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Transgenic Nicotiana benthamiana expressing the minor coat protein P74 of the phloem-limited Beet western yellows virus (BWYV) exhibited an unusual spatial pattern of post-transcriptional gene silencing (PTGS) when infected with BWYV or related viruses. Following infection, transgenic P74 and its mRNA accumulated to only low levels, 21 to 23 nucleotide RNAs homologous to the transgene appeared, and the transgene DNA underwent methylation. The infecting viral RNA, however, was not subject to significant silencing but multiplied readily and produced P74 in the phloem tissues, although the P74 encoded by the transgene disappeared from the phloem as well as the nonvascular tissues.

The current risk assessment framework for insecticides suffers from certain shortcomings in adequately addressing the effects of low doses on off-target species. To remedy this gap, a combination of behavioural assays and in vitro cellular approaches are required to refine the precision of toxicity assessment. The domestic honey bee has long been standing as an emblematic pollinator in ecotoxicology, and once more, it provides us with a practical testing model for this purpose. First, newly emerged bees (D1) were found more vulnerable than 6 days-old bees (D6) to deltamethrin, a widely used α-cyano-3-phenoxybenzyle pyrethroid. In D1 bees, the range of doses inducing mortality was shifted towards lower values (∼2-fold) with a correspondingly lower LD 50 (11 ng/bee). Moreover, at low doses that do not induce mortality in laboratory conditions, the locomotor behaviour of D1 bees was more impacted than in D6 bees. This was evidenced by an increase in immobility time and a decrease in locomotor performance across all tested doses for D1 bees (0.75, 1.5 and 3 ng/bee) during automated 21 h-long observations. Behavioural disorders are linked to deltamethrin's disruption of voltage-gated sodium channels (Na V s) functions, as quantified in cultured neuronal cells. In the presence of deltamethrin, patch-clamp experiments revealed a concentration- and a use-dependent slowing of Na V kinetics. Channel's deactivation is slowed by three orders of magnitude at 10 μM deltamethrin. Two additional phenoxybenzyle pyrethroids, including the commonly used cypermethrin, elicited quantitatively similar effects on Na V kinetics. The integration of in vitro cellular assays and behavioural assays may facilitate a deeper understanding and prediction of insecticides toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Low-cost and scalable technologies that allow people to measure microplastics in their local environment could facilitate a greater understanding of the global problem of marine microplastic pollution. A typical way to measure marine microplastic pollution involves imaging filtered seawater samples stained with a fluorescent dye to aid in the detection of microplastics. Although traditional fluorescence microscopy allows these particles to be manually counted and detected, this is a resource- and labour-intensive task. Here, we describe a novel, low-cost microscope for automated scanning and detection of microplastics in filtered seawater samples-the EnderScope. This microscope is based on the mechanics of a low-cost 3D printer (Creality Ender 3). The hotend of the printer is replaced with an optics module, allowing for the reliable and calibrated motion system of the 3D printer to be used for automated scanning over a large area (>20 × 20 cm). The EnderScope is capable of both reflected light and fluorescence imaging. In both configurations, we aimed to make the design as simple and cost-effective as possible, for example, by using low-cost LEDs for illumination and lighting gels as emission filters. We believe this tool is a cost-effective solution for microplastic measurement. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

In honey bees, circulation of blood (hemolymph) is driven by the peristaltic contraction of the heart vessel located in the dorsal part of the abdomen. Chlorantraniliprole (CHL) is an insecticide of the anthranilic diamide class which main mode of action is to alter the function of intracellular Ca 2+ release channels (known as RyRs, for ryanodine receptors). In the honey bee, it was recently found to be more toxic when applied on the dorsal part of the abdomen, suggesting a direct cardiotoxicity. In the present study, a short-term exposure of semi-isolated bee hearts to CHL (0.1-10 µM) induces alterations of cardiac contraction. These alterations range from a slow-down of systole and diastole kinetics, to bradycardia and cardiac arrest. The bees heart wall is made of a single layer of semi-circular cardiomyocytes arranged concentrically all along the long axis of tube lumen. Since the heart tube is suspended to the cuticle through long tubular muscles fibers (so-called alary muscle cells), the CHL effects in ex-vivo heart preparations could result from the modulation of RyRs present in these skeletal muscle fibers as well as cardiomyocytes RyRs themselves. In order to specifically assess effects of CHL on cardiomyocytes, for the first time, intact heart cells were enzymatically dissociated from bees. Exposure of cardiomyocytes to CHL induces an increase in cytoplasmic calcium, cell contraction at the highest concentrations and depletion of intracellular stores. Electrophysiological properties of isolated cardiomyocytes were described, with a focus on voltage-gated Ca 2+ channels responsible for the cardiac action potentials depolarization phase. Two types of Ca 2+ currents were measured under voltage-clamp. Exposure to CHL was accompanied by a decrease in voltage-activated Ca 2+ currents densities. Altogether, these results show that chlorantraniliprole can cause cardiac defects in honey bees., (© 2024. The Author(s).)

