Jérôme Mutterer

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers

The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an “in” and “out” diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

Oxidative Remodeling of Chromoplast Carotenoids: Identification of the Carotenoid Dioxygenase CsCCD and CsZCD Genes Involved in Crocus Secondary Metabolite Biogenesis

The accumulation of three major carotenoid derivatives—crocetin glycosides, picrocrocin, and safranal—is in large part responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried styles of Crocus. We have identified and functionally characterized the Crocus zeaxanthin 7,8(7′,8′)-cleavage dioxygenase gene (CsZCD), which codes for a chromoplast enzyme that initiates the biogenesis of these derivatives. The Crocus carotenoid 9,10(9′,10′)-cleavage dioxygenase gene (CsCCD) also has been cloned, and the comparison of substrate specificities between these two enzymes has shown that the CsCCD enzyme acts on a broader range of precursors. CsZCD expression is restricted to the style branch tissues and is enhanced under dehydration stress, whereas CsCCD is expressed constitutively in flower and leaf tissues irrespective of dehydration stress. Electron microscopy revealed that the accumulation of saffron metabolites is accompanied by the differentiation of amyloplasts and chromoplasts and by interactions between chromoplasts and the vacuole. Our data suggest that a stepwise sequence exists that involves the oxidative cleavage of zeaxanthin in chromoplasts followed by the sequestration of modified water-soluble derivatives into the central vacuole.

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins

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.

Mechanotransductive surfaces for reversible biocatalysis activation

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.

Arabidopsis SAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

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.

Transport of TMV Movement Protein Particles Associated with the Targeting of RNA to Plasmodesmata

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.

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Analysis of the supramolecular organization of enzymes in the lignin pathway shows that cytochrome P450s oligomerize and move along with the very mobile plant endoplasmic reticulum. Their expression favors relocalization of their soluble partner proteins nearer the membrane and association of sequential enzymes in the pathway. 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.

Characterization of Vitis vinifera NPR1 homologs involved in the regulation of Pathogenesis-Related gene expression

BackgroundGrapevine 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.ResultsTwo 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.ConclusionIn 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.

Cerebrospinal fluid‐contacting neurons in the rat spinal cord, a γ‐aminobutyric acidergic system expressing the P2X2 subunit of purinergic receptors, PSA‐NCAM, and GAP‐43 immunoreactivities: Light and electron microscopic study

Cerebrospinal fluid‐contacting neurons (CSFcNs) occur in various brain regions of lower vertebrates. In mammals, they are restricted to medullospinal areas, and little is known about their projection sites. In the present work, we investigated some morphofunctional characteristics of such neurons in the rat spinal cord by light and electron microscopic immunocytochemistry. CSFcNs expressing the P2X2 subunit of purinergic receptors were present throughout the spinal cord, though more numerous at lower thoracolumbar and sacral levels. These neurons coexpressed GAD and the polysialylated neural cell adhesion molecule (PSA‐NCAM), a marker of cellular plasticity. From low thoracic levels downward, tiny amyelinic axons (less than 200 nm in diameter) were tightly packed in bundles, which ran along the ependyma and extended ventrally, eventually concentrating against the walls of the ventral median fissure. In addition to P2X2, GAD, γ‐aminobutyric acid (GABA), and PSA, these axons expressed GAP‐43 immunoreactivity. Moreover, they were labelled along their entire lengths with antibodies against synaptotagmin and synaptophysin, but these failed to reveal intraspinal terminal fields. Taken together, our observations indicate the presence in the rat spinal cord of a highly plastic system of GABAergic CSFcNs that express the P2X2 subunit of purinergic receptors. The function of this original system remains open to question. In these neurons, the P2X2 receptors may confer a sensitivity to ATP either present in the CSF or released by nearby neurons of the central autonomic area. J. Comp. Neurol. 457:159–174, 2003.

Higher Plant Cells: Gamma-Tubulin and Microtubule Nucleation in the Absence of Centrosomes

The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells. Microsc. Res. Tech. 49:487–495, 2000.