and development, as initiated by a microarray analysis. This global profiling showed that a loss of TIC function causes a major reprogramming of gene expression that predicts numerous developmental, metabolic, and stress-related phenotypes. This led us to demonstrate that this mutant exhibits late flowering, a plastochron defect, and diverse anatomical phenotypes. We further observed a starch-excess phenotype and altered soluble carbohydrate levels. tic exhibited hypersensitivity to oxidative stress and abscisic acid, and this was associated with a striking resistance to drought. These phenotypes were connected to an increase in total glutathione levels that correlated with a readjustment of amino acids and polyamine pools. By comparatively analyzing our transcriptomic and metabolomic data, we concluded that TIC is a central element in plant homeostasis that integrates and coordinates developmental, metabolic, and environmental signals.

Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum.

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins.

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.

DELLA-interacting SWI3C core subunit of switch/sucrose nonfermenting chromatin remodeling complex modulates gibberellin responses and hormonal cross talk in Arabidopsis.

Switch (SWI)/Sucrose Nonfermenting (SNF)-type chromatin-remodeling complexes (CRCs) are involved in regulation of transcription, DNA replication and repair, and cell cycle. Mutations of conserved subunits of plant CRCs severely impair growth and development; however, the underlying causes of these phenotypes are largely unknown. Here, we show that inactivation of SWI3C, the core component of Arabidopsis (Arabidopsis thaliana) SWI/SNF CRCs, interferes with normal functioning of several plant hormone pathways and alters transcriptional regulation of key genes of gibberellin (GA) biosynthesis. The resulting reduction of GA4 causes severe inhibition of hypocotyl and root elongation, which can be rescued by exogenous GA treatment. In addition, the swi3c mutation inhibits DELLA-dependent transcriptional activation of GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptor genes. Down-regulation of GID1a in parallel with the DELLA repressor gene REPRESSOR OF GA1-3 1 in swi3c indicates that lack of SWI3C also leads to defects in GA signaling. Together with the recent demonstration of function of SWI/SNF ATPase BRAHMA in the GA pathway, these results reveal a critical role of SWI/SNF CRC in the regulation of GA biosynthesis and signaling. Moreover, we demonstrate that SWI3C is capable of in vitro binding to, and shows in vivo bimolecular fluorescence complementation interaction in cell nuclei with, the DELLA proteins RGA-LIKE2 and RGA-LIKE3, which affect transcriptional activation of GID1 and GA3ox (GIBBERELLIN 3-OXIDASE) genes controlling GA perception and biosynthesis, respectively. Furthermore, we show that SWI3C also interacts with the O-GlcNAc (O-linked N-acetylglucosamine) transferase SPINDLY required for proper functioning of DELLAs and acts hypostatically to (SPINDLY) in the GA response pathway. These findings suggest that DELLA-mediated effects in GA signaling as well as their role as a hub in hormonal cross talk may be, at least in part, dependent on their direct physical interaction with complexes responsible for modulation of chromatin structure.

Functional dissection of the PROPEP2 and PROPEP3 promoters reveals the importance of WRKY factors in mediating microbe-associated molecular pattern-induced expression.

· In Arabidopsis thaliana, small peptides (AtPeps) encoded by PROPEP genes act as damage-associated molecular patterns (DAMPs) that are perceived by two leucine-rich repeat receptor kinases, PEPR1 and PEPR2, to amplify defense responses. In particular, expression of PROPEP2 and PROPEP3 is strongly and rapidly induced by AtPeps, in response to bacterial, oomycete, and fungal pathogens, and microbe-associated molecular patterns (MAMPs). · The cis-regulatory modules (CRMs) within the PROPEP2 and PROPEP3 promoters that mediate MAMP responsiveness were delineated, employing parsley (Petroselinum crispum) protoplasts and transgenic A. thaliana plants harboring promoter-reporter constructs. By chromatin immunoprecipitation in vivo, DNA interactions with a specific transcription factor were detected. Furthermore, the PHASTCONS program was used to identify conserved regions of the PROPEP3 locus in different Brassicaceae species. · The major MAMP-responsive CRM within the PROPEP2 promoter is composed of several W boxes and an as1/OCS (activation sequence-1/octopine synthase) enhancer element, while in the PROPEP3 promoter the CRM is comprised of six W boxes. The WRKY33 transcription factor binds in vivo to these promoter regions in a MAMP-dependent manner. Both the position and orientation of the six W boxes are conserved within the PROPEP3 promoters of four other Brassicaceae family members. · WRKY factors are the major regulators of MAMP-induced PROPEP2 and PROPEP3 expression.

TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds.

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP-type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA-specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development.

Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1.

SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions.

The Arabidopsis class VIII myosin ATM2 is involved in endocytosis.

Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens-based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata.

Expression of AtWRKY33 encoding a pathogen- or PAMP-responsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements.

WRKY transcription factors regulate distinct parts of the plant defense transcriptome. Expression of many WRKY genes themselves is induced by pathogens or pathogen-mimicking molecules. Here, we demonstrate that Arabidopsis WRKY33 responds to various stimuli associated with plant defense as well as to different kinds of phytopathogens. Although rapid pathogen-induced AtWRKY33 expression does not require salicylic acid (SA) signaling, it is dependent on PAD4, a key regulator upstream of SA. Activation of AtWRKY33 is independent of de novo protein synthesis, suggesting that it is at least partly under negative regulatory control. We show that a set of three WRKY-specific cis-acting DNA elements (W boxes) within the AtWRKY33 promoter is required for efficient pathogen- or PAMP-triggered gene activation. This strongly indicates that WRKY transcription factors are major components of the regulatory machinery modulating immediate to early expression of this gene in response to pathogen attack.

Cell polarization, a crucial process in fungal defence.

Plant cells responding to fungal attack undergo large morphological alterations, along with rapid and extensive metabolic reprogramming. Cytological analysis of single infected plant cells revealed a large complexity of interdependent, rapid and dynamic changes of a multitude of cellular components. Among these changes are major rearrangements of the cytoskeleton, translocation of cytoplasm and of the cell nucleus to the fungal penetration site, and local apposition of barrier material around this site, which results in massive cell-wall reinforcement. If this first line of defence is overcome by the pathogen, in many cases, it is followed by hypersensitive plant cell death, which stops growth of the penetrating fungus and finally leads to its death. The speed and magnitude of the initial defence response appear to be crucial to plant disease resistance.

Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.

A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells.

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.

Differential gene expressions in arbuscular mycorrhizal-colonized tomato grown under heavy metal stress.

When tomato was grown in either "Breinigerberg" soil, which has a high content of Zn and of other heavy metals or in non-polluted soil enriched with up to 1 mM CdCl2, plants colonized with the arbuscular mycorrhizal fungus (AMF) Glomus intraradices grew distinctly better than non-mycorrhizal controls. An analysis of differential mRNA transcript formations was performed on several plant genes coding for products potentially involved in heavy metal tolerance. Northern blot analyses indicated that the mRNA from either roots or leaves was not differentially expressed in the case of LePCS1 (coding for phytochelatin synthase), Lemt1, Lemt3 and Lemt4 (for metallothioneins) or LeNramp2 (for a broad range heavy metal transporter) in both mycorrhizal and non-mycorrhizal plants, grown either with or without heavy metals. In contrast, Lemt2 was strongly expressed only in non-AMF-colonized roots, and only after growth in the Breinigerberg soil or in the presence of high CdCl2-concentrations. AMF colonization distinctly reduced the level of Lemt2 transcripts. This was also the case for the root specific LeNramp1 transporter, however, only after growth in the Breinigerberg soil, but not under Cd-stress. Likewise, the levels of LeNramp3 transcripts were reduced by the AMF colonization in roots, but not in leaves. Quantitative Real-Time RT-PCR-experiments performed with Lemt2, LeNramp1 and LeNramp3 largely corroborated the Northern analysis data. In situ hybridization experiments with Lemt2 and LeNramp1 showed that both genes were strongly expressed throughout the plant cells in non-colonized roots, whereas colonized roots revealed only few signals restricted to some parenchyma cells. All the data suggest that the transcript levels of some, but not all genes of the Nramp or mt family are elevated under heavy metal stress. AMF colonization results in a down-regulation of these genes, presumably due to the fact that the content of heavy metals is lower in mycorrhizal than in non-colonized roots. A suppression subtractive hybridization (SSH) Library from hyphae of the AMF G. intraradices grown in high versus low Zn++ provided none of the genes which were down-regulated at the plant side (mt or Nramp genes). In contrast, several gene sequences coding for enzymes potentially catalysing the detoxification of reactive oxygen species were found. Thus the fungal cells in the symbiosis may primarily have to cope with the heavy metal-induced oxidative stress.