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signaling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signaling activity in the SAM. We show that high GA signaling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organization of internodes, thus contributing to internode specification in the SAM., (© 2024. The Author(s).)

Emerging evidence indicates that in addition to its well-recognized functions in antiviral RNA silencing, dsRNA elicits pattern-triggered immunity (PTI), likely contributing to plant resistance against virus infections. However, compared to bacterial and fungal elicitor-mediated PTI, the mode-of-action and signaling pathway of dsRNA-induced defense remain poorly characterized. Here, using multicolor in vivo imaging, analysis of GFP mobility, callose staining, and plasmodesmal marker lines in Arabidopsis thaliana and Nicotiana benthamiana, we show that dsRNA-induced PTI restricts the progression of virus infection by triggering callose deposition at plasmodesmata, thereby likely limiting the macromolecular transport through these cell-to-cell communication channels. The plasma membrane-resident SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1, the BOTRYTIS INDUCED KINASE1/AVRPPHB SUSCEPTIBLE1-LIKE KINASE1 kinase module, PLASMODESMATA-LOCATED PROTEINs 1/2/3, as well as CALMODULIN-LIKE 41 and Ca2+ signals are involved in the dsRNA-induced signaling leading to callose deposition at plasmodesmata and antiviral defense. Unlike the classical bacterial elicitor flagellin, dsRNA does not trigger a detectable reactive oxygen species (ROS) burst, substantiating the idea that different microbial patterns trigger partially shared immune signaling frameworks with distinct features. Likely as a counter strategy, viral movement proteins from different viruses suppress the dsRNA-induced host response leading to callose deposition to achieve infection. Thus, our data support a model in which plant immune signaling constrains virus movement by inducing callose deposition at plasmodesmata and reveals how viruses counteract this layer of immunity., Competing Interests: Conflict of interest statement. The authors declare to have no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Biosolids are byproducts of wastewater treatment. With the increasing global population, the amounts of wastewater to be treated are expanding, along with the amounts of biosolids generated. The reuse of biosolids is now accepted for diversified applications in fields such as agriculture, engineering, agro-forestry. However, biosolids are known to be potential carriers of compounds that can be toxic to living beings or alter the environment. Therefore, biosolid reuse is subject to regulations, mandatory analyses are performed on heavy metals, persistent organic pollutants or pathogens. Conventional methods for the analysis of heavy metals and persistent organic pollutants are demanding, lengthy, and sometimes unsafe. Here, we propose mass spectrometry imaging as a faster and safer method using small amounts of material to monitor heavy metals and persistent organic pollutants in different types of biosolids, allowing for ecological and health risk assessment before reuse. Our methodology can be extended to other soil-like matrices., (© 2023. The Author(s).)

The combination of ever-increasing microscopy resolution with cytogenetical tools allows for detailed analyses of nuclear functional partitioning. However, the need for reliable qualitative and quantitative methodologies to detect and interpret chromatin sub-nuclear organization dynamics is crucial to decipher the underlying molecular processes. Having access to properly automated tools for accurate and fast recognition of complex nuclear structures remains an important issue. Cognitive biases associated with human-based curation or decisions for object segmentation tend to introduce variability and noise into image analysis. Here, we report the development of two complementary segmentation methods, one semi-automated ( iCRAQ ) and one based on deep learning ( Nucl.Eye.D ), and their evaluation using a collection of A. thaliana nuclei with contrasted or poorly defined chromatin compartmentalization. Both methods allow for fast, robust and sensitive detection as well as for quantification of subtle nucleus features. Based on these developments, we highlight advantages of semi-automated and deep learning-based analyses applied to plant cytogenetics., Competing Interests: The authors declare no conflict of interest.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vismione H (VH) is a fluorescent prenylated anthranoid produced by plants from the Hypericaceae family, with antiprotozoal activities against malaria and leishmaniosis. Little is known about its biosynthesis and metabolism in plants or its mode of action against parasites. When VH is isolated from Psorospermum glaberrimum , it is rapidly converted into madagascine anthrone and anthraquinone, which are characterized by markedly different fluorescent properties. To locate the fluorescence of VH in living plant cells and discriminate it from that of the other metabolites, an original strategy combining spectral imaging (SImaging), confocal microscopy, and non-targeted metabolomics using mass spectrometry, was developed. Besides VH, structurally related molecules including madagascine (Mad), emodin (Emo), quinizarin (Qui), as well as lapachol (Lap) and fraxetin (Fra) were analyzed. This strategy readily allowed a spatiotemporal characterization and discrimination of spectral fingerprints from anthranoid-derived metabolites and related complexes with cations and proteins. In addition, our study validates the ability of plant cells to metabolize VH into madagascine anthrone, anthraquinones and unexpected metabolites. These results pave the way for new hypotheses on anthranoid metabolism in plants.

In animal single cells in culture, nuclear geometry and stiffness can be affected by mechanical cues, with important consequences for chromatin status and gene expression. This calls for additional investigation into the corresponding physiological relevance in a multicellular context and in different mechanical environments. Using the Arabidopsis root as a model system, and combining morphometry and micro-rheometry, we found that hyperosmotic stress decreases nuclear circularity and size and increases nuclear stiffness in meristematic cells. These changes were accompanied by enhanced expression of touch response genes. The nuclear response to hyperosmotic stress was rescued upon return to iso-osmotic conditions and could even lead to opposite trends upon hypo-osmotic stress. Interestingly, nuclei in a mutant impaired in the functions of the gamma-tubulin complex protein 3 (GCP3) interacting protein (GIP)/MZT1 proteins at the nuclear envelope were almost insensitive to such osmotic changes. The gip1gip2 mutant exhibited constitutive hyperosmotic stress response with stiffer and deformed nuclei, as well as touch response gene induction. The mutant was also resistant to lethal hyperosmotic conditions. Altogether, we unravel a stereotypical geometric, mechanical, and genetic nuclear response to hyperosmotic stress in plants. Our data also suggest that chromatin acts as a gel that stiffens in hyperosmotic conditions and that the nuclear-envelope-associated protein GIPs act as negative regulators of this response., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Plants are exposed to the damaging effect of sunlight that induces DNA photolesions. In order to maintain genome integrity, specific DNA repair pathways are mobilized. Upon removal of UV-induced DNA lesions, the accurate re-establishment of epigenome landscape is expected to be a prominent step of these DNA repair pathways. However, it remains poorly documented whether DNA methylation is accurately maintained at photodamaged sites and how photodamage repair pathways contribute to the maintenance of genome/methylome integrities. Using genome wide approaches, we report that UV-C irradiation leads to CHH DNA methylation changes. We identified that the specific DNA repair pathways involved in the repair of UV-induced DNA lesions, Direct Repair (DR), Global Genome Repair (GGR) and small RNA-mediated GGR prevent the excessive alterations of DNA methylation landscape. Moreover, we identified that UV-C irradiation induced chromocenter reorganization and that photodamage repair factors control this dynamics. The methylome changes rely on misregulation of maintenance, de novo and active DNA demethylation pathways highlighting that molecular processes related to genome and methylome integrities are closely interconnected. Importantly, we identified that photolesions are sources of DNA methylation changes in repressive chromatin. This study unveils that DNA repair factors, together with small RNA, act to accurately maintain both genome and methylome integrities at photodamaged silent genomic regions, strengthening the idea that plants have evolved sophisticated interplays between DNA methylation dynamics and DNA repair., Competing Interests: The authors have declared that no competing interests exist.

The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis ( Arabidopsis thaliana ). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Chloromethane (CH 3 Cl) is a toxic gas mainly produced naturally, in particular by plants, and its emissions contribute to ozone destruction in the stratosphere. Conversely, CH 3 Cl can be degraded and used as the sole carbon and energy source by specialised methylotrophic bacteria, isolated from a variety of environments including the phyllosphere, i.e. the aerial parts of vegetation. The potential role of phyllospheric CH 3 Cl-degrading bacteria as a filter for plant emissions of CH 3 Cl was investigated using variants of Arabidopsis thaliana with low, wild-type and high expression of HOL1 methyltransferase previously shown to be responsible for most of CH 3 Cl emissions by A. thaliana. Presence and expression of the bacterial chloromethane dehalogenase cmuA gene in the A. thaliana phyllosphere correlated with HOL1 genotype, as shown by qPCR and RT-qPCR. Production of CH 3 Cl by A. thaliana paralleled HOL1 expression, as assessed by a fluorescence-based bioreporter. The relation between plant production of CH 3 Cl and relative abundance of CH 3 Cl-degrading bacteria in the phyllosphere suggests that CH 3 Cl-degrading bacteria co-determine the extent of plant emissions of CH 3 Cl to the atmosphere.

Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, we investigated the function of the low-duplicated CYP715 cytochrome P450 gene family that appeared early in seed plants and evolved under strong negative selection. Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower-insect interaction., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

Beet necrotic yellow vein virus (BNYVV) is a multipartite positive-strand RNA virus. BNYVV RNA-1 encodes a non-structural p237 polyprotein processed in two proteins (p150 and p66) by a cis-acting protease activity. BNYVV non-structural proteins are closely related to replication proteins of positive strand RNA viruses such as hepeviruses rather to other plant virus replicases. The p237 and dsRNA have been localized by TEM in ER structures of infected leaf cells whereas dsRNA was immunolabeled in infected protoplasts. The p150 contains domains with methyltransferase, protease, helicase and two domains of unknown function whereas p66 encompasses the RNA-dependent RNA-polymerase signature. We report the existing interactions between functional domains of the p150 and p66 proteins and the addressing of the benyvirus replicase to the endoplasmic reticulum. Yeast two-hybrid approach, colocalization with FRET-FLIM analyses and co-immunoprecipitation highlighted existing interactions that suggest the presence of a multimeric complex at the vicinity of the cellular membranous web., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The extracellular matrix (ECM) molecule tenascin-C (TNC) promotes tumor progression. This has recently been demonstrated in the stochastic murine RIP1-Tag2 insulinoma model, engineered to either express TNC abundantly or to be devoid of TNC. However, our knowledge about organization of the TNC microenvironment is scant. Here we determined the spatial distribution of TNC together with other ECM molecules in murine RIP1-Tag2 insulinoma and human cancer tissue (insulinoma and colorectal carcinoma). We found that TNC is organized in matrix tracks together with other ECM molecules of the AngioMatrix signature, a previously described gene expression profile that characterizes the angiogenic switch. Moreover, stromal cells including endothelial cells, fibroblasts and leukocytes were enriched in the TNC tracks. Thus, TNC tracks may provide niches for stromal cells and regulate their behavior. Given similarities of TNC rich niches for stromal cells in human insulinoma and colon cancer, we propose that the RIP1-Tag2 model may be useful for providing insights into the contribution of the tumor stroma specific ECM as promoter of cancer progression.

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.

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.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

We created FigureJ a new ImageJ plugin dedicated to scientific article figures preparation. Building a convincing figure is a demanding task that covers different steps ranging from content acquisition to figure assembly in editing software. Notions of image processing are required when it comes to even simple tasks such as cropping or resizing images and assembling them in a single figure. Scientific images are typically well handled in dedicated software but poorly supported in software used for laying out the final version of a figure for submission to review process., (© 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.)

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein-protein, and protein-membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.

The lateral diffusion of a protein (human serum albumin labeled with fluorescein isothiocyanate) within a highly hydrated polyelectrolyte film is studied. The film is built up with poly(L-lysine) as polycation and hyaluronate as polyanion. Fluorescence recovery after photobleaching is used to evaluate the mobility of the labeled protein. Spatial Fourier transformation is applied to the fluorescence intensity recorded at various times after bleaching of a narrow rectangular area within an image representative of the film. This approach necessitates no hypothesis on the intensity distribution at the end of the bleaching provided that the bleach has not appreciably changed the concentration ratios of the different diffusing species. Furthermore, under the hypothesis that molecules move according to Fick's law, we represent the Fourier transform by a weighted sum of exponentials each containing another diffusion coefficient and evaluate the proportion attached to each term of this sequence using the simulated annealing method. A criterion, combining goodness-of-fit and the entropy characterizing the diffusion coefficient spectrum, is proposed to avoid overinterpretation of the experimental data. The optimum spectrum of the diffusion coefficient is then extracted from the time evolution of the light intensity at various albumin concentrations within the films. It appears that the mobility, quantified by the amount of tracer molecules having a diffusion coefficient smaller than, e.g., 0.1 μm(2)/s, undergoes a transition between 20 and 2000 μg/mL of internal concentration. This suggests that the mutual interactions of the albumin molecules and the interactions between fluorescently labeled albumin and the film network become increasingly important in the reduction of the albumin mobility as the albumin concentration increases.

In plants and possibly other organisms, channelling of the reactive intermediates resulting from P450 oxygenation is thought to require the formation of supramolecular complexes associating membrane-bound and soluble enzymes. This implies a most probably loose membrane association of the soluble proteins. For the assessment of such membrane association in vivo, we propose an imaging strategy based on the accurate evaluation of fluorescent protein repartition and distance around endoplasmic reticulum (ER) tubules. It requires candidate protein fusion constructs with fluorescent reporters and transient expression in leaves of Nicotiana benthamiana. The method was tested with soluble eGFP/mRFP1, with various P450 and P450 reductase fluorescent fusions, and with anchored eGFP/mRFP1. It easily differentiated soluble and anchored proteins and detects subtle changes in ER tubules. The method was further assessed with a soluble protein previously shown to be loosely associated with the ER, the phenylalanine ammonia lyase PAL1 involved in the lignin biosynthetic pathway. This protein was found located in close vicinity to the ER. Taken together, these data indicate that the method proposed herein is suitable to monitor membrane association and relocalization of soluble proteins involved in the formation of metabolons., (© 2011 The Authors Journal compilation © 2011 FEBS.)

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.

Members of the Luteoviridae family occupy a very special position among plant viruses. Unlike most plant viruses that can infect almost all cell types, these viruses exhibit a specific tropism restricted to vascular tissues. The infection of these tissues is maintained by a piercing-sucking insect vector, an aphid that promotes viral plant-to-plant transmission by feeding on phloem sap. This review focuses on the movement in the phloem of viruses belonging to the Luteoviridae family underlining the roles of viral proteins in this process. A second part is dedicated to the unique mode of action of the silencing suppressor of the Polerovirus genus (one of the three Luteoviridae genera). Finally, several hypotheses are discussed in order to explain the phloem restriction of these peculiar viruses.

Lipid acyl hydrolases (LAH) have received recently increased attention in the context of plant defense. Multiple structurally unrelated gene families have been annotated in Arabidopsis as encoding potential lipid deacylating enzymes with numerous members being transcriptionally activated upon biotic stress. Confirming in silico predictions, experimental data have illustrated the wide subcellular distribution of LAHs indicating they likely interact with distinct membrane systems to initiate specific cellular responses. While recombinant LAHs are active in vitro on a small set of polar lipids, precise knowledge of in vivo substrates and hydrolysis products is generally lacking. Functional analysis of a few LAHs has revealed their roles in initiating oxylipin biosynthesis, cell death execution, signalling or direct antimicrobial activity. The picture emerging is that pathogenic challenge triggers a complex network of lipid hydrolysis events across the cellular compartments resulting in changes in membrane structures and release of signal precursors involved in the building-up of an adequate immune response., (© 2010 Landes Bioscience)

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Fibronectin, like other proteins involved in mechanotransduction, has the ability to exhibit recognition sites under mechanical stretch. Such cryptic sites are buried inside the protein structure in the native fold and become exposed under an applied force, thereby activating specific signalling pathways. Here, we report the design of new active polymeric nanoassembled surfaces that show some similarities to these cryptic sites. These nanoassemblies consist of a first polyelectrolyte multilayer stratum loaded with enzymes and capped with a second polyelectrolyte multilayer acting as a mechanically sensitive nanobarrier. The biocatalytic activity of the film is switched on/off reversibly by mechanical stretching, which exposes enzymes through the capping barrier, similarly to mechanisms involved in proteins during mechanotransduction. This first example of a new class of biologically inspired surfaces should have great potential in the design of various devices aimed to trigger and modulate chemical reactions by mechanical action with applications in the field of microfluidic devices or mechanically controlled biopatches for example.

Background: Grapevine protection against diseases needs alternative strategies to the use of phytochemicals, implying a thorough knowledge of innate defense mechanisms. However, signalling pathways and regulatory elements leading to induction of defense responses have yet to be characterized in this species. In order to study defense response signalling to pathogens in Vitis vinifera, we took advantage of its recently completed genome sequence to characterize two putative orthologs of NPR1, a key player in salicylic acid (SA)-mediated resistance to biotrophic pathogens in Arabidopsis thaliana., Results: Two cDNAs named VvNPR1.1 and VvNPR1.2 were isolated from Vitis vinifera cv chardonnay, encoding proteins showing 55% and 40% identity to Arabidopsis NPR1 respectively. Constitutive expression of VvNPR1.1 and VvNPR1.2 monitored in leaves of V. vinifera cv chardonnay was found to be enhanced by treatment with benzothiadiazole, a SA analog. In contrast, VvNPR1.1 and VvNPR1.2 transcript levels were not affected during infection of resistant Vitis riparia or susceptible V. vinifera with Plasmopara viticola, the causal agent of downy mildew, suggesting regulation of VvNPR1 activity at the protein level. VvNPR1.1-GFP and VvNPR1.2-GFP fusion proteins were transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, where they localized predominantly to the nucleus. In this system, VvNPR1.1 and VvNPR1.2 expression was sufficient to trigger the accumulation of acidic SA-dependent pathogenesis-related proteins PR1 and PR2, but not of basic chitinases (PR3) in the absence of pathogen infection. Interestingly, when VvNPR1.1 or AtNPR1 were transiently overexpressed in Vitis vinifera leaves, the induction of grapevine PR1 was significantly enhanced in response to P. viticola., Conclusion: In conclusion, our data identified grapevine homologs of NPR1, and their functional analysis showed that VvNPR1.1 and VvNPR1.2 likely control the expression of SA-dependent defense genes. Overexpression of VvNPR1 has thus the potential to enhance grapevine defensive capabilities upon fungal infection. As a consequence, manipulating VvNPR1 and other signalling elements could open ways to strengthen disease resistance mechanisms in this crop species.

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

The cell-to-cell movement of Tobacco mosaic virus through plasmodesmata (PD) requires virus-encoded movement protein (MP). The MP targets PD through the endoplasmic reticulum (ER)/actin network, whereas the intercellular movement of the viral RNA genome has been correlated with the association of the MP with mobile, microtubule-proximal particles in cells at the leading front of infection as well as the accumulation of the protein on the microtubule network during later infection stages. To understand how the associations of MP with ER and microtubules are functionally connected, we applied multiple marker three-dimensional confocal and time-lapse video microscopies to Nicotiana benthamiana cells expressing fluorescent MP, fluorescent RNA and fluorescent cellular markers. We report the reconstitution of MP-dependent RNA transport to PD in a transient assay. We show that transiently expressed MP occurs in association with small particles as observed during infection. The same MP accumulates in PD and mediates the transport of its messenger RNA transcript to the pore. In the cellular cortex, the particles occur at microtubule-proximal sites and can undergo ER-associated and latrunculin-sensitive movements between such sites. These and other observations suggest that the microtubule network performs anchorage and release functions for controlling the assembly and intracellular movement of MP-containing RNA transport particles in association with the ER.

Municipal solid waste incinerator bottom ash (MSWI BA) can be used in road construction where it can become exposed to microbial attack, as it can be used as a source of oligoelements by bacteria. The extent of microbial colonization of the bottom ash and the intensity of microbial processes can impact the rate of leaching of potentially toxic elements. As a consequence, our objective was to highlight the mutual interactions between MSWI bottom ash and Pseudomonas aeruginosa, a common bacteria found in the environment. Experiments were carried out for 133 days at 25 degrees C using a modified soxhlet's device and a culture medium, in a closed, unstirred system with weekly renewal of the aqueous phase. The solid products of the experiments were studied using a laser confocal microscopy, which showed that biofilms formed on mineral surfaces, possibly protecting them from leaching. Our results show that the total mass loss after 133 days is systematically higher in abiotic medium than in the biotic one in proportions going from 31 to 53% depending on element. Ca and Sr show that rates in biotic medium was approximately 19% slower than in abiotic medium during the first few weeks. However, in the longer term, both rates decreased to reach similar end values after 15 weeks. By taking into account the quantities of each tracer trapped in the layers we calculate an absolute alteration rate of MSWI BA in the biotic medium (531 microg m(-2) d(-1)) and in the abiotic one (756 microg m(-2) d(-1)).

In patients affected by Creutzfeldt-Jakob disease and in animals affected by transmissible spongiform encephalopathies, retinal functions are altered, and major spongiform changes are observed in the outer plexiform layer where photoreceptors have their synaptic terminals. In the present study, the prion protein PrP(c) was found to form aggregates in rod photoreceptor terminals from both rat and human retina, whereas no labeling was observed in cone photoreceptors. Discrete staining was also detected in the inner plexiform layer where the prion protein was located at human amacrine cell synapses. In mixed porcine retinal cell cultures, the PrP106-126 prion peptide triggered a 61% rod photoreceptor cell loss by apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling, whereas cone photoreceptors were not affected. Amacrine cells were also reduced by 47% in contrast to ganglion cells. Although this cell loss was associated with a 5.5-fold increase in microglial cells, the strict correlation between the PrP(c) prion protein expression and the peptide toxicity suggested that this toxicity did not rely on the release of a toxic compound by glial cells. These results provide new insights into the retinal pathophysiology of prion diseases and illustrate advantages of adult retinal cell cultures to investigate prion pathogenic mechanisms.

The alternate deposition of exponentially and linearly growing polyelectrolyte multilayers leads to the formation of multicompartment films. In this study, a new system consisting in nanometer-sized multilayer barriers deposited on or between multilayer compartments was designed to respond to mechanical stimuli and to act as nanovalves. The diffusion of polyelectrolytes through the barrier from one compartment to another can be switched on/off by tuning the mechanical stretching and thereby opening or closing nanopores in the barrier. This work represents a first step toward the design of chemically or biologically active films responding to mechanical stresses.

S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.

In a previous study (A. Hemmerlin, T.J. Bach, Plant Physiol. 123 (2000) 1257-1268), we have demonstrated that above a critical concentration, treatment with all-trans-farnesol induces cell-death in Nicotiana tabacum L. cv Bright Yellow-2 (TBY-2) cells. Now we used a fluorescent analog of farnesol (Fol(FLUO)), in which an isoprene unit is replaced by the fluorochrome 7-nitrobenz-2-oxa-1,3-diazol-4-yl, to visualize how cell integrity is affected. Fol(FLUO) exhibited the same toxicity as the natural compound and was shown to be readily taken up by TBY-2 cells, followed by integration into subcellular membrane structures. Although the plasma membrane seemed not to be labeled, Fol(FLUO) was associated with the tonoplast, endoplasmic reticulum, and Golgi apparatus or lipid bodies. Longer exposure times and increased Fol(FLUO) accumulation triggered the formation and proliferation of new membrane structures of as yet unknown function. Finally, at even higher and clearly cytotoxic concentrations of the analog, the cell contents became clearly disorganized, with cell swelling and ultimately plasmolysis.

Central nervous system neurons have traditionally been thought to express exclusively membrane transporters and/or vesicular transporters for their transmitter. Three vesicular glutamate transporters have recently been cloned: BNPI/VGLUT1 (a brain-specific sodium-dependent inorganic phosphate (Pi) transporter), and its homologs DNPI/VGLUT2 (differentiation-associated sodium-dependent Pi transporter) and VGLUT3. We investigated the subcellular distributions of these three vesicular transporters in rat and human retina. VGLUT1 was present in the outer and inner plexiform layers (OPL and IPL), as shown by punctate staining in both human and rat retina. In the OPL, it was colocalized with synaptophysin, consistent with its expression in glutamatergic photoreceptor terminals, and it was present in PKC-alpha-labeled glutamatergic bipolar cell terminals in the IPL. By contrast, VGLUT2 was present in horizontal cells and ganglion cells in rat and human retina. In human retina, VGLUT2 was also found in some amacrine cells, including GAD-immunopositive amacrine cells. VGLUT3 was present in glycine-releasing amacrine cells in rat retina but was restricted to a few ganglion cells in human retina. The distribution of VGLUT1 in excitatory synaptic terminal was consistent with its involvement in glutamate release at excitatory synapses, whereas the cellular distributions of VGLUT2 and VGLUT3 suggested that these molecules may be involved in functions other than glutamate release, such as glutamate storage for GABA synthesis in non-glutamatergic neurons.

A new gene-expression system based on RNA-5 of Beet necrotic yellow vein virus (BNYVV) was constructed to allow the expression of recombinant proteins in virally infected cells. Replication and expression levels of the RNA-5-based replicon containing the green fluorescence protein (GFP) gene were compared with those obtained with the well-characterized RNA-3-derived replicon (Rep-3). When RNA-3 and/or RNA-4 BNYVV RNAs were added to the inoculum, the expression levels of RNA-5-encoded GFP were considerably reduced. To a lesser extent, RNA-3-derived GFP expression was also affected by the presence of RNA-4 and -5. Both RNA-3- and RNA-5-derived molecules were able to express proteins within the same infected cells. Together with Rep-3, the RNA-5-derived replicon thus provides a new tool for the co-expression of different recombinant proteins. In Beta macrocarpa, Rep-5-GFP was able to move in systemic tissues in the presence of RNA-3 and thus provides a new expression system that is not restricted to the inoculated leaves.

The diffusion coefficient, D, and the proportion of mobile molecules, p, is measured for a fluorescently labeled polyelectrolyte in a multilayer film using fluorescence recovery after photobleaching (FRAP). The film was composed of poly(L-lysine) (PLL) and hyaluronan (HA). The labeled polyelectrolyte (PLL(FITC)) was either deposited on top of the film or embedded within it. A circular area of diameter approximately 60 microm was bleached using a confocal laser scanning microscope. Because molecules do already diffuse during the bleaching step, the initial light intensity profile is not characteristic of a uniformly bleached circular area. A formalism is developed in which a simple mathematical representation of a measured profile serves as starting profile. This radial distribution is introduced in the equation describing the time evolution of the labeled molecule concentration under the hypothesis that the recovery results from pure, two-dimensional Brownian diffusion of the mobile molecules according to Fick's law. The analysis of a series of images taken at successive times after bleaching (up to approximately one hour) leads to estimates of D of the order of 0.2 microm(2) s(-1) for labeled molecules deposited on top of the film and a 5-fold smaller value for the molecules embedded in the film. However, p is remarkably insensitive to the position in the multilayer architecture (p approximately 0.40)., (Copyright 2005 Wiley-Liss, Inc.)

Poly(L-lysine)/hyaluronan (PLL/HA) films were chemically cross-linked with a water soluble carbodiimide (EDC) in combination with a N-hydroxysulfo-succinimide (NHS) to induce amide formation. Fourier transform infrared spectroscopy confirms the conversion of carboxylate and ammonium groups into amide bonds. Quartz crystal microbalance-dissipation reveals that the cross linking reaction is accompanied by a change in the viscoelastic properties of the films leading to more rigid films. After the cross-linking reaction, both positively and negatively ending films exhibit a negative zeta potential. It is shown by fluorescence recovery after photobleaching measured by confocal laser scanning microscopy that cross-linking dramatically reduces the diffusion of the PLL chains in the network. Cross linking also renders the films highly resistant to hyaluronidase, an enzyme that naturally degrades hyaluronan. Finally, the adhesion of chondrosarcoma cells on the films terminating either with PLL or HA is also investigated. Whereas the non cross-linked films are highly resistant to cell adhesion, the cells adhere and spread well on the cross-linked films.